Exploration of "Strengthening Kidney Yin" Method to Alleviate Female Reproductive Damage Caused by Glycosides of Tripterygium wilfordii Polyglycoside / 中国实验方剂学杂志

Tripterygium wilfordii polyglycosides are one of the most commonly used Tripterygium wilfordii preparations， which have anti-inflammatory and immune-regulating effects. Their unique therapeutic effect on some autoimmune diseases and kidney diseases is almost irreplaceable by other similar drugs， but the possible reproductive damage is the bottleneck that hinders their clinical application. In clinical use， female patients often suffer from menstrual cycle disorders， decreased menstrual flow， even amenorrhea， infertility， and other symptoms， and the main toxic mechanism lies in damaging the reproductive and endocrine functions of the ovary and inhibiting the growth and development of follicles. Therefore， it is particularly necessary to understand the toxic and side effects of Tripterygium wilfordii polyglycosides on female reproduction and master the detoxification methods during clinical use. However， there is no clear solution to these problems. According to the theory of traditional Chinese medicine， "kidney governs reproduction"， and the relationship between kidney Yin， kidney essence， and female ovum is close. Therefore， by considering that the damage to the reproductive system caused by Tripterygium wilfordii polyglycosides belongs to the category of kidney deficiency， Yin damage， and essence deficiency， the "strengthening kidney Yin" method is proposed. It points out that the reproductive toxicity damage of Tripterygium wilfordii polyglycosides on the female can be effectively alleviated by tonifying kidney and Yin essence in clinical use. The relevant research on traditional Chinese medicine， classical prescription， test prescription， and acupuncture is summarized to verify the necessity of the "strengthening kidney Yin" method， so as to provide a theoretical basis for the safe and rational clinical use of Tripterygium wilfordii.

Digestive System Injury Induced by Tripterygium wilfordii and How to Reduce Toxicity and Enhance Efficacy： A Review / 中国实验方剂学杂志

Tripterygium wilfordii is widely used in the treatment of immune system disease and has a remarkable curative effect. Triptolide and Tripterygium glycosides are the most commonly used active ingredients in clinical practice， but their treatment window is narrow and there are many side effects. The damage involves the reproductive system， blood system， cardiovascular system， digestive system， etc. Based on clinical observations and literature summaries， the symptoms of adverse reactions mostly occur in the digestive system （liver and gastrointestinal tract）. Relevant scholars have launched a lot of studies of the manifestations of liver injury induced by T. wilfordii and the mechanism of liver injury. The mechanism is mainly related to liver cell apoptosis， induction of oxidative stress， immune injury， excessive autophagy of liver cells， abnormal fatty acid metabolism， and abnormal enzyme metabolism in liver tissues. This article reviewed and summarized relevant literature on gastrointestinal injury caused by T. wilfordii， but there are few studies on the manifestations and mechanisms of adverse reactions， which still need further research by scholars. In addition， this article also summarized the research on how to reduce toxicity and enhance efficacy of prescriptions prepared from T. wilfordii in the digestive system， mainly involving compatibility with western medicines （Methotrexate， Leflunomide， Iguratimod， etc.）， use along or combination with Chinese medicines （single Chinese medicine， Chinese medicine monomers， and Chinese medicine compounds）， acupuncture and moxibustion （electroacupuncture and moxibustion）， dosage form improvement （glycol plastid gel， self-dissolving microneedle， solid lipid nanoparticles， gastric floating sustained-release capsules， etc.）， processing （steaming， stir-frying， radish seed processing， money grass processing， licorice processing， etc.）， and other methods to reduce toxicity. To sum up， this article analyzed the manifestations， mechanisms， and methods of reducing toxicity and enhancing efficacy of T. wilfordii-induced liver injury and gastrointestinal injury by sorting out relevant literature， in order to provide a reference for the clinical application of T. wilfordii and some research ideas for the future in-depth study of T. wilfordii-induced digestive system injury.

Inflammatory and Immunomodulatory Effects of Tripterygium wilfordii Multiglycoside in Mouse Models of Psoriasis Keratinocytes / 中国结合医学杂志

OBJECTIVE@#To determine the role of Tripterygium wilfordii multiglycoside (TGW) in the treatment of psoriatic dermatitis from a cellular immunological perspective.@*METHODS@#Mouse models of psoriatic dermatitis were established by imiquimod (IMQ). Twelve male BALB/c mice were assigned to IMQ or IMQ+TGW groups according to a random number table. Histopathological changes in vivo were assessed by hematoxylin and eosin staining. Ratios of immune cells and cytokines in mice, as well as PAM212 cell proliferation in vitro were assessed by flow cytometry. Pro-inflammatory cytokine expression was determined using reverse transcription quantitative polymerase chain reaction.@*RESULTS@#TGW significantly ameliorated the severity of IMQ-induced psoriasis-like mouse skin lesions and restrained the activation of CD45+ cells, neutrophils and T lymphocytes (all P<0.01). Moreover, TGW significantly attenuated keratinocytes (KCs) proliferation and downregulated the mRNA levels of inflammatory cytokines including interleukin (IL)-17A, IL-23, tumor necrosis factor α, and chemokine (C-X-C motif) ligand 1 (P<0.01 or P<0.05). Furthermore, it reduced the number of γ δ T17 cells in skin lesion of mice and draining lymph nodes (P<0.01).@*CONCLUSIONS@#TGW improved psoriasis-like inflammation by inhibiting KCs proliferation, as well as the associated immune cells and cytokine expression. It inhibited IL-17 secretion from γ δ T cells, which improved the immune-inflammatory microenvironment of psoriasis.

