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Hongling Du,1– 3 Shunjie Zhang,1 Kezhu Yuan,4 Zhirui Yang,5 Mingquan Wu4 1Department of Pharmacy, West China Hospital, Sichuan University, Chengdu, Sichuan, People’s Republic of China; 2Department of Pharmacy, Sichuan Public Health Clinical Center, Chengdu, Sichuan, People’s Republic of China; 3State Key Laboratory of Southwestern Chinese Medicine Resources, Pharmacy School, Chengdu University of Traditional Chinese Medicine, Chengdu, Sichuan, People’s Republic of China; 4Department of Pharmacy, Sichuan Orthopedic Hospital, Chengdu, Sichuan, People’s Republic of China; 5Department of Nuclear Medicine, Chengdu Second People’s Hospital, Chengdu, Sichuan, People’s Republic of ChinaCorrespondence: Mingquan Wu, Department of Pharmacy, Sichuan Orthopedic Hospital, Chengdu, Sichuan, People’s Republic of China, Email 18782976939@163.comBackground: Rehmanniae Radix (RR) has received attention for its antithrombotic effect. However, few studies have independently explored the bioactive components responsible for its antithrombotic bioactivity and the potential mechanism. We aimed to reveal the antithrombotic mechanisms of RR by using metabolomics integrated with network pharmacology.Methods: A thrombosis model was established by intraperitoneal injection of type I carrageenan in rats, and antithrombotic function was evaluated at different doses of RR. Metabolomics was used to identify the differential metabolites in the serum. Network pharmacology was then applied to identify the potential targets for the antithrombotic activity of the RR. An integrated network of metabolomics and network pharmacology was constructed using Cytoscape. Finally, key targets were verified using molecular docking.Results: RR at 5.4 g/kg significantly alleviated the thrombosis. Thirteen potentially significant metabolites were involved in the therapeutic effects of RR against thrombosis, most of which were regulated for recovery after RR treatment. An integrated analysis of metabolomics and network pharmacology showed that the antithrombosis effect of RR was closely associated with the regulation of PLA2G2A, PTGS1, ALOX5, and CYP2C9. Molecular docking showed high affinity between the key targets and components of RR. We speculated that the components of RR, such as catalpol, ferulic acid methyl ester, and methyl 4-hydroxycinnamate, might act on key proteins, including PLA2G2A, PTGS1, and ALOX5, to exert antithrombosis effects.Conclusion: This study confirmed the antithrombotic effect of high-dose RR, revealed the antithrombotic mechanism and potential material basis, and laid the foundation for the antithrombotic clinical application of RR. Furthermore, it provides a successful case reference for screening natural herbal components and exploring their potential pharmacological mechanisms.Keywords: Rehmanniae radix, antithrombotic effects, metabolomics, network pharmacology

Jiayue Zhou,1,2 Yingxiang Wu,1,2 Zhiyan Lu,1,2 Yan Wang1,2 1Yunnan Provincial Key Laboratory of Entomological Biopharmaceutical R&D, Dali University, Dali, Yunnan Province, 671000, People’s Republic of China; 2National-Local Joint Engineering Research Center of Entomoceutics, Dali University, Dali, Yunnan Province, 671000, People’s Republic of ChinaCorrespondence: Yan Wang, Yunnan Provincial Key Laboratory of Entomological Biopharmaceutical R&D, Dali University, Dali, Yunnan Province, 671000, People’s Republic of China, Tel +86 0872-2257401, Email jessica9428@sina.comPurpose: Rodgersia sambucifolia Hemsl (also known as Yantuo) is a traditional Chinese medicine commonly utilized as a medicinal herb with its rhizomes, mainly used to regulate the immune function of the human body. However, relatively few studies have investigated its active components and potential mechanisms of action in vivo.Methods: First, the chemical composition in vitro was identified and analyzed using the UPLC-Q-TOF MS/MS technique. Cyclophosphamide (CTX) was then administered intraperitoneally to rats to establish an immunosuppression model. Physiological and biochemical parameters, organ indices, and histopathological findings were evaluated for efficacy. Subsequently, potential biomarkers in rat serum were identified using multivariate statistical analysis and enriched and topologized using online platforms such as MetaboAnalyst and KEGG to reveal the critical metabolic pathways and their roles in the immunomodulatory network. Finally, the integrated analysis of components in vivo and in vitro, along with metabolic pathways, was performed using network pharmacology and molecular docking technology to elucidate the mechanisms of their roles in organismal immunity.Results: A total of 28 chemical components in vitro were identified, while pharmacodynamic experiments confirmed the immunomodulatory effects of Yantuo, especially in the high-dose administration group. Metabolomics analysis showed that 37 potential immune-related biomarkers were identified in positive and negative ion modes, involving 16 metabolic pathways such as arginine biosynthesis, pyrimidine metabolism, and riboflavin metabolism. The results of network pharmacology and molecular docking indicated that Yantuo may affect 7-O-galloyl-catechin, Cynaroside, Quercetin-7-O-beta-D-glucopyranoside, and 1.6-bis-O-galloyl-beta-D-glucose through interactions with the immune system, with significant pathways of action including galactose metabolism, glycolysis/gluconeogenesis, pyrimidine metabolism, and riboflavin metabolism.Conclusion: In our experiments, we confirmed the organismal modulatory effect of Yantuo on immunocompromised rats, clarified the key components, target proteins, and pathways of its possible action, and provided possibilities for follow-up studies. Keywords: immunosuppression, Yantuo, metabolomics, network pharmacology, molecular docking

Changhong Xu,1,&ast; Yun Deng,1,&ast; Jiangwei Man,1,&ast; Huabin Wang,1 Tuanjie Che,2 Liyun Ding,3 Li Yang1 1Department of Urology, Institute of Urology, Gansu Province Clinical Research Center for Urinary System Disease, Lanzhou University Second Hospital, Lanzhou, Gansu, 730030, People’s Republic of China; 2Innovation Center of Functional Genomics and Molecular Diagnostics Technology of Gansu Province, Lanzhou, People’s Republic of China; 3School of Physical Science and Technology, Lanzhou University, Lanzhou, 730000, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Li Yang, Department of Urology, Institute of Urology, Gansu Province Clinical Research Center for Urinary System Disease, Lanzhou University Second Hospital, Lanzhou, Gansu, 730030, People’s Republic of China, Email ery_yangli@lzu.edu.cnObjective: This study investigates the targets, pathways, and mechanisms of Schisandrin B (Sch B) in alleviating renal ischemia-reperfusion injury (RIRI) using RNA sequencing and network pharmacology.Methods: The effects of Sch B on RIRI were assessed using hematoxylin-eosin (HE) and periodic acid-Schiff (PAS) staining, along with measurements of blood creatinine and urea nitrogen (BUN). Differential gene expression in mouse models treated with RIRI and Sch B+RIRI was analyzed through RNA-Seq. Key processes, targets, and pathways were examined using network pharmacology techniques. The antioxidant capacity of Sch B was evaluated using assays for reactive oxygen species (ROS), mitochondrial superoxide, and JC-1 membrane potential. Molecular docking was employed to verify the interactions between key targets and Sch B, and the expression of these targets and pathway was confirmed using qRT-PCR, Western blot, and immunofluorescence.Results: Sch B pre-treatment significantly reduced renal pathological damage, inflammatory response, and apoptosis in a mouse RIRI model. Pathological damage scores dropped from 4.33 ± 0.33 in the I/R group to 2.17 ± 0.17 and 1.5 ± 0.22 in Sch B-treated groups (p < 0.01). Creatinine and BUN levels were also reduced (from 144.6 ± 21.05 μmol/L and 53.51 ± 2.34 mg/dL to 50.44 ± 5.61 μmol/L and 17.18 ± 0.96 mg/dL, p < 0.05). Transcriptomic analysis identified four key targets (AKT1, ALB, ACE, CCL5) and the PI3K/AKT pathway. Experimental validation confirmed Sch B modulated these targets, reducing apoptosis and oxidative stress, and enhancing renal recovery.Conclusion: Sch B reduces oxidative stress, inflammation, and apoptosis by modulating key targets such as AKT1, ALB, ACE, and CCL5, while activating the PI3K/AKT pathway, leading to improved renal recovery in RIRI.Keywords: renal ischemia-reperfusion injury, schisandrin B, transcriptomics, network pharmacology, PI3K/AKT pathway

Xi Huang,1,2,&ast; Yu Peng,1,2,&ast; Lingfei Lu,1,&ast; Liwen Gao,1,2 Shanshan Wu,1,2 Jiandong Lu,1 Xinhui Liu1 1Department of Nephrology, Shenzhen Traditional Chinese Medicine Hospital, Guangzhou University of Chinese Medicine, Shenzhen, Guangdong, People’s Republic of China; 2The Fourth Clinical Medical College, Guangzhou University of Chinese Medicine, Shenzhen, Guangdong, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Xinhui Liu, Department of Nephrology, Shenzhen Traditional Chinese Medicine Hospital, Guangzhou University of Chinese Medicine, Shenzhen, Guangdong, 518033, People’s Republic of China, Email liuxinhui0317@163.comBackground: Huangqi-Danshen decoction (HDD) is a Chinese medicinal herb pair with good efficacy in treating chronic kidney disease, but its mechanism needs to be clarified.Aim: To uncover the underlying mechanism of HDD antagonizing renal fibrosis through network pharmacology (NP) analysis and experimental validation.Materials and Methods: The chemical components of water extract of HDD were analyzed by combining the ultra-high performance liquid chromatography coupled with Q-Exactive mass spectrum analysis (UHPLC-QE-MS) and HERB database. NP was used to identify core common targets of HDD components and renal fibrosis. Subsequently, male C57BL/6 mice were divided into Sham, unilateral ureteral obstruction (UUO) and UUO+HDD groups. Renal function, histopathology, Western blotting, and immunohistochemistry analyses were used to evaluate the protective effect of HDD on UUO mice. The effects of HDD on signaling pathways were validated in both UUO mice and transforming growth factor-β 1 (TGF-β 1)-induced HK-2 cells.Results: By combining UHPLC-QE-MS analysis and HERB database, 25 components were screened in HDD extract. There were 270 intersection targets of the 25 components and renal fibrosis. Based on their scores in protein-protein interaction analysis and degree values in component-pathway-target triadic network, 6 core common targets of the 25 components and renal fibrosis were identified, namely phosphoinositide 3-kinase (PI3K), signal transducer and activator of transcription 3 (Stat3), non-receptor tyrosine kinase Src (Src), epidermal growth factor receptor (EGFR), matrix metalloproteinase 9 (MMP9), and MMP2. HDD ameliorated renal tubular damage and collagen deposition and downregulated fibrosis-related proteins expression in UUO mice. Furthermore, HDD was demonstrated to reduce PI3K, Stat3, Src, EGFR, and MMP2 expressions, and enhance MMP9 expression in the kidney of UUO mice and in TGF-β 1-induced HK-2 cells.Conclusion: HDD can alleviate renal fibrosis which may be related to regulating the expression of essential proteins in the epithelial-mesenchymal transition and extracellular matrix production/degradation signaling pathways. Keywords: renal fibrosis, chronic kidney disease, Huangqi-Danshen decoction, network pharmacology, signaling pathway

Chunxia Yin,1,&ast; Taohua Lan,2,3,&ast; Yunshan Wu,4,5 Jing Cai,1 Haoxiang Li,1 Xiaolan Kuang,1 Lin Jiao,1 Xiaomin Ou,1 Hua Yang,2 Bo Liu,3– 5 Weihui Lu2,6,7 1The Second Clinical Medical College, Guangzhou University of Chinese Medicine, Guangzhou, People’s Republic of China; 2Department of Cardiology, Guangdong Provincial Hospital of Chinese Medicine, Guangzhou, People’s Republic of China; 3State Key Laboratory of Dampness Syndrome of Chinese Medicine, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou, People’s Republic of China; 4Guangzhou Key Laboratory of Chirality Research on Active Components of Traditional Chinese Medicine, Guangdong Provincial Hospital of Chinese Medicine, Guangzhou, People’s Republic of China; 5Guangdong Provincial Key Laboratory of Clinical Research on Traditional Chinese Medicine Syndrome, Guangzhou University of Chinese Medicine, Guangzhou, People’s Republic of China; 6State Key Laboratory of Traditional Chinese Medicine Syndrome, Guangdong Provincial Hospital of Chinese Medicine, Guangzhou University of Chinese Medicine, Guangzhou, People’s Republic of China; 7Chinese Medicine Guangdong Laboratory, Guangdong Hengqin, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Weihui Lu, Bo Liu, The Second Clinical Medical College, Guangzhou University of Chinese Medicine, Email weihui.lu@gzucm.edu.cn; doctliu@gzucm.edu.cnPurpose: This study was designed to evaluate the effect and mechanism of the Qushi Huatan (QSHT) decoction against coronary heart disease (CHD) through network pharmacology and experimental verification.Methods: In the present study, the active ingredients of the QSHT decoction were identified by ultra performance liquid chromatography/tandem mass spectrometry (UPLC/MS), then the potential ingredients and coronary heart disease targets were predicted using the SwissTarget Prediction database and the database of Genecards and OMIM database, respectively. A herb-compound-target network was constructed using Cytoscape. GO and KEGG enrichment analysis were performed using the ClusterProfiler data package of R software. Molecular docking was used to predict the core targets of QSHT against CHD. In addition, we used a myocardial infarction (MI) and high-fat diet ApoE−/− mice model to investigate the cardioprotective effects of QSHT. Western blotting and immunochemistry were used to verify the core targets and the signaling pathway.Results: A total of 68 active ingredients were found in the QSHT decoction. Network pharmacology indicated 28 targets and 147 signal pathways, including AKT1, HIF-1α, GSK-3β, TLR4 and NF-κB, those key targets were also verified by molecular docking. The results of GO and KEGG enrichment analysis showed that the targets of QSHT against CHD were largely associated with inflammatory and oxidative stress, and AKT/HIF-1α and TLR4/NF-κB pathways might be key functional pathways. In vivo, QSHT significantly improved cardiac function and attenuated fibrosis and inflammation. Furthermore, QSHT could significantly inhibit the expression of HIF-1α, TLR4, phosphorylation of AKT1, GSK-3β and NF-κB after MI in ApoE−/− mice.Conclusion: Based on network pharmacology, molecular docking and experimental verification, this study demonstrated that QSHT could improve cardiac function and attenuate cardiac fibrosis by regulating TLR4/NF-κB and AKT/HIF-1α signaling pathway in post- MI and high-fat diet ApoE−/− mice.Keywords: network pharmacology, molecular docking, Qushi Huatan decoction, coronary heart disease

Yujie Li,1 Yadong Yin,2 Juan Xiong,3 Zhipeng Zhang,4 Linglong Li,3 Baoshun Zhang,4 Feng Zhang,3 Dehong Mao3 1College of Traditional Chinese Medicine, Chongqing Medical University, Chongqing, People’s Republic of China; 2Xijing Hospital, Air Force Medical University, Xi’an, People’s Republic of China; 3Department of Otorhinolaryngology, Yongchuan Chinese Medicine Hospital Affiliated to Chongqing Medical University, Chongqing, People’s Republic of China; 4College of Pharmaceutical Sciences, Southwest University, Chongqing, People’s Republic of ChinaCorrespondence: Feng Zhang; Dehong Mao, Email 164382044@163.com; 19m001@hospital.cqmu.edu.cnBackground: Yujiang Paidu Decoction (YJPD) has demonstrated clinical efficacy in the treatment of chronic rhinosinusitis. However, the effects and mechanisms of the YJPD on chronic rhinosinusitis with nasal polyps (CRSwNP) remain unclear.Purpose: This study aimed to elucidate the potential mechanism of action of YJPD in the treatment of CRSwNP based on network pharmacology, transcriptomics and experiments.Methods: A CRSwNP mouse model was established using ovalbumin (OVA) and staphylococcus aureus enterotoxin B (SEB) for 12 weeks and the human nasal epithelial cell (HNEpC) model was induced with IL-13 in vitro. Behavioral tests, scanning electron microscopy (SEM), micro-CT and pathological change of nasal tissues were observed to investigate the therapeutic effects of YJPD. Network pharmacology and transcriptomics were launched to explore the pharmacological mechanisms of YJPD in CRSwNP treatment. Finally, an ELISA, immunofluorescence, RT-qPCR, Western blotting and Tunel were performed for validation.Results: Different doses of YJPD intervention effectively alleviated rubbing and sneezing symptoms in CRSwNP mice. Additionally, YJPD significantly reduced abnormal serological markers, structural damage of the nasal mucosa, inflammatory cell infiltration, goblet cell increases, and inhibited OVA-specific IgE levels and the secretion of Th2 cytokines such as IL-4, IL-5, and IL-13. Moreover, transcriptomics and network pharmacology analyses indicated that YJPD may exert anti-inflammatory and anti-apoptotic effects by inhibiting the MAPK/AP-1 signaling pathway. The experimental findings supported this conclusion, which was further corroborated by similar results observed in IL13-induced HNEpCs in vitro.Conclusion: YJPD could alleviate inflammatory status and epithelial apoptosis by inhibiting aberrant activation of MAPK/AP-1 signaling pathway. This finding provides a strong basis for using YJPD as a potential treatment in CRSwNP. Keywords: Yujiang paidu decoction, chronic rhinosinusitis with nasal polyps, network pharmacology, transcriptomics, inflammation

