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Background: A balanced protein homeostasis network helps cholangiocarcinoma (CCA) maintain their oncogenic growth, and disrupting proteostasis therapeutically will induce proteotoxic stress. Phosphatase and tensin homolog (PTEN) have been reported to be involved in proteostasis, and PTEN-associated pathways are commonly altered in CCA. Celastrol, a triterpene from plants, exhibits cytotoxic effects in various types of cancer. However, the underlying mechanisms remain unclear., Purpose: We investigated the therapeutic effect of celastrol in CCA and identified the molecular characteristics of tumors that were sensitive to celastrol. The target of celastrol was explored. We then evaluated the candidate combination therapeutic strategy to increase the effectiveness of celastrol in celastrol-insensitive CCA tumors., Methods: Various CCA cells were categorized as either celastrol-sensitive or celastrol-insensitive based on their response to celastrol. The molecular characteristics of cells from different groups were determined by RNA-seq. PTEN status and its role in proteasome activity in CCA cells were investigated. The CMAP analysis, molecular docking, and functional assay were performed to explore the effect of celastrol on proteasome activities. The correlation between PTEN status and clinical outcomes, as well as proteasomal activity, were measured in CCA patients. The synergistic therapeutic effect of autophagy inhibitors on celastrol-insensitive CCA cells were measured., Results: Diverse responses to celastrol were observed in CCA cells. PTEN expression varied among different CCA cells, and its status could impact cell sensitivity to celastrol: PTEN high tumor cells were resistant to celastrol, while PTEN low cells were more sensitive. Celastrol induced proteasomal dysregulation in CCA cells by directly targeting PSMB5. Cells with low PTEN status transcriptionally promoted proteasome subunit expression in an AKT-dependent manner, making these cells more reliant on proteasomal activities to maintain proteostasis. This caused the PTEN low CCA cells sensitive to celastrol. A negative correlation was found between PTEN levels and the proteasome signature in CCA patients. Moreover, celastrol treatment could induce autophagy in PTEN high CCA cells. Disrupting the autophagic pathway in PTEN high CCA cells enhanced the cytotoxic effect of celastrol., Conclusion: PTEN status in CCA cells determines their sensitivity to celastrol, and autophagy inhibitors could enhance the anti-tumor effect in PTEN high CCA., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

Objective: To explore the possible regulatory mechanism of microRNA (miRNA) in moxibustion treatment for decreased ovarian reserve (DOR)., Methods: The DOR model was constructed by intragastrical Tripterygium glycoside suspension administration, and moxibustion therapy was simultaneously given. The morphological ovarian changes were observed by hematoxylin and eosin staining. The miRNA expression profile was detected by RNA sequencing, and bioinformatics analysis was performed. Cytoscape software 3.6.1 was used to establish a regulatory network and differentially expressed miRNAs were verified by reverse transcription quantitative polymerase chain reaction (RT-qPCR)., Results: Decreased number of mature follicles, increased atresia follicles, and abnormal granulosa cell morphology were observed in the model group compared with the control group. The moxibustion group demonstrated increased mature follicles, decreased atretic follicles, and significantly decreased abnormal morphology of granulosa cells compared with the model group. Additionally, RNA sequencing results manifested significantly up-regulated miRNA expressions (miR-92b-3p, miR-26-5p_R + 1_1ss10TC, miR-206-3p, miR-9993b-3p_1ss6GA, miR-7857-3p_R-1, miR-219a-2-3p_1ss10GC, miR-3968-p5_1ss10AT, and PC-5p-6478_1795) and down-regulated miR-664-2-5p_R + 1 in the model group, compared with the control group, and the moxibustion group reversed abnormal disorder levels of these miRNAs. Moreover, these differentially expressed miRNAs were mainly involved in the phosphatidylinositol-3-kinase / protein kinase B signaling pathway and nuclear factor erythropoietin-2-related factor 2 / heme oxygenase 1 signaling pathway. Finally, network and RT-qPCR verification revealed miR-9993b-3p_1ss6GA as the most critical miRNA., Conclusion: This experiment proved the effectiveness of moxibustion in improving the ovarian reserve of rats by regulating miRNA expression, especially miR-9993b-3p_1ss6GA.

Background: Chronic myeloid leukemia (CML) is driven primarily by the constitutively active BCR-ABL fusion oncoprotein. Although the development of tyrosine kinase inhibitors has markedly improved the prognosis of CML patients, it remains a significant challenge to overcome drug-resistant mutations, such as the T315I mutation of BCR-ABL, and achieve treatment-free remission in the clinic., Purpose: The identification of new intervention targets beyond BCR-ABL could provide new perspectives for future research and therapeutic intervention. A network pharmacology analysis was conducted to identify the most promising natural product with anti-CML activity. Celastrol was selected for further analysis to gain insights into its mechanism of action (MoA), with the aim of identifying potential new intervention targets for BCR-ABL T315I-mutant CML., Methods: Transcriptomic and proteomic analyses were conducted to systematically investigate the molecular MoA of celastrol in K562 T315I cells. To identify the target proteins of celastrol, mass spectrometry-coupled cellular thermal shift assay (MS-CETSA) was carried out, followed by validations with genetic knockdown and overexpression, cell proliferation assay, comet assay, Western blotting, celastrol probe-based in situ labeling and pull-down assay, molecular docking, and biolayer interferometry., Results: Our multi-omics analyses revealed that celastrol primarily induces DNA damage accumulation and the unfolded protein response in K562 T315I cells. Among the twelve most potential celastrol targets, experimental evidence demonstrated that the direct interaction of celastrol with YY1 and HMCES increases the levels of DNA damage, leading to cell death., Conclusion: This study represents the first investigation utilizing a proteome-wide label-free target deconvolution approach, MS-CETSA, to identify the protein targets of celastrol. This study also develops a new systems pharmacology strategy. The findings provide new insights into the multifaceted mechanisms of celastrol and, more importantly, highlight the potential of targeting proteins in DNA damage and repair pathways, particularly YY1 and HMCES, to combat drug-resistant CML., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

The long-term use of tripterygium glycosides (TG) can lead to male reproductive damage. Research indicates that zinc and selenium exhibit a synergistic effect in the male reproductive system, with the combined preparation demonstrating superior therapeutic effects compared to individual preparations. The purpose of this study was to explore the specific mechanism by which zinc and selenium mitigate reproductive toxicity induced by TG in male rats. Rats were randomly assigned to three groups: control group (C group), model group (M group, receiving TG at 30 mg/kg/day), and model + zinc + selenium group (ZS group). The ZS group was also given TG gavage for the first 4 weeks. Starting from the fifth week until the conclusion of the eighth week, the ZS group received an additional protective treatment of 10 mg/kg/day Zn and 0.1 mg/kg/day Se 4 h after TG administration. Following euthanasia, blood samples, rat testis, and epididymis tissues were collected for further experiments. Combined zinc-selenium treatment corrects the imbalance of zinc-selenium homeostasis in testicular tissue induced by TG. This is achieved by upregulating the expression of metal transcription factor (MTF1) and zinc transporters ZIP8 and ZIP14 and downregulating the expression of ZnT10. Improvement of zinc and selenium homeostasis enhanced the expression of zinc-containing enzymes (ADH, LDH, and ALP) and selenoproteins (GPx1 and SELENOP) in the testis. At the same time, zinc and selenium mitigate TG-induced reproductive damage by promoting the activity of antioxidant enzymes and upregulating the expression of proteins associated with the oxidative stress pathway, including Nrf2, Keap1, HO-1, PI3K, and p-AKT., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Oligoasthenozoospermia is the most common type of semen abnormality in male infertile patients. Betaine (BET) has been proved to have pharmacological effects on improving semen quality. BET also belongs to endogenous physiological active substances in the testis. However, the physiological function of BET in rat testis and its pharmacological mechanism against oligoasthenozoospermia remain unclear., Purpose: This research aims to prove the therapeutic effect and potential mechanism of BET on oligoasthenozoospermia rat model induced by Tripterygium wilfordii glycosides (TWGs)., Methods: The oligoasthenozoospermia rat model was established by a continuous gavage of TWGs (60 mg/kg) for 28 days. Negative control group, oligoasthenozoospermia group, positive drug group (levocarnitine, 300 mg/kg), and 200 mg/kg, 400 mg/kg, and 800 mg/kg BET groups were created for exploring the therapeutic effect of BET on the oligoasthenozoospermia rat model. The therapeutic effect was evaluated by HE and TUNEL staining. Immunofluorescence assay of DNMT3A, PIWIL1, PRMT5, SETDB1, BHMT2, and METTL3, methylation capture sequencing, Pi-RNA sequencing, and molecular docking were used to elucidate potential pharmacological mechanisms., Results: It is proved that BET can significantly restore testicular pathological damage induced by TWGs, which also can significantly reverse the apoptosis of spermatogenic cells. The spermatogenic cell protein expression levels of DNMT3A, PIWIL1, PRMT5, SETDB1, BHMT2, and METTL3 significantly decreased in oligoasthenozoospermia group. 400 mg/kg and 800 mg/kg BET groups can significantly increase expression level of the above-mentioned proteins. Methylation capture sequencing showed that BET can significantly increase the 5mC methylation level of Spata, Spag, and Specc spermatogenesis-related genes. Pi-RNA sequencing proved that the above-mentioned genes produce a large number of Pi-RNA under BET intervention. Pi-RNA can form complexes with PIWI proteins to participate in DNA methylation of target genes. Molecular docking indicated that BET may not directly act as substrate for methyltransferase and instead participates in DNA methylation by promoting the methionine cycle and increasing S-adenosylmethionine synthesis., Conclusion: BET has a significant therapeutic effect on oligoasthenozoospermia rat model induced by TWPs. The mechanism mainly involves that BET can increase the methylation level of Spata, Specc, and Spag target genes through the PIWI/Pi-RNA pathway and up-regulation of methyltransferases (including DNA methyltransferases and histone methyltransferases)., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

Celastrol (Cel), extracted from Tripterygium wilfordii Hook , is a potential antiobesity drug, except for its adverse reactions in clinic. In the present study, we synthesized a promising celastrol-chitosan conjugate (Cel-CS1K) and evaluated its antiobesity effect and biological safety in diet-induced obese mice. Cel-CS1K showed higher drug loading (over 10 wt %), good solubility (18-19 mg/mL) in water, slower peak time ( T max = 4 h), and clearance ( T 1/2 = 8.97 h) in rats. Cel-CS1K effectively attenuated the cytotoxicity, celastrol-induced apoptosis, and fat accumulation of hepatocytes. Cel-CS1K reduced body weight and dietary amount same as the free Cel but with lower toxicity in blood, liver, and testis. Cel-CS1K improved the glucose homeostasis, HDL-C level, insulin sensitivity, and leptin sensitivity, while it significantly reduced the gene expression levels of LDL-C, TG, and TC in obese mice. Furthermore, the adipose-related gene expression levels provided evidence in support of a role for Cel-CS1K in losing weight by the multimode regulation. Overall, Cel-CS1K provides a translatable therapeutic strategy for the treatment of diet-induced obese humans.