Diterpenoids of Tripterygium wilfordii decrease inflammatory response of macrophage by inhibiting multiple signaling pathways / 中国药理学通报

Aim To study the anti-inflammatory activ¬ity of diterpenes from Tripterygium wilfordii on lipopo- lysaccharide ( LPS)-induced macrophage and its mech¬anism. Methods MTT assay was used to detect the cytotoxicity of compounds. The Griess method was used to detect the NO on LPS-induced RAW264. 7 cells. ELISA was applied to determine the contents of inter- leukin 6 (IL-6) , tumor necrosis factor a ( TNF-a ) , interleukin lp (IL-lfj) and interleukin 18 (IL-18) in cell culture supernatant. Western blot was used to de¬tect IkBcx, .INK, ERK, p38, STAT3 and their phos-phorylation in LPS-induced RAW264.7, as well as the effect on COX-2, iNOS, NLRP3, caspase-1 , cleaved- caspase-1. Flow cytometry was employed to detect the effects of compounds on the phagocytosis of RAW 264. 7 cells. Results Hypoglicin II (1) and ent-pimara-8 (14) , 15-diene-19-ol (6) , two diterpenoid compounds from Tripterygium wilfordii could effectively inhibit the expression of inflammatory mediators ( COX-2 and iN- OS) and inflammatory cytokines (IL-6, IL-lp, IL- 18) in LPS-induced RAW264. 7 cells. Further re¬search found that the phosphorylation of IkBcx , JNK, ERK, P38, STAT3 and NLRP3 was all inhibited; however, there was no significant effect on the expres¬sion of IkBcx, JNK, ERK, P38 and STAT3. At the same time, they also inhibited the phagocytosis of mac-rophages. Conclusions The anti-inflammatory mecha¬nism of Tripterygium wilfordii diterpenoids 1 and 6 might be through inhibiting the production of NLRP3 inflammatory bodies, inflammatory mediators (COX-2 and iNOS) and inflammatory cytokines (IL-6, IL-lp and IL-18) , which is closely related to inhibiting the activation of MAPK, NF-kB and STAT3 pathway.

Exploration on "Efficacy-toxicity" Association Mechanisms of Tripterygium wilfordii Polyglycoside Tablets Against Rheumatoid Arthritis Based on Multi-omics Integrated Regulatory Network / 中国实验方剂学杂志

ObjectiveTo explore the "efficacy-toxicity" association mechanisms of Tripterygium wilfordii polyglycoside tablets （TWPT） by establishing and analyzing an interaction network associated with the clinical efficacy of TWPT in the treatment of rheumatoid arthritis （RA） and TWPT-induced liver injury. MethodOn the basis of the TWPT efficacy-related gene expression profile and TWPT-induced liver injury-related protein expression profile which were both obtained from our clinical cohorts， the "efficacy-toxicity" association network of TWPT was constructed， and the key network targets were identified by calculating the topological values of the nodes， including the degree， closeness and betweenness. After that， the biological functions and pathways of the key network targets were investigated by enrichment analysis. ResultA total of 119 differentially expressed genes （58 up-regulated and 61 down-regulated） between RA patients with TWPT well and weak response were identified as TWPT efficacy-related genes by clinical transcriptomics， and 49 differentially expressed proteins （36 up-regulated and 13 down-regulated） were demonstrated to be TWPT-induced liver injury-related proteins by clinical proteomics. In addition， the clinical symptom enrichment analysis indicated that the TWPT efficacy-related genes were significantly associated with various clinical symptoms of arthralgia in traditional Chinese medicine and clinical phenotypes of modern medicine， and most of the TWPT-induced liver injury-related proteins were involved in digestive system abnormalities. Therefore， the aforementioned multi-omics data represented the main clinical symptoms of TWPT treating RA and inducing liver injury. Mechanically， the "efficacy-toxicity" association network revealed that both TWPT efficacy-related genes and TWPT-induced liver injury-related core proteins were involved in the "immune-inflammatory" imbalance， especially playing an important role in neutrophil degranulation， complement cascade reaction， and immune-inflammatory response mediated by protein post-translational modification. Notably， the above genes and proteins were also enriched in various signaling pathways related to cell proliferation and cell cycle regulation， such as RAS and mitogen-activated protein kinase （MAPK） signaling pathway， and in several liver functional processes， such as glycogen metabolism and redox reaction. ConclusionThis study systematically explained the "efficacy-toxicity" association characteristics and molecular mechanisms of TWPT by applying a research strategy integrating clinical phenomics， transcriptomics and proteomics， laying a good data foundation for exploring the "efficacy enhancing and toxicity-reducing" mechanisms of TWPT.

Meta-analysis of Effect of Tripterygium wilfordii Polyglycoside Tablets Combined with Methotrexate and/or Leflunomide on Autoantibodies in Rheumatoid Arthritis / 中国实验方剂学杂志

