Effects of triptolide on ovarian oxidative stress and mitophagy of granulosa cells in rats / 新乡医学院学报

Objective To observe the effects of different concentration and intervention time of triptolide(TP)on ovarian oxidative stress and mitophagy in rats.Methods Fifty 3-month-old female Sprague-Dawley rats were observed for 2 consecutive estrus cycles,and 25 rats with normal estrus cycles were selected and divided into the blank control group,experiment group 1,experiment group 2,experiment group 3 and experiment group 4 according to random number table method,with 5 rats in each group.Rats in experiment group 1 were administered intragastrically 400 μg·kg-1 TP once a day for 30 d.Rats in experiment group 2 were given 400 μg·kg-1 TP once a day for 40 d.Rats in experiment group 3 were given 500 μg·kg-1 TP once a day for 30 d.Rats in the experiment group 4 were given 500 μg·kg-1 TP once a day for 40d.Rats in the blank control group were given 10 mL·kg-1 distilled water once a day for 40 d.Serum anti-mullerian hormone(AMH),estradiol(E2)and follicle-stimulating hormone(FSH)levels were detected by enzyme-linked immunosorbent assay;hematoxylin-eosin staining was used to observe the pathological changes of ovarian tissue;malondialdehyde(MDA)level and superoxide dismutase(SOD)activity in ovarian tissue were detected by microplate reader.The morphological and structural changes of mitochondria in ovarian granulosa cells of rats in each group were observed by transmission electron microscope,and the apoptosis rate of ovarian granulosa cells was detected by flow cytometer.Results Compared with the blank control group,the levels of AMH and E2 in serum and SOD activity in ovarian tissue of rats in the experiment groups 1,2,3 and 4 significantly decreased,while the levels of FSH in serum and MDA in ovarian tissue significantly increased(P＜0.05).Compared with the experiment group 1,the levels of AMH and E2 in serum and SOD activity in ovarian tissue of rats in experiment groups 2,3 and 4 significantly decreased,while the levels of FSH in serum and MDA in ovarian tissue significantly increased(P＜0.05).There was no significant difference in the levels of AMH,E2 and FSH in serum,SOD activity and MDA level in ovarian tissue of rats among experiment groups 2,3 and 4(P＞0.05).The apoptosis rate of ovarian granulosa cells in experiment groups 1,2,3 and 4 was significantly higher than that in the blank control group(P＜0.05);the apoptosis rate of ovarian granulosa cells in experiment groups 2,3 and 4 was significantly higher than that in experiment group 1(P＜0.05);the apoptosis rate of ovarian granulosa cells in experiment groups 3 and 4 was significantly higher than that in experiment group 2(P＜0.05);the apoptosis rate of ovarian granulosa cells in experiment group 4 was significantly higher than that in experiment group 3(P＜0.05).In the blank control group,the ovarian tissue capsule was intact,and the number and development of primordial,primary and secondary follicles in the cortex were normal,with rare atretic follicles and less corpus luteum.The fibrous connective tissue in the medullary area was closely arranged,and no obvious edema or necrosis was observed.In experiment groups 1,2,3 and 4,the number of follicles in ovarian tissue decreased,the number of atretic follicles increased,and necrosis and shedding of follicles in the granular layer and cystic dilatation of follicles were observed.The pathological changes of ovarian tissue in experiment group 1 were relatively light,with fewer atretic follicles and less cell necrosis in the granulosa cell layer.The degree of ovarian tissue lesions in experiment group 4 was the most severe,with more atretic follicles and cell necrosis and shedding in the granulosa cell layer.The degree of ovarian tissue lesions in experiment groups 2 and 3 was less than that in experiment group 4,with fewer atretic follicles,necrosis of granulosa cell layer and follicle cystic dilatation.In the blank control group,the ovarian granulosa cells were normal in morphology and structure,with irregular polygon nuclei,uniform distribution of chromatin,mainly euchromatin,clear and complete nuclear membrane,mitochondria and other organelles with complete and clear structure visible in the cytoplasm.Different degrees of mitophagy were observed in the cytoplasm of granulosa cells of rats in experiment groups 1,2,3 and 4;the mitophagy of rats in experiment groups 2,3 and 4 was more severe than that in experiment group 1;that in experiment groups 3 and 4 was more severe than experiment group 2;the mitophagy of rats in experiment group 3 was similar to that in experiment group 4.Under electron microscopy,more mitochondrial autophagosomes were found in the cytoplasm of ovarian granulosa cells in experiment groups 3 and 4.Most mitochondria were mildly swollen,some mitochondrial crista structures disappeared,and the rough endoplasmic reticulum showed cystic dilatation.Conclusion The ovarian dysfunc-tion model in rats can be successfully established by TP intragastric administration.The ovarian injury of rats is related to TP dose and intervention time.TP-induced ovarian oxidative stress may be an important factor triggering ovarian hypofunction,and it may work through oxidative stress affecting ovarian endocrine function and inducing granulosa cell apoptosis and other patho-physiological processes.With the aggravation of oxidative stress damage,the mitophagy increases.After reaching a certain de-gree,mitophagy will not continue to increase with the increase of TP intervention time.

