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Chengxi Zhang,1,2 Xiaoqian Li,1,2 Dan Gao,1,2 Huahe Zhu,3 Shun Wang,1,2 Bo Tan,4 Aidong Yang1,2 1School of Traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, People’s Republic of China; 2Center for Traditional Chinese Medicine and Epidemic Disease, Shanghai Institute of Infectious Disease and Biosecurity, Shanghai, People’s Republic of China; 3Department of Integrative Medicine, Huashan Hospital, Fudan University, Shanghai, People’s Republic of China; 4Laboratory of Clinical Pharmacokinetics, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai, People’s Republic of ChinaCorrespondence: Aidong Yang; Bo Tan, Tel +86-21-135 6482 0109 ; +86-21-137 6418 9001, Fax +86-21-51322141 ; +86-21-20256699, Email aidongy@126.com; tbot@163.comPurpose: Tingli Dazao Xiefei Decoction (TDXD) is a Traditional Chinese Medicine (TCM) formula used to treat acute lung injury (ALI). However, the precise mechanism of TDXD in treating ALI remains unclear. We investigated the therapeutic mechanism of TDXD against ALI using a complementary approach combining network pharmacology, molecular docking, and in vitro and in vivo experiments.Material and Methods: Potential drug targets of TDXD and relevant target genes associated with ALI were retrieved from Chinese medicines and disease genes databases. Bioinformatics technology was employed to screen potential active ingredients and core targets. Validation experiments were conducted using a lipopolysaccharide (LPS)-induced ALI mouse (C57BL/6J) model, LPS-induced inflammatory RAW264.7 cells, and molecular docking between active compounds of TDXD and potential targets.Results: Network pharmacology suggested that the mechanism of TDXD against ALI involved phosphoinositide 3-kinase (PI3K) / protein kinase B (AKT) / phosphatase and tensin homolog (PTEN) and Janus kinase 2 (JAK2) / signal transducer and activator of transcription 3 (STAT3) pathways. Quercetin, β-sitosterol, kaempferol, isorhamnetin, and L-stepholidine were identified as the main active compounds of TDXD that exerted anti-ALI effects. Molecular docking indicated that these compounds exhibited good binding capabilities (≤ − 5kcal/mol) to key targets in PI3K/AKT/PTEN and JAK2/STAT3 signaling pathways. In the animal model, TDXD alleviated injuries and inflammatory responses in lung tissues, accompanied by inhibition of expression of tumor necrosis factor-α (TNF-α), Interleukin-6 (IL-6), STAT3, and Suppressor of Cytokine Signaling 3 (SOCS3) mRNA, and key proteins in PI3K/AKT/PTEN and JAK2/STAT3 pathways (all P values < 0.05). Cell based experiments showed that TDXD dose-dependently inhibited the expression of essential proteins in PI3K/AKT/PTEN and JAK2/STAT3 pathways (P < 0.05).Conclusion: This study revealed that the mechanism of TDXD in ALI treatment might involve simultaneous regulation of PI3K/AKT/PTEN and JAK2/STAT3 pathways.Keywords: network pharmacology, molecular docking, lipopolysaccharide, PI3K/AKT/PTEN, JAK2/STAT3

Yilun Lu,1– 3,&ast; Ding Li,1– 3,&ast; Yueyue Huang,1– 3 Yuanyuan Sun,1– 3 Hongmin Zhou,1– 3 Fanrong Ye,1– 3 Hongjing Yang,1– 3 Tingting Xu,1– 3 Shichao Quan,3– 6 Jingye Pan1– 5 1Department of Intensive Care Unit, the First Affiliated Hospital of Wenzhou Medical University, Wenzhou, People’s Republic of China; 2Key Laboratory of Intelligent Treatment and Life Support for Critical Diseases of Zhejiang Province, Wenzhou, People’s Republic of China; 3Wenzhou Key Laboratory of Critical Care and Artificial Intelligence, Wenzhou, People’s Republic of China; 4Collaborative Innovation Center for Intelligence Medical Education, Wenzhou, People’s Republic of China; 5Zhejiang Engineering Research Center for Hospital Emergency and Process Digitization, Wenzhou, People’s Republic of China; 6Department of General Medicine, the First Affiliated Hospital of Wenzhou Medical University, Wenzhou, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Jingye Pan, Department of Intensive Care Unit, the First Affiliated Hospital of Wenzhou