ObjectiveTo evaluate the effect of Tripterygium wilfordii polyglycoside tablets （TWPT） combined with conventional synthetic disease-modifying antirheumatic drugs （csDMARDs） including methotrexate （MTX） and/or leflunomide （LEF） on autoantibodies in rheumatoid arthritis （RA） patients. MethodPubMed， EMBASE， Web of Science， Cochrane Library， China National Knowledge Infrastructure （CNKI）， VIP， Wanfang Data， and China Biomedical Literature Service System （SinoMed） were searched for randomized controlled trials （RCTs） of TWPT combined with MTX and/or LEF in the treatment of RA patients from database inception to December 1， 2021. Primary outcome indicators included rheumatoid factor （RF） and anti-citrullinated protein antibody （ACPA）， and secondary outcome indicators included immunoglobulin （IgA， IgG， and IgM） and adverse drug events （ADE）. ResultThirty-one RCTs， involving 2 643 adult patients， were included， including 20 RCTs of TWPT combined with MTX， 10 of TWPT combined with LEF， and one of TWPT combined with MTX and TWPT. The follow-up time ranged from two weeks to 13 months. Compared with csDMARDs alone， TWPT combined with other drugs significantly improved serum RF of RA patients ［SMD=-2.45， 95% CI ［-2.97， -1.93］， P<0.000 01］， anti-CCP ［SMD=-1.41， 95% CI （-2.35， -0.48）， P=0.003］， IgM ［SMD=-1.90， 95% CI （-3.03， -0.76）， P=0.001］， and IgA ［SMD=-1.18， 95% CI （-2.23， -0.12）， P=0.03］. There were no significant effects on IgG ［SMD=-1.02， 95% CI （-2.04， 0.01）， P=0.05］ and ADE ［RR=0.87， 95% CI （0.66， 1.15）， P=0.32］. ConclusionThe results of this study show that compared with csDMARDs alone， TWPT combined with csDMARDs can effectively improve the levels of autoantibodies in RA patients without increasing the incidence of ADE. However， due to the limited quality and quantity of the included RCTs， the relevant conclusions are only used as a reference for the clinical diagnosis and treatment of RA， and more high-quality studies are still needed to further confirm their efficacy.

Mechanism of Glycyrrhizae Radix et Rhizoma Alleviating Tripterygium wilfordii Polyglycoside Tablets-induced Liver Injury / 中国实验方剂学杂志

ObjectiveTo investigate the protective effect of cytochrome P4502D6 （CYP2D6） and cytochrome P4503A4 （CYP3A4）， key enzymes of drug metabolism in liver， on acute liver injury in water extract of Glycyrrhizae Radix et Rhizoma （WEOGRR）. MethodHealthy male Kunming mice were divided into normal group， model group， WEOGRR low-， medium- and high-dose groups （5， 10， 15 g·kg-1·d-1） and positive drug group （diammonium glycyrrhizinate， 75 mg·kg-1·d-1）， with 10 in each group. One week after preventive administration， acute liver injury model was induced by single intragastric administration of 270 mg·kg-1 Tripterygium Glycosides tablets， and samples were collected after 18 h. The pathological changes of liver were observed by hematoxylin-eosin （HE） staining. Serum liver function indexes including alanine aminotransferase （ALT）， aspartate aminotransferase （AST）， γ-glutamyl transpeptadase （γ-GT）， alkaline phosphatase （ALP）， and total bilirubin （TBIL） as well as the levels of oxidative stress indexes including malondialdehyde （MDA） and superoxide dismutase （SOD） in hepatocytes were determined by biochemical method. Real-time polymerase chain reaction （Real-time PCR） and Western blot were performed to detect the mRNA and protein expression levels of CYP2D6 and CYP3A4， respectively. ResultCompared with normal group， model group had significant hepatocyte swelling and inflammatory cell infiltration （P<0.01）， increased AST， ALT， γ-GT， ALP and TBIL （P<0.05）， elevated MDA and decreased SOD （P<0.01） as well as down-regulated mRNA and protein expression levels of CYP2D6 and CYP3A4 （P<0.05）. Compared with the model group， the normal group had intact liver structure without obvious abnormality， and the WEOGRR groups and positive drug group presented alleviated hepatocyte swelling and inflammatory cell infiltration （P<0.01）， reduced AST， ALT， γ-GT， ALP and TBIL （P<0.01）， lowered MDA and increased SOD （P<0.01） as well as up-regulated expression levels of CYP2D6 and CYP3A4 （P<0.01）. ConclusionThe protective effect of WEOGRR on acute liver injury induced by Tripterygium glycosides tablets may be related to reducing the contents of AST， ALT， γ-GT， ALP and TBIL in serum， inhibiting MDA and increasing the activity of SOD in liver cells， and enhancing the activities of CYP2D6 and CYP3A4， thus accelerating the metabolism of toxic substances.

Effect of Tripterygium wilfordii Polyglycoside on Expression of NFAT2/COX-2 in Kidney Tissues of Rats with Diabetic Nephropathy / 中国实验方剂学杂志

ObjectiveTo explore the underlying mechanism of Tripterygium wilfordii polyglycoside tablets （TWPT） in the prevention and treatment of kidney injury in diabetic nephropathy （DN） through the nuclear factor of activated T-cells 2（NFAT2）/cyclooxygenase-2（COX-2） pathway. MethodForty-two male SD rats of SPF grade were selected and randomly divided into a normal group （n=8） and an experimental group （n=34） after one week of adaptive feeding. The rats in the normal group were fed conventionally. The DN model was established in rats of the experimental group by intraperitoneal injection of streptozotocin （STZ） following one week of feeding on a high-fat and high-glucose diet. After the death and failure cases during modeling were eliminated， the remaining 24 model rats were randomly divided into model group， valsartan （8.33 mg·kg-1·d-1） group， and TWPT （5 mg·kg-1·d-1） group. Rats in normal group and model group were given equal amounts of normal saline by gavage. After six weeks， body weight was measured and urine samples were collected. Blood samples were collected from the abdominal aorta， and then the rats were sacrificed for sampling. Biochemical indicators， such as serum blood urea nitrogen （BUN）， serum creatinine （SCr）， alanine aminotransferase （ALT）， blood lipid， blood glucose， and 24-hour urine total protein （24 h UTP）， were determined. Hematoxylin-eosin （HE） staining and Masson staining were used to observe the pathology of the kidney. Enzyme-linked immunosorbent assay （ELISA） was used to detect NFAT2 and COX-2 expression levels in the serum. Western blot and Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR）were adopted to detect NFAT2， COX-2 protein and mRNA expression in kidney tissues， respectively. ResultCompared with the normal group， the model group showed elevated 24 h UTP， BUN， SCr， CHO， TG， and FBG， increased serum NFAT2 and COX-2 production and expression （P<0.01）， and elevated protein and mRNA expression of NFAT2 and COX-2 in kidney tissues （P<0.01）. In addition， the pathology of the kidney showed enlarged glomeruli， mild proliferation of mesangial cells， and widened mesangial stroma. Compared with the model group， the TWPT group showed decreased 24 h UTP， BUN， SCr， CHO， TG， and FBG （P<0.05，P<0.01）， basically normal glomerular morphology， decreased expression of serum NFAT2 and COX-2 （P<0.01）， and down-regulated protein and mRNA expression of NFAT2 and COX-2 in kidney tissues （P<0.01）. ConclusionTWPT can alleviate 24 h UTP in DN model rats， protect renal function， and improve renal pathology， and its mechanism of action may be related to the down-regulation of NFAT2/COX-2 expression in the serum and kidney tissues.

