Betaine alleviates spermatogenic cells apoptosis of oligoasthenozoospermia rat model by up-regulating methyltransferases and affecting DNA methylation.

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).

[The Miao medicine Sidaxue alleviates rheumatoid arthritis in rats possibly by downregulating matrix metalloproteinases].

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-1ß 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-1ß, 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-1ß/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.

Network pharmacology­based investigation of potential targets of triptonodiol acting on non-small-cell lung cancer.

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.

DCTPP1, a reliable Q-biomarker for comprehensive evaluation of the quality of tripterygium glycoside tablets based on chemical references.

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 IC50. 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.

Anti-inflammatory sesquiterpene polyol esters from the stem and branch of Tripterygium wilfordii.

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 IC50 value of 8.77 µmol·L-1. Moreover, compound 7 showed the strongest inhibitory effect with the secretion of IL-6 at 27.36%.

Toxic effects of Tripterygium glycoside tablets on the reproductive system of male rats by metabolomics, cytotoxicity, and molecular docking.

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.

Tripterygium wilfordii protects against an animal model of autoimmune hepatitis.

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.

Network Pharmacology and Experimental Validation to Explore That Celastrol Targeting PTEN is the Potential Mechanism of Tripterygium wilfordii (Lév.) Hutch Against IgA Nephropathy.

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.

Cross-talk between the RAS-ERK and mTOR signalings-associated autophagy contributes to tripterygium glycosides tablet-induced liver injury.

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.

Two new chemical constituents from the stem and branch of Tripterygium wilfordii.

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 IC50 value ranging from 3.51 µM to 30.40 µM.

Screening of major hepatotoxic components of Tripterygium wilfordii based on hepatotoxic injury patterns.

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 CCK8 assay. TNF-α and IL-1ß 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-1ß mRNA levels in macrophages, while triptophenolide, hypodiolide and wilforine significantly reduced TNF-α and IL-1ß 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.

Pharmacognostic Profile and Screening of Anti-Proliferative Potential of Methanolic Extract of Tripterygium wilfordii Plant on WRL-68 Cell Line and Function of Polycystin-1.

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 (IC50) 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.

[Spectral characteristics of sesquiterpene pyridine alkaloids from Tripterygium plants].

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-ß-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.

Undescribed abietane-type diterpenoids and oleanane-type triterpenoids from the stem and branch of Tripterygium wilfordii.

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 IC50 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).

Potential shared therapeutic and hepatotoxic mechanisms of Tripterygium wilfordii polyglycosides treating three kinds of autoimmune skin diseases by regulating IL-17 signaling pathway and Th17 cell differentiation.

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.

Diverse dihydroagarofuran sesquiterpene derivatives from the stem and branch of Tripterygium wilfordii.

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).

Cytotoxic terpenoids from Tripterygium hypoglaucum against human pancreatic cancer cells SW1990 by increasing the expression of Bax protein.

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 IC50 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 IC50 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.

Effect of major components of Tripterygium wilfordii Hook. f on the uptake function of organic anion transporting polypeptide 1B1.

Organic anion transporting polypeptide 1B1 (OATP1B1), which is specifically expressed at the basolateral membrane of human hepatocytes, is well recognized as the key determinant in the pharmacokinetics of a wide variety of drugs and considered as an important drug-drug interaction (DDI) site. Triptergium wilfordii Hook. f. (TWHF) is a traditional Chinese medicine that has a long history in treating diseases and more pharmacological effects were demonstrated recently. Components of TWHF mainly belong to the groups of alkaloids, diterpenoids, and triterpenoids. However, whether TWHF constituents are involved in herb-drug interaction (HDI) remains largely unknown. In the present study, we investigated the effect of four major components of TWHF, i.e. Triptolide (TPL), Celastrol (CL), and two alkaloids Wilforine (WFR) and Wilforgine (WFG) on the function of OATP1B1. It was found that co-incubation of these compounds greatly inhibited the uptake function of OATP1B1, with WFG (IC50 = 3.63 ± 0.61 µM) and WFR (IC50 = 3.91 ± 0.30 µM) showing higher inhibitory potency than TPL (IC50 = 184 ± 36 µM) and CL (IC50 = 448 ± 81 µM). Kinetic analysis revealed that co-incubation of WFG or WFR led to the reduction of both Km and Vmax of the DCF uptake. On the other hand, pre-incubation of WFG or WFR increased Km value of OATP1B1; while CL affected both Km and Vmax. In conclusion, co- and pre-incubation of the tested TWHF components inhibited OATP1B1 activity in different manners. Although co-incubation of WFG and WFR did not seem to directly compete with the substrates, pre-incubation of these alkaloids may alter the substrate-transporter interaction.

Three new sesquiterpenoid alkaloids from the roots of Tripterygium wilfordii and its cytotoxicity.

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 IC50 value in the range of 0.26-9.67 µΜ and compounds 1-5 showed potent cytotoxicity against LN-229 cell with IC50 values in the range of 0.50-7.38 µΜ.

Tripterygium glycoside ameliorates neuroinflammation in a mouse model of Aß25-35-induced Alzheimer's disease by inhibiting the phosphorylation of IκBα and p38.

Alzheimer's disease (AD) is acommon neurodegenerative disease in the aged population. Tripterygium glycoside (TG) has been reported to protect the nervous system. However, the effect of TG on AD is still unknown. We aimed to explore the effect of TG on AD. Thirty-two C57BL/6J mice were randomly selected and assigned to the normal control, AD model, AD+donepezil, and AD+TG groups. PC12 cells were assigned to the normal control, AD cell model, and AD+TG groups. The alterations in spatial memory and learning abilities of mice were measured by Morris water maze. Neuronal damage in mice was detected using Nissl staining. The expression levels of Aß25-35, p-Tau, and CD11b in brain tissues were detected using immunohistochemistry. The expression levels of IL-1ß, TNF-α, NO, p-P38, P38, p-IκBα, Caspase1, COX2, and iNOS were measured using ELISAs, qRT-PCR, and western blotting.TG significantly improved the spatial memory and learning abilities of AD mice. Compared toAD model group, significantly lower expression levels of Aß25-35, p-Tau, and CD11b were observed in AD+TG group (p < 0.05). The neuron density significantly increased in AD+TG group (p < 0.05). Significantly lower expression levels of IL-1ß, TNF-α, NO, caspase-1, COX2, iNOS, p-IκBα and p-P38 MAPK were detected in AD+TG group (p < 0.05). In summary, TG may exert aneuroprotective effect by suppressing the release of inflammatory factors and microglial activity and inhibiting the phosphorylation of IκBα and p38 MAPK. These findings may improve our understanding of the mechanism of TG intervention in AD.