Medical University, Wenzhou, People’s Republic of China, Email wmupanjingye@126.com Shichao Quan, Department of General Medicine, the First Affiliated Hospital of Wenzhou Medical University, Wenzhou, People’s Republic of China, Email generalpractice@qq.comPurpose: Sepsis is an aggressive and life-threatening organ dysfunction induced by infection. Excessive inflammation and coagulation contribute to the negative outcomes for sepsis, resulting in high morbidity and mortality. In this study, we explored whether Eupatilin could alleviate lung injury, reduce inflammation and coagulation during sepsis.Methods: We constructed an in vitro sepsis model by stimulating RAW264.7 cells with 1 μg/mL lipopolysaccharide (LPS) for 6 hours. The cells were divided into control group, LPS group, LPS+ Eupatilin (Eup) group, and Eup group to detect their cell activity and inflammatory cytokines and coagulation factor levels. Cells in LPS+Eup and Eup group were pretreated with Eupatilin (10μM) for 2 hours. In vivo, mice were divided into sham operation group, cecal ligation and puncture (CLP) group and Eup group. Mice in the CLP and Eup groups were pretreated with Eupatilin (10mg/kg) for 2 hours by gavage. Lung tissue and plasma were collected and inflammatory cytokines, coagulation factors and signaling were measured.Results: In vitro, tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and tissue factor (TF) expression in LPS-stimulated RAW264.7 cells was downregulated by Eupatilin (10μM). Furthermore, Eupatilin inhibited phosphorylation of the JAK2/STAT3 signaling pathway and suppressed p-STAT3 nuclear translocation. In vivo, Eupatilin increased the survival rate of the mice. In septic mice, plasma concentrations of TNF-α, IL-1β and IL-6, as well as TF, plasminogen activator inhibitor 1 (PAI-1), D-dimer, thrombin-antithrombin complex (TAT) and fibrinogen were improved by Eupatilin. Moreover, Eupatilin alleviated lung injury by improving the expression of inflammatory cytokines and TF, fibrin deposition and macrophage infiltration in lung tissue.Conclusion: Our results revealed that Eupatilin may modulate inflammation and coagulation indicators as well as improve lung injury in sepsis via the JAK2/STAT3 signaling pathway.Keywords: Eupatilin, sepsis, inflammation, coagulation, JAK2/STAT3

Xinmei Yang,1– 3 Xijun Wu,4 Xiaosen Wu,5 Lei Huang,1,2 Jingrui Song,1,2 Chunmao Yuan,1,2 Zhixu He,3,6 Yanmei Li1,2 1State Key Laboratory for Functions and Applications of Medicinal Plants, Guizhou Medical University, Guiyang, 550014, People’s Republic of China; 2The Key Laboratory of Chemistry for Natural Products of Guizhou Province and Chinese Academy of Sciences, Guizhou Medical University, Guiyang, 550014, People’s Republic of China; 3Stem Cell and Tissue Engineering Research Center, Guizhou Medical University, Guiyang, 550004, People’s Republic of China; 4Department of Laboratory, The Affiliated Jinyang Hospital of Guizhou Medical University, Guiyang, 550023, People’s Republic of China; 5FuRong Tobacco Research Station, Xiangxi Autonomous Prefecture Tobacco Company Yongshun Branch, Yongshun, 416700, People’s Republic of China; 6Department of Pediatrics, Affiliated Hospital of Guizhou Medical University, Guiyang, 550001, People’s Republic of ChinaCorrespondence: Yanmei Li, State Key Laboratory for Functions and Applications of Medicinal Plants, Guizhou Medical University, Guiyang, 550014, People’s Republic of China, Tel/Fax +86 85183805081, Email liyanmei518@hotmail.com Zhixu He, Stem Cell and Tissue Engineering Research Center, Guizhou Medical University, Guiyang, 550004, People’s Republic of China, Tel/Fax +86 13595019670, Email hzx@gmc.edu.cnPurpose: Chronic myelogenous leukemia (CML) is a hematological malignancy with increased proliferation of cells of the myeloid series. This can disrupt normal hematopoiesis. The 1-(2-(dimethylamino)acetyl)-rocaglaol (MQ-16) is a new synthetic flavagline compound that showed promising activity in chronic myeloid leukemia K562 cells. This study aims to analyze