Asthenospermia is a predominant cause of male infertility, and antioxidant supplements can be effective in treating asthenospermia. We demonstrate the antioxidant potential of traditional Chinese medicine, the Yishenhuoxue (YSHX) formula, in treating polyglycosides of Tripterygium wilfordii (GTW)-induced asthenospermia in rats. Fifty male rats were randomly divided into the normal, model, and treatment groups. HE staining was used to evaluate the improvement of spermatogenic function of rats, and TBA reaction, qRT-PCR, Western Blot and other methods were used to determine the changes of oxidative stress indicators and to evaluate the improvement of antioxidant capacity of rats by YSHX. Comparison with the model group showed significant improvement in pathological damage caused by GTW to seminiferous tubules. MDA and NO content in rat testes decreased, especially in middle- and high-dosage groups. No significant changes were observed in SOD and CAT activity or mRNA expression. GSH-Px activity and GSH mRNA expression were significantly higher in the low-dosage group than in the model group. Compared to the model group, GR activity was significantly lower in the middle and high dosage groups, while the mRNA expression was higher. The PKC-beta level increased, while p-ERK1/2, NF-κB, and the ratio of p-ERK1/2*(ERK1/2)-1 decreased significantly in the treatment groups. Therefore, YSHX can alleviate GTW-induced testicular damage, enhance GSH-Px activity, regulate GSH redox cycling, and mitigate oxidative stress injury. Furthermore, YSHX can promote PKC-beta expression and inhibit the phosphorylation of ERK1/2 and NF-κB. Using YSHX may be an effective way to increase sperm motility via the PKC-ERK1/2-NF-ĸB axis.

Tripterygium glycosides (TG) is extracted from the roots of Chinese herbal medicine named Tripterygium wilfordii Hook F (TwHF). TG tablets are the representative TwHF-based agents with anti-inflammatory and immunomodulatory activities for treating rheumatoid arthritis. Although the curative effect of TG is remarkable, the clinical application is limited by a variety of organ toxicity. One of the most serious side-effects induced by TG is damage of the male reproductive system and the toxic mechanism is still not fully elucidated. TG-induced testicular injury was observed in male mice by treated with different concentrations of TG. The results showed that TG induced a significant decrease in testicular index. Pathological observation showed that spermatogenic cells were obviously shed, arranged loosely, and the spermatogenic epithelium was thin compared with control mice. In addition, the toxic effect of TG on mouse spermatogonia GC-1 cells was investigated. The results displayed that TG induced significant cytotoxicity in mouse GC-1 cells. To explore the potential toxic components that triggered testicular injury, the effects of 8 main components of TG on the viability of GC-1 cells were detected. The results showed that celastrol was the most toxic component of TG to GC-1 cells. Western blot analysis showed that LC3-II and the ratio of LC3-II/LC3-I were significantly increased and the expression level of p62 were decreased in both TG and celastrol treated cells, which indicated the significant activation of autophagy in spermatogonia cells. Therefore, autophagy plays an important role in the testicular injury induced by TG, and inhibition of autophagy is expected to reduce the testicular toxicity of TG., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Tripterygium glycosides (TG) can seriously damage male reproductive function, and the reproductive system is difficult to restore after stopping the administration of TG in male rats. Zinc (Zn) is one of the most important trace elements in the human body and plays an important role in maintaining male fertility. The aim of this study was to investigate whether zinc supplementation could improve the testicular reproductive damage induced by TG toxicity in rats and to investigate its mechanism of action. The results showed that zinc sulfate (ZnSO 4 ) could improve testicular tissue structure and semen parameters, promote testosterone synthesis, increase zinc-containing enzyme activity, increase zinc concentration in serum and testicular tissues, and maintain zinc homeostasis in male rats induced by TG toxicity. Zinc supplementation activated relevant signalling molecules in the KEAP1-NRF2/ARE pathway and alleviated TG-induced oxidative stress. Therefore, this study concluded that zinc supplementation could improve reproductive damage by regulating zinc homeostasis and the expression of genes related to oxidative stress., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Objective: To explore the inhibitory effect of Sidaxue , a traditional Miao herbal medicine formula, on articular bone and cartilage destruction and synovial neovascularization in rats with collagen-induced arthritis (CIA)., Methods: In a SD rat model of CIA, we tested the effects of daily gavage of Sidaxue at low, moderate and high doses (10, 20, and 40 g/kg, respectively) for 21 days, with Tripterygium glycosides (GTW) as the positive control, on swelling in the hind limb plantar regions by arthritis index scoring. Pathologies in joint synovial membrane of the rats were observed with HE staining, and serum TNF-α and IL-1β levels were detected with ELISA. The expressions of NF-κB p65, matrix metalloproteinase 1 (MMP1), MMP2 and MMP9 at the mRNA and protein levels in the synovial tissues were detected using real-time PCR and Western blotting. Network pharmacology analysis was conducted to identify the important target proteins in the pathways correlated with the therapeutic effects of topical Sidaxue treatment for RA, and the core target proteins were screened by topological analysis., Results: Treatment with GTW and Sidaxue at the 3 doses all significantly alleviated plantar swelling, lowered arthritis index scores, improved cartilage and bone damage and reduced neovascularization in CIA rats ( P <0.05), and the effects of Sidaxue showed a dose dependence. Both GTW and Sidaxue treatments significantly lowered TNF-α, IL-1β, NF-κB p65, MMP1, MMP2, and MMP9 mRNA and protein expressions in the synovial tissues of CIA rats ( P <0.05). Network pharmacological analysis identified MMPs as the core proteins associated with topical Sidaxue treatment of RA., Conclusion: Sidaxue alleviates articular bone and cartilage damages and reduces synovial neovascularization in CIA rats possibly by downregulating MMPs via the TNF-α/IL-1β/NF-κB-MMP1, 2, 9 signaling pathway, and MMPs probably plays a key role in mediating the effect of Sidaxue though the therapeutic pathways other than oral administration.

Background: The sustainable supply of medicinal plants is important, and cultivating and domesticating them has been suggested as an optimal strategy. However, this can lead to a loss of genetic diversity. Tripterygium wilfordii Hook. f. is a medicinal plant commonly used in traditional Chinese medicine, but its wild populations are dwindling due to excessive harvesting. To protect the species and meet the increasing demand, it is urgent to cultivate it on a large scale. However, distinguishing between T. wilfordii and T. hypoglaucum, two similar species with different medicinal properties, is challenging. Therefore, it is crucial to understand the genetic diversity and population structure of these species for their sustainable utilization., Results: In this study, we investigated the genetic diversity and population structure of the two traditional medicinal semiwoody vines plant species, Tripterygium wilfordii and T. hypoglaucum, including wild and cultivated populations using chloroplast DNA (cpDNA) sequences and microsatellite loci. Our results indicated that the two species maintain a high level of genetic divergence, indicating possible genetic bases for the different contents of bioactive compounds of the two species. T. wilfordii showed lower genetic diversity and less subdivided population structures of both markers than T. hypoglaucum. The potential factors in shaping these interesting differences might be differentiated pollen-to-seed migration rates, interbreeding, and history of population divergence. Analyses of cpDNA and microsatellite loci supported that the two species are genetically distinct entities. In addition, a significant reduction of genetic diversity was observed for cultivated populations of the two species, which mainly resulted from the small initial population size and propagated vegetative practice during their cultivation., Conclusion: Our findings indicate significant genetic divergence between T. wilfordii and T. hypoglaucum. The genetic diversity and population structure analyses provide important insights into the sustainable cultivation and utilization of these medicinal plants. Accurate identification and conservation efforts are necessary for both species to ensure the safety and effectiveness of crude drug use. Our study also highlighted the importance of combined analyses of different DNA markers in addressing population genetics of medicinal plants because of the contrasts of inheritance and rates of gene flow. Large-scale cultivation programs should consider preserving genetic diversity to enhance the long-term sustainability of T. wilfordii and T. hypoglaucum. Our study proposed that some populations showed higher genetic diversity and distinctness, which can be considered with priority for conservation and as the sources for future breeding and genetic improvement., (© 2024. The Author(s).)

Huobahua, namely, Tripterygium hypoglaucum (Levl.) Hutch, known as a traditional Chinese herbal medicine, especially its underground parts, has been widely developed into several Tripterygium agents for the treatment of rheumatoid arthritis and other autoimmune diseases. It has sparked wide public concern about its safety, such as multi-organ toxicity. However, the toxic characteristics and damage mechanism of Huobahuagen extract (HBHGE) remain unclear. In the present study, subchronic oral toxicity study of HBHGE (10.0 g crude drug/kg/day for 12 weeks) was performed in male rats. Hematological, serum biochemical, and histopathological parameters, urinalysis, and plasma metabolic profiling were assessed. The single-dose subchronic toxicity results related to HBHGE exhibited obvious toxicity to the testis and epididymis of male rats. Furthermore, plasma metabolomics analysis suggested that a series of metabolic disorders were induced by oral administration of HBHGE, mainly focusing on amino acid (glutamate, phenylalanine, and tryptophan) metabolisms, pyrimidine metabolism, glutathione metabolism, and steroid hormone biosynthesis. Moreover, it appeared that serum testosterone in male rats treated with HBHGE for 12 weeks, decreased significantly, and was susceptible to the toxic effects of HBHGE. Taken together, conventional pathology and plasma metabolomics for preliminarily exploring subchronic toxicity and underlying mechanism can provide useful information about the reduction of toxic risks from HBHGE and new insights into the development of detoxification preparations., (© 2023 John Wiley & Sons Ltd.)