Jingjing Wei,1,&ast; Zhaoyang Liu,1,2,&ast; Mingming Li,1 Lingyun Du,1 Xia Zhu,1 Yi Leng,1 Changyu Han,1 Qingqing Xu,1 Chunhong Zhang1 1Department of Dermato-Venereology, the Second Hospital, Cheeloo College of Medicine, Shandong University, Jinan, People’s Republic of China; 2Department of Dermato-Venereology, Tianjin Medical University General Hospital, Tianjin, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Chunhong Zhang, Department of Dermato-Venereology, the Second Hospital, Cheeloo College of Medicine, Shandong University, 247 Beiyuan Dajie Street, Jinan, People’s Republic of China, Tel +86-531-85875027, Fax +86-531-88962544, Email aliu133@163.comBackground: Psoriasis is an immune-mediated chronic inflammatory disease. Qingre Lishi Decoction (QRLSD) has achieved great clinical effect in the treatment of psoriasis. However, the potential bioactive components and the mechanisms are yet unclear.Aim: To analyze the serum parameters of rats fed with QRLSD, screen out the active components of QRLSD, and explore the potential targets and pathway of QRLSD in the treatment of psoriasis.Materials and Methods: The active components of serum containing QRLSD were analyzed using ultra-high performance liquid chromatography quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF/MS). The targets of QRLSD in the treatment of psoriasis were predicted by network pharmacology and molecular docking. In vitro experiments verified the underlying mechanism.Results: By UPLC-Q-TOF/MS, 15 prototype components and 22 metabolites were identified in serum containing QRLSD. Subsequently, 260 chemical composition targets and 218 psoriasis targets were overlapped to obtain 23 intersection targets, including LGALS3, TNF, F10, DPP4, EGFR, MAPK14, STAT3 and others. TNF, IL-10, GAPDH, STAT3, EGFR, ITGB1, LGALS3 genes were identified as potential drug targets in the PPI network analyzed by CytoHubba. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses indicated that QRLSD may improve psoriasis by regulating immune and inflammatory pathways, the cytokine mediated signal transduction pathways and other signaling pathways. Molecular docking results showed that the main active components of the serum containing QRLSD had higher affinities for TNF and LGALS3. In vitro experiments confirmed that QRLSD may decrease levels of inflammatory cytokines by suppressing the NF-κB signaling pathway activated by TNF-α in human keratinocytes.Conclusion: This study explores the potential compounds, targets and signaling pathways of QRLSD in the treatment of psoriasis, which will help clarify the efficacy and mechanism of QRLSD.Keywords: qingre lishi decoction, psoriasis, UPLC-Q-TOF/MS, network pharmacology, molecular docking, NF-κB signaling pathway

Qingyang Liu,1,&ast; Jian Wang,2,&ast; Chunhui Ding,3 Ying Chu,1 Fengying Jiang,1 Yunxia Hu,4 Haifeng Li,2 Qiubo Wang1 1Department of Clinical Laboratory, Wuxi Ninth People’s Hospital Affiliated to Soochow University, Wuxi, Jiangsu, 214000, People’s Republic of China; 2Department of Orthopedics, Wuxi Ninth People’s Hospital Affiliated to Soochow University, Wuxi, Jiangsu, 214000, People’s Republic of China; 3Department of Pharmacy, Wuxi Ninth People’s Hospital Affiliated to Soochow University, Wuxi, Jiangsu, 214000, People’s Republic of China; 4Department of Rheumatology and Immunology, The Affiliated Wuxi People’s Hospital of Nanjing Medical University, Wuxi, Jiangsu, 214000, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Qiubo Wang, Department of Clinical Laboratory, Wuxi Ninth People’s Hospital Affiliated to Soochow University, No. 999 Liang Xi Road, Binhu District, Wuxi, Jiangsu, 214000, People’s Republic of China, Email wangqiubo2020@suda.edu.cn Haifeng Li, Department of orthopedics, Wuxi Ninth People’s Hospital Affiliated to Soochow University, No. 999 Liang Xi Road, Binhu District, Wuxi, Jiangsu, 214000, People’s Republic of China, Email lihaifeng2008@suda.edu.cnPurpose: Sinomenine (SIN) is commonly used in Traditional Chinese Medicine (TCM) as a respected remedy for rheumatoid arthritis (RA). Nevertheless, the therapeutic mechanism of SIN in RA remains incompletely understood. This study aimed to delve into the molecular mechanism of SIN in the treatment of RA.Methods: The potential targets of SIN were predicted using the TCMSP server, STITCH database, and SwissTarget Prediction. Differentially expressed genes (DEGs) in RA were obtained from the GEO database. Enrichment analyses and molecular docking were conducted to explore the potential mechanism of SIN in the treatment of RA. In vitro and in vivo studies were conducted to validate the intervention effects of SIN on rheumatoid arthritis, as determined through network pharmacology analyses.Results: A total of 39 potential targets associated with the therapeutic effects of SIN in RA were identified. Enrichment analysis revealed that these potential targets are primarily enriched in PI3K-Akt signaling pathway, and the molecular docking suggests that SIN may act on specific proteins in the pathway. Experimental results have shown that exposure to SIN inhibits cytokine secretion, promotes apoptosis, reduces metastasis and invasion, and blocks the activation of the PI3K-Akt signaling pathway in RA fibroblast-like synoviocytes (RA-FLS). Moreover, SIN treatment alleviated arthritis-related symptoms and regulated the differentiation of CD4+ T cells in the spleen of collagen-induced arthritis (CIA) mice.Conclusion: By utilizing network pharmacology, molecular modeling, and in vitro/in vivo validation, this study demonstrates that SIN can alleviate RA by inhibiting the PI3K-Akt signaling pathway. These findings enhance the understanding of the therapeutic mechanisms of SIN in RA, offering a stronger theoretical foundation for its future clinical application. Keywords: sinomenine, rheumatoid arthritis, network pharmacology, PI3K-Akt signaling pathway

Rui Shen,1,2,&ast; Kebin Cheng,3,&ast; Guanyi Li,1 Zhendong Pan,4 Xijier Qiaolongbatu,1 Yuting Wang,1 Cui Ma,1 Xucong Huang,1 Li Wang,1 Wenjing Li,1 Yuanyuan Wang,2 Lili Jing,1 Guorong Fan,2 Zhenghua Wu2 1School of Pharmaceutical Sciences, Shanghai Jiao Tong University, Shanghai, 200240, People’s Republic of China; 2Department of Clinical Pharmacy, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200080, People’s Republic of China; 3Department of Respiratory and Critical Care Medicine, Shanghai Pulmonary Hospital, School of Medicine, Tongji University, Shanghai, 200433, People’s Republic of China; 4Department of Clinical Pharmacy, Eye and ENT Hospital, Fudan University, Shanghai, 200031, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Zhenghua Wu, Department of Clinical Pharmacy, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, No. 85 Wujin Road, Shanghai, 200080, People’s Republic of China, Tel +86-159-0161-7923, Email wuzhenghua@sjtu.edu.cnPurpose: Alisma orientale (AO, Alisma orientale (Sam). Juzep) has been widely employed for the treatment of macular edema (ME) in traditional Chinese medicine due to its renowned water-relief properties. Nonetheless, the comprehensive investigation of AO in alleviating ME remained unexplored. This study aims to identify the active components of AO that target the eye and investigate its pharmacological effects and mechanisms on ME.Methods: The study commenced with UPLC-Triple-TOF/MS analysis to identify the primary constituents of AO. Zebrafish eye tissues were then analyzed after a five-day administration of AO to detect absorbed components and metabolites. Subsequently, network pharmacology, molecular docking, and molecular dynamics simulations were employed to predict the mechanisms of ME treatment via biological target pathways. In vivo experiments were conducted to corroborate the pharmacological actions and mechanisms.Results: A total of 7 compounds, consisting of 2 prototype ingredients and 5 metabolites (including isomers), were found to traverse the blood-eye barrier and localized within eye tissues. Network pharmacology results showed that AO played a role in the treatment of ME mainly by regulating the pathway network of PI3K-AKT and MAPK with TNF-α centered. Computational analyses suggested that 11-dehydro-16-oxo-24-deoxy-alisol A, a metabolite of alisol A, mitigates edema through TNF-α inhibition. Furthermore, zebrafish fundus confocal experiments and HE staining of eyes confirmed the attenuating effects of alisol A on fundus angiogenesis and ocular edema, representing the first report of AO’s ME-inhibitory effects.Conclusion: In this study, computational analyses with experimental validation were used to understand the biological activity and mechanism of alisol A in the treatment of ME. The findings shed light on the bioactive constituents and pharmacological actions of AO, offering valuable insights and a theoretical foundation for its clinical application in managing ME. Keywords: Alisma orientale, macular edema, network pharmacology, zebrafish, TNF-&#x03B1

Yanan Li,1,2,&ast; Jingyi Hu,1,2,&ast; Ryan Au,1– 3 Cheng Cheng,1,2 Feng Xu,1,2 Weiyang Li,1,2 Yuguang Wu,1,2 Yuan Cui,1,2 Lei Zhu,1,2 Hong Shen1,2 1Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing, 210029, People’s Republic of China; 2Jiangsu Provincial Hospital of Chinese Medicine, Nanjing, 210029, People’s Republic of China; 3Academy of Chinese Culture and Health Sciences, Oakland, CA, 94612, USA&ast;These authors contributed equally to this workCorrespondence: Hong Shen; Lei Zhu, Jiangsu Provincial Hospital of Chinese Medicine (Affiliated Hospital of Nanjing University of Chinese Medicine), 155 Hanzhong Road, Qinhuai District, Nanjing, Jiangsu Province, People’s Republic of China, Tel\Fax +86-025- 86617141, Email shenhong999@njucm.edu.cn; zhulei5100@njucm.edu.cnBackground: Qingchang Tongluo Decoction (QTF) is clinically used for the treatment of intestinal fibrosis in Crohn’s Disease (CD). However, the role of QTF in CD-associated fibrosis and its potential pharmacological mechanism remains unclear.Purpose: The objective of this study was to elucidate the potential mechanism of QTF in treating CD-associated fibrosis, employing a combination of bioinformatics approaches — encompassing network pharmacology and molecular docking — complemented by experimental validation.Methods: To investigate the material basis and potential protective mechanism of QTF, a network pharmacology analysis was conducted. The core components and targets of QTF underwent molecular docking analysis to corroborate the findings obtained from network pharmacology. In vitro, a colon fibrotic model was established by stimulating IEC-6 cells with 10 ng/mL of transforming growth factor(TGF-β 1). In vivo, an intestinal fibrosis model was induced in BALB/c mice by TNBS. The role of QTF in inhibiting the TGF-β 1/Smad signaling pathway was investigated through RT-qPCR, Western blotting, immunohistochemistry staining, and immunofluorescence staining.Results: Network pharmacology analysis revealed that QTF could exert its protective effect. Bioinformatics analysis suggested that Flavone and Isoflavone might be the key components of the study. Additionally, AKT1, IL-6, TNF, and VEGFA were identified as potential therapeutic targets. Furthermore, experimental validation and molecular docking were employed to corroborate the results obtained from network pharmacology. RT-qPCR, Immunofluorescence, and Western blotting results demonstrated that QTF significantly improved colon function and inhibited pathological intestinal fibrosis in vivo and in vitro.Conclusion: Through the application of network pharmacology, molecular docking, and experimental validation, QTF could be confirmed to inhibit the proliferation of intestinal fibroblasts associated with CD and reduce the expression of Collagen I and VEGFA. This effect is achieved through the attenuation of ECM accumulation, primarily via the inhibition of the TGF-β 1/Smad signaling pathway. Keywords: Qingchang Tongluo Decoction, Crohn’s Disease, intestinal fibrosis, network pharmacology, Pharmacology

Banglan Cai,1,2,&ast; Manman Qi,2,3,&ast; Xue Zhang,1,2 Denghai Zhang1– 3 1School of Basic Medicine, Ningxia Medical University, Yinchuan, People’s Republic of China; 2Shanghai Health Commission Key Laboratory of Artificial Intelligence (AI)-Based Management of Inflammation and Chronic Diseases, Shanghai Pudong Gongli Hospital, Shanghai, People’s Republic of China; 3School of Medicine, Shanghai University, Shanghai, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Denghai Zhang; Xue Zhang, Shanghai Health Commission Key Laboratory of Artificial Intelligence (AI)-Based Management of Inflammation and Chronic Diseases, Shanghai Pudong Gongli Hospital, Shanghai, 200135, People’s Republic of China, Tel +86-18916173857 ; +86-13524805404, Email Denghai_zhang@163.com; 2021021020@nxmu.edu.cnBackground: As a traditional Chinese medicine monomer derived from Tripterygium wilfordii Hook.f. with potential anticancer activity, celastrol can induce ferroptosis in hepatic stellate cells and inhibit their activation to alleviate liver fibrosis. Activation of ferroptosis can effectively inhibit Hepatocellular carcinoma (HCC). Whether celastrol inhibits HCC by inducing ferroptosis remains to be studied.Purpose: To explore the potential targets of celastrol against HCC through ferroptosis based on network pharmacology and to verify the anticancer effect of celastrol on HepG2 cells.Methods: We collected celastrol targets, HCC, and ferroptosis-related genes through online databases, and got their intersection targets. Subsequently, we obtained a protein-protein interaction (PPI) network, and performed gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis to gain key genes for further study. They were verified in vitro and were performed molecular docking. The changes in cell proliferation and ferroptosis characteristics of HepG2 cells after celastrol treatment were detected.Results: 31 core target genes were screened for PPI network and enrichment analysis. The most significantly related KEGG pathway was chemical carcinogenesis-reactive oxygen species. The mRNA and protein levels of GSTM1 were significantly decreased after celastrol treatment. Molecular docking demonstrated the interaction between celastrol and GSTM1. Ferroptosis was induced and cell proliferation was inhibited by celastrol in HCC cells.Conclusion: Celastrol induces ferroptosis in HCC via regulating GSTM1 expression and may serve as a novel therapeutic compound with clinical potential in HCC treatment. Keywords: network pharmacology, celastrol, hepatocellular carcinoma, ferroptosis, reactive oxygen species

Benchi Cai,&ast; Qitong Wang,&ast; Lifan Zhong, Fang Liu, Xinyu Wang, Tao Chen Hainan Affiliated Hospital of Hainan Medical University, Haikou, Hainan, 570311, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Tao Chen, Email chentao@hnyxy15.wecom.workBackground: Parkinson’s disease (PD) is the most prevalent movement disorder. Curcumin, a polyphenol with hydrophobic properties, has been proved against Parkinson. Our previous study suggested that curcumin’s effectiveness in treating Parkinson’s disease may be linked to the gut-brain axis, although the specific mechanism by which curcumin exerts neuroprotective effects in the brain remains unknown.Methods: The therapeutic efficacy of curcumin was evaluated using behavioral tests, immunofluorescence of tyrosine hydroxylase (TH). Network pharmacology and transcriptomics predicted the mechanisms of curcumin in PD. Activation of the phosphatidylinositol 3-kinase PI3K/AKT pathway was confirmed by quantitative polymerase chain reaction (qPCR) and immunofluorescence.Results: Curcumin restored the dyskinesia and dopaminergic neurons damage of MPTP-induced mice. Curcumin against Parkinson’s disease by regulating inflammation, oxidative stress, and aging. The mechanisms of these were associated with activation of PI3K / AKT pathway.Conclusion: In conclusion, the neuroprotective mechanisms of curcumin activate PI3K / AKT pathway in Parkinson’s disease was revealed by our study.Keywords: Parkinson’s disease, curcumin, network pharmacology, transcriptomics, PI3K / AKT pathway