Effect and Mechanism of Cuscutae Semen Total Flavonoids Combined with Tripterygium wilfordii Polyglycoside Tablets on Ovarian Germline Stem Cells of Physiological Mice by Notch Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo observe the effect of Cuscutae Semen total flavonoids combined with Tripterygium wilfordii polyglycoside tablets （TWPT） on ovarian germline stem cells of female physiological mice through neurogenic locus notch homolog （Notch） signaling pathway. MethodSixty female Kunming mice （5 weeks old） were randomly divided into normal group， Tripterygium wilfordii polyglycoside tablets low-， high-dose groups （13.65 mg·kg-1·d-1 and 27.3 mg·kg-1·d-1， 1 and 2 times clinical equivalent dose）， Cuscutae Semen total flavonoids low- and high-dose groups （150 mg·kg-1·d-1 and 300 mg·kg-1·d-1）， and combination group （13.65 mg·kg-1·d-1 TWPT and 150 mg·kg-1·d-1 Cuscutae Semen total flavonoids）， with 10 in each group. After 3 weeks of continuous administration， the uterus/brain and ovarian/brain indexes were calculated， and the pathological changes of ovarian tissue were observed under light microscope. The content of estradiol in serum was determined by enzyme linked immunosorbent assay （ELISA）. Immunofluorescence assay was performed to observe the expressions of germline stem cell markers in ovarian epithelium， including mouse vasa homologue （Mvh）， octamer-binding transcription factor 4 （Oct4）， tyrosine-protein kinase receptor （c-kit）， Nanog， Notch signaling pathway molecules， neurogenic locus notch homolog protein 1 （Notch1）， hes family BHLH transcription factor 1（Hes1）， and jagged canonical Notch ligand 1 （JAG1）. ResultCompared with the normal group， low and high doses of TWPT had no significant effect on the uterus/brain and ovary/brain indexes and the uterus and ovary morphologies of mice， while only the number of atretic follicles was increased （P<0.01）. The expressions of ovarian germline stem cell markers and Notch signaling pathway molecules had a decreasing trend in TWPT low-dose group， while the expressions of Mvh， c-kit， and Nanog were down-regulated （P<0.05， P<0.01） and the expressions of Notch1 and Hes1 were also reduced （P<0.01） in TWPT high-dose group. However， the above indexes were increased in Cuscutae Semen total flavonoids low-dose group （P<0.05， P<0.01）. Compared with the low does of TWPT group， the combination group had a decrease in the increased number of atretic follicles （P<0.01）， an improvement in the down-regulated expressions of Mvh and Nanog （P<0.01）， and an increase in the expressions of Notch1 and Hes1 （P<0.05， P<0.01）. ConclusionOvarian germline stem cells are the source target of the reproductive toxicity of TWPT. Cuscutae Semen total flavonoids participate in the regulation of the germline stem cell pathways to alleviate the reproductive toxicity caused by TWPT， and its mechanism of action may be related to the Notch signaling pathway.

Systematic Review on Safety of Chinese Medicines Combined with Tripterygium wilfordii Preparations in Treatment of Rheumatoid Arthritis and Exploration on Underlying Network Regulatory Mechanisms of Enhancing Efficacy and Reducing Toxicity / 中国实验方剂学杂志