the underlying mechanisms of MQ-16 against CML.Methods: Growth, cell cycle progression, and apoptosis were assessed in K562 cells following MQ-16 exposure by MTT assay and flow cytometry. The effect of MQ-16 on DNA strands between nucleosomes was examined by 1% agarose gel electrophoresis. PI3K/Akt/mTOR, JAK2/STAT3, and mitogen-activated protein kinase (MAPK) pathway-related proteins were detected in MQ-16–treated K562 cells by Western blot.Results: MQ-16 significantly inhibited the proliferation of K562 cells and arrested the cell cycle at the G2/M phase in a time- and concentration-dependent manner. MQ-16 induced mitochondria-dependent apoptosis by downregulating the anti-apoptotic proteins Bcl-2 and Bcl-xL and induced time- and concentration-dependent DNA fragmentation. In addition, MQ-16 affected the expression of PI3K/Akt/mTOR, JAK2/STAT3, and MAPK pathway-related proteins.Conclusion: In summary, MQ-16 appears to be a promising chemotherapeutic drug for treating CML.Keywords: CML, flavagline, cycle arrest, apoptosis, PI3K/Akt/mTOR, JAK2/STAT3, MAPK

Fangxiao Cheng,1 Geheng Yuan,1 Jiao He,2 Yimin Shao,1 Junqing Zhang,1 Xiaohui Guo1 1Department of Endocrinology, Peking University First Hospital, Beijing 100034, People’s Republic of China; 2Department of Endocrinology, Baoding First Central Hospital, Baoding 071000, Hebei Province, People’s Republic of ChinaCorrespondence: Geheng Yuan; Xiaohui GuoDepartment of Endocrinology, Peking University First Hospital, No. 8 Xishiku Street, Xicheng District, Beijing 100034, People’s Republic of ChinaTel|Fax +86 010 83575103Email 139197109@qq.com; bdyyguoxiaohui@sina.comPurpose: Dipeptidyl peptidase 4 (DPP4) is one of the newly identified adipokines, which acts as paracrine in adipose tissue and as endocrine hormones in the liver, muscles and central nervous system. Expression of DPP4 was significantly upregulated in obese patients upon insulin resistance (IR) conditions, but the mechanism underlying the dysregulation of DPP4 remains unclear. This study aimed to investigate the DPP4 expression in adipose tissue and adipocytes under IR conditions or with liraglutide intervention, and explore the potential molecular mechanisms.Methods: Obesity-associated IR animal and cell models were, respectively, constructed by using high-fat diet and palmitic acid (PA) stimulation. Expression of DPP4 in adipose tissues and adipocytes was estimated by quantitative real-time RT-PCR and Western-blot. Effects of the AMPK/JAK2/STAT3 pathway on DPP4 were examined by regulating the activity of AMPK and the JAK2/STAT signaling. The therapeutic efficacy of liraglutide in the IR models was evaluated, and its regulatory effects on DPP4 expression and the underlying molecular mechanisms were explored.Results: The expression of DPP4 was markedly upregulated in both the animal and cell IR models. In the adipocyte, DPP4 expression was found to be suppressed by the activation of AMPK, and this inhibition effect was mediated by the JAK2/STAT3 signaling. Moreover, liraglutide could alleviate the obesity-induced IR, and led to the downregulation of DPP4 in IR animal and cell models. Liraglutide intervention resulted in the activation of AMPK and deactivation of the JAK2/STAT3 signaling in the adipocytes.Conclusion: Taken together, the expression of DPP4 is upregulated in adipose tissues and adipocytes upon IR conditions, but is reduced after liraglutide intervention. The dysregulation of DPP4 in the adipocytes may be performed by the AMPK/JAK2/STAT3 pathway.Keywords: obesity, adipocyte, DPP4, AMPK, JAK2/STAT3, liraglutide, insulin resistance