Jilei Li,1,&ast; Wenchao Dan,2,&ast; Chenchen Zhang,3 Nian Liu,3 Yichong Wang,1 Jixiang Liu,3 Shengsheng Zhang1 1Digestive Disease Center, Beijing Hospital of Traditional Chinese Medicine, Capital Medical University, Beijing, 100010, People’s Republic of China; 2Department of Dermatology, Beijing Hospital of Traditional Chinese Medicine, Capital Medical University, Beijing, 100010, People’s Republic of China; 3Beijing University of Chinese Medicine, Beijing, 100029, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Shengsheng Zhang, Digestive Disease Center, Beijing Hospital of Traditional Chinese Medicine, Capital Medical University, Beijing, 100010, Tel +8613801088329, Email zhangshengsheng@bjzhongyi.comPurpose: This study aimed to delineate the molecular processes underlying the therapeutic effects of berberine on UC by employing network pharmacology tactics, molecular docking, and dynamic simulations supported by empirical validations both in vivo and in vitro.Patients and Methods: We systematically screened potential targets and relevant pathways affected by berberine for UC treatment from comprehensive databases, including GeneCards, DisGeNET, and GEO. Molecular docking and simulation protocols were used to assess the interaction stability between berberine and its principal targets. The predictions were validated using both a DSS-induced UC mouse model and a lipopolysaccharide (LPS)-stimulated NCM460 cellular inflammation model.Results: Network pharmacology analysis revealed the regulatory effect of the TLR4/NF-κB/HIF-1α pathway in the ameliorative action of berberine in UC. Docking and simulation studies predicted the high-affinity interactions of berberine with pivotal targets: TLR4, NF-κB, HIF-1α, and the HIF inhibitor KC7F2. Moreover, in vivo analyses demonstrated that berberine attenuates clinical severity, as reflected by decreased disease activity index (DAI) scores, reduced weight loss, and mitigated intestinal inflammation in DSS-challenged mice. These outcomes include suppression of the proinflammatory cytokines IL-6 and TNF-α and downregulation of TLR4/NF-κB/HIF-1α mRNA and protein levels. Correspondingly, in vitro findings indicate that berberine decreases cellular inflammatory injury and suppresses TLR4/NF-κB/HIF-1α signaling, with notable effectiveness similar to that of the HIF-1α inhibitor KC7F2.Conclusion: Through network pharmacology analysis and experimental substantiation, this study confirmed that berberine enhances UC treatment outcomes by inhibiting the TLR4/NF-κB/HIF-1α axis, thereby mitigating inflammatory reactions and improving colonic pathology. Keywords: berberine, ulcerative colitis, TLR4/NF-κB/HIF-1α, network pharmacology, molecular docking, experimental validation, inhibitor

Lina Du,1 Dezhi Yang,2 Lan Wu,1 Li Mei,1 Sarula Wu,1 Yasula Ba,1 Yongchang Bao,1 Rigugaqiqige Su,1 Lin Song1 1College of Mongolian Medical, Inner Mongolia Medical University, Hohhot, Inner Mongolia, People’s Republic of China; 2Innovative Mongolian Medical Engineering Research Center, Inner Mongolia International Mongolian Hospital, Hohhot, Inner Mongolia, People’s Republic of ChinaCorrespondence: Lin Song, Inner Mongolia Medical University, Tel +15849163188, Email songlinwpsword@163.comObjective: To explored the potential molecular mechanism of Sugemule-4 decoction (MMS-4D) in treating insomnia.Methods: DL-4-chlorophenylalanine (PCPA) + chronic unpredictable mild stress stimulation (CUMS) was used to induce an insomnia model in rats. After the model was successfully established, MMS-4D was intervened at low, medium, and high doses for 7 days. The open-field test (OFT) was used to preliminarily evaluate the efficacy. The potential mechanism of MMS-4D in treating insomnia was investigated using gut microbiota, serum metabolomics, and network pharmacology (NP). Experimental validation of the main components of the key pathways was carried out using ELISA and Western blot.Results: The weights of the insomnia-model rats were significantly raised (p ≤ 0.05), the total exercise distance in the OFT increased (p ≤ 0.05), the rest time shortened, and the number of standing times increased (p ≤ 0.05), after treatment with MMS-4D. Moreover, there was a substantial recovery in the 5-HT, DA, GABA, and Glu levels in the hypothalamus tissue and the 5-HT and GABA levels in the colon tissue of rats. The expression of DAT and DRD1 proteins in the hippocampus of insomnia rats reduced after drug treatment. MMS-4D may treat insomnia by regulating different crucial pathways including 5-HT -, DA -, GABA -, and Glu-mediated neuroactive light receiver interaction, cAMP signaling pathway, serotonergic, glutamatergic, dopaminergic, and GABAergic synapses.Conclusion: This study revealed that MMS-4D can improve the general state and behavioral changes of insomnia model rats. Its mechanism may be related to the reversal of abnormal pathways mediated by 5-HT, DA, GABA, and Glu, such as Serotonergic synapse, Dopaminergic synapse, Glutamatergic synapse, and GABAergic synapse. Keywords: Mongolian medicine sugemule-4 decoction, insomnia, metabolomics, gut microbiota, network pharmacology, molecular mechanism

Bingjie Ma,1 Pengyi Hou,2 Ran Liu,3 Aijun Cang,1 Limei Zhao4 1Department of Pharmacy, People’s Hospital of Liaoning Province, Shenyang, 110010, People’s Republic of China; 2SCIEX Analytical Instrument Trading Co, Shanghai, 200000, People’s Republic of China; 3School of Food and Drug, Shenzhen Polytechnic, Shenzhen, 518000, People’s Republic of China; 4Department of Pharmacy, Shengjing Hospital of China Medical University, Shenyang, 110010, People’s Republic of ChinaCorrespondence: Limei Zhao, Department of Pharmacy, Shengjing Hospital of China Medical University, Shenyang, 110010, People’s Republic of China, Email lmzhao19@163.comBackground: Chemotherapy-induced myelosuppression (CIM) is a common adverse reaction with a high incidence rate that seriously affects human health. Shengyu Decoction (SYD) is often used to treat CIM. However, its pharmacodynamic basis and therapeutic mechanisms remain unclear.Purpose: This study aimed to clarify the active components and mechanisms of SYD in CIM.Methods: LC-QTOF/MS was used to identify the absorbable components of SYD. A series of network pharmacology methods have been applied to explore hub targets and potential mechanisms. Molecular docking was used to identify the binding ability of potential active ingredients and hub targets. Finally, in vitro experiments were performed to validate these findings.Results: In this study, 33 absorbable prototype components were identified using LC-QTOF/MS. A total of 62 possible targets of SYD in myelosuppression were identified. KEGG pathway enrichment analyses showed that some signaling pathways such as PI3K-Akt and HIF-1 may be the mechanisms by which it functions. Among them, we verified the PI3K-Akt pathway. 6 Hub proteins were screened by Protein–protein interaction (PPI) network analysis. Molecular docking results showed that four absorbable components in SYD showed good binding with six Hub targets. The effectiveness of the four predicted compounds and the mechanism were verified in vitro. It has also been shown that the active component could promote the proliferation of bone marrow stromal cells (BMSCs) and block apoptosis of BMSCs, which may be related to the PI3K-Akt pathway. This result is consistent with the network pharmacology approach and molecular docking predictions.Conclusion: Our results provided not only the candidate active component of SYD, but also a new insights into mechanism of SYD in the treatment of CIM. Keywords: Shengyu decoction, SYD, myelosuppression, network pharmacology, molecular docking, active components, mechanism

Yue Lu,1– 3,&ast; Simin Pan,4,&ast; Wenzhen Li,5 Yao Qi,6,7 Li Li,1– 3 Yu-Hong Yan,1– 3 Jianan Wei,1– 3 Dan-Ni Yao,1– 3 Jingjing Wu,1– 3 Hao Deng,1– 3 Shuyan Ye,1– 3 Haiming Chen,1– 3 Qubo Chen,1– 3 Hengjun Gao,6,7 Ling Han,1– 3 Chuanjian Lu1– 3 1State Key Laboratory of Dampness Syndrome of Chinese Medicine, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine (Guangdong Provincial Hospital of Chinese Medicine), Guangzhou, People’s Republic of China; 2Guangdong Provincial Key Laboratory of Clinical Research on Traditional Chinese Medicine Syndrome, Guangzhou, People’s Republic of China; 3Guangdong-Hong Kong-Macau Joint Laboratory on Chinese Medicine and Immune Disease Research, Guangzhou University of Chinese Medicine, Guangzhou, People’s Republic of China; 4The Second Clinical College, Guangzhou University of Chinese Medicine, Guangzhou, People’s Republic of China; 5The Clinical College of Acupuncture Rehabilitation, Guangzhou University of Chinese Medicine, Guangzhou, People’s Republic of China; 6Shanghai Molecular Medicine Engineering Technology Research Center, Shanghai, People’s Republic of China; 7Shanghai National Engineering Research Center of Biochip, Shanghai, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Ling Han; Chuanjian Lu, Email linghan99@gzucm.edu.cn; lcj@gzucm.edu.cnBackground: Psoriasis is a widespread chronic, immune-mediated skin disease with frequent recurrences, and is extremely harmful to the physical and mental health of patients, causing enormous suffering and exerting considerable economic burdens on the health care system as a whole. In more than a decade of clinical use, the optimized formula of Yinxieling (PSORI-CM01) has consistently demonstrated its effectiveness for treating psoriasis. However, its underlying mechanism remains largely unexplored.Methods: The network pharmacology analysis was conducted to predict the mechanism and protective effect of PSORI-CM01 in treating psoriasis. Subsequently, we collected blood samples from 21 patients with psoriasis as part of a randomized, double-blind, and double-dummy clinical trial for microRNA expression profiling. Finally, it was experimentally confirmed that PSORI-CM01 improved psoriasis by regulating miR-20a-3p and miR-3184-3p expression.Results: As a result of the network pharmacology analysis, PSORI-CM01 improved psoriasis through the regulation of autophagy, cellular apoptosis, cellular proliferation, and anti-inflammatory processes. In the target-miRNA regulatory network, these key targets were mainly associated with the regulation of hsa-miR-20a-3p, hsa-miR-155-5p, has-miR-3184-3p, hsa-miR-328-3p and hsa-miR-124-3p. Based on the microRNA expression profiling results, the PSORI-CM01 treatment group exhibited five up-regulated genes and 16 down-regulated genes compared with the healthy control group. In particular, miR-20a-3p and miR-3184-3p were the primary differentially expressed microRNAs, and they were significantly enriched in the signaling pathways involving autophagy, apoptosis, proliferation, and anti-inflammation. Further experiments confirmed that PSORI-CM01 effectively regulates miR-20a-3p and miR-3184-3p, resulting in increased autophagy.Conclusion: We demonstrated by combining network pharmacology and clinical studies of miRNA expression profiles in PBMCs that PSORI-CM01 effectively modulated miR-20a-3p and miR-3184-3p, leading to an increase in autophagy and a decrease in keratinocyte proliferation.Keywords: PSORI-CM01, Psoriasis vulgaris, MicroRNA expression profile, network pharmacology, autophagy

Lisheng Chen,1,2 Tingting He,3 Ruilin Wang,3 Honghong Liu,4 Xin Wang,1,2 Haotian Li,2 Manyi Jing,2 Xuelin Zhou,5 Shizhang Wei,6 Wenjun Zou,1 Yanling Zhao1,2 1College of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu, People’s Republic of China; 2Department of Pharmacy Department, The Fifth Medical Center of Chinese PLA General Hospital, Beijing, People’s Republic of China; 3Division of Integrative Medicine, The Fifth Medical Center of General Hospital of PLA, Beijing, People’s Republic of China; 4Integrated TCM & Western Medicine Department, The Fifth Medical Center of Chinese PLA General Hospital, Beijing, People’s Republic of China; 5Department of Pharmacology, School of Basic Medical Sciences, Capital Medical University, Beijing, People’s Republic of China; 6Department of Anatomy, Histology and Embryology, School of Basic Medical Sciences, Peking University, Beijing, People’s Republic of ChinaCorrespondence: Yanling Zhao, Department of Pharmacy, The Fifth Medical Center of Chinese PLA General Hospital, Beijing, People’s Republic of China, Email zhaoyl2855@126.com Wenjun Zou, College of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu, People’s Republic of China, Email zouwenjun@163.comBackground: The Zuojin Pill (ZJP) is widely used for treating chronic atrophic gastritis (CAG) in clinical practice, effectively ameliorating symptoms such as vomiting, pain, and abdominal distension in patients. However, the underlying mechanisms of ZJP in treating CAG has not been fully elucidated.Purpose: This study aimed to clarify the characteristic function of ZJP in the treatment of CAG and its potential mechanism.Methods: The CAG model was established by alternant administrations of ammonia solution and sodium deoxycholate, as well as an irregular diet. Therapeutic effects of ZJP on body weight, serum biochemical indexes and general condition were analyzed. HE staining and AB-PAS staining were analyzed to characterize the mucosal injury and the thickness of gastric mucosa. Furthermore, network pharmacology and molecular docking were used to predict the regulatory mechanism and main active components of ZJP in CAG treatment. RT-PCR, immunohistochemistry, immunofluorescence and Western blotting were used to measure the expression levels of apoptosis-related proteins, gastric mucosal barrier-associated proteins and PI3K/Akt signaling pathway proteins.Results: The results demonstrated that ZJP significantly improved the general state of CAG rats, alleviated weight loss and gastric histological damage and reduced the serum biochemical indicators. Network pharmacology and molecular docking found that ZJP in treating CAG by inhibiting inflammation, suppressing apoptosis, and protecting the gastric mucosal barrier via the PI3K/Akt signaling pathway. Further experiments confirmed that ZJP obviously modulated the expression of key proteins involved in gastric mucosal cell apoptosis, such as Bax, Bad, Apaf-1, cleaved-caspase-3, cleaved-caspase-9, Cytochrome C, Bcl-2, and Bcl-xl. Moreover, ZJP significantly reversed the protein expression of Occludin, ZO-1, Claudin-4 and E-cadherin.Conclusion: Our study revealed that ZJP treats CAG by inhibiting the PI3K/Akt signaling pathway. This research provided a scientific basis for the rational use of ZJP in clinical practice.Keywords: Zuojin Pill, chronic atrophic gastritis, network pharmacology, molecular docking, PI3K/Akt signaling pathway

Lang Liu,1,&ast; Binghua Zhang,1,&ast; Zhenwei Zhou,2 Jie Yang,1 Ailin Li,1 Yongji Wu,1 Zeyu Peng,1 Xiangyan Li,2 Zhonghua Liu,3 Xiangyang Leng,1 Changwei Zhao,4 Haisi Dong,2 Wenhai Zhao4 1College of Traditional Chinese Medicine, Changchun University of Chinese Medicine, Changchun, People’s Republic of China; 2Northeast Asia Institute of Traditional Chinese Medicine, Changchun University of Chinese Medicine, Changchun, People’s Republic of China; 3Department of Orthopaedics, The Third Affiliated Hospital of Changchun University of Chinese Medicine, Changchun, People’s Republic of China; 4Affiliated Hospital of Changchun University of Chinese Medicine, Changchun University of Chinese Medicine, Changchun, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Haisi Dong, Northeast Asia Institute of Traditional Chinese Medicine, Changchun University of Chinese Medicine, Changchun, Jilin, 130117, People’s Republic of China, Tel/Fax +86 431-18604361976, Email adonghaisi@163.com Wenhai Zhao, Affiliated Hospital of Changchun University of Chinese Medicine, Changchun University of Chinese Medicine, Changchun, Jilin, 130021, People’s Republic of China, Tel/Fax +86 431-86177836, Email zwh9899@163.comBackground: Knee osteoarthritis (KOA) is a persistent degenerative condition characterized by the deterioration of cartilage. The Chinese herbal formula Radix Rehmanniae Praeparata- Angelica Sinensis-Radix Achyranthis Bidentatae (RAR) has often been used in effective prescriptions for KOA as the main functional drug, but its underlying mechanism remains unclear. Therefore, network pharmacology and verification experiments were employed to investigate the impact and mode of action of RAR in the treatment of KOA.Methods: The destabilization of the medial meniscus model (DMM) was utilized to assess the anti-KOA effect of RAR by using gait analysis, micro-computed tomography (Micro-CT), and histology. Primary chondrocytes were extracted from the rib cartilage of a newborn mouse. The protective effects of RAR on OA cells were evaluated using a CCK‐8 assay. The antioxidative effect of RAR was determined by measuring reactive oxygen species (ROS), superoxide dismutase (SOD), and glutathione (GSH) production. Furthermore, network pharmacology and molecular docking were utilized to propose possible RAR targets for KOA, which were further verified through experiments.Results: In vivo, RAR significantly ameliorated DMM-induced KOA characteristics, such as subchondral bone sclerosis, cartilage deterioration, gait abnormalities, and the degree of knee swelling. In vitro, RAR stimulated chondrocyte proliferation and the expression of Col2a1, Comp, and Acan. Moreover, RAR treatment significantly reduced ROS accumulation in an OA cell model induced by IL-1β and increased the activity of antioxidant enzymes (SOD and GSH). Network pharmacology analysis combined with molecular docking showed that Mapk1 might be a key therapeutic target. Subsequent research showed that RAR could downregulate Mapk1 mRNA levels in IL-1β-induced chondrocytes and DMM-induced rats.Conclusion: RAR inhibited extracellular matrix (ECM) degradation and oxidative stress response via the MAPK signaling pathway in KOA, and Mapk1 may be a core target.Keywords: knee osteoarthritis, RAR, cartilage degeneration, network pharmacology, Mapk1