ObjectiveTo systematically evaluate the safety of Chinese medicines combined with Tripterygium wilfordii polyglycoside tablets/Tripterygium wilfordii tablets （TWPT/TWT） in the treatment of rheumatoid arthritis （RA）， and to explore the network regulatory mechanisms of enhancing efficacy and reducing toxicity of commonly used combination regimes. MethodThe literature involving the adverse reactions of TWPT/TWT in treating RA was searched and collected from three Chinese databases （CNKI， Wanfang Data， VIP） and three English databases （PubMed， Cochrane Library， Embase） from the inception of the databases to July 2021. All studies were assessed by the Cochrane risk of bias tool， and the data were extracted and analyzed by Stata 15.0. Furthermore， Integrative Pharmacology-based Research Platform of Traditional Chinese Medicine 2.0 （TCMIP v2.0，http：//www.tcmip.cn/） was used to construct a "drug target-symptom gene of efficacy and toxicity" interaction network， to explore the underlying network regulatory mechanisms of enhancing efficacy and reducing toxicity of common T. wilfordii preparation combinations. ResultA total of 2 132 articles on Chinese medicines combined with TWPT/TWT in the treatment of RA were retrieved， and 18 of them were finally included. The systematic review showed that the adverse reactions of TWPT/TWT against RA mainly occurred in the digestive system， blood system， and reproductive system， of which digestive system had the highest incidence of damages. However， the combination with Chinese medicines effectively alleviated the adverse reactions caused by TWPT/TWT ［RR （95% CI）=0.45 （0.30， 0.66）， P<0.01］. In addition， the subgroup analysis indicated that the age of RA patients， course of disease， combination regimen， medication dosage and duration of treatment all affected the occurrence of adverse reactions of TWPT/TWT. It was found in clinical studies that total glucosides of paeony （TGP） and TWPT/TWT was most widely combined， and the effect of TGP in reducing TWPT/TWT-induced hepatotoxicity was also more significant than that of other Chinese medicines. Moreover， taking this combination regime as an example， this paper explored the "efficacy-toxicity" association mechanisms of TGP-TWPT/TWT against RA. The "drug target-symptom gene of efficacy and toxicity" interaction network revealed that the core network targets of TGP-TWPT/TWT enhanced efficacy and reduced toxicity mainly through regulating immunity-inflammation-related pathways， metabolic pathways and cell signal transduction. Especially， interleukin-4 （IL-4） and interleukin-13 （IL-13）， which were involved in the "immunity-inflammation" module， were the common targets of TGP-TWPT/TWT to enhance efficacy and reduce toxicity. The endogenous sterols， bile acids and bile salts， insulin secretion and other metabolic pathways in the "body metabolism" module were closely associated with the mechanisms of TWPT/TWT inducing hepatotoxicity and TGP reducing hepatotoxicity. While cell function regulation pathways， such as stem cell factor （SCF）/tyrosine kinase receptor （KIT） signaling pathway and phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt）signaling pathway were involved in both anti-RA effects and hepatotoxicity of TWPT/TWT. ConclusionClinical application of suitable Chinese medicines combined with TWPT/TWT in the treatment of RA can effectively improve the rheumatism and reduce the adverse reactions of TWPT/TWT， and TGP-TWPT/TWT has the most significant toxicity-reducing effect. Further biological network-based investigation indicates that the toxicity-reducing mechanism of TGP-TWPT/TWT may be related to the regulation of interleukin signaling pathway and bile acid metabolism pathway， and the synergistic efficacy-enhancing effect of the combination may be achieved by acting on interleukin signaling pathway and cell function regulation pathway.

Study on the effects and mechanism of total glucosides of paeony on enhancing efficacy and reducing toxicity of Tripterygium wilfordii polyglycoside in the treatment of eczema / 中国药房

OBJECTIVE To investigate the effects of total glucosides of paeony （TGP） on enhancing efficacy and reducing toxicity of Tripterygium wilfordii polyglycoside （TWP） in the treatment of eczema. METHODS Totally 50 SD male rats were collected to establish eczema model by sensitizing with 2，4-dinitrofluorobenzene-acetone olive oil solution （volume ratio was 4∶1） on the abdominal area and provoking on the back and ear. Model rats were randomly divided into model group， loratadine group （0.9 mg/kg）， TWP group （9.45 mg/kg）， TGP group （162 mg/kg） and compatibility group （TWP 9.45 mg/kg+TGP 162 mg/kg）， with 10 rats in each group. Other 10 rats were collected to set as normal group. Three days after the first sensitization， administration groups were given relevant medicine intragastrically， and normal group and model group were given constant volume of 0.1% CMC-Na solution intragastrically， once a day， for consecutive 21 d. Twenty-four hours later after the final administration， the general condition of rats in each group was observed， and the eczema area and severity index （EASI） were scored； ear swelling degree of rats was measured， and the skinhistomorphology observation and pathological score were performed； protein expressions of p38 mitogen-activated 13938427612@126.com protein kinase （p38 MAPK）， phosphorylated p38 MAPK （p- MAPK） and phosphorylation level of p38 MAPK in rat skin tissue were detected； the levels of inflammatory indexes （interleukin-4， interferon- γ）， liver and kidney function indexes [glutamic-pyruvic transaminase （GPT）， glutamic-oxaloacetic transaminase （GOT）， serum creatinine （SCr） and blood urea nitrogen （BUN）] and oxidant stress indexes [total superoxide dismutase （T-SOD） and malondialdehyde （MDA）] were measured. RESULTS Compared with normal group， EASI score， ear swelling degree， pathological score， protein expressions of p38 MAPK and p-p38 MAPK， phosphorylation level of p38 MAPK， the levels of inflammatory indexes and BUN were all increased significantly in model group （P＜0.05）. Compared with model group， EASI scores， ear swelling degree， pathological scores， protein expressions of p38 MAPK and p-p38 MAPK， phosphorylation levels of p38 MAPK and levels of inflammatory indexes were all improved significantly in administration groups （P＜0.05）. The levels of GPT， GOT， SCr and BUN were increased significantly in TWP group， while the serum levels of GOT and SCr in TGP group and serum level of SCr in loratadine group were all decreased significantly （P＜0.05）. The levels of T-SOD in liver and kidney tissue were all decreased significantly in TWP group and compatibility group， while the levels of MDA were increased significantly （P＜0.05）. The compatibility group showed more obvious effect in improving the ear swelling degree， pathological score， p38 MAPK expression and its phosphorylation level and levels of inflammatory indexes， and could reverse the abnormality of liver and kidney indexes caused by TWP （P＜0.05）. CONCLUSIONS The combination of TGP and TWP has the effects of anti-inflammatory， synergistic and hepatorenal detoxification in eczema model rats. Its mechanism may be associated with down-regulating the expression of serum proinflammatory indexes and inhibiting the activation of p38 MAPK pathway.