Ethnopharmacological Relevance: Tripterygium glycosides (TG), which are extracted from the traditional Chinese medicine, Tripterygium wilfordii Hook F. (TwHF), has promising applications in the treatment of renal diseases; however, since its active components exerts bidirectional kidney toxicity, its clinical application is severely restricted., Aim of the Study: Recent investigations have demonstrated definite toxicity-reducing effects from glycyrrhizic acid glycosides (GA) when combined with TG; however, the mechanism remains unclear. To our knowledge, this is the first study to investigate the specific molecular mechanism by which GA alleviates TG-induced renal toxicity from the perspective of tight junctions., Materials and Methods: Dynamic analyses, which investigated the changes in kidney toxicity biomarkers for different combinations and concentrations of TG and GA, were conducted for three weeks on SD rats and renal tissue structural changes were examined after three weeks of administration. Additionally, the transcription and translation levels of the relevant tight junctions and RhoA/ROCK1/MLC signalling proteins were analysed in HK-2 cells., Results: Our study showed that TG can cause transient tubulotoxicity at certain doses, and that the combined application of GA and TG can repair tight junction structures by regulating the key factors in the RhoA/ROCK1/MLC signalling pathway, thus reducing TG-induced nephrotoxicity., Conclusions: Overall, this study provides a new strategy to reduce TG-induced toxicity by protecting renal tight junctions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Tripterygium wilfordii has been widely used for the treatment of rheumatoid arthritis, which is frequently accompanied by severe gastrointestinal damage. The molecular mechanism underlying the gastrointestinal injury of Tripterygium wilfordii are yet to be elucidated., Methods: Transmission electron microscopy, and pathological and biochemical analyses were applied to assess intestinal bleeding. Metabolic changes in the serum and intestine were determined by metabolomics. In vivo (time-dependent effect and dose-response) and in vitro (double luciferase reporter gene system, DRATs, molecular docking, HepG2 cells and small intestinal organoids) studies were used to identify the inhibitory role of celastrol on intestinal farnesoid X receptor (FXR) signaling. Fxr-knockout mice and FXR inhibitors and agonists were used to evaluate the role of FXR in the intestinal bleeding induced by Tripterygium wilfordii., Results: Co-treatment with triptolide + celastrol (from Tripterygium wilfordii) induced intestinal bleeding in mice. Metabolomic analysis indicated that celastrol suppressed intestinal FXR signaling, and further molecular studies revealed that celastrol was a novel intestinal FXR antagonist. In Fxr-knockout mice or the wild-type mice pre-treated with pharmacological inhibitors of FXR, triptolide alone could activate the duodenal JNK pathway and induce intestinal bleeding, which recapitulated the pathogenic features obtained by co-treatment with triptolide and celastrol. Lastly, intestinal bleeding induced by co-treatment with triptolide and celastrol could be effectively attenuated by the FXR or gut-restricted FXR agonist through downregulation of the duodenal JNK pathway., Conclusions: The synergistic effect between triptolide and celastrol contributed to the gastrointestinal injury induced by Tripterygium wilfordii via dysregulation of the FXR-JNK axis, suggesting that celastrol should be included in the quality standards system for evaluation of Tripterygium wilfordii preparations. Determining the mechanism of the FXR-JNK axis in intestinal bleeding could aid in the identification of additional therapeutic targets for the treatment of gastrointestinal hemorrhage diseases. This study also provides a new standard for the quality assessment of Tripterygium wilfordii used in the treatment of gastrointestinal disorders., Competing Interests: Declaration of Competing Interest The authors declared no conflict of interest, (Copyright © 2023. Published by Elsevier GmbH.)

Context: Qingluotongbi formula (QLT) is a Chinese medicine compound consisting of Tripterygium wilfordii Hook. f. (Celastraceae, TW), Panax notoginseng (Burkill) F.H.Chen (Araliaceae, PN), Rehmannia glutinosa (Gaertn.) DC. (Orobanchaceae, RG), Sinomenium acutum (Thunb.) Rehder & E.H. Wilson (Menispermaceae, SA), and Bombyx mori L. (Bombycidae, BM)., Objective: This study investigated the protective effect and possible mechanism of QLT against TW-induced liver injury in mice., Materials and Methods: To establish the model of TW-induced liver injury in mice, C57BL/6J mice were randomly divided into 4 groups: control group, low-dose TW group, middle-dose TW group, and high-dose TW group. To observe the effects of QLT and its individual ingredients against TW-induced liver injury, C57BL/6J mice were randomly divided into 7 groups: control group, TW group, QLT group, PN group, RG group, SA group, BM group.After administration for 7 days, C57BL/6J mice were tested for biochemical indicators and liver pathological changes. Then, we evaluated the mitochondrial function and analysed the gene and protein expression related to the peroxisome proliferator-activated receptor alpha (PPARα)/peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) pathway by quantitative real-time PCR (qRT-PCR) and Western blotting., Results: Compared with the control group (0.30 ± 0.35), TW significantly increased mice liver histological score (L, 0.95 ± 1.14; M, 1.25 ± 1.16; H, 4.00 ± 1.13). QLT and its ingredients significantly improved the pathology scores (CON, 0.63 ± 0.74; TW, 4.19 ± 1.53; QLT, 1.56 ± 0.62; PN, 1.94 ± 0.68; RG, 2.75 ± 1.39; SA, 4.13 ± 0.99; BM, 4.13 ± 0.99). Western blot and qRT-PCR analysis revealed that QLT and its ingredients reversed TW-induced suppression of PPARα/PGC1-α pathway.Discussion and conclusions: These findings provide valuable information for compound compatibility studies and TW clinical applications.

Extensive phytochemical investigation of the seeds of Tripterygium wilfordii led to the identification of 54 polyesterified dihydro-β-agarofuran-type sesquiterpenoids, including 27 previously undescribed ones, named Tripwilin I-XXVII (1-27). Comprehensive spectroscopic and single-crystal X-ray diffraction analyses, along with electronic circular dichroism (ECD) calculations were used for the structural elucidation of the new compounds. Biological assay revealed that 37 compounds among the isolates exhibited significant inhibition against osteoclastogenesis induced by receptor activator of nuclear factor-κB ligand (RANKL) at 10 µM. Further investigation indicated that Triptogelin C-3 (54), with the most potent osteoclastogenesis inhibitory activity, regulated the osteoclast marker genes (MMP-9, c-Fos, CTSK, and TRAP) and proteins in a dose-dependent manner in vitro. Besides, celaforin D-1 (28), 1α,6β,15-triacetoxy-8α,9α-dibenzoyloxy-2α-hydroxydihydro-β-agarofuran (34), triptogelin A-2 (37), and chiapen D (49) showed moderate suppressive effects on the proliferation of T and B lymphocytes with IC 50 values ranging between 8.1 ± 0.8 and 19.0 ± 0.9 μM., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: Triptonodiol is a very promising antitumor drug candidate extracted from the Chinese herbal remedy Tripterygium wilfordii Hook. F., and related studies are underway., Methods: To explore the mechanism of triptonodiol for lung cancer treatment, we used network pharmacology, molecular docking, and ultimately protein validation. Gene ontology (GO) analysis and Kyoto Encyclopedia of Gene and Genome (KEGG) pathway enrichment analysis were performed through the David database. Molecular docking was performed using PyMoL2.3.0 and AutoDock Vina software. After screening, the major targets of triptonodiol were identified for the treatment of lung cancer. Target networks were established, Protein-protein interaction (PPI) network topology was analyzed, then KEGG pathway enrichment analysis was performed. Useful proteins were screened by survival analysis, and Western blot analysis was performed., Results: Triptonodiol may regulate cell proliferation, drug resistance, metastasis, anti-apoptosis, etc., by acting on glycogen synthase kinase 3 beta (GSK3B), protein kinase C (PKC), p21-activated kinase (PAK), and other processes. KEGG pathway enrichment analysis showed that these targets were associated with tumor, erythroblastic oncogene B (ErbB) signaling, protein phosphorylation, kinase activity, etc. Molecular docking showed that the target protein GSK has good binding activity to the main active component of triptonodiol. The protein abundance of GSK3B was significantly downregulated in non-small-cell lung cancer cells H1299 and A549 treated with triptonodiol for 24 h., Conclusion: The cellular-level studies combined with network pharmacology and molecular docking approaches provide new ideas for the development and therapeutic application of triptonodiol, and identify it as a potential GSK inhibitor., (© 2023. The Author(s).)

We aimed to explore novel biomarkers involved in alterations of metabolism and gene expression related to the hepatotoxic effects of Tripterygium glycosides tablet (TGT) in rats. Rats were randomly divided into groups based on oral administration of TGTs for 6 weeks: control, low-dose (9.5 mg/kg), and high-dose (18.9 mg/kg). Serum samples and total liver RNA were subjected to metabonomic and transcriptomic analyses. Thirteen metabolites were significantly up-regulated by liver injury induced by Tripterygium glycosides. Five potential biomarkers were more sensitive than Alanine aminotransferase (ALT) for accurate and timely prediction of hepatic damage. The four metabolic pathways most obviously regulated by hepatotoxicity were D-glutamine and D-glutamate metabolism, alanine, aspartate and glutamate metabolism, ether lipid metabolism, and tryptophan metabolism. Transcriptomics revealed significant differences in 1792 mRNAs and 400 long non-coding (lnc) RNAs. Dysregulated lncRNAs in the TGT - induced hepatotoxicity group were associated with genes involved in amino acid metabolism using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis. Up-regulated expression of Ehhadh, Gpt, and Got1, and down-regulated expression of dopa decarboxylase (Ddc), Cyp1a2, Ido2, Aldh1b1, and asparagine synthetase (Asns) was validated by quantitative real-time PCR. This multiomics study has elucidated the relationship between amino metabolism and liver injury, revealing potential biomarkers.