Xiangjun Guo,1,2 Zhongyi Yan,2 Gongming Zhang,2 Xiang Wang,2 Yun Pan,2 Mao Huang1 1Department of Respiratory and Critical Care Medicine, The First Affiliated Hospital of Nanjing Medical University, Nanjing, People’s Republic of China; 2Department of Respiratory and Critical Care Medicine, The First People’s Hospital of Lianyungang, Lianyungang, Jiang su, People’s Republic of ChinaCorrespondence: Mao HuangDepartment of Respiratory and Critical Care Medicine, The First Affiliated Hospital of Nanjing Medical University, Nanjing, People’s Republic of ChinaTel +86 1381388616Email huangmao6116@126.comPurpose: Recent studies have shown that STIP1 is associated with proliferation and migration in numerous types of tumors; however, the role of STIP1 in lung adenocarcinoma is still poorly understood. Therefore, the aim of this study was to evaluate the role of STIP1 in lung adenocarcinoma, in vitro and in vivo.Methods: The expression of STIP1 in lung adenocarcinoma was assessed by immunohistochemistry, RT-qPCR, and Western blot. The effects of STIP1 on the proliferation of lung adenocarcinoma cells were detected by the cell counting kit-8 assay; the effect of STIP1 on adhesion of lung adenocarcinoma cells was detected by Giemsa staining, while the cell scratch and Transwell assays were employed to examine the effect of STIP1 on the migratory ability of lung adenocarcinoma cells. Finally, apoptosis was evaluated by Hoechst staining and flow cytometry.Results: The expression level of STIP1 in lung adenocarcinoma tissue was significantly higher than that in adjacent normal tissue (P

Ying Wang,1 Meng Xue,1 Yangyang Xia,1 Qian Jiang,1 Zhihua Huang,1,2 Cheng Huang1,21Department of Physiology, Gannan Medical University, Ganzhou 341000, People’s Republic of China; 2Pain Medicine Research Institute, Gannan Medical University, Ganzhou 341000, People’s Republic of ChinaBackground: Neuropathic pain with complicated mechanism severely disrupts patient quality of life. The novel approaches and more effective management should be further investigated. It was reported that alpha-7 nicotinic acetylcholine receptor (α7nAChR) and janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling in dorsal root ganglion (DRG) contributed to the pathogenesis of neuropathic pain. Our previous study has shown that electroacupuncture (EA) alleviated neuropathic pain via activating α7nAChR in the spinal cord. However, whether the effect of 2 Hz EA on spared nerve injury (SNI)-induced neuropathic pain is mediated through modulation of α7nAChR and JAK2/STAT3 pathway in the DRG remains unclear.Materials and methods: The SNI-induced neuropathic pain rat model was used in this study. After application of 2 Hz EA treatment to SNI rats on day 3, 7, 14 and 21 post-surgery, the expression levels of α7nAChR, JAK2/STAT3 and some cytokines in DRG were determined by qRT-PCR and Western blot analysis.Results: We found that SNI induced significant down-regulation of α7nAChR mRNA and protein expression. SNI also obviously elicited the decrease in anti-inflammatory cytokine IL-10 protein expression. The enhancement of p-JAK2, p-STAT3, pro-inflammatory cytokines IL-1β and IL-6 protein levels induced by SNI were also observed. However, 2 Hz EA treatment to SNI rats distinctly improved α7nAChR and IL-10 levels and reduced p-JAK2, p-STAT3, IL-1β and IL-6 expression in the DRG.Conclusion: Our present study suggested that 2 Hz EA treatment indeed activated α7nAChR, suppressed JAK2/STAT3 signaling and re-balanced the relationship between pro-inflammatory and anti-inflammatory cytokines in DRG of SNI rat, which provided insight into our understanding of the mechanism for 2 Hz EA to attenuate neuropathic pain.Keywords: neuropathic pain, electroacupuncture, α7nAChR, JAK2/STAT3, dorsal root ganglion

Fayong Ke,1,2,* Zheng Wang,1,2,* Xiaoling Song,1,2 Qiang Ma,1,2 Yunping Hu,2 Lin Jiang,2 Yijian Zhang,2 Yingbin Liu,1,2 Yong Zhang,1 Wei Gong1,2 1Department of General Surgery, Xinhua Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, People’s Republic of China; 2Institute of Biliary Disease Research, School of Medicine, Shanghai Jiao Tong University, Shanghai, People’s Republic of China *These authors contributed equally to this work Background: Cholangiocarcinoma (CCA) is the most common biliary tract malignancy in the world with high resistance to current chemotherapies and extremely poor prognosis. The main objective of this study was to investigate the