Anti-inflammatory sesquiterpene polyol esters from the stem and branch of Tripterygium wilfordii / 中国天然药物

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%.

Effect of multi-glycosides of Tripterygium wilfordii on renal injury in diabetic kidney disease rats through NLRP3/caspase-1/GSDMD pyroptosis pathway / 中国中药杂志

This study investigated the effect of multi-glycosides of Tripterygium wilfordii(GTW) on renal injury in diabetic kidney disease(DKD) rats through Nod-like receptor protein 3(NLRP3)/cysteine-aspartic acid protease-1(caspase-1)/gsdermin D(GSDMD) pyroptosis pathway and the mechanism. To be specific, a total of 40 male SD rats were randomized into the normal group(n=8) and modeling group(n=34). In the modeling group, a high-sugar and high-fat diet and one-time intraperitoneal injection of streptozotocin(STZ) were used to induce DKD in rats. After successful modeling, they were randomly classified into model group, valsartan(Diovan) group, and GTW group. Normal group and model group were given normal saline, and the valsartan group and GTW group received(ig) valsartan and GTW, respectively, for 6 weeks. Blood urea nitrogen(BUN), serum creatinine(Scr), alanine ami-notransferase(ALT), albumin(ALB), and 24 hours urinary total protein(24 h-UTP) were determined by biochemical tests. The pathological changes of renal tissue were observed based on hematoxylin and eosin(HE) staining. Serum levels of interleukin-1β(IL-1β) and interleukin-18(IL-18) were detected by enzyme-linked immunosorbent assay(ELISA). Western blot was used to detect the expression of pyroptosis pathway-related proteins in renal tissue, and RT-PCR to determine the expression of pyroptosis pathway-related genes in renal tissue. Compared with the normal group, the model group showed high levels of BUN, Scr, ALT, and 24 h-UTP and serum levels of IL-1β and IL-18(P<0.01), low level of ALB(P<0.01), severe pathological damage to kidney, and high protein and mRNA levels of NLRP3, caspase-1, and GSDMD in renal tissue(P<0.01). Compared with the model group, valsartan group and GTW group had low levels of BUN, Scr, ALT, and 24 h-UTP and serum levels of IL-1β and IL-18(P<0.01), high level of ALB(P<0.01), alleviation of the pathological damage to the kidney, and low protein and mRNA levels of NLRP3, caspase-1, and GSDMD in renal tissue(P<0.01 or P<0.05). GTW may inhibit pyroptosis by decreasing the expression of NLRP3/caspase-1/GSDMD in renal tissue, thereby relieving the inflammatory response of DKD rats and the pathological injury of kidney.

Mechanism of Tripterygium wilfordii in treating renal cell carcinoma using network pharmacology / 国际生物医学工程杂志

Objective:To explore the mechanism of Tripterygium wilfordii in treating renal cell carcinoma using network pharmacology and experimental validation. Methods:The traditional Chinese medicine systems pharmacology (TCMSP) database and analysis platform was utilized to screen the active ingredients of T. wilfordii and predict the targets using the Swiss database. Renal cell carcinoma related targets were collected from DisGeNET, GeneCards, and OMIM databases, and intersecting targets were obtained through Venny 2.1.0. Protein-protein interaction (PPI) networks were mapped using the STRING database and Cytoscape software, and gene ontology (GO) and kyoto encyclopedia of genes and genomics (KEGG) enrichment analyses were performed. Component-target-pathway networks were constructed using Cytoscape software. To induce the subcutaneous transplantation tumor model of renal cell carcinoma, nude mice were randomly divided into a control group and a treatment group, with 5 mice in each group. In the treatment group, mice were gastrically instilled with 615 mg/ml of T. wilfordii solution (1 845 mg T. wilfordii granules dissolved into 3 ml water) 2.46 g/kg, 10 μl/time, once daily for 21 d. In the control group, mice were gastrically instilled with an equal amount of saline. Tumor volume was measured once every 5 days, and the expression of serine/threonine protein kinase 1 (AKT1), signal transduction and transcription activator 3 (STAT3), tumor necrosis factor (TNF), tumor protein p53 (TP53), JUN, mitogen-activated protein kinase 8 (MAPK8), and MAPK14 was detected by enzyme-linked immunoassay. Results:Twenty-eight active pharmaceutical ingredients and 117 potential targets of T. wilfordii were screened; 13 425 related disease targets were identified; and finally, 113 drug-disease intersecting targets were obtained. In the PPI network, AKT1, STAT3, TNF, TP53, JUN, MAPK8, and MAPK14 were the core targets. GO analysis showed that BP mainly included nuclear receptor activity, ligand-activated transcription factor activity, RNA polymerase-specific DNA-binding transcription factor binding, nuclear steroid receptor activity, and adrenergic receptor activity, etc. CC mainly included the response to hormones, the cellular response to lipids, the positive regulation of cell migration, the response to TNF, the inflammatory response, the cellular response to hormonal stimulation, the response to hypoxia, the response to metal ions, etc. MF involves membrane rafts, membrane microregions, the outer side of the plasma membrane, the lateral side of the membrane, plasma membrane rafts, presynaptic membranes, vesicles, transcriptional regulatory complexes, post-synaptic membranes, synaptic membranes, etc. KEGG analysis showed that T. wilfordii treatment of RCC involves signaling pathways such as lipid and atherosclerosis, advanced glycosylation end product-receptor for advanced glycosylation end products (AGE-RAGE), TNF, Toll-like receptor, phosphatidylinositol 3-kinase-protein kinase B (PI3K-Akt), and MAPK. The experimental validation results showed that the tumor volume was reduced after treatment with tretinoin ( P < 0.05), the expression of TP53 protein was increased ( P < 0.001), and the expression of AKT1, STAT3, TNF, JUN, MAPK8, and MAPK14 proteins were all reduced (all P < 0.001). Conclusions:In this study, the target and signaling pathways of T. wilfordii treatment of renal cell carcinoma were initially predicted, providing a reference basis for further research on its protective mechanism and clinical application.