Background: As first-line clinical drugs, tripterygium glycoside tablets (TGTs) often have inconsistent efficacy and toxic side effects, mainly due to inadequate quality control. Therefore, clinically relevant quality standards for TGTs are urgently required., Purpose: Based on chemical substances and considering pharmacological efficacy, we aimed to develop an effective quality evaluation method for TGTs., Methods: Representative commercial samples of TGTs were collected from different manufacturers, and qualitative UHPLC/LTQ-Orbitrap-MS and quantitative UHPLC-MS/MS analysis methods were successfully applied to evaluate their quality similarities and differences based on their chemical properties. Then the anti-immunity, anti-inflammatory and antitumor activities of TGTs and related monomers were evaluated using Jurkat, RAW264.7, MIA PaCa-2, and PANC-1 as cellular models. Subsequently, we predicted and verified small molecule-DCTPP1 interactions via molecular docking using the established DCTPP1 enzymatic activity assay. Finally, we performed a gray relational analysis to evaluate the chemical characteristics and biological effects of TGTs produced by different manufacturers., Results: We collected 24 batches of TGTs (D01-D24) from 5 manufacturers (Co. A, Co. B, Co. C, Co. D, Co. E) for quality evaluation. The chemical composition analysis revealed significant differences in the substance bases of the samples. The D02, D18-D20 samples from Co. B constituted a separate group that differed from other samples, mainly in their absence of diterpenoids and triterpenoids, including triptolide, triptophenolide, and triptonide. In vitro anti-immunity, antitumor and anti-inflammatory tests using the same TGT concentration revealed that, except for D02, D18-D20, the remaining 20 samples exhibited different degrees of anti-immunity, antitumor and anti-inflammatory activity. Our experiments verified that triptolide, triptophenolide, and triptonide were all DCTPP1 inhibitors, and that TGTs generally exhibited DCTPP1 enzyme inhibitory activity. Moreover, the inhibitory activity of D02, D18-D20 samples from Co. B was much lower than that of the other samples, with a nearly tenfold difference in IC 50 . Further comprehensive analysis revealed a high correlation between DCTPP1 enzyme inhibition activity and the anti-immunity and antitumor and anti-inflammatory activities of these samples., Conclusion: The established DCTPP1 enzymatic activity assay proved suitable for quantitative pharmacological and pharmaceutical analysis to complement the existing quality control system for TGTs and to evaluate their effectiveness., Competing Interests: Declaration of Competing Interest The authors declare that they have no known conflicts of interest associated with this publication, and they have received no significant financial support for the research that could have influenced its outcome., (Copyright © 2023 Elsevier GmbH. All rights reserved.)

Tripterygium wilfordii (TW), a well-known traditional Chinese medicine, was widely used in the treatment of autoimmune disorders and inflammatory diseases. However, the clinical use of TW was limited by severe toxicities, such as hepatotoxicity and nephrotoxicity. Our previous studies indicated that roasting was an effective approach for reducing TW-induced toxicity. After roasting, celastrol was completely decomposed, partially converted into 1-hydroxy-2,5,8-trimethyl-9-fluorenone and the total alkaloids content were significantly reduced. However, the detoxication mechanisms of roasting on TW were poorly unknown. This study aimed to explore the toxicity and detoxification mechanisms of TW after roasting based on urine metabolomics. Promising biomarkers were evaluated by multiple comparison analyses. Sixteen toxicity biomarkers were identified between control group and total extract group. Twelve toxicity biomarkers were identified between control group and total alkaloids group. Eight toxicity biomarkers were identified between control group and celastrol group. These metabolites were mainly involved in seven metabolic pathways, summarized as pentose and glucuronate interconversions, lipid metabolism (sphingolipid metabolism, glycerophospholipid metabolisms, fatty acid biosynthesis and steroid hormone biosynthesis) and amino acid metabolism (taurine and hypotaurine metabolism, tryptophan metabolism). After roasting, the toxicities of total extract, total alkaloids and celastrol were relieved by ameliorative serum parameters and pathological changes in hepatic and renal tissues which revealed that the reduction of celastrol and total alkaloids played important roles in the detoxification of roasting on TW. Furthermore, roasting regulated the levels of fourteen potential biomarkers in the total extract group, ten potential biomarkers in the total alkaloids group and seven candidate biomarkers in the celastrol group to normal levels. Biological pathway analysis revealed that roasting may ameliorate TW-induced metabolic disorders in pentose and glucuronate interconversions, lipid metabolism and amino acid metabolism. This study provided evidence for the application of roasting in TW., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Sesquiterpene pyridine alkaloids (SPAs) are bioactive analogues derived from the genus Tripterygium and have anti-inflammatory and anti-rheumatic properties. Attributed to the similar sesquiterpene structures, the total SPAs showed severe peak overlap in 1D NMR and HPLC, leading to difficulties in identification and quantification. Interestingly, the application of band-selective HSQC NMR that specifically excited the region corresponding to the H-3 of SPAs prompted a signal separation of the total SPAs. Based on the high resolution, 23 SPAs were identified from the band-selective HSQC spectrum. The coupling constants (J CH , J HH ) and relaxation times (T 1 , T 2 ) of SPAs were measured, and it was found that they caused less than 1% attenuation of the HSQC signals, so the HSQC signals of SPAs had almost uniform responses. The concentrations of 23 SPAs were determined by standard curve method, using wilforgine as the calibration. In addition, we extended the pulse length-based concentration determination (PULCON) as a more efficient external standard method to the band-selective HSQC spectrum, and the results showed that the concentrations of alkaloids determined by PULCON were consistent with those measured by standard curve method. The developed quantification approach was validated according to the <761> of United States Pharmacopoeia (USP), demonstrating that the established band-selective HSQC approach is reliable for the rapid quantification of analogues in botanical extracts., Competing Interests: Declaration of competing interest The authors declared no potential conflicts of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

A chemical investigation of 95% ethanol extract from the stem and branch of Tripterygium wilfordii has resulted in the isolation and characterization of two new compounds, one neolignan ( 1 ) and one phenylalanine derivative ( 2 ), as well as four known compounds ( 3-6 ). The structures of the new compounds were determined based on extensive spectroscopic analyses. The absolute configuration of compound 1 was defined by X-ray crystallographic analyses and electronic circular dichroism calculation. In addition, compounds 2 and 4-6 exhibited inhibitory effects against NO production in LPS-induced RAW 264.7 macrophages with the IC 50 value ranging from 3.51 μM to 30.40 μM.

Ethnopharmacological Relevance: Tripterygium wilfordii tablets (TWT) is widely used to treat autoimmune diseases such as rheumatoid arthritis. Celastrol, one main active ingredient in TWT, has been shown to produce a variety of beneficial effects, including anti-inflammatory, anti-obesity, anti-cancer, and immunomodulatory. However, whether TWT could protect against Concanavalin A (Con A)-induced hepatitis remains unclear., The Aim of the Study: This study aims to investigate the protective effect of TWT against Con A-induced hepatitis and elucidate the underlying mechanism., Materials and Methods: Metabolomic analysis, pathological analysis, biochemical analysis, qPCR and Western blot analysis and the Pxr-null mice were used in this study., Results: The results indicated that TWT and its active ingredient celastrol could protect against Con A-induced acute hepatitis. Plasma metabolomics analysis revealed that metabolic perturbations related to bile acid and fatty acid metabolism induced by Con A were reversed by celastrol. The level of itaconate in the liver was increased by celastrol and speculated as an active endogenous compound mediating the protective effect of celastrol. Administration of 4-octanyl itaconate (4-OI) as a cell-permeable itaconate mimicker was found to attenuate Con A-induced liver injury through activation of the pregnane X receptor (PXR) and enhancement of the transcription factor EB (TFEB)-mediated autophagy., Conclusions: Celastrol increased itaconate and 4-OI promoted activation of TFEB-mediated lysosomal autophagy to protect against Con A-induced liver injury in a PXR-dependent manner. Our study reported a protective effect of celastrol against Con A-induced AIH via an increased production of itaconate and upregulation of TFEB. The results highlighted that PXR and TFEB-mediated lysosomal autophagic pathway may offer promising therapeutic target for the treatment of autoimmune hepatitis., Competing Interests: Declaration of competing interest The authors have declared no conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background: Tripterygium glycoside tablets (TGT) is the most common preparation from Tripterygium wilfordii Hook F, which is widely used in clinical for treating rheumatoid arthritis (RA) and other autoimmune diseases. However, its serious reproductive toxicity limits its application., Purpose: This study aimed to elucidate the toxic effects of TGT on the reproductive system of male RA rats and its potential toxic components and mechanism., Methods: Collagen-induced arthritis (CIA) rat model was established, and TGT suspension was given at low, medium, and high doses. Gonadal index, pathological changes, and the number of spermatogenic cells were used to evaluate the toxic effects of TGT on the reproductive system. Non-targeted metabolomics of testicular tissue was conducted by UHPLC-QTOF/MS. Combined with network toxicology, the key targets of TGT-induced reproductive toxicity were screened and RT-qPCR was used to validation. In vitro toxicity of 19 components of TGT was evaluated using TM3 and TM4 cell lines. Molecular docking was used to predict the interaction between toxic components and key targets., Results: TGT reduced testicular and epididymis weight. Pathology analysis showed a lot of deformed and atrophic spermatogenic tubules. The number of spermatogenic cells decreased significantly (P<0.0001). A total of 58 different metabolites including platelet-activating factor (PAF), lysophosphatidylcholine (Lyso PC), phosphatidylinositol (PI), glutathione (GSH), and adenosine monophosphate (AMP) were identified by testicular metabolomics. Glycerophospholipid metabolism, ether lipid metabolism, and glutathione metabolism were key pathways responsible for the reproductive toxicity of TGT. Ten key reproductive toxicity targets were screened by network toxicology. The cytotoxicity test showed that triptolide, triptonide, celastrol, and demethylzeylasteral could significantly reduce the viability of TM3 and TM4 cells. Alkaloids had no apparent toxic effects. Molecular docking showed that the four toxic components had a good affinity with 10 key targets. All binding energies were less than -7 kcal/mol. The RT-qPCR results showed the Cyp19a1 level was significantly up-regulated. Pik3ca and Pik3cg levels were significantly down-regulated., Conclusion: Through testicular metabolomics, we found that TGT may cause reproductive toxicity through CYP19A1, PIK3CA, and PIK3CG three target, which was preliminarily revealed. This study laid the foundation for elucidating the toxicity mechanism of TGT and evaluating its safety and quality., Competing Interests: Declaration of Competing Interest The authors declare that there are no conflicts of interest., (Copyright © 2023 Elsevier GmbH. All rights reserved.)