Tripterine promotes pyroptosis of liver cancer cells by regulating ATF4/caspase-3/GSDME signaling pathway / 中国药理学通报

Aim To explore the new mechanism of triptolide (TRI) inhibiting the progression of hepatocellular carcinoma (HCC) . Methods Different concentrations (0, 0 . 5, 2, and 8 jjunol • L~) of TRI were administered to act on liver cancer cells, and then the cell phenotypes and possible mechanisms were explored using experimental methods such as CCK-8, cell cloning, Transwell, and protein immunoblotting; siRNA was used to interfere with the target gene GSDME and its role was determined. Finally, the mechanism of TRI inhibiting the growth of HCC cells in vivo was validated using a transplanted tumor model. Results TRI could inhibit the proliferation, cloning, and invasion of HCC cells, and promote cell apoptosis. Immunoblotting results showed that the expression of GSDME was significantly upregulated in HepG2 or He-pal-6 hepatocellular carcinoma after TRI treatment, while the expression of cleaved caspase-3 and PARP also significantly increased. Knocking out GSDME could partially reverse TRI-induced cell apoptosis. At the same time, cells knocked down by GSDME had stronger cloning and migration abilities, and the apoptosis rate was reduced compared to the TRI treatment group alone. In vivo experiments showed that TRI inhibited HCC tumor growth, and the TRI + siGSDME group had a faster tumor growth rate than the TRI treatment group alone did. In addition, after TRI stimulation, p-eIF2a and ATF4 in HepG2 and Hepal-6 cells significantly increased. The immunofluorescence results showed a dose-dependent increase in the number of ATF4 positive cells in HepG2 and Hepal-6 cells after TRI stimulation. Conclusion The inhibitory effect of TRI on the growth and invasion of liver cancer cells may be related to its regulation of the ATF4/caspase-3/GSDME signaling pathway and promotion of liver cancer cell apoptosis.