Research on efficacy-toxicity of Tripterygium wilfordii Hook.F.In treatment of lupus nephritis based on network pharmacology / 中国药理学通报

Aim To explore the efficacy-toxicity of Tripterygium wilfordii Hook.f.in intervention of lupus nephritis by the method of network pharmacology.Methods Firstly, the active components were searched and the action targets of Tripterygium wilfordii Hook.f.were predicted through the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP).Secondly, the target genes of LN were collected through CoolGeN, OMIM and Gene Cards databases.And then, it was mapped to Tripterygium wilfordii Hook.f.targets and the drug component-disease target interaction network was constructed with Cytoscape software, and STRING database was used to analyze the protein interaction network.Finally, the common targets were analyzed by GO and KEGG pathway enrichment analysis using DAVID database to explore the potential mechanism of Tripterygium wilfordii Hook.f.in the treatment of LN.Results A total of 52 active components of Tripterygium wilfordii Hook.f.and 38 targets for the treatment of LN were screened.Most of the components had potential therapeutic effects on LN, but the effects of triptolide, tripterine, kaempferol and β-sitosterol may not be conducive to the improvement of LN.The results of KEGG analysis showed that efficacy-toxicity mainly involved NOD-like receptor signaling pathway, p53 signaling pathway, Toll-like receptor signal pathway and so on.Conclusions Tripterygium wilfordii Hook.f.plays the efficacy-toxicity effect on LN by regulating immune inflammation, cell proliferation and apoptosis, and its overall intervention effect needs further experimental study.

Mechanism of Tripterygium wilfordii in Treatment of Triple Negative Breast Cancer Based on Network Pharmacology and Experimental Validation / 中国实验方剂学杂志

ObjectiveTo explore the active ingredients， therapeutic targets， and relative signaling pathways of Tripterygium wilfordii in the treatment of triple negative breast cancer （TNBC） based on network pharmacology， and to verify the mechanism through in vitro cell model. MethodThe active ingredients of T. wilfordii were screened from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform （TCMSP）. The targets of TNBC were obtained from DisGeNET and GeneCards. Venny was used to identify the potential therapeutic targets of T. wilfordii against TNBC. Protein-protein interaction （PPI） network was constructed with String database. Gene ontology （GO） annotation and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway enrichment were carried out with DAVID to predict the mechanisms of potential targets. The molecular docking between triptolide and key targets were performed with AutoDock Vina. The effect of triptolide （0， 5， 10， 20， 30， 40， 50， 60， 80 nmol·L-1） on the proliferation of MDA-MB-231 cells was determined through methyl thiazolyl tetrazolium （MTT） assay. The effect of triptolide （0， 12.5， 25， 50 nmol·L-1） on the apoptosis of MDA-MB-231 cells was detected with Hoechst 33342 staining. Western blot was performed to detect the effect of triptolide （0， 25， 50 nmol·L-1） on the expression levels of key targets. ResultT. wilfordii had 23 active ingredients related to 55 potential targets of TNBC. GO and KEGG enrichment revealed that the potential targets were associated with 103 biological processes， 15 cellular components， and 35 molecular functions， and were involved in 140 signaling pathways including atherosclerosis and apoptosis. The results of molecular docking demonstrated that triptolide could bind with the targets including threonine kinase 1 （Akt1）， vascular endothelial growth factor A （VEGFA）， cellular tumor antigen p53 （p53）， transcription factor AP-1 （JUN）， signal transducer and activator of transcription 3 （STAT3）， tumor necrosis factor （TNF）， mitogen-activated protein kinase 8 （MAPK8）， prostaglandin G/H synthase 2 （PTGS2）， and Caspase-3. According to the results of MTT assay， triptolide （20， 30， 40， 50， 60， 80 nmol·L-1） inhibited the proliferation of MDA-MB-231 cells compared with blank control （P<0.05， P<0.01）. Hoechst 33342 staining showed that triptolide （12， 25， 50 nmol·L-1） induced the apoptosis of MDA-MB-231 cells compared with black control （P<0.05， P<0.01）. Western blot showcased that 50 nmol·L-1 triptolide down-regulated the relative expression levels of p-Akt， TNF-α， and VEGFA， while 25 and 50 nmol·L-1 triptolide up-regulated the relative expression level of p53 in a dose-dependent manner compared with the blank control （P<0.05， P<0.01）. ConclusionT. wilfordii has multiple ingredients， targets， and pathways in the treatment of TNBC. It may regulate p53， VEGFA， TNF-α and other key targets to induce cell apoptosis and suppress angiogenesis and inflammatory response， which provides a scientific basis for the further investigation and clinical application of T. wilfordii.