Background and Aims: Drug-induced liver injury (DILI) remains a critical issue and a hindrance to clinical application of Tripterygium Glycosides Tablet (TGT) despite its favorable therapeutic efficacy in rheumatoid arthritis. Herein, we aimed to elucidate the molecular mechanisms underlying TGT-induced hepatotoxicity., Methods: Chemical profiling of TGT was identified by UPLC-Q/TOF-MS/MS and its putative targets were predicted based on chemical structure similarity calculation. Following "DILI-related gene-TGT putative target" interaction network construction, a list of key network targets was screened according to nodes' topological importance and functional relevance. Both in vivo and in vitro experiments were performed to determine drug hepatotoxicity and the underlying mechanisms., Result: A total of 49 chemical components and 914 putative targets of TGTs were identified. Network calculation and functional modularization screened RAS-ERK and mTOR signalings-associated autophagy to be one of the candidate targets of TGT-induced hepatotoxicity. Experimentally, TGT significantly activated RAS-ERK axis, elevated the number of autophagosomes and the expression of LC3II protein, but reduced the expression of p62 protein and suppressed mTOR phosphorylation in the liver tissues of TGT-induced acute liver injury mice and chronic liver injury mice in vivo and AML12 cells in vitro. Moreover, TGT and mL-098 (an activator of RAS) co-treatment reduced AML12 cell viability via regulating autophagy and TGT-induced liver injury-related indicators more dramatically than TGT treatment alone, whereas Salirasib (an inhibitor of RAS) had an opposite effect., Conclusion: RAS-ERK-mTOR cross-talk may play a crucial role in TGT-induced hepatocyte autophagy, offering a promising target for developing novel therapeutics to combat TGT-induced hepatotoxicity., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Purpose: Accumulating clinical evidence showed that Tripterygium hypoglaucum (Lév.) Hutch (THH) is effective against IgA nephropathy (IgAN), but the mechanism is still unclear. This study is to evaluate the renal protective effect and molecular mechanism of THH against IgAN via network pharmacology, molecular docking strategy and experimental validation., Methods: Several databases were used for obtaining the active ingredients of THH, the corresponding targets, as well as the IgAN-related genes. The critical active ingredients, functional pathways, and potential for the combination of the hub genes and their corresponding active components were determined through bioinformatics analysis and molecular docking. The IgAN mouse model was treated with celastrol (1 mg/kg/d) for 21 days, and the aggregated IgA1-induced human mesangial cell (HMC) was treated with various concentrations of celastrol (25, 50 or 75 nM) for 48 h. The immunohistochemistry and Western blot techniques were applied to evaluate the protein expression of the predicted target. The cell counting kit 8 (CCK8) was used to detect HMC proliferation., Results: A total of 17 active ingredients from THH were screened, covering 165 IgAN-related targets. The PPI network identified ten hub targets, including PTEN. The binding affinity between the celastrol and PTEN was the highest (-8.69 kJ/mol). The immunohistochemistry showed that celastrol promoted the expression of PTEN in the glomerulus of IgAN mice. Furthermore, the Western blot techniques showed that celastrol significantly elevated the expression of PTEN and inhibited PCNA and Cyclin D1 in vitro and in vivo. The CCK8 assay determined that celastrol decreased HMC proliferation in a concentration-dependent manner., Conclusion: This study suggests that activating PTEN by celastrol may play a pivotal role in THH alleviating IgAN renal injury., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2023 Zhao et al.)

The stem and branch extract of Tripterygium wilfordii (Celastraceae) afforded seven new dihydroagarofuran sesquiterpene polyesters [tripterysines A-G (1-7)] and eight known ones (8-15). The chemical structures of these new compounds were established based on combinational analysis of HR-ESI-MS and NMR techniques. The absolute configurations of tripterysines A-C (1-3) and E-G (5-7) were determined by X-ray crystallographic analysis and circular dichroism spectra. All the compounds were screened for their inhibitory effect on inflammation through determining their inhibitory effect on nitric oxide production in LPS-induced RAW 264.7 cells and the secretion of inflammatory cytokines TNF-α and IL-6 in LPS-induced BV2 macrophages. Compound 9 exhibited significant inhibitory activity on NO production with an IC 50 value of 8.77 μmol·L -1 . Moreover, compound 7 showed the strongest inhibitory effect with the secretion of IL-6 at 27.36%., (Copyright © 2023 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.)

Objective: Tripterygium glycoside (TG) is a fat-soluble extract of Tripterygium wilfordii, with anti-inflammatory properties associated with TLR signaling pathways. This study constructed a targeted delivery system for experimental colitis, namely, eudragit (EuL)-coated chitosan (Ch)-TG conjugate microspheres (Ch-TG-MS/EuL), and evaluated its therapeutic efficacy and underlying mechanisms., Methods: Ch-TG-MS was fabricated using emulsification cross-linking technique and then coated with EuL to create Ch-TG-MS/EuL. Drug release properties were assessed using a dialysis model. Additionally, the therapeutic benefits of Ch-TG-MS/EuL on colonic inflammation and its specific effect on TLR4/NF-κB signaling in intestinal mucosa were evaluated in vivo using a DSS-induced murine colitis model., Results: The Ch-TG-MS/EuL microspheres appeared as yellow powders with a slightly enlarged shape, rough surface, and adhesions. The Ch-TG-MS/EuL formulations also exhibited high entrapment efficiency and drug loading rate. High-performance liquid chromatography revealed that Ch-TG-MS/EuL exhibited a less intense peak than free TG, confirming that the drug is contained within the formulation. Free TG displayed explosive release within the first 5 h of administration, while Ch-TG-MS/EuL prevented the pre-mature release of TG and exhibited controllable release up to 24 h. In vivo, noticeable amelioration of intestinal mucosal tissue destruction was achieved with Ch-TG-MS/EuL compared to free TG. Additionally, immunohistochemical and western blotting results revealed that Ch-TG-MS/EuL markedly down-regulated the expression of intestinal mucosal TLR4, MyD88, and NF-κB p65. Hence, Ch-TG-MS/EuL may ameliorate the colon inflammatory response by inhibiting the hyperactivation of TLR4/NF-κB signaling., Conclusion: Novel Ch-TG-MS/EuL preparation may represent a colonic delivery system for UC therapeutics by inhibiting TLR4/NF-κB hyperactivation., Data Availability: All experimental data supporting the conclusions of this study are available from the corresponding author on reasonable request., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Background: Tripterygium wilfordii Hook. F. (TwHF), a traditional Chinese medicine, is widely used in the treatment of rheumatoid arthritis. Due to multiorgan toxicity, particularly hepatotoxicity, the application of TwHF is restricted. To clarify the hepatotoxic substances, zebrafish, hepatocytes and macrophages were used for screening based on hepatotoxic injury patterns. This study provides a basis for further elucidation of the hepatotoxic mechanism of TwHF., Methods: First, 12 compounds were selected according to the chemical categories of TwHF. The fluorescence area and fluorescence intensity of zebrafish livers were observed and calculated. The viability of two hepatocyte lines was detected by CCK 8 assay. TNF-α and IL-1β mRNA expression in bone marrow-derived macrophages was used to evaluate macrophage activation, a factor of potential indirect hepatotoxicity. Finally, the hepatotoxic characteristics of 4 representative components were verified in mice in vivo., Results: Parthenolide, triptolide, triptonide, triptobenzene H, celastrol, demethylzeylasteral, wilforlide A, triptotriterpenic acid A and regelidine significantly reduced the fluorescence area and fluorescence intensity of zebrafish livers. The viability of L-02 or AML-12 cells was significantly inhibited by parthenolide, triptolide, triptonide, celastrol, demethylzeylasteral, and triptotriterpenic acid A. Parthenolide, triptolide, triptonide, celastrol, demethylzeylasteral and triptobenzene H significantly increased TNF-α and IL-1β mRNA levels in macrophages, while triptophenolide, hypodiolide and wilforine significantly reduced TNF-α and IL-1β mRNA levels. Triptotriterpenic acid A, celastrol and triptobenzene H at a dose of 10 mg/kg significantly increased the levels of mouse serum alanine aminotransferase and aspartate aminotransferase and aggravated liver inflammation., Conclusions: Parthenolide, triptolide, triptonide, celastrol, demethylzeylasteral, triptotriterpenic acid A and triptobenzene H might be the main hepatotoxic components of TwFH. Among them, only triptotriterpenic acid A presents direct hepatotoxicity. Triptobenzene H exerts indirect liver damage by activating macrophages. Parthenolide, triptolide, triptonide, celastrol, and demethylzeylasteral can directly and indirectly cause liver injury., (© 2023. The Author(s).)

Tripterygium wilfordii Hook F from the family Celastraceae is a traditional Chinese medicine (TCM) whose principal chemical constituents are terpenoids, including sesquiterpene alkaloids and diterpenoids, which have unique and diverse structures and remarkable biological activities. In order to advance pharmacological research and guide the preparation of monomer compounds derived from T. wilfordii , a systematic approach to efficiently discover new compounds or their derivatives is needed. Herein, compound separation and identification were performed by offline reversed-phase × supercritical fluid chromatography coupled mass spectrometry (RP × SFC-Q-TOF-MS/MS) and Global Natural Product Social (GNPS) molecular networking. The 2D chromatography system exhibited a high degree of orthogonality and significant peak capacity, and SFC has an advantage during the separation of sesquiterpene alkaloid isomers. Feature-based molecular networking offers the great advantage of quickly detecting and clustering unknown compounds, which greatly assists in intuitively judging the type of compound, and this networking technique has the potential to dramatically accelerate the identification and characterization of compounds from natural sources. A total of 324 compounds were identified and quantitated, including 284 alkaloids, 22 diterpenoids and 18 triterpenoids, which means that there are numerous potential new compounds with novel structures to be further explored. Overall, feature-based molecular networking provides an effective method for discovering and characterizing novel compounds and guides the separation and preparation of targeted natural products.