Study of Triptolide on Liver Injury and Detoxification Strategy / 中国实验方剂学杂志

As a commonly used traditional Chinese medicine in clinical practice， Tripterygium wilfordii has the functions of dispelling wind and removing dampness， detoxicating and destroying parasites， detumescence， pain relief， promoting blood circulation， and dredging collateral. Modern pharmacological studies show that it also has other functions such as anticancer， anti-inflammation， and immunosuppression. It has been widely used to treat autoimmune diseases， renal diseases， and tumors. T. wilfordii contains a variety of chemical components， among which triptolide （TP） can cause varying degrees of damage to human digestive， circulatory， reproductive， and other systems， with liver injury being the most common one， which greatly limits the development of TP in new drug research and industrial application. Therefore， the authors focused on the research hotspot of TP-induced liver injury and summarized relevant Chinese and international literature regarding the clinical manifestations， injury mechanisms， and detoxification strategies of TP-induced liver injury. This helps to provide a scientific basis for the clinical drug safety and scientific drug supervision of TP. The clinical manifestations of TP-induced liver injury are mostly abnormal transaminases， loss of appetite， nausea and vomiting， anorexia， yellow staining of skin and sclera， and yellow urine. The mechanisms of the above clinical manifestations involve apoptosis， oxidative stress， influence on cytochrome P450 superfamily， macrophage polarization， regulation of biological clock gene Clock， etc. Among them， cell apoptosis is related to neurogenic locus notch homolog protein 1 （Notch1）， dynamin-related protein 1 （Drp1）-cytochrome C （Cyt C）， phosphatidylinositide 3-kinases （PI3K）/protein kinase B （Akt）， mitogen-activated protein kinase （MAPK）， tumor suppressor protein 53 （p53）， Fas cell surface death receptor （Fas）/Caspase-8， and other signaling pathways. Oxidative stress is related to inhibition of nuclear factor-erythroid 2-related factor 2 （NRF2） signaling pathway， promotion of cytochrome P450 2E1 （CYP2E1） expression， and excessive accumulation of reactive oxygen （ROS）. The influence of the cytochrome P450 superfamily is manifested as reducing the substrate affinity， activity， and expression of cytochrome P450 3A （CYP3A）， cytochrome P450 2C9 （CYP2C9）， cytochrome P450 2C19 （CYP2C19）， and cytochrome P450 1A2 （CYP1A2）. Promoting the transformation of macrophages into the M1 type is related to the secretion of inflammatory factors and the accumulation of endotoxin， and the internal rhythmic regulation of the biological clock gene Clock， is related to the expression of cytochrome P450 3A11 （CYP3A11） metabolic enzyme. The detoxification strategies in the clinical application include herbs-processing detoxification strategy and drug-pairing detoxification. The traditional Chinese medicines and monomers that are helpful for detoxification include Glycyrrhiza uralensis， Paeonia lactiflora， Lysimachia christinae， Rehmannia glutinosa， saffron， and paeoniflorin. The reviews and discussion about these topics can help to provide more references for related research and clinical application of TP.

Improvement effect and mechanism of triptolide on sciatica rats by regulating cGAS/STING signaling pathway / 中国药房

OBJECTIVE To investigate the improvement effect and mechanism of triptolide （TP） on sciatica rats. METHODS Sciatica rat model was prepared and then randomly divided into model group （normal saline）， indomethacin group （positive control， 7.5 mg/kg）， TP low-dose and high-dose groups （TP-L group and TP-H group， 50， 100 μg/kg TP）， and high-dose TP+ stimulator of interferon gene （STING） activator group （TP-H+DMXAA group， 100 μg/kg TP+25 mg/kg DMXAA）， with 12 rats in each group. Another 12 unligated rats were selected as sham operation group （normal saline）. After 14 days of intraperitoneal administration， the paw mechanical withdrawal threshold （PWT） and paw withdrawal thermal latency （PWL） were detected； the pathological changes， morphology of sciatic nerve and the number of microglia in sciatic nerve were observed. The levels of interleukin-1β （IL-1β） and tumor necrosis factor-α （TNF-α）， mRNA and protein expression levels of cyclic guanosine monophosphate- adenosine monophosphate synthase （cGAS） and STING in sciatic nerve were detected. RESULTS Compared with sham operation group， PWT and PWL of rats in model group were obviously reduced and shortened， the number of Nissl bodies was obviously decreased， while the number of microglia， sciatic neuropathology score， the levels of IL-1β and TNF-α， mRNA and protein expressions of cGAS and STING were obviously increased （P＜0.05）， and sciatic nerve injury was serious. Compared with model group， the changes of various indexes in indomethacin group， TP-L group and TP-H group were opposite to the above （P＜0.05）， and sciatic nerve injury was reduced. STING activator DMXAA weakened the inhibitory effect of TP on the activity of microglia and inflammatory response in sciatica rats （P＜0.05）. CONCLUSIONS TP may reduce the activity of microglia and inflammatory response by down-regulating the cGAS/STING signaling pathway， thus alleviating sciatica in rats.