inhibitory effects of cryptotanshinone (CTS), a natural compound isolated from Salvia miltiorrhiza Bunge, on CCA both in vitro and in vivo and to explore the underlying mechanisms of CTS-induced apoptosis and cell cycle arrest. Methods: The anti-tumor activity of CTS on HCCC-9810 and RBE cells was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay and colony forming assays. Cell cycle changes were detected by flow cytometric analysis. Apoptosis was detected by annexin V/propidium iodide double staining and Hoechst 33342 staining assays. The efficacy of CTS in vivo was evaluated using a HCCC-9810 xenograft model in athymic nude mice. The expression of key proteins involved in cell apoptosis and signaling pathway in vitro was analyzed by Western blot analysis. Results: CTS induced potent growth inhibition, S-phase arrest, apoptosis, and colony-forming inhibition in HCCC-9810 and RBE cells in a dose-dependent manner. Intraperitoneal injection of CTS (0, 10, or 25 mg/kg) for 4 weeks significantly inhibited the growth of HCCC-9810 xenografts in athymic nude mice. CTS treatment induced S-phase arrest with a decrease of cyclin A1 and an increase of cyclin D1 protein level. Bcl-2 expression was downregulated remarkably, while Bax expression was increased after apoptosis occurred. Additionally, the activation of JAK2/STAT3 and PI3K/Akt/NFκB was significantly inhibited in CTS-treated CCA cells. Conclusion: CTS induced CCA cell apoptosis by suppressing both the JAK2/STAT3 and PI3K/Akt/NFκB signaling pathways and altering the expression of Bcl-2/Bax family, which was regulated by these two signaling pathways. CTS may serve as a potential therapeutic agent for CCA. Keywords: cholangiocarcinoma, cryptotanshinone, apoptosis, JAK2/STAT3, PI3K/Akt/NFκB

Objective: Diabetes mellitus (DM) implicates oxidative stress, apoptosis, and inflammation, all of which may contribute liver injury. Aerobic exercise is assured to positively regulate metabolism in the liver. This project was designed to investigate whether and how aerobic exercise improves DM-induced liver injury., Methods: Seven-week-old male db/db mice and age-matched m/m mice were randomly divided into a rest control group or a group that received 12 weeks of aerobic exercise by treadmill training (10 m/min). Haematoxylin and eosin (HE) staining, electron microscopy, Oil Red O staining and TUNEL assays were used to evaluate the histopathological changes in mouse liver. The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TRIG), cholesterol (CHOL) were analyzed by serum biochemical analysis. Interleukin-6 (IL-6), tumour necrosis factor-α (TNF-α), and tissue levels of malondialdehyde (MDA) and superoxide dismutase (SOD) were analyzed via ELISA. Nuclear factor E2-associated factor-2 (Nrf2), nuclear factor κB (NF-κB) and JAK2/STAT3 pathway-related proteins were measured by immunofluorescence, Western blotting and q-PCR. F4/80 expression in liver tissues was assessed by immunohistochemistry., Results: In diabetic mice, exercise training significantly decreased the levels of serum TRIG, CHOL, IL-6, TNF-α, ALT and AST; prevented weight gain, hyperglycaemia, and impaired glucose and insulin tolerance. Morphologically, exercise mitigated the diabetes-induced increase in liver tissue microvesicles, inflammatory cells, F4/80 (macrophage marker) levels, and TUNEL-positive cells. In addition, exercise reduced the apoptosis index, which is consistent with the results for caspase-3 and Bax. Additionally, exercise significantly increased SOD activity, decreased MDA levels, activated Nrf2 and decreased the expression of NF-kB, phosphorylated JAK2 and STAT3 proteins in the livers of diabetic mice., Conclusion: This study demonstrated that aerobic exercise reversed liver dysfunction in db/db mice with T2DM by reducing oxidative stress, apoptosis and inflammation, possibly by enhancing Nrf2 expression and inhibiting the JAK2/STAT3 cascade response., Competing Interests: The authors declare no conflicts of interest in this work., (© 2023 Sun et al.)