Yan Ma,1,2,&ast; Qian Cui,3,&ast; Wenjing Zhu,1,4,&ast; Mei Wang,1,2 Li Zhai,5 Wenmin Hu,1,2 Dongdong Liu,6 Min Liu,5 Yongchun Li,2 Meng Li,2 Wei Han1,2 1Qingdao Key Laboratory of Common Diseases, Qingdao Municipal Hospital, School of Medicine and Pharmacy, Ocean University of China, Qingdao, Shandong, 260071, People’s Republic of China; 2Department of Respiratory and Critical Care Medicine, Qingdao Municipal Hospital, University of Health and Rehabilitation Sciences, Qingdao, 266071, People’s Republic of China; 3Department of Respiratory and Critical Care Medicine, Shenzhen Luohu People’s Hospital, Shenzhen, 518000, People’s Republic of China; 4NMPA Key Laboratory for Quality Research and Evaluation of Traditional Marine Chinese Medicine, Qingdao, 266071, People’s Republic of China; 5Department of Pharmacy, Qingdao Municipal Hospital, University of Health and Rehabilitation Sciences, Qingdao, 266071, People’s Republic of China; 6Department of Respiratory and Critical Care Medicine, Shanting District People’s Hospital, Zaozhuang, 277200, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Wei Han; Meng Li, Department of Respiratory and Critical Care Medicine, Qingdao Municipal Hospital, University of Health and Rehabilitation Sciences, Qingdao, 266071, People’s Republic of China, Email hanw@uor.edu.cn; cqdpd@126.comPurpose: Lung adenocarcinoma currently ranks the leading causes of cancer-related mortality worldwide. Many anti-inflammation herbs, like tetramethylpyrazine, have shown their anti-tumor potentials. Here, we evaluated the role of a novel chalcone derivative of tetramethylpyrazine ((E) -1- (E) -1- (2-hydroxy-5-chlorophenyl) -3- (3,5,6-trimethylpyrazin-2-yl) -2-propen-1, HCTMPPK) in lung adenocarcinoma.Methods: The effects of HCTMPPK on cell proliferation, apoptosis, and invasion were investigated by in-vitro assays, including CCK-8, colony formation assay, flow cytometry, transwell assay, and wound-healing assay. The therapeutic potential of HCTMPPK in vivo was evaluated in xenograft mice. To figure out the target molecules of HCTMPPK, a network pharmacology approach and molecular docking studies were employed, and subsequent experiments were conducted to confirm these candidate molecules.Results: HCTMPPK effectively suppressed the proliferative activity and migration, as well as enhanced the apoptosis of A549 cells in a concentration-dependent manner. Consistent with this, tumor growth was inhibited by HCTMPPK significantly in vivo. Regarding the mechanisms, HCTMPPK down-regulated Bcl-2 and MMP-9 and up-regulating Bax and cleaved-caspase-3. Subsequently, we identified 601 overlapping DEGs from LUAD patients in TCGA and GEO database. Then, 15 hub genes were identified by PPI network and CytoHubba. Finally, MELK was verified to be the HCTMPPK targeted site, through the molecular docking studies and validation experiments.Conclusion: Overall, our study indicates HCTMPPK as a potential MELK inhibitor and may be a promising candidate for the therapy of lung cancer.Keywords: HCTMPPK((E) -1- (E) -1- (2-hydroxy-5-chlorophenyl) -3- (3,5,6-trimethylpyrazin-2-yl) -2-propen-1), chalcone, TMP, lung adenocarcinoma, network pharmacology, AutoDock

Shu Jiao Zhang,1,2,&ast; Yi Fei Zhang,1,2,&ast; Xue Hui Bai,1,2 Meng Qi Zhou,1,2 Ze Yu Zhang,1,2 Shuai Xing Zhang,1,2 Zi Jing Cao,1,2 Lin Wang,1,2 Shao Wei Ding,1,2 Hui Juan Zheng,1,2 Yu Ning Liu,1– 3 Guo Yong Yu,1 Wei Jing Liu1– 3 1Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing, People’s Republic of China; 2Key Laboratory of Chinese Internal Medicine of Ministry of Education, Beijing Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing, People’s Republic of China; 3Renal Research Institution of Beijing University of Chinese Medicine, Beijing University of Chinese Medicine, Beijing, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Guo Yong Yu, Dongzhimen Hospital, Beijing University of Chinese Medicine, No. 5 Haiyuncang, Beijing, Dongcheng District, 100700, People’s Republic of China, Email 18901133535@163.com Wei Jing Liu, Renal Research Institution of Beijing University of Chinese Medicine, and Key Laboratory of Chinese Internal Medicine of Ministry of Education and Beijing, Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing, People’s Republic of China, Tel/Fax +86-010-84013190, Email 1977@hotmail.comBackground: Acteoside, an active ingredient found in various medicinal herbs, is effective in the treatment of diabetic kidney disease (DKD); however, the intrinsic pharmacological mechanism of action of acteoside in the treatment of DKD remains unclear. This study utilizes a combined approach of network pharmacology and experimental validation to investigate the potential molecular mechanism systematically.Methods: First, acteoside potential targets and DKD-associated targets were aggregated from public databases. Subsequently, utilizing protein-protein interaction (PPI) networks, alongside GO and KEGG pathway enrichment analyses, we established target-pathway networks to identify core potential therapeutic targets and pathways. Further, molecular docking facilitated the confirmation of interactions between acteoside and central targets. Finally, the conjectured molecular mechanisms of acteoside against DKD were verified through experimentation on unilateral nephrectomy combined with streptozotocin (STZ) rat model. The underlying downstream mechanisms were further investigated.Results: Network pharmacology identified 129 potential intersected targets of acteoside for DKD treatment, including targets such as AKT1, TNF, Casp3, MMP9, SRC, IGF1, EGFR, HRAS, CASP8, and MAPK8. Enrichment analyses indicated the PI3K-Akt, MAPK, Metabolic, and Relaxin signaling pathways could be involved in this therapeutic context. Molecular docking revealed high-affinity binding of acteoside to PIK3R1, AKT1, and NF-κB1. In vivo studies validated the therapeutic efficacy of acteoside, demonstrating reduced blood glucose levels, improved serum Scr and BUN levels, decreased 24-hour urinary total protein (P< 0.05), alongside mitigated podocyte injury (P< 0.05) and ameliorated renal pathological lesions. Furthermore, this finding indicates that acteoside inhibits the expression of pyroptosis markers NLRP3, Caspase-1, IL-1β, and IL-18 through the modulation of the PI3K/AKT/NF-κB pathway.Conclusion: Acteoside demonstrates renoprotective effects in DKD by regulating the PI3K/AKT/NF-κB signaling pathway and alleviating pyroptosis. This study explores the pharmacological mechanism underlying acteoside’s efficacy in DKD treatment, providing a foundation for further basic and clinical research.Keywords: DKD, acteoside, network pharmacology, experimental verification

Ziyan Wang,1,2,&ast; Chengxin Liu,1,2,&ast; Jiaming Wei,2,3 Hui Yuan,1,2 Min Shi,2,3 Fei Zhang,2,3 Qinghua Zeng,2,3 Aisi Huang,2,3 Lixin Du,4 Ya Li,4 Zhihua Guo2,3 1First Clinical College of Chinese Medicine, Hunan University of Chinese Medicine, Changsha, 410208, People’s Republic of China; 2Hunan Key Laboratory of Colleges and Universities of Intelligent Traditional Chinese Medicine Diagnosis and Preventive Treatment of Chronic Diseases of Hunan Universities of Chinese Medicine, Hunan University of Chinese Medicine, Changsha, 410208, People’s Republic of China; 3School of Traditional Chinese Medicine, Hunan University of Chinese Medicine, Changsha, 410208, People’s Republic of China; 4School of Pharmacy, Hunan University of Chinese Medicine, Changsha, 410208, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Ya Li; Zhihua Guo, Email 003872@hnucm.edu.cn; 004294@hnucm.edu.cnObjective: This study aims to explore the mechanism of action of Yixintai in treating chronic ischemic heart failure by combining bioinformatics and experimental validation.Materials and Methods: Five potential drugs for treating heart failure were obtained from Yixintai (YXT) through early mass spectrometry detection. The targets of YXT for treating heart failure were obtained by a search of online databases. Gene ontology (GO) functional enrichment analysis and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analyses were conducted on the common targets using the DAVID database. A rat heart failure model was established by ligating the anterior descending branch of the left coronary artery. A small animal color Doppler ultrasound imaging system detected cardiac function indicators. Hematoxylin-eosin (HE), Masson’s, and electron microscopy were used to observe the pathological morphology of the myocardium in rats with heart failure. The network pharmacology analysis results were validated by ELISA, qPCR, and Western blotting.Results: A total of 107 effective targets were obtained by combining compound targets and eliminating duplicate values. PPI analysis showed that inflammation-related proteins (TNF and IL1B) were key targets for treating heart failure, and KEGG enrichment suggested that NF-κB signaling pathway was a key pathway for YXT treatment of heart failure. Animal model validation results indicated the following: YXT can significantly reduce the content of intestinal microbiota metabolites such as trimethylamine oxide (TMAO) and improve heart failure by improving the EF and FS values of heart ultrasound in rats and reducing the levels of serum NT-proBNP, ANP, and BNP to improve heart failure. Together, YXT can inhibit cardiac muscle hypertrophy and fibrosis in rats and improve myocardial ultrastructure and serum IL-1β, IL-6, and TNF-α levels. These effects are achieved by inhibiting the expressions of NF-κB and PKC.Conclusion: YXT regulates the TMAO/PKC/NF-κB signaling pathway in heart failure.Keywords: heart failure, yixintai, TMAO, PKC/NF-κB pathway, traditional Chinese medicine, network pharmacology

Shipeng Chen,1– 3 Mengna Li,1– 3 Changning Xue,1– 3 Xiangting Zhou,1– 3 Jianxia Wei,1– 3 Lemei Zheng,1– 3 Yumei Duan,1– 3 Hongyu Deng,1 Faqing Tang,1 Wei Xiong,1– 3 Bo Xiang,1– 3 Ming Zhou1– 3 1NHC Key Laboratory of Carcinogenesis, Hunan Key Laboratory of Oncotarget Gene, Hunan Cancer Hospital and the Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha, 410013, People’s Republic of China; 2Cancer Research Institute, Central South University, Changsha, 410078, People’s Republic of China; 3The Key Laboratory of Carcinogenesis and Cancer Invasion of the Chinese Ministry of Education, Central South University, Changsha, 410078, People’s Republic of ChinaCorrespondence: Ming Zhou; Bo Xiang, Email zhouming2001@163.com; xiangbolin@csu.edu.cnPurpose: Cinobufotalin injection has obvious curative effects on liver cancer patients with less toxicity and fewer side effects than other therapeutic approaches. However, the core ingredients and mechanism underlying these anti-liver cancer effects have not been fully clarified due to its complex composition.Methods: Multidimensional network analysis was used to screen the core ingredients, key targets and pathways underlying the therapeutic effects of cinobufotalin injection on liver cancer, and in vitro and in vivo experiments were performed to confirm the findings.Results: By construction of ingredient networks and integrated analysis, eight core ingredients and ten key targets were finally identified in cinobufotalin injection, and all of the core ingredients are tightly linked with the key targets, and these key targets are highly associated with the cell cycle-related pathways, supporting that both cinobufotalin injection and its core ingredients exert anti-liver cancer roles by blocking cell cycle-related pathways. Moreover, in vitro and in vivo experiments confirmed that either cinobufotalin injection or one of its core ingredients, cinobufagin, significantly inhibited cell proliferation, colony formation, cell cycle progression and xenograft tumor growth, and the key target molecules involved in the cell cycle pathway such as CDK1, CDK4, CCNB1, CHEK1 and CCNE1, exhibit consistent changes in expression after treatment with cinobufotalin injection or cinobufagin. Interestingly, some key targets CDK1, CDK4, PLK1, CHEK1, TTK were predicted to bind with multiple of core ingredients of cinobufotalin injection, and the affinity between one of the critical ingredients cinobufagin and key target CDK1 was further confirmed by SPR assay.Conclusion: Cinobufotalin injection was confirmed to includes eight core ingredients, and they play therapeutic effects in liver cancer by blocking cell cycle-related pathways, which provides important insights for the mechanism of cinobufotalin injection antagonizing liver cancer and the development of novel small molecule anti-cancer drugs. Keywords: cinobufotalin injection, liver cancer, cinobufagin, network pharmacology, cell cycle, core ingredients

Yutao Peng,1 Chanjuan Zhou2 1Department of Function, The First Affiliated Hospital, Hengyang Medical School, University of South China, Hengyang, Hunan, People’s Republic of China; 2Department of Clinical Psychology, The First Affiliated Hospital, Hengyang Medical School, University of South China, Hengyang, Hunan, People’s Republic of ChinaCorrespondence: Chanjuan Zhou, Department of Clinical psychology, The First Affiliated Hospital, Hengyang Medical School, University of South China, 69 Chuan Shan Road, Hengyang, Hunan, 421001, People’s Republic of China, Tel +86 13212694904, Email zhouchanjuan_86@163.comAim: Scutellaria baicalensis, a traditional Chinese medicinal herb renowned for its anti-inflammatory, antioxidant, and anti-tumor properties, has shown promise in alleviating cognitive impairment associated with Alzheimer’s disease. Nonetheless, the exact neuroprotective mechanism of Scutellaria baicalensis against Alzheimer’s disease remains unclear. In this study, network pharmacology was employed to explore the possible mechanisms by which Scutellaria baicalensis protects against Alzheimer’s disease.Methods: The active compounds of Scutellaria baicalensis were retrieved from the TCMSP database, and their corresponding targets were identified. Alzheimer’s disease-related targets were obtained through searches in the GeneCards and OMIM databases. Cytoscape 3.6.0 software was utilized to construct a regulatory network illustrating the “active ingredient-target” relationships. Subsequently, the target genes affected by Scutellaria baicalensis in the context of Alzheimer’s disease were input into the String database to establish a PPI network. GO analysis and KEGG analysis were conducted using the DAVID database to predict the potential pathways associated with these key targets. Following this, the capacity of these active ingredients to bind to core targets was confirmed through molecular docking. In vitro experiments were then carried out for further validation.Results: A total of 36 active ingredients from Scutellaria baicalensis were screened out, which corresponded to 365 targets. Molecular docking results demonstrated the robust binding abilities of Baicalein, Wogonin, and 5,2’-Dihydroxy-6,7,8-trimethoxyflavone to key target proteins (SRC, PIK3R1, and STAT3). In vitro experiments showed that the active components of Scutellaria baicalensis can inhibit STAT3 expression by downregulating the PIK3R1/SRC pathway in Neuro 2A cells.Conclusion: In summary, these findings collectively suggest that Scutellaria baicalensis holds promise as a viable treatment option for Alzheimer’s disease.Keywords: Scutellaria baicalensis, Alzheimer’s disease, network pharmacology, molecular docking

Teng Fan,1– 3,&ast; Yuanyuan Huang,1– 3,&ast; Zeyu Liu,1,2,&ast; Jinsheng Huang,1,2 Bin Ke,1– 3 Yuming Rong,1,2 Huijuan Qiu,1,2 Bei Zhang1– 3 1TCM&VIP Department, Sun Yat-sen University Cancer Center, Guangzhou, People’s Republic of China; 2State Key Laboratory of Oncology in South China, Sun Yat-sen University Cancer Center, Guangzhou, People’s Republic of China; 3Integrated Traditional Chinese and Western Medicine Research Center, Sun Yat-sen University Cancer Center, Guangzhou, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Bei Zhang, TCM&VIP Department, Sun Yat-sen University Cancer Center, 651 Dongfeng Road East, Guangzhou, 510060, People’s Republic of China, Tel/Fax +86-20-87342708, Email zhangbei@sysucc.org.cnBackground: The ChaiShao Shugan Formula (CSSGF) is a traditional Chinese medicine formula with recently identified therapeutic value in triple-negative breast cancer (TNBC). This study aimed to elucidate the underlying mechanism of CSSGF in TNBC treatment.Methods: TNBC targets were analyzed using R and data were from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. The major ingredients and related protein targets of CSSGF were explored via the Traditional Chinese Medicine Systems Pharmacology database, and an ingredient–target network was constructed via Cytoscape to identify hub genes. The STRING database was used to construct the PPI network. GO and KEGG enrichment analyses were performed via R to obtain the main targets. The online tool Kaplan‒Meier plotter was used to identify the prognostic genes. Molecular docking was applied to the core target genes and active ingredients. MDA-MB-231 and MCF-7 cell lines were used to verify the efficacy of the various drugs.Results: A total of 4562 genes were screened as TNBC target genes. The PPI network consisted of 89 nodes and 845 edges. Our study indicated that quercetin, beta-sitosterol, luteolin and catechin might be the core ingredients of CSSGF, and EGFR and c-Myc might be the latent therapeutic targets of CSSGF in the treatment of TNBC. GO and KEGG analyses indicated that the anticancer effect of CSSGF on TNBC was mainly associated with DNA binding, transcription factor binding, and other biological processes. The related signaling pathways mainly involved the TNF-a, IL-17, and apoptosis pathways. The molecular docking data indicated that quercetin, beta-sitosterol, luteolin, and catechin had high affinity for EGFR, JUN, Caspase-3 and ESR1, respectively. In vitro, we found that CSSGF could suppress the expression of c-Myc or promote the expression of EGFR. In addition, we found that quercetin downregulates c-Myc expression in two BC cell lines.Conclusion: This study revealed the effective ingredients and latent molecular mechanism of action of CSSGF against TNBC and confirmed that quercetin could target c-Myc to induce anti-BC effects.Keywords: triple-negative breast cancer, chaishao shugan formula, network pharmacology, molecular docking, c-Myc