Evaluation of toxin-induced injury and protective effect of triptolide based on a cardiovascular chip model / 药学实践与服务

Objective To construct a cardiovascular chip model for evaluating the damage of vascular glycocalyx induced by four marine toxins: okadaic acid (OA), conotoxin (CTX), tetrodotoxin (TTX) and gymnodimine (GYM), and explore the protective effect of triptolide on toxin-induced injury. Methods Human umbilical vein endothelial cells（HUVEC) were inoculated into a three-channel microfluidic chip. CCK-8 method and immunofluorescence staining were used to analyze the damage of cell viability and glycocalyx tissue induced by low, middle and high concentrations of marine toxin, as well as the protective effect of triptolide on toxin-induced injury. Results The cells in the cardiovascular chip grew well and had structurally intact glycocalyx. Compared with the control group, the activity of HUVEC cells were inhibited in group of the medium and high concentration of OA and high concentration of GYM (P<0.05). The activity of cells had not been inhibited by CTX and TTX significantly , but all the four toxins caused serious damage to the glycocalyx tissue (P<0.01). After pre-protection with triptolide, the toxicity of the four toxins to HUVEC cells and the damage rate of glycocalyx decreased significantly. Conclusion The four marine biotoxins could damage the activity and glycocalyx of HUVEC cells in a dose-dependent manner, while triptolide has a protective effect on HUVEC cells injured by toxin.

Triptolide induces apoptosis of human melanoma A375 cells through the inositol-requiring enzyme 1/c-Jun N-terminal kinase signaling pathway / 中华皮肤科杂志

Objective:To investigate the effect of triptolide on the apoptosis of human melanoma A375 cells through the inositol-requiring enzyme 1 (IRE1) /c-Jun N-terminal kinase (JNK) signaling pathway, and to explore its possible mechanisms.Methods:Cultured A375 cells were treated with triptolide at different concentrations of 0, 50, 100, 200 nmol/L (experimental control group, 50, 100, 200 nmol/L triptolide groups, respectively), and a blank control group (DMEM high-glucose medium without cells) was set up. Methyl thiazol tetrazolium (MTT) assay was performed to evaluate the cell viability at 24, 48, and 72 hours after the start of treatment, flow cytometry to detect cell apoptosis at 24 hours after the start of treatment, and real-time fluorescence-based quantitative PCR (RT-qPCR) and Western blot analysis were conducted to determine mRNA and protein expression of IRE1, JNK, and c-Jun, respectively. After pretreatment with the JNK inhibitor SP600125 for 72 hours, some A375 cells were then treated with 100 nmol/L triptolide for 24 hours (SP600125 + 100 nmol/L triptolide group), and the A375 cells only treated with 100 nmol/L triptolide served as control group (100 nmol/L triptolide group). Effects of triptolide on the mRNA expression of IRE1, JNK, and c-Jun in A375 cells, as well as on cell apoptosis, were investigated. Statistical analysis was performed using two-way analysis of variance, one-way analysis of variance, and Dunnett′s test.Results:After the treatment with different concentrations of triptolide for different durations, the cell viability was significantly lower in all triptolide groups than in the experimental control group ( Ftriptolide concentration = 18.36, P = 0.002), and gradually decreased over time ( F time = 8.54, P = 0.018). After 24-hour treatment, the apoptosis rate of A375 cells significantly differed among the 4 groups treated with different concentrations of triptolide ( F = 5 234.97, P < 0.001) ; additionally, the apoptosis rate was significantly higher in the 50, 100, and 200 nmol/L triptolide groups (16.99% ± 0.33%, 30.78% ± 0.40%, 38.91% ± 0.51%, respectively) than in the experimental control group (4.33% ± 0.02%, all P < 0.05), and gradually increased with the rising concentrations of triptolide. The mRNA expression levels of IRE1, JNK, and c-Jun were all significantly higher in the 50, 100, and 200 nmol/L triptolide groups than in the experimental control group (all P < 0.05), and gradually increased with the increase of triptolide concentration. Moreover, the protein expression levels of IRE1, JNK, c-Jun, p-JNK, and p-c-Jun in A375 cells in the triptolide groups also showed the same trend. After pretreatment with the JNK inhibitor SP600125 for 72 hours, the apoptosis rate was significantly lower in the SP600125 + 100 nmol/L triptolide group (21.88% ± 0.55%) than in the 100 nmol/L triptolide group without SP600125 pretreatment (30.78% ± 0.40%, t = -22.51, P < 0.001), and the mRNA expression levels of IRE1, JNK, and c-Jun were also significantly decreased in the SP600125 + 100 nmol/L triptolide group compared with the 100 nmol/L triptolide group (all P < 0.05) . Conclusion:Triptolide may induce apoptosis of human melanoma A375 cells by activating the IRE1/JNK signaling pathway.