Background: Although thunder god vine ( Tripterygium wilfordii ) has been widely used for treatment of idiopathic membranous nephropathy (IMN), the pharmacological mechanisms underlying its effects are still unclear. This study investigated potential therapeutic targets and the pharmacological mechanism of T. wilfordii for the treatment of IMN based on network pharmacology., Methods: Active components of T. wilfordii were obtained from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform. IMN-associated target genes were collected from the GeneCards, DisGeNET, and OMIM databases. VENNY 2.1 was used to identify the overlapping genes between active compounds of T. wilfordii and IMN target genes. The STRING database and Cytoscape 3.7.2 software were used to analyze interactions among overlapping genes. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses of the targets were performed using Rx64 4.0.2 software, colorspace, stringi, DOSE, clusterProfiler, and enrichplot packages., Results: A total of 153 compound-related genes and 1485 IMN-related genes were obtained, and 45 core genes that overlapped between both categories were identified. The protein-protein interaction network and MCODE results indicated that the targets TP53, MAPK8, MAPK14, STAT3, IFNG, ICAM1, IL4, TGFB1, PPARG, and MMP1 play important roles in the treatment of T. wilfordii on IMN. Enrichment analysis showed that the main pathways of targets were the AGE signaling pathway, IL-17 signaling pathway, TNF signaling pathway, and Toll-like receptor signaling pathway., Conclusion: This study revealed potential multi-component and multi-target mechanisms of T. wilfordii for the treatment of IMN based on network pharmacological, and provided a scientific basis for further experimental studies.

Tripterygium glycoside tablet (TGT) has been used clinically to alleviate diabetic nephropathy (DN) for decades. However, the mechanism of its anti-DN has not been fully clarified. The aim of this study was to elucidate molecular mechanism of TGT in repairing renal function injury. The results of biochemical parameters and renal histopathology implied that TGT intervention could attenuate creatinine, albumin excretion rate and histological injury of kidney in DN mouse model. Moreover, UHPLC-QTOF-MS/MS-based untargeted metabolomic analysis indicated that 11 metabolites in kidney of mice with DN were restored after TGT treatment, and the most prominent metabolic alteration was triglyceride (TG) metabolism. Mechanistically, TGT effectively improved the function of impaired kidney by promoting TG catabolism via modulation of adipose triglyceride lipase in DN mice. Our findings identified the link between circulating metabolites and DN, suggesting that it might be a possibility to intervene in DN by targeting metabolism., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Tripterygium Glycosides Tablets (TGT) has shown obvious anti-rheumatoid arthritis (RA) effects accompanied by hepatotoxicity. Despite that many studies looked at TGT's anti-RA or hepatotoxic mechanism and substance basis, the results were still insufficient. Furthermore, the anti-RA and hepatotoxicity investigations of TGT were undertaken separately, neglecting the relationship between efficacy and toxicity. Herein, an integrated approach combining metabolomics, network pharmacology, serum pharmacochemistry, and molecular docking was adopted to elucidate the mechanism and substance basis of Tripterygium Glycosides Tablets (TGT) on anti-rheumatoid arthritis and hepatotoxicity simultaneously. The results showed that 33 components in TGT were absorbed into rat serum. Two toxic targets (PRKCA, FASN), three effective targets (PLA2G10, PTGES, PLA2G1B), and four effective and toxic targets (PTGS1, PTGS2, PLA2G2A, ALOX5) were obtained by metabolomics combined with network analysis and network pharmacology. A component-target-RA-hepatotoxicity network was constructed and five hepatotoxic components (1-desacetylwilforgine, wilfordconine, wilforgine, wilformine, wilfornine D), eight effective-toxic components (14-oxo-19-(4 → 3) abeo-abieta-3,8,12-tetraen-19,18-olide, 7-oxo-18(4 → 3) abeo-abieta-3,8,11,13-tetraen-18-oic acid, hypoglaulide, triptotriterpenic acid A, wilforol F, wilforlide B, triptoquinone B, wilforlide A); and 23 non-effective and non-toxic components were acquired and validated by molecular docking. In addition, our research revealed that glycerophospholipid metabolism and ether lipid metabolism were correlated to both hepatotoxicity and anti-RA of TGT. While in sphingolipid metabolism, ceramidases regulated ceramide-sphingosine and phytoceramide-phytosphingosine reaction were found to be correlated to hepatotoxicity, sphinganine-1-phosphate lyase (SPL) regulated sphingosine 1-phosphate (S1P)-phosphoethanolamine and sphinganine 1-phosphate-phosphoethanolamine were found to be attributed to anti-RA effects., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Tripterygium wilfordii Hook. f. is a well-known traditional Chinese medicine used to treat autoimmune diseases. Sesquiterpene pyridine alkaloids (SPAs) are a major class of components found in this herb that have piqued the interest of researchers due to their complex and diverse structures as well as significant biological activities. In this study, ten new SPAs, wilfordatine A-J ( 1 - 10 ), were isolated from the roots of T. wilfordii , along with ten known analogues ( 11 - 20 ). Their structures were primarily elucidated by extensive 1D and 2D NMR spectroscopic analysis. To search for more immunosuppressive ingredients related to the clinical efficacy of T. wilfordii , the total alkaloids (TA) and compounds 4 , 5 , and 9 - 16 were tested for their inhibitory effects on nuclear factor-kappa B (NF-κB) pathway in Lipopolysaccharide (LPS) induced HEK293/NF-κB-Luc cells. Among them, TA, compounds 5 , 11 , and 16 showed potent immunosuppressive activity, with IC 50 values of 7.25 μg/mL, 8.75 μM, 0.74 μM, and 15.66 μM, respectively, and no influence on the cell viability at a concentration of 100 μg/mL (TA) or 100 μM ( 5 , 11 , and 16 ). Accordingly, TA, 5 , 11 , and 16 , especially 11 , were identified as promising candidates for further investigation into their potential use as immunosuppressive agents.

Ethnopharmacological Relevance: Tripterygium wilfordii polyglycosides (TWP) are extracted from Tripterygium wilfordii Hook. f., which has the significant effects of anti-inflammation and immunosuppression and has been widely used to treat autoimmune diseases in traditional Chinese medicine., Aim of Study: In Chinese clinical dermatology, TWP was generally used for the treatment of autoimmune skin diseases including psoriasis (PSO), systemic lupus erythematosus (SLE) and pemphigus (PEM). However, the potential hepatotoxicity (HPT) induced by TWP was also existing with the long-term use of TWP. This study aims to explore the potential shared therapeutic mechanism of TWP treating PSO, SLE, PEM and the possible hepatotoxic mechanism induced by TWP., Materials and Methods: Network pharmacology was used to predict the potential targets and pathways in this study. The main bioactive compounds in TWP was screened according to TCMSP, PubChem, ChEMBL databases and Lipinski's Rule of Five. The potential targets of these chemical constituents were obtained from PharmMapper, SEA and SIB databases. The related targets of PSO, SLE, PEM and HPT were collected from GeneCards, DrugBank, DisGeNET and CTD databases. The target network construction was performed through STRING database and Cytoscape. GO enrichment, KEGG enrichment and molecular docking were then performed, respectively. In particular, imiquimod (IMQ)-induced PSO model was selected as the representative for the experimental verification of effects and shared therapeutic mechanisms of TWP., Results: 41 targets were considered as the potential shared targets of TWP treating PSO, SLE and PEM. KEGG enrichment indicated that IL-17 signaling pathway and Th17 cell differentiation were significant in the potential shared therapeutic mechanism of TWP. The animal experimental verification demonstrated that TWP could notably ameliorate skin lesions (P˂0.001), decrease inflammatory response (P˂0.05, P˂0.01, P˂0.001) and inhibit the differentiation of Th1/Th17 cells (P˂0.05, P˂0.01) compared to PSO model group. The molecular docking and qPCR validation then showed that TWP could effectively act on MAPK14, IL-2, IL-6 and suppress Th17 cell differentiation and IL-17 signaling pathway. The possible hepatotoxic mechanism of TWP indicated that there were 145 hepatotoxic targets and it was also associated with IL-17 signaling pathway and Th17 cell differentiation, especially for the key role of ALB, CASP3 and HSP90AA1. Meanwhile, the potential correlations between efficacy and hepatotoxicity of TWP showed that 28 targets were shared by therapeutic and hepatotoxic mechanisms such as IL-6, IL-2, MAPK14, MMP9, ALB, CASP3 and HSP90AA1. These significant relevant targets were also involved in IL-17 signaling pathway and Th17 cell differentiation., Conclusions: There were shared disease targets in PSO, SLE and PEM, and TWP could treat them by potential shared therapeutic mechanisms of suppressing IL-17 signaling pathway and Th17 cell differentiation. The possible hepatotoxicity induced by TWP was also significantly associated with the regulation of IL-17 signaling pathway and Th17 cell differentiation. Meanwhile, the potential correlations between efficacy and hepatotoxicity of TWP also mainly focused on IL-17 signaling pathway and Th17 cell differentiation, which provided a potential direction for the study of the mechanism of "You Gu Wu Yun" theory of TWP treating autoimmune skin diseases in the future., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Objective: To investigate the effectiveness and safety of tripterygium glycosides (TG) tablet for the treatment of Lupus nephritis (LN)., Methods: Several databases were systematically searched including PubMed, Embase, Cochrane, Wiley, China National Knowledge Infrastructure Database, SinoMed and Wanfang Library till June 20, 2020. Revman5.3 was utilized to analyze the data according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Statement., Results: In total, 8 randomized controlled trials involving 583 participants were identified. Meta-analyses showed that, compared with glucocorticoids (GC) alone, the combination with TG tablet provided a statistically significant improvement in total remission (TR) ( = 1.27, 95% : 1.08-1.50, = 0.004), complete remission (CR) ( = 1.61, 95% : 1.05-2.47, = 0.03) and C3 levels ( = 0.27, 95% : 0.14-0.39, < 0.000 1), C4 levels ( = 0.12, 95% : 0.07-0.17, < 0.000 01). No significant differences were seen in TR, CR, proteinuria, serum creatinine, C3 and C4 (TR: = 1.00, 95% : 0.87-1.16, = 0.95; CR: = 1.10, 95% : 0.78-1.56, = 0.58; proteinuria levels: = -0.06, 95% : -0.13 to 0.01, = 0.10; serum creatinine levels: = -0.01, 95%: -7.36 to 7.35, = 1.00; C3 levels: = 0.01, 95%: -0.06 to 0.07, = 0.84; C4 levels: = -0.01, 95%: -0.03 to 0.01, = 0.49) between azathioprine (AZA) / leflomit (LEF) + GC and TG tablet + GC. Adverse events (hepatic dysfunction, nausea, vomitting) showed no statistical differences between the TG tablet + GC group and the GC group. There were more new onset of irregular menstruation in the TG tablet + GC group than those in the AZA + GC ( = 3.57, 95% : 1.40-9.11, = 0.008) /LEF+ GC ( = 6.69, 95% : 2.42-18.46, = 0.000 2) group, but leucopenia lower than those in AZA + GC group ( = 0.38, 95% : 0.17-0.85, = 0.02) and alopecia ( = 0.14, 95% : 0.03-0.77, = 0.02) and rash ( = 0.09, 95% : 0.01-0.69, = 0.02) lower than those in LEF + GC group., Conclusions: This review indicates that TG tablet maybe effective in LN treatment. Nevertheless, adverse events cannot be ignored. Large sample, multi-center, high-quality clinical studies are needed to verify the exact effects and safety of TG tablet in treatment of LN.