Yinghong Li,1,&ast; Fanwu Wu,1,&ast; Jianbo Zhang,1 Ye Xu,1 Hong Chang,1 Yueyue Yu,1 Chunhua Jiang,1 Xiujuan Gao,1 Huijuan Liu,1 Zhen Chen,2 Chenxi Wu,1 Ji-An Li1,3 1Hebei Key Laboratory of Integrated Traditional Chinese and Western Medicine for Diabetes and Its Complications, College of Traditional Chinese Medicine, North China University of Science and Technology, Tangshan, People’s Republic of China; 2Oriental Herbs KFT, Budapest, Hungary; 3School of Public Health, North China University of Science and Technology, Tangshan, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Chenxi Wu; Ji-An Li, Tel +86-15930951086, Fax +86-03158805765, Email chenxi.wu@ncst.edu.cn; JianLi@ncst.edu.cnPurpose: Type 2 diabetes mellitus (T2DM) is associated with reduced insulin uptake and glucose metabolic capacity. Potentilla discolor Bunge (PDB) has been used to treat T2DM; however, the fundamental biological mechanisms remain unclear. This study aimed to understand the active ingredients, potential targets, and underlying mechanisms through which PDB treats T2DM.Methods: Components and action targets were predicted using network pharmacology and molecular docking analyses. PDB extracts were prepared and validated through pharmacological intervention in a Cg>InRK1409A diabetes Drosophila model. Network pharmacology and molecular docking analyses were used to identify the key components and core targets of PDB in the treatment of T2DM, which were subsequently verified in animal experiments.Results: Network pharmacology analysis revealed five effective compounds made up of 107 T2DM-related therapeutic targets and seven protein–protein interaction network core molecules. Molecular docking results showed that quercetin has a strong preference for interleukin-1 beta (IL1B), IL6, RAC-alpha serine/threonine-protein kinase 1 (AKT1), and cellular tumor antigen p53; kaempferol exhibited superior binding to tumor necrosis factor and AKT1; β-sitosterol demonstrated pronounced binding to Caspase-3 (CASP3). High-performance liquid chromatography data quantified quercetin, kaempferol, and β-sitosterol at proportions of 0.030%, 0.025%, and 0.076%, respectively. The animal experiments revealed that PDB had no effect on the development, viability, or fertility of Drosophila and it ameliorated glycolipid metabolism disorders in the diabetes Cg>InRK1409A fly. Furthermore, PDB improved the body size and weight of Drosophila, suggesting its potential to alleviate insulin resistance. Moreover, PDB improved Akt phosphorylation and suppressed CASP3 activity to improve insulin resistance in Drosophila with T2DM.Conclusion: Our findings suggest that PDB ameliorates diabetes metabolism disorders in the fly model by enhancing Akt activity and suppressing CASP3 expression. This will facilitate the development of key drug targets and a potential therapeutic strategy for the clinical treatment of T2DM and related metabolic diseases. Keywords: Potentilla discolor Bunge, T2DM, network pharmacology, molecular docking, Drosophila

Xuqing Zhou,1,2,&ast; Xu Wang,1,2,&ast; Jiaqi Li,3,&ast; Mengxue Zhang,4 Yi Yang,1 Shihui Lei,1 Ying He,3 Hua Yang,3 Desheng Zhou,4 Chun Guo1,2 1Experiment Center of Medical Innovation, the First Hospital of Hunan University of Chinese Medicine, Changsha, Hunan, 410007, People’s Republic of China; 2The First Clinical College of Traditional Chinese Medicine, Hunan University of Chinese Medicine, Changsha, Hunan, 410007, People’s Republic of China; 3College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha, Hunan, 410128, People’s Republic of China; 4Department of Neurology, the First Hospital of Hunan University of Chinese Medicine, Changsha, Hunan, 410007, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Chun Guo, Email chunguosm@163.comBackground: Annao Pingchong decoction (ANPCD) is a traditional Chinese decoction which has definite effects on treating intracerebral hemorrhage (ICH) validated through clinical and experimental studies. However, the impact of ANPCD on oxidative stress (OS) after ICH remains unclear and is worth further investigating.Aim: To investigate whether the therapeutic effects of ANPCD on ICH are related to alleviating OS damage and seek potential targets for its antioxidant effects.Materials and Methods: The therapeutic candidate genes of ANPCD on ICH were identified through a comparison of the target genes of ANPCD, target genes of ICH and differentially expressed genes (DEGs). Protein–protein interaction (PPI) network analysis and functional enrichment analysis were combined with targets-related literature to select suitable antioxidant targets. The affinity between ANPCD and the selected target was verified using macromolecular docking. Subsequently, the effects of ANPCD on OS and the selected target were further investigated through in vivo experiments.Results: Forty-eight candidate genes were screened, in which silent information regulator sirtuin 1 (SIRT1) is one of the core genes that has antioxidant effects and ICH significantly affected its expression. The good affinity between 6 compounds of ANPCD and SIRT1 was also demonstrated by macromolecular docking. The results of in vivo experiments demonstrated that ANPCD significantly decreased modified neurological severity scoring (mNSS) scores and serum MDA and 8-OHdG content in ICH rats, while significantly increasing serum SOD and CAT activity, complicated with the up-regulation of ANPCD on SIRT1, FOXO1, PGC-1α and Nrf2. Furthermore, ANPCD significantly decreased the apoptosis rate and the expression of apoptosis-related proteins (P53, cytochrome c and caspase-3).Conclusion: ANPCD alleviates OS damage and apoptosis after ICH in rats. As a potential therapeutic target, SIRT1 can be effectively regulated by ANPCD, as are its downstream proteins. Keywords: intracerebral hemorrhage, Annao Pingchong decoction, oxidative stress, network pharmacology, vivo experiments, SIRT1

Ji Guo,1– 3 Shengqi Yang,1– 4 Weifeng Zhai,1– 3,5 Yue Xie,1– 4 Zhan Shen,1– 4 Jianpo Zhang,1– 3 Yongwei Jia1– 3,5 1Guanghua Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai, 200052, People’s Republic of China; 2Shanghai Guanghua Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai, 200052, People’s Republic of China; 3Institute of Arthritis Research in Integrative Medicine, Shanghai Academy of Traditional Chinese Medicine, Shanghai, 200052, People’s Republic of China; 4Shanghai University of Traditional Chinese Medicine, Shanghai, 201203, People’s Republic of China; 5Department of Orthopedics, Shanghai University of Medicine & Health Sciences Affiliated Zhoupu Hospital, Shanghai, 201318, People’s Republic of ChinaCorrespondence: Ji Guo; Yongwei Jia, Email 153498661@qq.com; spinejia@163.comBackground: Intervertebral disc degeneration (IVDD) is a pathophysiological process that leads to severe back pain or neurological deficits. The Bushen Huoxue Formula (BSHXF) is a traditional herbal remedy widely used to treat diseases related to IVDD. However, its pharmacological mechanism needs further exploration.Objective: This study aimed to elucidate the mechanisms through which BSHXF treats IVDD-related diseases by integrating metabolomics with network pharmacology.Methods: Network pharmacology was utilized to identify potential targets of BSHXF against IVDD. Additionally, an animal model of needle puncture-induced disc degeneration was established to assess the effect of BSHXF. Mice were randomly assigned to the sham group, model group, and BSHXF group. Various techniques, including PCR, CCK-8 assay, MRI, histological examinations, and immunohistochemical analyses, were employed to evaluate degenerative and oxidative stress conditions in mouse disc tissue and cultured nucleus pulposus (NP) cells. UHPLC-HRMS/MS was used to differential distinct metabolites in the disc tissue from different groups, and MetaboAnalyst 5.0 was employed to enrich the metabolic pathways.Results: Through network pharmacology, 15 core proteins were identified through protein-protein interaction (PPI) network construction. Functional enrichment analysis highlighted the critical role of BSHXF in addressing IVDD by influencing the response to oxidative stress. Furthermore, experimental evidence demonstrated that BSHXF significantly improved the pathological progression of IVDD and increased oxidative stress markers SOD-1 and GPX1, both in the disc degeneration model and cultured NP cells. Metabolomics identified differential metabolites among the three groups, revealing 15 metabolic pathways between the sham and model groups, and 13 metabolic pathways enriched between the model and BSHXF groups.Conclusion: This study, integrating network pharmacology and metabolomics, suggests that BSHXF can alleviate IVDD progression by modulating oxidative stress. Key metabolic pathways associated with BSHXF-mediated reduction of oxidative stress include the citrate cycle, cysteine and methionine metabolism, alanine, aspartate and glutamate metabolism, glycine, serine and threonine metabolism, D-glutamine and D-glutamate metabolism, glutathione metabolism, and tryptophan metabolism. While this research demonstrates the therapeutic potential of BSHXF in reducing oxidative stress levels in IVDD, further research is needed to thoroughly understand its underlying mechanisms.Keywords: metabolomics, network pharmacology, intervertebral disc degeneration, oxidative stress, Bushen Huoxue formula

Xiang-Yu Ji,1– 3 Cheng-Jing Lei,2,3 Shuang Kong,2,3 Han-Fei Li,2,3 Si-Yu Pan,2,3 Yu-Jing Chen,2,3 Fan-Rong Zhao,2,3 Tian-Tian Zhu1– 3 1Department of Pharmacy, the First Affiliated Hospital of Xinxiang Medical University, Xinxiang, Henan, People’s Republic of China; 2College of Pharmacy, Xinxiang Medical University, Xinxiang, Henan, People’s Republic of China; 3Henan International Joint Laboratory of Cardiovascular Remodeling and Drug Intervention, Xinxiang, Henan, People’s Republic of ChinaCorrespondence: Tian-Tian Zhu, College of Pharmacy, Xinxiang Medical University, Xinxiang, Henan, 453003, People’s Republic of China, Tel +86 15225990135, Email zhutt@xxmu.edu.cnPurpose: The underlying causes of pulmonary arterial hypertension (PAH) often remain obscure. Addressing PAH with effective treatments presents a formidable challenge. Studies have shown that Hydroxysafflor yellow A (HSYA) has a potential role in PAH, While the mechanism underlies its protective role is still unclear. The study was conducted to investigate the potential mechanisms of the protective effects of HSYA.Methods: Using databases such as PharmMapper and GeneCards, we identified active components of HSYA and associated PAH targets, pinpointed intersecting genes, and constructed a protein-protein interaction (PPI) network. Core targets were singled out using Cytoscape for the development of a model illustrating drug-component-target-disease interactions. Intersection targets underwent analysis for Gene Ontology (GO) functions and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment. Selected components were then modeled for target interaction using Autodock and Pymol. In vivo validation in a monocrotaline-induced PAH (MCT-PAH) animal model was utilized to substantiate the predictions made by network pharmacology.Results: We associated HSYA with 113 targets, and PAH with 1737 targets, identifying 34 mutual targets for treatment by HSYA. HSYA predominantly affects 9 core targets. Molecular docking unveiled hydrogen bond interactions between HSYA and several PAH-related proteins such as ANXA5, EGFR, SRC, PPARG, PGR, and ESR1.Conclusion: Utilizing network pharmacology and molecular docking approaches, we investigated potential targets and relevant human disease pathways implicating HSYA in PAH therapy, such as the chemical carcinogenesis receptor activation pathway and the cancer pathway. Our findings were corroborated by the efficacious use of HSYA in an MCT-induced rat PAH model, confirming its therapeutic potential.Keywords: network pharmacology, molecular docking, hydroxy-safflower yellow A, pulmonary arterial hypertension

Yi-Ling Jiang, Yi Xun Department of Oncology, The First Affiliated Hospital, Hengyang Medical School, University of South China, Hengyang, Hunan, People’s Republic of ChinaCorrespondence: Yi Xun, Department of Oncology, The First Affiliated Hospital, Hengyang Medical School, University of South China, Hengyang, Hunan, People’s Republic of China, Tel +8618773486975, Email 469239341@qq.comPurpose: This study aimed to investigate the effect of Salvia miltiorrhiza on colorectal cancer, as well as the mechanisms involved.Methods: The active compounds of Salvia miltiorrhiza and the associated genes in colorectal cancer were sourced from publicly available databases. Targets associated with colorectal cancer were identified by searching the GeneCards and OMIM databases. Subsequently, the Cytoscape 3.6.0 software was employed to create a regulatory network that illustrates the relationships among active ingredients, colorectal cancer, and their corresponding targets. The String database was utilized to generate a PPI network. Molecular docking studies, conducted with AutoDock Vina, verified the binding capabilities of these active components to core targets. The findings from network pharmacology analysis were corroborated through in vitro experiments.Results: In this study, we identified 39 active components derived from Salvia miltiorrhiza that are predicted to target 544 genes associated with colorectal cancer through network pharmacology. Through a combined analysis of network pharmacology, we isolated three key targets: SRC, IL6, and INS. Molecular docking results convincingly demonstrated Salvia miltiorrhiza’s strong binding affinity to these targets. Additionally, in vitro experiments confirmed that Salvia miltiorrhiza effectively inhibited the progression of colorectal cancer via regulating the INS/SRC/IL6 pathway.Conclusion: Salvia miltiorrhiza emerges as a compelling herbal intervention for colorectal cancer. This study lays the foundation for potential future clinical trials assessing the efficacy of Salvia miltiorrhiza in the management of colorectal cancer.Keywords: Salvia miltiorrhiza, network pharmacology, traditional Chinese medicine, docking technology, colorectal cancer

Long Lv,1,&ast; Jinghu Du,1,&ast; Daorong Wang,2 Zeqiang Yan2 1Department of General Surgery, Xiangyang Central Hospital, Affiliated Hospital of Hubei University of Arts and Science, Xiangyang, Hubei, 441021, People’s Republic of China; 2Department of Gastroenterology, Xiangyang Central Hospital, Affiliated Hospital of Hubei University of Arts and Science, Xiangyang, Hubei, 441021, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Daorong Wang; Zeqiang Yan, Department of Gastroenterology, Xiangyang Central Hospital, Affiliated Hospital of Hubei University of Arts and Science, Xiangyang, Hubei, 441021, People’s Republic of China, Tel +13581425716 ; +13597505970, Email 415004128@qq.com; yzqi48959@163.comBackground: Gastric cancer (GC) is a common fatal malignancy. The aim of this study was to explore and validate the tumor-suppressive role and mechanism of Radix Bupleuri in GC.Methods: The active constituents of Radix Bupleuri were screened using TCMSP database. SwissTargetPrediction database was used to predict potential target genes of the compounds. GeneCards, TTD, DisGeNET, OMIM, and PharmGKB databases were used to search for GC-related targets. STRING database and Cytoscape 3.10 software were used for protein–protein interaction network construction and screening of core targets. DAVID database was used for GO and KEGG analyses. Core targets were validated using molecular docking. Cell proliferation and apoptosis were detected using CCK-8 and flow cytometry after GC cells were treated with isorhamnetin. The mRNA and protein expression levels of genes were detected using qRT PCR and Western blot. The metastasis potential of GC cells was evaluated in a nude mouse model.Results: A total of 371 potential targets were retrieved by searching the intersection of Radix Bupleuri and GC targets. Petunidin, 3’,4’,5’,3,5,6,7-Heptamethoxyflavone, quercetin, kaempferol, and isorhamnetin were identified as the main bioactive compounds in Radix Bupleuri. SRC, HSP90AA1, AKT1, and EGFR, were core targets through which Radix Bupleuri suppressed GC. The tumor-suppressive effect of Radix Bupleuri on GC was mediated by multiple pathways, including PI3K-AKT, cAMP, and TNF signaling. The key compounds of Radix Bupleuri had good binding affinity with the core target. Isorhamnetin, a key component of Radix Bupleuri, could inhibit proliferation and metastasis, and induces apoptosis of GC cells. In addition, isorhamnetin could also reduce the mRNA expression of core targets, and the activation of PI3K/AKT pathway.Conclusion: This study identified potential targets and pathways of Radix Bupleuri against GC through network pharmacology and molecular docking, providing new insights into the pharmacological mechanisms of Radix Bupleuri in GC treatment. Keywords: gastric cancer, Radix Bupleuri, network pharmacology, molecular docking