CiteSpace knowledge map analysis of research progress on Tripterygium wilfordii toxicity / 中国中药杂志

This study systematically searched CNKI and Web of Science(WoS) for the research papers on the toxicity of Tripterygium wilfordii included from database inception to August 31, 2021, and visually displayed the authors, research institutions, keywords, and other contents using bibliometrics and CiteSpace 5.8.3. Furthermore, the current situation and research progress on T. wilfordii safety were also analyzed based on information extraction to find the research hotspot, evolution path, and development trend, and to provide references for future research. A total of 1 876 Chinese papers and 243 English papers were included in the study. The analysis of authors showed that WANG Qi and ZHANG Luyong had the most publications in Chinese and English papers, respectively. According to the analysis of research institutions, National Institutes for Food and Drug Control and China Pharmaceutical University possessed the largest number of Chinese and English papers, respectively, but there was less cooperation between them. The analysis of keywords in Chinese and English papers showed that the research contents of the safety of T. wilfordii mainly focused on clinical monitoring, mechanism, dosage form improvement, quality standard, component analysis, monomer research, efficiency and toxicity reduction, etc. Metabonomics, tripterine, and the underlying mechanism of toxicity were the research hotspots in the future. At present, the research on the toxicity of T. wilfordii is still under development. It is necessary to highlight the in-depth research and strengthen the inter-group and inter-region cooperation of authors or institutions to provide references for the research on the toxicity of T. wilfordii.

Preliminary exploration of detoxification mechanism of processing methods on cardiotoxicity induced by radix Tripterygium wilfordii in mice via Nrf2/HO-1 pathway / 中国中药杂志

This study aims to investigate the detoxification effects of different processing methods on the cardiotoxicity induced by radix Tripterygium wilfordii, and preliminarily explore the detoxification mechanism via the nuclear factor E2-related factor 2(Nrf2)/heme oxygenase 1(HO-1) pathway. The raw and processed products [stir-fried product, product stir-fried with Lysimachiae Herba(JQC), product stir-fried with Phaseoli Radiati Semen(LD), product stir-fried with Paeoniae Radix Alba(BS), product stir-fried with Glycyrrhizae Radix et Rhizoma(GC), and product stir-fried with vinegar(CZ)] of radix T. wilfordii were administrated to mice by gavage at a dose of 2 g·kg~(-1)(based on crude drugs) for 28 days. Twenty-four hours after the last administration, we measured the serum biochemical indexes of mice to evaluate the detoxification effect. Furthermore, we determined the expression of key proteins of Nrf2/HO-1 pathway in mouse heart tissue by Western blot and some oxidation/antioxidation-related indexes by corresponding kits to explore the detoxification mechanism. The administration of the raw product elevated the levels of serum creatine kinase, lactate dehydrogenase, and malondialdehyde, a product of cardiac lipid peroxidation(P<0.01), down-regulated the protein levels of Nrf2 and HO-1(P<0.01), and reduced the levels of total superoxide dismutase, glutathione, glutathione peroxidase, and glutathione S-transferase(P<0.01). However, after the administration of the products stir-fried with JQC, LD, BS, GC, and CZ, the abnormalities of the above indexes induced by the raw product were recovered(P<0.05 or P<0.01). In particular, the product stir-fried with JQC showed the best performance. Taken all together, the cardiotoxicity induced by radix T. wilfordii could be attenuated by stir-frying with JQC, LD, BS, GC, and CZ, and the stir-frying with JQC showed the best detoxification effect. The mechanism might be associated with the cardiac antioxidant defense and oxidative damage mitigation mediated by the up-regulated Nrf2.

Mechanistic analysis for the origin of diverse diterpenes in Tripterygium wilfordii

Tripterygium wilfordii is a valuable medicinal plant rich in biologically active diterpenoids, but there are few studies on the origins of these diterpenoids in its secondary metabolism. Here, we identified three regions containing tandemly duplicated diterpene synthase genes on chromosomes (Chr) 17 and 21 of T. wilfordii and obtained 11 diterpene synthases with different functions. We further revealed that these diterpene synthases underwent duplication and rearrangement at approximately 2.3-23.7 million years ago (MYA) by whole-genome triplication (WGT), transposon mediation, and tandem duplication, followed by functional divergence. We first demonstrated that four key amino acids in the sequences of TwCPS3, TwCPS5, and TwCPS6 were altered during evolution, leading to their functional divergence and the formation of diterpene secondary metabolites. Then, we demonstrated that the functional divergence of three TwKSLs was driven by mutations in two key amino acids. Finally, we discovered the mechanisms of evolution and pseudogenization of miltiradiene synthases in T. wilfordii and elucidated that the new function in TwMS1/2 from the terpene synthase (TPS)-b subfamily was caused by progressive changes in multiple amino acids after the WGT event. Our results provide key evidence for the formation of diverse diterpenoids during the evolution of secondary metabolites in T. wilfordii.

Identification and functional characterization of 2,3-oxidosqualene cyclase genes family in Tripterygium wilfordii / 药学学报

italic>Tripterygium wilfordii Hook. f. is a valuable medicinal plant, with anti-tumor, anti-inflammatory, immunosuppressive and other pharmacological activities. Triterpenoids are one of the main active components that exert pharmacological effects. However, the content of triterpenoids dominated by triptolide is very low in Tripterygium wilfordii, and the analysis of the biosynthetic pathway of triterpenoids in Tripterygium wilfordii provides an effective new idea for obtaining these compounds. 2,3-Oxidosqualene cyclases (OSCs) are the key enzyme that catalyzes the formation of triterpene skeleton diversity. Based on the genome and transcriptome data of Tripterygium wilfordii, 16 OSC genes were identified and analyzed. Phylogenetic analysis showed that 16 TwOSC proteins could be mainly classified as four groups. They are β-amyrin synthase group, friedelin synthase group, multifunctional amyrin synthase and cycloartenol synthase group. TwOSC6 was successfully cloned. Functional characterization analysis revealed that TwOSC6 can catalyze the formation of α-amyrin and β-amyrin. This indicates that TwOSC6 is a multifunctional amyrin synthase. This provides new gene resources for the diversity of Tripterygium wilfordii triterpenoids, as well as new gene elements for biosynthesis triterpenoids.