Six undescribed abietane-type diterpenoids (tripterydinoids A-F) and five undescribed oleanane-type triterpenoids (tripterytrinoids A-E) were obtained and determined from the stem and branch of Tripterygium wilfordii Hook. f. (Celastraceae). Tripterydinoids A-C possessed the abietane-type diterpenoid skeleton with rare 8, 9-epoxy ring. The structures of undescribed compounds were established by extensive spectroscopic studies [HRESIMS, 1D/2D-NMR and electronic circular dichroism (ECD) calculation]. The absolute configurations of tripterydinoids A, B, E and tripterytrinoid A were defined by X-ray crystallographic analyses. Bioactivity screening indicated that tripterydinoids A-C exhibited potent inhibitory effects against NO release in LPS-activated RAW 264.7 macrophages with IC 50 values of 6.93, 4.46 and 2.98 μM, respectively. Meanwhile, tripterydinoids A-D and tripterytrinoids B, C showed moderate and selective cytotoxicities against five human tumor cell lines (A375, Huh7, MCF-7, HCT-116 and NCI-H460)., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Three undescribed acylated sucroses (1-3), one undescribed butenolide analog (4) along with three known compounds (5-7) were isolated from the aqueous EtOH extract of the dried leaves of Tripterygium wilfordii. Their structures were elucidated on the basis of spectroscopic analyses, electron circular dichroism (ECD) techniques, and saccharide hydrolysis. All the isolated compounds were tested for their anti-tyrosinase effects. Among them, 6 exhibited similar inhibitory effects on tyrosinase with IC 50 values of 0.073 mM comparing to arbutin. Additionally, the possible mechanism of the interaction between 6 and the active site of tyrosinase was explored by molecular docking., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Objective: Valsartan has been studied to exert effects on kidney disease. However, the concrete function of valsartan in combination with tripterygium glycosides in chronic nephritis remained largely unknown. The study was designed to unravel the impacts of valsartan and tripterygium glycosides in chronic nephritis through the Toll-like Receptor 4 (TLR4) pathway., Methods: The renal function indicators such as serum creatinine (Scr), blood urea nitrogen (BUN) and β 2 microglobulin ( β 2-MG), 24 h urine protein ( Upro) levels, and blood lipid indicators such as total cholesterol (TC), low-density lipoprotein (LDL-C), triacylglycerol (TG) and high-density lipoprotein (HDL-C), inflammatory factors (e.g., IL-1 β and IL-8), and the proportion of T lymphocyte subpopulations (CD4+ and CD8+) were detected in chronic nephritis patients before and after treatment with valsartan alone or valsartan combined with tripterygium glycosides. Symptoms of adverse reactions were recorded. TLR4 expression in the patients' serum was examined., Results: Compared to patients before treatment, after treatment with valsartan alone or valsartan combined with tripterygium glycosides, the renal function indicators Scr, BUN, and 24 h levels were reduced, and TC, TG, and LDL-C levels were reduced, while HDL-C levels were elevated; inflammatory responses (IL-1 β and IL-8) were mitigated; CD4+ ratio and CD4+/CD8+ ratio increased yet CD8+ ratio decreased; TLR4 expression was silenced after treatment. All of the changes were more obvious in patients after being treated with valsartan combined with tripterygium glycosides., Conclusion: Valsartan in combination with tripterygium glycosides protects against chronic nephritis via suppressing the Toll-like Receptor 4 pathway., Competing Interests: The authors have no conflicts of interest., (Copyright © 2022 Jiabao Dong et al.)

Tripterygium glycosides tablets (TGTs) are widely used in clinical practice to treat rheumatoid arthritis and other autoimmune diseases, with significant beneficial effects but also high toxicity, necessitating rigorous quality evaluation and control. In current study, a rapid resolution liquid chromatography tandem electrospray ionization triple quadrupole mass spectrometry (RRLC-ESI-MS/MS) method was developed and validated for the quantitative analysis of 14 components of ten batches of TGTs produced by different manufacturers, including four diterpenoids, three triterpenoids, and seven sesquiterpene alkaloids. Meanwhile, the NO inhibition effects of these TGTs were evaluated in LPS-induced RAW264.7 cells for their downstream anti-inflammatory activities, as well as their cytotoxicity. The results indicate that the TGTs from different manufacturers showed poor quality consistency, as evidenced by large variations in chemical profiles and biological effects, which may increase the risks associated with clinical use. To improve the quality status of TGTs, it is crucial to identify indicator components whose characterization can accurately reflect the efficacy and toxicity of TGTs from which they were derived. Our study reveals that triptolide, triptoquinone B, celastrol, and demethylzelaysteral considerably contributed to the anti-inflammatory activity and/or cytotoxicity of TGTs, implying that they should be further investigated as candidate indicator components for TGT quality control.

Sesquiterpene pyridine alkaloids are important components in Tripterygium plants, possessing a wide range of pharmacological activities, such as anti-inflammation immunosuppression, anti-tumor, anti-virus, and deinsectization, and are of great research value. They are composed of highly oxidized dihydro-β-furansquiterpene and pyridine dicarboxylic acid through ester bonds. According to the structural characteristics of pyridine dicarboxylic acid fragments, they can be divided into various structural subtypes. Up to now, more than 110 sesquiterpene pyridine alkaloids have been isolated and identified from Tripterygium plants. This study reviewed the structural features and spectral(i.e., UV, IR, MS, and NMR) characteristics of sesquiterpene pyridine alkaloids and summarized the structural elucidation process in detail to provide references for their further research and development.

Three new sesquiterpenoid alkaloids, cangorin K ( 1 ), dimacroregelines C ( 2 ) and D ( 3 ), as well as two known sesquiterpenoids ( 4 - 5 ), were isolated from the roots of Tripterygium wilfordii Hook. f. The structures of new compounds were characterised by extensive 1D and 2D NMR spectroscopic analyses, as well as HRESIMS data, and the known compounds were established by 1 D NMR spectra referring to the literatures. Cytotoxicity evaluation of these compounds against two human tumour lines (SMMC7721, LN229) was investigated by CCK-8 assay and displayed that compounds 1 - 4 showed potent cytotoxicity against SMMC7721 cell with IC 50 value in the range of 0.26-9.67 μΜ and compounds 1 - 5 showed potent cytotoxicity against LN-229 cell with IC 50 values in the range of 0.50-7.38 μΜ.