Tengfei Chen,1,&ast; Sihan Li,1,&ast; Dongyin Lian,1 Qin Hu,2 Hongping Hou,1 Delian Niu,1 Han Li,1 Ling Song,1 Yunhang Gao,1 Ying Chen,1 Xiaoru Hu,3 Jianrong Li,1 Zuguang Ye,1 Bo Peng,1 Guangping Zhang1 1Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, People’s Republic of China; 2College of Life Sciences and Bio-Engineering, Beijing University of Technology, Beijing, People’s Republic of China; 3National Institute for Food and Drug Control, Beijing, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Bo Peng; Guangping Zhang, Tel +8610 64056575, Email bpeng@icmm.ac.cn; gpzhang@icmm.ac.cnPurpose: This study aimed to investigate the main pharmacological action and underlying mechanisms of Jin Gu Lian Capsule (JGL) against rheumatoid arthritis (RA) based on network pharmacology and experimental verification.Methods: Network pharmacology approaches were performed to explore the core active compounds of JGL, key therapeutic targets, and signaling pathways. Molecular docking was used to predict the binding affinity of compounds with targets. In vivo experiments were undertaken to validate the findings from network analysis.Results: A total of 52 targets were identified as candidate JGL targets for RA. Sixteen ingredients were identified as the core active compounds, including, quercetin, myricetin, salidroside, etc. Interleukin-1 beta (IL1B), transcription factor AP-1 (JUN), growth-regulated alpha protein (CXCL1), C-X-C motif chemokine (CXCL)3, CXCL2, signal transducer and activator of transcription 1 (STAT1), prostaglandin G/H synthase 2 (PTGS2), matrix metalloproteinase (MMP)1, inhibitor of nuclear factor kappa-B kinase subunit beta (IKBKB) and transcription factor p65 (RELA) were obtained as the key therapeutic targets. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed that the efficacy of JGL was functionally involved in regulating immune-mediated inflammation, in which IL-17/NF-κB signaling was recommended as one of the main pathways. Molecular docking suggested that the core active compounds bound strongly to their respective targets. Experimentally, JGL treatment mitigated inflammation, showed analgesic activity, and ameliorated collagen-induced arthritis. Enzyme-linked immunosorbent assay showed that JGL effectively reduced the serum levels of cytokines, chemokines, and MMPs. Immunohistochemistry staining showed that JGL markedly reduced the expression of the targets in IL-17/NF-κB pathway including IL-17A, IL-17RA, NF-κB p65, C-X-C motif ligand 2, MMP1 and MMP13.Conclusion: This investigation provided evidence that JGL may alleviate RA symptoms by partially inhibiting the immune-mediated inflammation via IL-17/NF-κB pathway. Keywords: rheumatoid arthritis, Jin Gu Lian capsules, network pharmacology, experimental validation, immune-mediated inflammation

Yuan Cui,1,2 Jingyi Hu,2 Yanan Li,1,2 Ryan Au,1,2 Yulai Fang,2 Cheng Cheng,1 Feng Xu,1,2 Weiyang Li,1,2 Yuguang Wu,1,2 Lei Zhu,2 Hong Shen2 1Nanjing University of Chinese Medicine, Nanjing, People’s Republic of China; 2Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing, People’s Republic of ChinaCorrespondence: Lei Zhu; Hong Shen, Tel/Fax +86-25- 86617141, Email shenhong999@njucm.edu.cn; zhulei5100@njucm.edu.cnBackground: Baiyu Decoction (BYD), a clinical prescription of traditional Chinese medicine, has been proven to be valuable for treating ulcerative colitis (UC) by enema. However, the mechanism of BYD against UC remains unclear.Purpose: A combination of bioinformatics methods including network pharmacology and molecular docking and animal experiments were utilized to investigate the potential mechanism of BYD in the treatment of UC.Materials and Methods: Firstly, the representative compounds of each herb in BYD were detected by liquid chromatography-mass spectrometry. Subsequently, we predicted the core targets and potential pathways of BYD for treating UC through network pharmacology. And rat colitis model was established with dextran sodium sulfate. UC rats were subjected to BYD enema administration, during which we recorded body weight changes, disease activity index, and colon length to assess the effectiveness of BYD. Besides, quantitative real-time PCR, western blotting, ELISA and immunofluorescence were used to detect intestinal inflammatory factors, intestinal barrier biomarkers and TOLL-like receptor pathway in rats. Finally, the core components and targets of BYD were subjected to molecular docking so as to further validate the results of network pharmacology.Results: A total of 41 active compositions and 203 targets related to BYD-UC were subjected to screening. The results of bioinformatics analysis showed that quercetin and kaempferol may be the main compounds. Additionally, AKT1, IL-6, TP53, TNF and IL-1β were regarded as potential therapeutic targets. KEGG results explained that TOLL-like receptor pathway might play a pivotal role in BYD protecting against UC. In addition, animal experiments and molecular docking validated the network pharmacology results. BYD enema treatment can reduce body weight loss, lower disease activity index score, reverse colon shortening, relieve intestinal inflammation, protect intestinal barrier, and inhibit TOLL-like receptor pathway in UC rats. Besides, molecular docking suggested that quercetin and kaempferol docked well with TLR4, AKT1, IL-6, TP53.Conclusion: Utilizing network pharmacology, animal studies, and molecular docking, enema therapy with BYD was confirmed to have anti-UC efficacy by alleviating intestinal inflammation, protecting the intestinal barrier, and inhibiting the TOLL-like receptor pathway. Researchers should focus not only on oral medications but also on the rectal administration of medications in furtherance of the cure of ulcerative colitis. Keywords: network pharmacology, ulcerative colitis, enema therapy, TLR4/MyD88/NF-κB pathway

Qingsong Liu,1,2,&ast; Lunyu Li,2,&ast; Dan Zheng,1 Songlin Jin,2 Xiaotian Guan,2 Zeting Fu,2 Zhigang Xiong,1 Haili Ding3 1Sichuan Provincial People’s Hospital, University of Electronic Science and Technology of China, Chengdu, People’s Republic of China; 2School of Sports Medicine and Health, Chengdu Sport University, Chengdu, People’s Republic of China; 3Insititute of Sports Medicine and Health, Chengdu Sport University, Chengdu, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Haili Ding, No. 2, Gymnasium Road, Chengdu, Sichuan, People’s Republic of China, Tel +86 13688467479, Email dingdingtang111@163.comPurpose: This study examined the underlying mechanisms of SJD’s anti-inflammatory and analgesic effects on acute GA flares.Methods: This study used pharmacology network and molecular docking methods. The active ingredients of ShuiJingDan (SJD) were obtained from the Traditional Chinese Medicine Systems Pharmacology Analysis Platform (TCMSP), and the relevant targets of GA were obtained from the Online Mendelian Inheritance in Man (OMIM) database and Therapeutic Target Database (TTD). The core drug group–target–disease Venn diagram was formed by crossing the active ingredients of SJD and the relevant targets. Gene Ontology (GO) analysis was conducted for functional annotation, DAVID was used for Kyoto Encyclopedia of Genes, and Genomes pathway enrichment analysis, and R was used to find the core targets. The accuracy of SJD network pharmacology analysis in GA treatment was verified by molecular docking simulations. Finally, a rat GA model was used to further verify the anti-inflammatory mechanism of SJD in the treatment of GA.Results: SJD mainly acted on target genes including IL1B, PTGS2, CXCL8, EGF, and JUN, as well as signal pathways including NF-κB, Toll-like receptor (TLR), IL-17, and MAPK. The rat experiments showed that SJD could significantly relieve ankle swelling, reduce the local skin temperature, and increased the paw withdrawal threshold. SJD could also reduce synovial inflammation, reduced the concentrations of interleukin-1β (IL-1β), IL-8, and COX-2 in the synovial fluid, and suppressed the expression of IL1B, CXCL8, and PTGS2 mRNA in the synovial tissue.Conclusion: SJD has a good anti-inflammatory effect to treat GA attacks, by acting on target genes such as IL-1β, PTGS2, and CXCL8.Keywords: ShuiJingDan, gouty arthritis, network pharmacology, molecular docking, IL-1β, COX-2

Dong Zhang,1,2 Siwei Duan,3 Zhangyou He,4 Zeming Zhu,3 Zhiping Li,3 Qincheng Yi,4 Tiantian Cai,1,2 Juanjuan Li,1,2 Nan Chen,1,2 Shaoju Guo1,2 1Gastrointestinal Ward, The Fourth Clinical Medical College of Guangzhou University of Chinese Medicine, Shenzhen, People’s Republic of China; 2Gastrointestinal Ward, Shenzhen Hospital of Traditional Chinese Medicine, Shenzhen, People’s Republic of China; 3Institute of Gastroenterology, Science and Technology Innovation Center of Guangzhou University of Chinese Medicine, Guangzhou, People’s Republic of China; 4Institute of Gastroenterology, Guangzhou University of Chinese Medicine, Guangzhou, People’s Republic of ChinaCorrespondence: Shaoju Guo, Gastrointestinal Ward, The Fourth Clinical Medical College of Guangzhou University of Chinese Medicine, Shenzhen, People’s Republic of China, Tel +86-0755-82571384, Email gsj1080@163.comPurpose: This study was conducted to explore the mechanism of Sijunzi Decoction (SJZ) in the treatment of ulcerative colitis (UC).Methods: The study aimed to investigate the active components and targets of SJZ in the treatment of UC by screening databases such as TCMSP, GeneCards, OMIM, Distinct, TTD, and Drugbank. An online Venn tool, Cytoscape 3.7.2, and Autodock Tools were used to analyze the components and targets. The study also used a mouse model of UC to further investigate the effects of SJZ. HE staining, immunofluorescence, ELISA, qPCR, and Western blot were used to detect various indices.Results: Eighty-three active components and 112 action targets were identified from SJZ, including 67 targets for treating UC-related NETs. The five core targets identified were AKT1, JUN, IL1B, PTGS2, and TNF, and molecular docking studies indicated that the five targets were well-docked with ginsenoside Rh2, isoflavones, and formononetin. Animal experiments demonstrated that SJZ could alleviate various parameters such as weight, colon length, spleen index, disease activity index, and intestinal pathology of the UC mice. Immunofluorescence and Western blot showed that SJZ could reduce the expression of IL1B and TNF in intestinal neutrophils while increasing the expression of Occludin. Cellular immunofluorescence suggests that SJZ can reduce the expression of TNF and IL1B in NETs. The qPCR results also suggested that SJZ could inhibit TNF signal. Furthermore, ELISA results suggested that SJZ could inhibit the expression of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) while promoting the expression of anti-inflammatory cytokines (IL-10, IL-37, TGF-β).Conclusion: SJZ treats UC by reducing the content of intestinal NETs, with primary targets on the NETs being IL1B and TNFand suppress TNF signal. The practical components of SJZ may be ginsenoside Rh2, isoflavones, and formononetin.Graphical Abstract: Keywords: ulcerative colitis, Sijunzi decoction, network pharmacology, IL1B, TNF

Yan Lan,1,2,&ast; Yu-Kun Zheng,3,&ast; Liu-Yi Wu,3 Zi-Jun Zhou,3 Ruo-Xin Guan,3 Heng Xu,3 Ji-Yuan Tu,3 Xin Gu,3 Rui Wang,3 Nan Jiang,3 Yuan Wu,4 Cheng-Ren Shu,1,2 Zhong-Shi Zhou3 1Department of Pharmacy, Huangshi Central Hospital, Affiliated Hospital of Hubei Polytechnic University, Huangshi, Hubei, People’s Republic of China; 2Hubei Key Laboratory of Kidney Disease Pathogenesis and Intervention, Hubei, People’s Republic of China; 3College of Pharmacy, Hubei University of Chinese Medicine, Wuhan, Hubei, People’s Republic of China; 4Department of Radiation Oncology, Hubei Cancer Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Cheng-Ren Shu, Department of Pharmacy, Huangshi Central Hospital, NO. 141, Tianjin Road, Huangshi, Hubei, People’s Republic of China, Tel +86-27-13986590438, Email 397112615@qq.com Zhong-Shi Zhou, College of Pharmacy, Hubei University of Chinese Medicine, No. 1, Huangjiahu West Road, Hongshan District, Wuhan City, Hubei Province, People’s Republic of China, Tel +86-27-15871745189, Email monkeyzzs@126.comObjective: Traditional Chinese medicine Polygonum cuspidatum (PC) has significant effects on reducing pain. In this study, we investigated the analgesic effects of the alcohol extract of PC on three types of inflammatory pain and explored its mechanism.Methods: Potential targets for the analgesic effects of the main active components of PC alcohol extract were screened by network pharmacology and molecular docking. Three different inflammatory pain mouse models (acetic acid twisting, formalin foot swelling, and xylene ear swelling) were used to study the analgesic effects of PC. The expression of latent signaling pathways in L4-6 spinal cord tissues in formalin foot swelling mice was evaluated using real-time qPCR (RT-qPCR), Western blot (WB), and immunohistochemistry (IHC) analyses.Results: Network pharmacology analysis shows that PC analgesic mechanism is related to the MAPK/ERK signaling pathway. The five main active components of PC have good docking ability with JNK and p38. PC alcohol extract significantly reduced the pain behavior and alleviated inflammatory reactions in three mouse models, inhibited the mRNA and protein phosphorylation levels of JNK, ERK, p38, and CREB in spinal cord tissues.Conclusion: PC alcohol extract can inhibit inflammation and alleviate pain, which is related to its inhibition of the MAPK/ERK signaling pathway in spinal cord. Thus, PC alcohol extract is a promising candidate for pain treatment.Keywords: Polygonum cuspidatum, network pharmacology, MAPK/ERK, analgesic

Yicheng Guo,1 Huiqun Gan,1 Shigui Xu,1 Guosheng Zeng,2 Lili Xiao,2 Zhijun Ding,2 Jie Zhu,3 Xinglong Xiong,3 Zhiyuan Fu1 1Department of Pharmacy, Dermatology Hospital of Jiangxi Province, Nanchang, People’s Republic of China; 2Jiangxi Provincial Clinical Research Center for Skin Diseases, Nanchang, People’s Republic of China; 3Candidate Branch of National Clinical Research Center for Skin Diseases, Nanchang, People’s Republic of ChinaCorrespondence: Zhiyuan Fu, Department of Pharmacy, Dermatology Hospital of Jiangxi Province, 388, Yinbing Road, Nanchang, Jiangxi, People’s Republic of China, Tel +86 13970827820, Email fuzhiyuan0774@yeah.netPurpose: This study aims to confirm the efficacy of Xijiao Dihuang decoction (XJDHT), a classic prescription, in treating psoriasis and to explore the potential therapeutic mechanism.Methods: For pharmacodynamic analysis, a mouse model of imiquimod cream (IMQ)-induced psoriasis was constructed. Active ingredients and genes of XJDHT, as well as psoriasis-related targets, were obtained from public databases. Intersecting genes (IGEs) of XJDHT and psoriasis were collected by Venn Diagram. A protein–protein interaction (PPI) network of IGEs is constructed through the STRING database. The Molecular Complex Detection (MCODE) and Cytohubba plug-ins of Cytoscape software were used to identified hub genes. In addition, we conducted enrichment analysis of IGEs using the R package clusterProfiler. Hub genes were validated via external GEO databases. The influence of XJDHT on Hub gene expression was examined by qPCR and ELISA, and molecular docking was used to evaluate the binding efficacy between active ingredients and hub genes.Results: The results revealed that XJDHT possesses 92 potential genes for psoriasis, and 8 Hub genes were screened. Enrichment analysis suggested that XJDHT ameliorate psoriasis through multiple pathways, including AGE-RAGE, HIF-1, IL-17 and TNF signaling pathway. Validation data confirmed the differential expression of IL6, VEGFA, TNF, MMP9, STAT3, and TLR4. Molecular docking revealed a strong affinity between active ingredients and Hub genes. The efficacy of XJDHT in improving psoriatic lesions in model mice was demonstrated by PASI score and HE staining, potentially attributed to the down-regulation of VEGFA, MMP9, STAT3, TNF, and IL-17A, as evidenced by ELISA and qPCR.Conclusion: This study employed network pharmacology and in vitro experiments to identify the potential mechanisms underlying the therapeutic effects of XJDHT on psoriasis, providing a new theoretical basis for its clinical application in the treatment of psoriasis.Keywords: psoriasis, Xijiao Dihuang decoction, network pharmacology, molecular docking, angiogenesis

Yuanyuan Zhong,1,2 Bin Wang,1,2 Wenjing Chen,1,2 Hongying Zhang,1,2 Jing Sun,1,2 Jingcheng Dong1,2 1Department of Integrative Medicine, Huashan Hospital, Fudan University, Shanghai, People’s Republic of China; 2Institute of Integrated Traditional Chinese and Western Medicine, Huashan Hospital, Fudan University, Shanghai, People’s Republic of ChinaCorrespondence: Jingcheng Dong; Jing Sun, Email jcdong2004@126.com; sjing0610@163.comPurpose: To investigate the effectiveness of modified Bu-Shen-Yi-Qi decoction (MBSYQ) in the treatment of osteoporosis associated with chronic obstructive pulmonary disease (COPD) and its underlying mechanisms of action.Methods: Disease targets, active ingredients and targets were predicted by TTD, CTD, DisGeNET, HERB (BenCaoZuJian as its Chinese name), and multiple-TCM databases; In addition, the screened targets were performed via the online platforms DAVID 6.8 and Metascape for GO and KEGG pathway enrichment analysis; The relationship between the MBSYQ and core targets were verified by molecular docking technique. Then we established a COPD-associated osteoporosis rat model by passive 24-week cigarette exposure. We assessed the efficacy of MBSYQ by lung histopathology assessment and distal femur/the first lumbar vertebra (L1) microstructural assay. In addition, we performed tibial RNA sequencing, which was validated by RT-PCR and Western blot.Results: Screening revealed that the 350 active compounds of MBSYQ anchored 228 therapeutic targets for COPD-related osteoporosis; KEGG pathway enrichment analysis showed that the key targets mainly regulated MAPK and PI3K/AKT signaling pathways. In vivo studies showed that MBSYQ treatment alleviated pathological alterations in lung tissue, and reversed the bone loss and microstructure damage in the femur/L1 of model rats. The RNA seq indicated that MBSYQ could upregulate genes associated with anti-oxidative stress and aerobic respiration. The GSEA analysis displayed that MAPK and PI3K/AKT pathways were inhibited by CS exposure and activated by MBSYQ.Conclusion: MBSYQ is effective in the prevention and treatment of COPD-related osteoporosis, partially achieved by improving oxygen metabolism and activating MAPK and PI3K/AKT pathways.Keywords: COPD, osteoporosis, modified Bu-Shen-Yi-Qi formulae, network pharmacology, transcriptomics

Rizky Yulion,1 Lili Andriani,2 Siti Hamidatul Aliyah3 1Departement of Pharmacy, Sekolah Tinggi Ilmu Kesehatan Harapan Ibu Jambi, Jambi City, Jambi Province, Indonesia; 2Chemistry Doctoral Program, Faculty of Mathematics and Natural Sciences, Andalas University, Padang, West Sumatra, Indonesia; 3Centre for Biomedical Research, Research Organization for Health, National Research and Innovation Agency (BRIN), Cibinong Science Centre, Bogor, West Java, IndonesiaCorrespondence: Siti Hamidatul Aliyah, Centre for Biomedical Research, Research Organization for Health, National Research and Innovation Agency (BRIN), Cibinong Science Centre, Cibinong, Bogor, West Java, Indonesia, Email siti.hamidatul.aliyah@brin.go.id

Yonghua Ye,1 Yuping Yang,1 Li Yan,1 Li Zhou,2 Shihui Yu,1 Quan Du,3 Xiaoqiao Dong,3 Qiang Yuan,1 Letian Shan,2,4 Le Guo1 1College of Pharmaceutical Sciences, Zhejiang Chinese Medical University, Hangzhou, People’s Republic of China; 2The First Affiliated Hospital, Zhejiang Chinese Medical University, Hangzhou, People’s Republic of China; 3Department of Neurosurgery, Affiliated Hangzhou First People’s Hospital, Zhejiang University School of Medicine, Hangzhou, People’s Republic of China; 4Cell Resource Bank and Integrated Cell Preparation Center of Xiaoshan District, Hangzhou Regional Cell Preparation Center (Shangyu Biotechnology Co., Ltd), Hangzhou, People’s Republic of ChinaCorrespondence: Letian Shan, The First Affiliated Hospital, Zhejiang Chinese Medical University, Hangzhou, People’s Republic of China, Tel +15355015568, Email letian.shan@zcmu.edu.cn Le Guo, College of Pharmaceutical Sciences, Zhejiang Chinese Medical University, Hangzhou, People’s Republic of China, Tel +18986648306, Email le.guo@zcmu.edu.cnPurpose: To explore the pharmacological effects and mechanisms of Qinghao Biejia decoction (QBD) against non-small-cell lung cancer (NSCLC) based on network pharmacology and to verify the anticancer effect of artemisinin B (ART B), the active ingredient of QBD, on H1299 cells.Methods: Ultra-performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry (UPLC-QTOF-MS/MS) was applied to explore the chemoprofile of QBD. A zebrafish xenograft model was used to determine the anti-cancer efficacy of QBD. Cell counting kit-8 assay, terminal deoxyribonucleotide transferase-mediated-dUTP nick-end labeling assay; immunofluorescence, and flow cytometry were used to evaluate the in vitro anti-proliferative and pro-apoptotic effects of QBD and ART B on H1299 cells. Subsequently, the related targets and action mechanisms of both QBD and ART B predicted by network pharmacological analyses were experimentally validated by real-time PCR and Western blot assays on H1299 cells.Results: UPLC-QTOF-MS/MS identified a total of 69 compounds (such as ART B, mangiferin, and artemisinic acid) in QBD. The in vivo data showed that QBD significantly inhibited the growth of H1299 cells in xenograft larval zebrafish from 125 to 500 μg/mL. The in vitro data showed that QBD induced apoptosis of H1299 cells, accompanied by down-regulating the expression of BCL-2 and up-regulating the expression of BIM, PUMA, BAX, c-PARP, γ-H2A.X, c-CASP3, and c-CASP8. Alike QBD, ART B exerted similar anti-proliferative and pro-apoptotic effects on H1299 cells. Moreover, ART B inhibited expressions of BCL2L1, AKT1, AKT2, MMP-2, and EGFR, and up-regulated ALB expression. Mechanistically, ART B promoted apoptosis of H1299 cells by inhibiting PI3K/Akt signaling pathway.Conclusion: This study revealed the anti-NSCLC efficacy of QBD. ART B, the effective component of QBD, plays an anti-NSCLC role by down-regulating the PI3K-Akt signaling pathway. It suggests that QBD and ART B are promising drug candidates for NSCLC treatment.Graphical Abstract: Keywords: Qinghao Biejia decoction, non-small-cell lung cancer, larval zebrafish, network pharmacology, artemisinin B

Jiasheng Deng,1,&ast; Le Qin,2,&ast; Zhipin Zhou3,&ast; 1School of Pharmacy, Guangxi University of Chinese Medicine, Nanning, Guangxi, People’s Republic of China; 2Department of Pharmacy, Affiliated Hospital of Youjiang Medical University for Nationalities, Baise, Guangxi, People’s Republic of China; 3Department of Pharmacy, Liuzhou People’s Hospital, Liuzhou, Guangxi, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Zhipin Zhou, Department of Pharmacy, Liuzhou People’s Hospital, 8 Wenchang Road, Cheng Zhong District, Liuzhou, Guangxi, People’s Republic of China, Tel +86 − 18776921947, Email gxzhouzhipin@126.comAim: Many studies have demonstrated the hepatoprotective or anti-fibrotic effects of Isodon ternifolius, but its pharmacological basis and mechanism remain unclear. In this study, we used in vitro models to validate the predicted results and revealed the potential mechanism of action and active ingredients through network pharmacology methods and molecular docking.Methods: The chemical components of Isodon ternifolius were identified by literatures. Potential targets of Isodon ternifolius were predicted by Swiss Target Prediction. The disease targets were collected through the databases of Gene Card. Common targets of Isodon ternifolius and liver fibrosis were obtained by online tool Venny 2.1. PPI protein interaction network was obtained using String database, and target protein interaction network was drawn using Cytoscape software. Signaling pathway enrichment analysis was performed on drug-disease targets with of DAVID database.Results: Twenty-one potential active ingredients and 298 potential targets were predicted by Swiss Target Prediction platform. Ninety pathways related to liver fibrosis were obtained by KEGG enrichment. The TLR4, MAPK and PI3K-Akt signaling pathways are mostly associated with liver fibrosis. Molecular docking techniques were used to validate the core target proteins TNF, Akt1, MAPK1, EGFR and TLR4 binding to the ingredients of Isodon ternifolius, which showed that a multitude of ingredients of Isodon ternifolius were able to bind to the above target proteins, especially 2α-hydroxy oleanolic acid and (-)-Lambertic acid. Our experimental validation results showed that Isodon ternifolius inhibited the activation of PI3K-Akt and ERK1/2 signaling pathways.Conclusion: Through a network pharmacology approach and in vitro cell assay, we predicted and validated the active compounds of Isodon ternifolius and its potential targets for LF treatment. The results suggest that the mechanism of Isodon ternifolius treating LF by inhibiting angiogenesis may be related to the ERK1/2 and PI3K/Akt signaling pathways.Keywords: Chinese medicine, Isodon ternifolius (D. Don), network pharmacology, molecular docking, signaling pathway, liver fibrosis

Hai-bo Liu,1,&ast; Min Yang,2,&ast; Wan Li,2 Ting Luo,2 Yang Wu,2 Xiang-yu Huang,2 Yao-lei Zhang,3 Tao Liu,2 Yong Luo2,&ast; 1Department of Biomedical Engineer, General Hospital of Western Theater Command, Chengdu, Sichuan, People’s Republic of China; 2Department of Traditional Chinese Medicine, General Hospital of Western Theater Command, Chengdu, Sichuan, People’s Republic of China; 3Basic Medical Laboratory, General Hospital of Western Theater Command, Chengdu, Sichuan, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Yong Luo, Department of Traditional Chinese Medicine, General Hospital of Western Theater Command, Chengdu, Sichuan, 610083, People’s Republic of China, Tel +86 28-86571619, Email yongl019@sina.comPurpose: Dispelling dampness, relieving turbidity and dredging collaterals decoction (DED), is a traditional Chinese medicine used in the treatment of hyperuricemia. We aimed to explore the effect and mechanism of DED in the treatment of hyperuricemia.Methods: The effects of DED (9.48, 4.74, and 2.37 g/kg/d) on potassium oxonate (750 mg/kg/d)-induced hyperuricemia in rats were evaluated by serum uric acid (UA), creatinine (CRE), blood urea nitrogen (BUN), and renal pathological changes. Network pharmacology was used to identify the effective components and targets of DED, and the key targets and signaling pathways for its effects on hyperuricemia were screened. Molecular docking was used to predict the action of DED. H&E, immunohistochemistry, WB, and PCR were used to validate the network pharmacology results.Results: DED can effectively alleviate hyperuricemia, inhibit UA, CRE, BUN, and xanthine oxidase (XOD) activity, and reduce renal inflammatory cell infiltration and glomerular atrophy. The experiment identified 27 potential targets of DED for hyperuricemia, involving 9 components: wogonin, stigmasterol 3-O-beta-D-glucopyranoside, 3β-acetoxyatractylone, beta-sitosterol, stigmasterol, diosgenin, naringenin, astilbin, and quercetin. DED can relieve hyperuricemia mainly by inhibiting RAGE, HMGB1, IL17R, and phospho-TAK1, and by regulating the AGE-RAGE and IL-17 signaling pathways.Conclusion: DED can alleviate hyperuricemia by inhibiting XOD activity and suppressing renal cell apoptosis and inflammation via the AGE-RAGE signaling pathway and IL-17 signaling pathway. This study provides a theoretical basis for the clinical application of DED.Graphical Abstract: Keywords: dispelling dampness, relieving turbidity, dredging collaterals, hyperuricemia, network pharmacology

Lei Yu, Yun Gai Department of Oncology I, Seventh People’s Hospital of Shanghai University of Traditional Chinese Medicine, Shanghai City, People’s Republic of ChinaCorrespondence: Yun Gai, Department of Oncology I, Seventh People’s Hospital of Shanghai University of Traditional Chinese Medicine, No. 358, DaTong Road, PuDong New Area, Shanghai City, 200137, People’s Republic of China, Tel +86- 13917898986, Email gaiyun_sh7@yeah.netPurpose: This study reported the efficacy and underlying mechanism of agrimonolide (AM) in treating colon cancer.Methods: Colon cancer-AM-related targets were screened from online database. AM targets for colon cancer were identified by Venn diagram. Main molecular function, biological process, cellular component and pathways associated with AM targets for colon cancer were analyzed by GO and KEGG enrichment analysis. Relationship of the 10 core targets of AM for colon cancer with the top 15 BP and KEGG pathways was analyzed by Cytoscape software. A “component-target-pathway” network was constructed to select the hub genes of AM for colon cancer. AM effects on colon cancer cell viability, proliferation, invasion, migration and apoptosis were researched by CCK-8, colony formation, Transwell invasion, wound healing and flow cytometry assays. Tumor-bearing nude mice models were constructed and given AM treatment. Hub gene expression in cells/tissues was detected by Western blot.Results: A total of 107 targets were selected as AM targets for colon cancer. The 10 core targets were related to the top 15 biological process terms and KEGG pathways. PI3K, AKT and mTOR were selected as the hub genes of AM for colon cancer. AM weakened colon cell proliferation, invasion, migration and apoptosis inhibition, and suppressed colon cell in vivo growth. AM up-regulated Caspase-3 and BAX proteins, down-regulated C-Myc, Cyclin D1 and BCL-2 proteins, and inactivated the PI3K/AKT/mTOR pathway both in vitro and in vivo.Conclusion: AM suppressed colon cancer progression through inactivating the PI3K/AKT/mTOR pathway. It may be useful for colon cancer treatment.Keywords: colon cancer, agrimonolide, network pharmacology, PI3K/AKT/mTOR pathway, in vivo

Ruye Wang,1,&ast; Ying Zhao,2,&ast; Chenyun Miao,3 Yun Chen,1 Ning Ren,1 Liuqin Yang,1 Wei Cheng,4 Qin Zhang,1,5 Xiaohong Fang1 1Department of TCM Gynecology, Hangzhou TCM Hospital of Zhejiang Chinese Medical University, Hangzhou, People’s Republic of China; 2School of Public Health, Zhejiang Chinese Medical University, Hangzhou, People’s Republic of China; 3School of Life Sciences, Zhejiang Chinese Medical University, Hangzhou, People’s Republic of China; 4Department of Orthopedics, Hangzhou TCM Hospital of Zhejiang Chinese Medical University, Hangzhou, People’s Republic of China; 5Research Institute of Women’s Reproductive Health, Zhejiang Chinese Medical University, Hangzhou, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Qin Zhang; Xiaohong Fang, Department of Medical TCM Gynecology, Hangzhou TCM Hospital of Zhejiang Chinese Medical University, Hangzhou, Zhejiang, 310007, People’s Republic of China, Email zhaqin01@163.com; fxh13527806@163.comPurpose: The aim of this study is to examine, using network pharmacology analysis and experimental validation, the pharmacological processes by which Yulin Formula (YLF) reduces cyclophosphamide-induced diminished ovarian reserve (DOR).Methods: First, information about the active components, associated targets, and related genes of YLF and DOR was gathered from open-access databases. The primary targets and pathways of YLF to reduce DOR were predicted using studies of functional enrichment from the Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Ontology (GO), and Protein-Protein Interaction (PPI) networks. Second, we built a cyclophosphamide-induced diminished ovarian reserve (DOR) rat model to verify the primary target proteins implicated in the predicted signaling pathway to explore the mechanism of improve ovarian function of YLF.Results: 98 targets met the targets of the 82 active ingredients in YLF and DOR after searching the intersection of the active ingredient targets and DOR targets. Fourteen targets, including AKT and Caspase-3 among others, were hub targets, according to the PPI network study. The PI3K/AKT pathway was revealed to be enriched by numerous targets by the GO and KEGG enrichment studies, and it was used as a target for in vivo validation. Animal studies showed that YLF administration not only reduced the number of atretic follicles, the proportion of TUNEL-positive ovarian cells, the rate of apoptosis of granulosa cells (GCs) and the proportion of abnormal mitochondria in DOR rats, but also reversed the high expression of Caspase-3, Caspase-9, BAX, cytochrome C, PI3K and P-AKT, improving the ovarian reserve in cyclophosphamide (CTX)-induced DOR rats.Conclusion: Our research results predicted the active ingredients and potential targets of YLF-interfering DOR by an integrated network pharmacology approach, and experimentally validated some key target proteins participated in the predicted signaling pathway. A more comprehensive understanding of the pharmacological mechanism of YLF for DOR treatment was obtained.Keywords: diminished ovarian reserve, Yulin Formula, network pharmacology, pharmacological mechanisms, apoptosis, PI3K/AKT

Lei Zhang,1,&ast; Honglin Li,1,&ast; Liang Zhang,2 Zhihui Zu,1 Dinglin Xu,1 Jianwei Zhang1,2 1The First Clinical College, Shandong University of Traditional Chinese Medicine, Jinan, People’s Republic of China; 2Gynecology, Obstetrics and Reproductive Center, Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Jianwei Zhang, Affiliated Hospital of Shandong University of Traditional Chinese Medicine, 42, Wenhua West Road, Jinan, Shandong Province, People’s Republic of China, Tel +8653168901404, Email zhangjianwei1970@sina.comPurpose: Yangjing Zhongyu Tang (YJZYT) is a classic Chinese prescription for infertility treatment and exerts therapeutic effects via activity on the thin endometrium (TE). However, the major components and underlying mechanisms of YJZYT actions remain to be established. The main objectives of this study were to clarify the effects of YJZYT on the TE and provide insights into the related mechanisms based on network pharmacology and molecular docking analyses.Methods: Network pharmacology was employed to explore the main bioactive components and targets of YJZYT. TE-related genes were obtained from the Genecards database and screened for intersections with YJZYT. The Cytoscape 3.8.2 was used to build a “compounds-disease-targets” network and molecular docking analysis performed on key targets. The mechanism of action of YJZYT was further validated in vivo using a rat model.Results: A total of 98 YJZYT active ingredients, 2409 thin endometrium-associated genes, and 186 common targets were obtained. Through topological analysis, 10 core objectives were screened. Data from the PPI network suggest that AKT1, TNF, VEGFA, IL-6, TP53, INS, ESR1, MMP9, ALB, and ACTB serve as key targets in the action of YJZYT on TE. PI3K-Akt, TNF, apoptosis, IL-17 and MAPK were established as the main functional pathways. Molecular docking analysis revealed high affinity of the active ingredients of YJZYT, specifically, ursolic acid, palbinone, stigmasterol, and beta-sitosterol, for TNF, VEGFA, IL-6, AKT, and MMP9. YJZYT improved endometrial recovery, promoted endometrial angiogenesis, and upregulated protein expression of VEGF, PI3K, AKT, and p-AKT in the TE rat model.Conclusion: Network pharmacological and animal studies facilitated the prediction and validation of the active components and key targets of YJZYT potentially contributing to TE. Preliminary evidence from in vivo experiments showed that YJZYT promotes angiogenesis and thin endometrial repair via regulation of the PI3K/AKT pathway, providing a reference for further research.Keywords: network pharmacology, molecular docking, Yangjing Zhongyu Tang, thin endometrium, PI3K/AKT signaling pathway