Tripterygium wilfordii genus preparations (TWGP) are widely used in traditional Chinese medicines for the treatment of autoimmune diseases and immune diseases with definite therapeutic effects but high toxicity. The aim of this study is to identify and compare chemical compounds in three types of commercial TWGP using UHPLC-QTOF-MS/MS and molecular networking (MN) technology. First, the mass fragmentation pathways of 10 compounds were investigated, which included two sesquiterpene alkaloids, four diterpenoids, and four triterpenoids. The chemical compounds were then identified using UHPLC-QTOF-MS/MS and a conventional database. Following that, molecular network technology was used to further identify the GNPS platform. Finally, metabonomic data analysis was used to compare 92 commercial TWGP samples from 13 manufacturers. A total of 103 compounds were identified, with the molecular network detecting 40 of them. Moreover, the quality of commercial Tripterygium glycoside tablets varies greatly and 26 compounds, including triptolide, wilforine, wilforgine, and demethylzeylasteral, were discovered to be the main differential compounds in tripterygium glycosides tablets. This was the first time MN technology was used for compound analysis in TWGP, laying the foundation for classifying effective and toxic substances and TWGP quality control., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: The HIV-1 infected immunological non-responders (INRs) are characterized by poor immune reconstitution after long-term treatment. Tripterygium Wilfordii Hook F (TwHF) pill is a traditional Chinese patent drug with extensive immunosuppressive effects and has been clinically proven efficacy in treating INRs., Purpose: The therapeutic mechanism of TwHF pills in the treatment of INRs was investigated by the combined multi-omics analysis on clinical samples and network pharmacology approach., Methods: Clinically, the peripheral blood mononuclear cells (PBMC) samples of TwHF-treated INRs from different time points were collected to conduct the transcriptomic and proteomic profiling. Key effector pathways of TwHF were enriched and analyzed by the ingenuity pathway analysis (IPA). Computationally, the TwHF-related compounds were obtained from traditional Chinese medicine databases, and literature search and structural prediction were performed to identify TwHF-related targets. Integrated with the INR-related targets, the 'TwHF-compounds-targets-INR' network was constructed to analyze core effector targets by centrality measurement. Experimentally, the effects of TwHF compounds on the T cells activation and expression of identified targets were evaluated with in vitro cell culture., Results: 33 INRs were included and treated with TwHF pills for 17 (IQR, 12-24) months. These patients experienced rapid growth in the CD4 + T cell counts and decreased T cell activation. The multi-omics analysis showed that the interferon (IFN)-signaling pathway was significantly inhibited after taking TwHF pills. The network pharmacology predicted the central role of the signal transducer and activator of transcription 1 (STAT1) in the 'TwHF-compounds-targets-INR' network. Further bioinformatic analysis predicted STAT1 would regulate over 58.8% of identified down-regulated genes. Cell experiments validated that triptolide (TPL) would serve as the major bioactivity compound of TwHF pills to inhibit the immune cell activation, the production of IFN-γ, the expression of downstream IFN-stimulated genes, and the phosphorylation of STAT1., Conclusion: Our research is the first to systemic verify the mechanisms of TwHF in treating INRs. The IFN signaling pathway and the STAT1 would be the major effector targets of TwHF pills in treating INRs. The TPL would be the major bioactive compound to inhibit the IFN response and the phosphorylation of STAT1. Our observations suggest the basis for further application of TPL analogous in treating INRs., (Copyright © 2022 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Ten new dihydroagarofuran sesquiterpene polyol esters, tripterdines A-J (1-10) were isolated from the stem and branch of Tripterygium wilfordii. The structures of new compounds were elucidated on the basis of extensive spectroscopic analyses, including UV, IR, HRESIMS, NMR, and CD exciton chirality method. The structures of compounds 1, 3, and 6 were confirmed by X-ray crystallographic analyses. The anti-inflammatory and cytotoxic activities were assessed for all the compounds (1-10). Compounds 3, 5 and 10 exhibited potent anti-inflammatory activities with the secretion level of TNF-α ranging from 43.86% to 51.27%, and the IL-6 ranging from 32.44% to 50.64%. In addition, compounds 1, 3, 7 and 9 showed weak cytotoxicities against three human tumor cell lines (Huh7, MCF-7 and HCT-116)., (Copyright © 2022. Published by Elsevier B.V.)

Objective: To conduct qualitative and quantitative analyses of Tripterygium hypoglaucum in Yinning Tablets, a compound preparation of traditional Chinese herbal medicine., Methods: Thin-layer chromatography (TLC) was used for qualitative analysis of Tripterygium hypoglaucum in Yining Tablets and the analytical protocols were optimized. High-performance liquid chromatography (HPLC) was used to quantitatively analyze the content of triptolide (the main active ingredient of Tripterygium hypoglaucum) in Yinning Tablets., Results: The results of TLC analysis showed that the test sample of Yinning Tablets and the positive control samples both produced clear, well separated spots without obvious interference in the blank samples. Assessment of the influences of the thin-layer plates from different manufacturers, temperature and humidity on the test results demonstrated good durability of the test. HPLC analysis of triptolide showed a good linear relationship within the concentration range of 1-100 μg/mL (regression equation: A =22.219C-19.165, r =0.9999); the contents of triptolide in 3 batches of Yinning tablets were 0.34, 0.34, and 0.28 μg per tablet, all within the range of 0.28-0.34 μg per tablet. It was finally determined that each Yinning tablet should not contain more than 0.6 μg of triptolide., Conclusion: TLC and HPLC are simple, accurate, durable and specific for qualitative and quantitative analyses of Tripterygium hypoglaucum in Yinning Tablets.

Ethnopharmacological Relevance: Tripterygium hypoglaucum (Kunmingshanhaitang in Chinese) is a plant of the genus Tripterygium which have been used as anti-tumor folk medicines in Yi and Bai ethnic groups in Yunnan province, China for hundreds of years. Terpenoids from T. hypoglaucum presented therapeutic effects on multiple tumors. But there were few studies about pancreatic cancer treatment of these terpenoids. Pancreatic cancer is an aggressive malignancy and lacked of specific drugs. Currently, anti-tumor drugs have poor therapeutic effect and prognosis for pancreatic cancer., Aim of the Study: This study aimed to elucidate the terpenoids from T. hypoglaucum and illuminate their anti-pancreatic cancer bioactivities., Material and Methods: Terpenoids were obtained through sequential chromatographic methods including silica gel, MCI gel, Sephadex LH-20, and preparative HPLC. Their structures were determined by HRESIMS, 1D and 2D NMR spectroscopic analysis. The absolute configurations of some new diterpenoids were assigned through comparison of experimental and calculated circular dichroism spectra. The cytotoxicity of isolates was measured using the MTT method on human pancreatic cancer cells SW1990. The effects on expressions of AKT, Erk1/2, p-AKT, p-Erk1/2, and Bax proteins in human pancreatic cancer cells SW1990 of these compounds were determined by western blotting assays., Results: Eleven new (compounds 1∼11) and fourteen known terpenoids (compounds 12∼25) were isolated from the underground parts of T. hypoglaucum. These compounds were belonged to abietane diterpenoids, isoprimara diterpenoids, ent-kaurane diterpenoids, oleanane triterpenoids, and friedelane triterpenoids. Compounds 5, 7, 8, 9, 16, 18, 22, 24, and 25 possessed significant cytotoxicity against SW1990 cells with IC 50 values of 19.28 ± 4.39, 9.91 ± 2.23, 27.32 ± 5.89, 56.43 ± 6.92, 0.16 ± 0.05, 0.58 ± 0.15, 0.81 ± 0.04, 0.48 ± 0.11, and 10.01 ± 1.39 μM respectively. After compounds 16, 22, and 24 been treated with the pancreatic cancer cells in medium and high doses, the protein expressions of AKT, p-AKT, Erk, and p-Erk were not remarkably reduced and the expressions of Bax protein were significantly increased., Conclusion: This study indicated that terpenoids from T. hypoglaucum could inhibit human pancreatic cancer cells SW1990. Especially, compounds 16, 22, and 24 possessed significant cytotoxicity against SW1990 cells with low IC 50 values and could increase the expressions of Bax protein. These compounds shared a wide variety of structural characteristics which provided us more candidate molecules for the development of anti-pancreatic cancer drugs and further prompted us to investigate their anti-pancreatic mechanisms., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Ethnopharmacological Relevance: Tripterygium glycosides tablets (TGT) and Tripterygium wilfordii tablets (TWT) have been used to treat autoimmune diseases clinically, however, the side effects of TWT are higher than TGT, especially for hepatotoxicity., The Aim of the Study: This study aims to determine the mechanism of TWT-induced liver injury., Materials and Methods: We performed metabolomic analysis of samples from mice with liver injury induced by TGT and TWT. Ppara-null mice were used to determine the role of PPARα in TWT-induced liver injury., Results: The results indicated that TWT induced the accumulation of medium- and long-chain carnitines metabolism, which was associated with the disruption of PPARα-IL6-STAT3 axis. PPARα agonists fenofibrate could reverse the liver injury from TWT and TP/Cel, and its protective role could be attenuated in Ppara-null mice. The toxicity difference of TWT and TGT was due to the different ratio of triptolide (TP) and celastrol (Cel) in the tablet in which TP/Cel was lower in TWT than TGT. The hepatotoxicity induced by TP and Cel also inhibited PPARα and upregulated IL6-STAT3 axis, which could be alleviated following by PPARα activation., Conclusions: These results indicated that PPARα plays an important role in the hepatotoxicity of Tripterygium wilfordii, and PPARα activation may offer a promising approach to prevent hepatotoxicity induced by the preparations of Tripterygium wilfordii., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Tripterygium wilfordii is a medicinal plant that plays a crucial role in health care programs, especially in developing countries, and had anti-tumor, anti-inflammatory, anti-fertility, anti-bacterial, and other therapeutic effects. This study was designed to determine the anti-proliferative effects of methanolic extract of T. wilfordii on the WRL-68 cell line and the function of polycystin-1 (PC-1). The half-maximal inhibitory concentration (IC 50 ) values were recorded in WRL-68 and AsPC-1 cell lines as 193 µg/ml and 149.2 µg/ml, respectively, at 2-2.55 and 2-2.2 µg/ml methanolic plant concentrations. The maximum cytotoxic activities of the extract on the growth inhibition of WRL-68 and AsPC-1 were generally observed at 97.64% and 95.94% at extract concentrations of 50 µg/ml and 25 µg/ml, respectively. The pharmacognostic profile of T. wilfordii extract was found to be alkaloids, tannins, terpenoides, flavonoids, glycosides, and phenols. The extracts of T. wilfordii were tested through gas chromatography-mass spectrometry showing four peaks representing mostly of 3-Oxobutanol; ethyl acetate; acetic acid ethyl ester; chlorbromuron; 1-(methylthio)-, (E)-; n-Hexadecanoic acid; tetradecanoic acid; and 9-Octadecenoic acid. Therefore, the results of this study revealed that the methanolic extract of T. wilfordii was more potential in inducing anti-proliferative activity of WRL-68 and AsPC-1 human cell lines than the control. In addition, the current study was the first study that reported the anti-proliferative potential of T. wilfordii in the treatment of human embryonic liver WRL-68 cancer cells., Competing Interests: The authors declare that they have no conflict of interest.

In this paper, the safety of Tripterygium wilfordii polyglycoside (TW) preparation was evaluated by combining literature research and evidence-based evaluation research, so as to provide evidence-based safety information of Tripterygium wilfordii polyglycoside preparation (nephroptosis) for government decision making and clinical application. In this paper, we propose a network structure inspired by the LSTM gate mechanism. All the research methods of the included references are evaluated by internationally recognized evaluation tools or standards. Prevalence was analyzed according to the type of intervention (e.g., time of administration) and route of administration. The results of this experiment provide methods and suggestions for the evaluation of traditional Chinese medicine nephroptosis in the future., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2022 Wuqiang Qi et al.)