Lijuan Wang,1,&ast; Huoliang Liu,2,&ast; Yi Wang,1 XiaoFan Hong,1 Xiaoyan Huang,1,3,4 Miaoru Han,1 Dan Wang,1 Wenjun Shan,1 Ping Li,1,3 Haowen Gu,1 Bo Liu,3,5 Kun Bao1,3,4,6 1Second Clinical Medical College, Guangzhou University of Chinese Medicine, Guangzhou, People’s Republic of China; 2The Affiliated TCM Hospital of Guangzhou Medical University, Guangzhou, People’s Republic of China; 3State Key Laboratory of Dampness Syndrome of Chinese Medicine, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine (Guangdong Provincial Hospital of Chinese Medicine), Guangzhou, People’s Republic of China; 4Guangdong-Hong Kong-Macau Joint Laboratory on Chinese Medicine and Immune Disease Research, Guangzhou, People’s Republic of China; 5Guangzhou Key Laboratory of Chirality Research on Active Components of Traditional Chinese Medicine, Guangzhou, People’s Republic of China; 6Guangdong Provincial Key Laboratory of Chinese Medicine for Prevention and Treatment of Refractory Chronic Disease, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine (Guangdong Provincial Hospital of Chinese Medicine), Guangzhou, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Bo Liu; Kun Bao, Email doctliu@gzucm.edu.cn; baokun@aliyun.comBackground: Nephrotic syndrome (NS) and its numerous complications remain the leading causes of morbidity and mortality globally. Sanqi Qushi granule (SQG) is clinically effective in NS. However, its potential mechanisms have yet to be elucidated.Methods: A network pharmacology approach was employed in this study. Based on oral bioavailability and drug-likeness, potential active ingredients were picked out. After acquiring overlapping targets for drug genes and disease-related genes, a component-target-disease network and protein–protein interaction analysis (PPI) were constructed using Cytoscape, followed by GO and KEGG enrichment analyses. Adriamycin was injected into adult male Sprague-Dawley (SD) rats via the tail vein to establish NS model. Kidney histology, 24-hr urinary protein level, creatinine (Cr), blood urea nitrogen (BUN), triglyceride (TG), total cholesterol (TC), and low-density lipoprotein (LDL-C) level were assessed. Western blotting, immunohistochemistry, and TUNEL staining were applied.Results: In total, 144 latent targets in SQG acting on NS were screened by a network pharmacology study, containing AKT, Bax, and Bcl-2. KEGG enrichment analysis suggested that PI3K/AKT pathway was enriched primarily. In vivo validation results revealed that SQG intervention ameliorated urine protein level and podocyte lesions in the NS model. Moreover, SQG therapy significantly inhibited renal cells apoptosis and decreased the ratio of Bax/Bcl-2 protein expression. Moreover, we found that Caspase-3 regulated the PI3K/AKT pathway in NS rats, which mediated the anti-apoptosis effect.Conclusion: By combining network pharmacology with experimental verification in vivo, this work confirmed the treatment efficacy of SQG for NS. SQG protected podocyte from injury and inhibited kidney apoptosis in NS rats via the PI3K/AKT pathway at least partially.Keywords: Sanqi Qushi granule, nephrotic syndrome, podocyte injury, kidney apoptosis, network pharmacology, PI3K/AKT pathway

Wei-guang Yang,&ast; Ao Sun,&ast; Rong Zhu, Nan Liu, Wei-jie He, Lin-lin Liu Department of Nephrology, The First Affiliated Hospital of China Medical University, Shen Yang, Liao Ning, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Lin-lin Liu, Department of Nephrology, The First Affiliated Hospital of China Medical University, 155 Nan Jing North Street in He Ping District, Shen Yang, Liao Ning, People’s Republic of China, Email catherine-ll@126.comBackground: Artemisinin (ART) is a safe and effective antimalarial drug. In recent years, antimalarial drugs have demonstrated a good therapeutic efficacy in IgA nephropathy, suggesting that this may become a new treatment option.Purpose: We aimed to evaluate the effect and mechanism of artemisinin in IgA nephropathy.Methods: In this study, CMap database was used to predict the artemisinin therapeutic effect for IgA nephropathy. A network pharmacology approach was applied to explore the unknown mechanism of artemisinin in IgA nephropathy. We used molecular docking to predict the binding affinity of artemisinin with the targets. A mouse model of IgA nephropathy was established to investigate the therapeutic effect of artemisinin on IgA nephropathy. In vitro, the cell counting Kit-8 assay was used to evaluate the cytotoxicity of artemisinin. Flow cytometry and PCR assays were used to detect the effects of artemisinin on oxidative stress and fibrosis in lipopolysaccharide (LPS)-stimulated mesangial cells. Western blot and immunofluorescence were used to detect the expression of pathway proteins.Results: CMap analysis showed artemisinin may reverse the expression levels of differentially expressed genes in IgA nephropathy. Eighty-seven potential targets of artemisinin in the treatment of IgA nephropathy were screened. Among them, 15 hub targets were identified. Enrichment analysis and GSEA analysis indicated that response to reactive oxygen species is the core biological process. AKT1 and EGFR had the highest docking affinity with artemisinin. In vivo, artemisinin could improve renal injury and fibrosis in mice. In vitro, artemisinin attenuated LPS-induced oxidative stress and fibrosis promoted AKT phosphorylation and Nrf2 nuclear translocation.Conclusion: Artemisinin reduced the level of fibrosis and oxidative stress with IgA nephropathy through the AKT/Nrf2 pathway, which provided an alternative treatment for IgAN.Keywords: IgA nephropathy, artemisinin, network pharmacology, AKT, Nrf2

Li Chen,1 Wang-Yang Xu,2 Hao Chen,1 Yan-Quan Han,1 Yu-Ting Zhang1 1The First Affiliated Hospital, Anhui University of Traditional Chinese Medicine, Hefei, People’s Republic of China; 2The College of Pharmacy, Anhui University of Traditional Chinese Medicine, Hefei, People’s Republic of ChinaCorrespondence: Hao Chen; Yanquan Han, The First Affiliated Hospital, Anhui University of Traditional Chinese Medicine, Hefei, People’s Republic of China, Email chenhao071522@163.com; hyquan2003@163.comPurpose: Gandouling Tablets (GDL), a proprietary Chinese medicine, have shown a preventive effect against Wilson’s disease (WD)-induced neuronal damage in previous studies. However, the potential mechanisms need additional investigation. Combining metabonomics and network pharmacology revealed the GDL pathway against WD-induced neuronal damage.Methods: The WD rat model with a high copper load was developed, and nerve damage was assessed. Total metabonomics was used to identify distinct hippocampus metabolites and enriched metabolic pathways in MetaboAnalyst. The GDL’s possible targets against WD neuron damage were then determined by network pharmacology. Cytoscape constructed compound metabonomics and pharmacology networks. Moreover, molecular docking and Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) validated key targets.Results: GDL reduced WD-induced neuronal injury. Twenty-nine GDL-induced metabolites may protect against WD neuron injury. According to network pharmacology, we identified three essential gene clusters, of which genes in cluster 2 had the most significant impact on the metabolic pathway. A comprehensive investigation identified six crucial targets, including UGT1A1, CYP3A4, CYP2E1, CYP1A2, PIK3CB, and LPL, and their associated core metabolites and processes. Four targets reacted strongly with GDL active components. GDL therapy improved five targets’ expression.Conclusion: This collaborative effort revealed the mechanisms of GDL against WD neuron damage and a way to investigate the potential pharmacological mechanisms of other Traditional Chinese Medicine (TCM).Graphical Abstract: Keywords: GDL, metabonomics, network pharmacology, mechanisms

Mingsheng Lyu,1,2 Jingbo Qin,3 Shuaiyang Huang,2 Dongmei Shao,4 Guirui Huang,2 Fan Yang,5,6 Xuefeng Gong,7 Shiyu Zhang,2 Zhijie Zhang,2 Ji Wang,6 Hongsheng Cui2 1Center of Respiratory Disease, Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing, People’s Republic of China; 2Department of Respiratory, The Third Affiliated Hospital, Beijing University of Chinese Medicine, Beijing, People’s Republic of China; 3Department of Geratology, Hospital of Chengdu University of Traditional Chinese Medicine, Chengdu, People’s Republic of China; 4Department of Infectious Disease, Shunyi Hospital, Beijing Hospital of Traditional Chinese Medicine, Beijing, People’s Republic of China; 5College of Traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing, People’s Republic of China; 6National Institute of Traditional Chinese Medicine (TCM) Constitution and Preventive Medicine, Beijing University of Chinese Medicine, Beijing, People’s Republic of China; 7Department of Traditional Chinese Medicine, Beijing Chaoyang Hospital, Capital Medical University, Beijing, People’s Republic of ChinaCorrespondence: Hongsheng Cui, Department of Respiratory, The Third Affiliated Hospital, Beijing University of Chinese Medicine, Beijing, People’s Republic of China, Email hshcui@sina.com; Ji Wang, National Institute of Traditional Chinese Medicine Constitution and Preventive Medicine, Beijing University of Chinese Medicine, Beijing, People’s Republic of China, Email doctorwang2009@126.comBackground: Tuo-Min-Ding-Chuan Decoction (TMDCD) is an effective traditional Chinese medicine (TCM) formula granule for allergic asthma (AA). Previous studies proved its effects on controlling airway inflammations, while the specific mechanism was not clear.Methods: We conducted a network pharmacology study to explore the molecular mechanism of TMDCD against AA with the public databases of TCMSP. Then, HUB genes were screened with the STRING database. DAVID database performed GO annotation and KEGG functional enrichment analysis of HUB genes, and it was verified with molecular docking by Autodock. Then, we built a classic ovalbumin-induced allergic asthma mice model to explore the mechanism of anti-inflammation effects of TMDCD.Results: In the network pharmacology study, we found out that the potential mechanism of TMDCD against AA might be related to NOD-like receptor (NLR) signaling pathway and Toll-like receptor (TLR) signaling pathway. In the experiment, TMDCD showed remarkable effects on alleviating airway inflammations, airway hyperresponsiveness (AHR), and airway remodeling in the asthmatic mice model. Further molecular biology and immunohistochemistry experiments suggested TMDCD could repress TLR4-NLRP3 pathway-mediated pyroptosis-related gene transcriptions to inhibit expressions of target proteins.Conclusion: TMDCD could alleviate asthmatic mice model airway inflammations by regulating TLR4-NLRP3 pathway-mediated pyroptosis.Keywords: Tuo-Min-Ding-Chuan Decoction, allergic asthma, TLR4, NLRP3, pyroptosis, network pharmacology, traditional Chinese medicine

Peigen Wu,1– 3 Churui Chang,3 Guanglin Zhu,4 Lixiang Zhai,5 Xu Zhang,3 Qiuchan Huan,1,3 Zhengxian Gao,1,3 Huan Deng,2 Yue Liang,1 Haitao Xiao2,3 1Department of Pharmacy, Peking University Shenzhen Hospital, Shenzhen, People’s Republic of China; 2School of Pharmaceutical Sciences, Health Science Center, Shenzhen University, Shenzhen, People’s Republic of China; 3School of Pharmaceutical Sciences, Guizhou Medical University, University Town, Guizhou, People’s Republic of China; 4Traditional Chinese Medicine Hospital of Qijiang, Chongqing, People’s Republic of China; 5School of Chinese Medicine, Hong Kong Baptist University, Kowloon, Hong Kong Special Administrative Region, People’s Republic of ChinaCorrespondence: Haitao Xiao, School of Pharmaceutical Sciences, Health Science Center, Shenzhen University, Shenzhen, 518060, People’s Republic of China, Email xhaitao@szu.edu.cn Yue Liang, Department of Pharmacy, Peking University Shenzhen Hospital, Shenzhen, People’s Republic of China, Email bmqueen@163.comPurpose: To explore the potential mechanism of glycosidic fraction of Picrorhiza scrophulariiflora Pennell (GPS) extract for the treatment of colitis using UPLC-QTOF-MS analysis, network pharmacology and experimental research.Methods: The active components of GPS extract were identified by UPLC-QTOF-MS analysis and extracted their targets from the databases, which was used for network pharmacology analysis. Kyoto Encyclopedia of genes and genomes (KEGG) pathway analysis was performed to discover potential therapeutic mechanisms, and the network pharmacology results were then validated by in vivo and in vitro experiments.Results: The results showed that GPS extract significantly alleviated the clinical signs of colitis, including body weight, disease activity index, colon shortening, and colon tissue damage, and inhibited the transcription and production of colonic IL-1β and IL-6 in DSS-induced colitis mice. In vitro, GPS extract also significantly suppressed nitric oxide (NO) production, iNOS expression, IL-1β and IL-6 transcription of LPS-activated RAW 264.7 cells. Network pharmacology integrated with experimental validation identified that GPS extract significantly suppressed Akt, p38, ERK, and JNK phosphorylation in vivo and in vitro, and luteolin, apocynin, caffeic acid, caffeic acid methyl ester, luteoloside, picroside II, aucubin, cinnamic acid, vanillic acid, and sweroside were the main components responsible for the anti-inflammatory effect of GPS. These findings demonstrate that the potential anti-inflammatory effect of GPS extract against colitis is achieved through suppressing PI3K/Akt and MAPK pathways, and that the abovementioned active components mainly exerted its anti-inflammatory effect.Conclusion: The therapeutic effect of GPS extract on colitis is related to PI3K/Akt and MAPK pathways, which is a promising remedy for colitis therapy.Graphical Abstract: Keywords: Picrorhiza scrophulariiflora, colitis, bioactive compounds, molecular mechanism, network pharmacology

Su Yuan,1,2 Biao Zuo,2,3 Si-Cong Zhou,1,2 Meng Wang,2,3 Kai-Yue Tan,2,3 Zhi-Wei Chen,2,3 Wen-Fu Cao1– 3 1Department of Combination of Chinese and Western Medicine, the First Affiliated Hospital of Chongqing Medical University, Chongqing, People’s Republic of China; 2Chongqing Key Laboratory of Traditional Chinese Medicine for Prevention and Cure of Metabolic Diseases, Chongqing, People’s Republic of China; 3College of Traditional Chinese Medicine, Chongqing Medical University, Chongqing, People’s Republic of ChinaCorrespondence: Wen-Fu Cao, The First Affiliated Hospital of Chongqing Medical University, No. 1, Youyi Road, Yuanjiagang, Yuzhong District, Chongqing, 400016, People’s Republic of China, Email caowenfu20220401@163.comPurpose: Our study aims to reveal the pharmacological mechanism of Astragaloside IV in the treatment of pulmonary fibrosis(PF) through network pharmacology and experimental validation.Methods: We first determined the in vivo anti-pulmonary fibrosis effect of Astragaloside IV by HE, MASSON staining, and lung coefficients, then used network pharmacology to predict the signaling pathways and molecularly docked key pathway proteins, and finally validated the results by in vivo and in vitro experiments.Results: In in vivo experiments, we found that Astragaloside IV improved body weight (P < 0.05), increased lung coefficients (P < 0.05), and reduced lung inflammation and collagen deposition in mice with pulmonary fibrosis. The network pharmacology results showed that Astragaloside IV had 104 cross-targets with idiopathic pulmonary fibrosis, and the results of KEGG enrichment analysis indicated that cellular senescence could be an important pathway for Astragaloside IV in the treatment of pulmonary fibrosis. Astragaloside IV also bound well to senescence-associated proteins, according to molecular docking results. The results of both in vivo and in vitro experiments showed that Astragaloside IV significantly inhibited senescence protein markers such as P53, P21, and P16 and delayed cellular senescence (P < 0.05). In in vivo experiments, we also found that Astragaloside IV reduced the production of SASPs (P < 0.05), and in in vitro experiments, Astragaloside IV also reduced the production of ROS. In addition, by detecting epithelial-mesenchymal transition(EMT)-related marker protein expression, we also found that Astragaloside IV significantly inhibited the development of EMT in both in vivo and in vitro experiments (P < 0.05).Conclusion: Our research found that Astragaloside IV could alleviate bleomycin-induced PF by preventing cellular senescence and EMT.Keywords: astragaloside IV, pulmonary fibrosis, network pharmacology, cellular senescence, epithelial-mesenchymal transition