Protective effect and mechanism of Astragalus membranaceus and Angelica sinensis compatibility against triptolide-induced hepatotoxicity by regulating Keap1/Nrf2/PGC-1α / 中国中药杂志

This paper aims to investigate the protective effect and mechanism of Astragalus membranaceus and Angelica sinensis before and after compatibility against triptolide(TP)-induced hepatotoxicity. The experiment was divided into a blank group, model group, Astragalus membranaceus group, Angelica sinensis group, and compatibility groups with Astragalus membranaceus/Angelica sinensis ratio of 1∶1, 2∶1, and 5∶1. TP-induced hepatotoxicity model was established, and corresponding drug intervention was carried out. The levels of alanine transaminase(ALT), aspartate transaminase(AST), and alkaline phosphatase(ALP) in serum were detected. Pathological injuries of livers were detected by hematoxylin-eosin(HE) staining. The levels of malondialdehyde(MDA), superoxide dismutase(SOD), glutathione peroxidase(GSH-Px), and reduced glutathione(GSH) in the liver were measured. Wes-tern blot method was used to detect the expression of nuclear factor erythroid 2-related factor 2(Nrf2), Kelch-like ECH-associated protein 1(Keap1), peroxisome proliferator-activated receptor gamma, coactivator-1 alpha(PGC-1α), heme oxygenase-1(HO-1), and NAD(P)H quinone dehydrogenase 1(NQO1) in livers. Immunofluorescence was used to detect the expression of Nrf2 and PGC-1α in livers. The results indicated that Astragalus membranaceus/Angelica sinensis ratio of 2∶1 and 5∶1 could significantly reduce the levels of serum AST, ALT, and ALP, improve the pathological damage of liver tissue, increase the levels of GSH and GSH-Px, and reduce the content of MDA in liver tissue. Astragalus membranaceus/Angelica sinensis ratio of 1∶1 and 2∶1 could significantly improve the level of SOD. Astragalus membranaceus and Angelica sinensis before and after compatibility significantly increased the protein expression of HO-1 and NQO1, improved the protein expression of Nrf2 and PGC-1α, and decreased the protein expression of Keap1 in liver tissue. The above results confirmed that the compatibility of Astragalus membranaceus and Angelica sinensis had antioxidant effects by re-gulating Keap1/Nrf2/PGC-1α, and the Astragalus membranaceus/Angelica sinensis ratio of 2∶1 and 5∶1 had stronger antioxidant effect and significantly reduced TP-induced hepatoto-xicity.

A single-cell landscape of triptolide-associated testicular toxicity in mice / 药物分析学报

Triptolide is a key active component of the widely used traditional Chinese herb medicine Tripterygium wilfordii Hook.F.Although triptolide exerts multiple biological activities and shows promising efficacy in treating inflammatory-related diseases,its well-known safety issues,especially reproductive toxicity has aroused concerns.However,a comprehensive dissection of triptolide-associated testicular toxicity at single cell resolution is still lacking.Here,we observed testicular toxicity after 14 days of triptolide exposure,and then constructed a single-cell transcriptome map of 59,127 cells in mouse testes upon triptolide-treatment.We identified triptolide-associated shared and cell-type specific differentially expressed genes,enriched pathways,and ligand-receptor pairs in different cell types of mouse testes.In addition to the loss of germ cells,our results revealed increased macrophages and the inflammatory response in triptolide-treated mouse testes,suggesting a critical role of inflammation in triptolide-induced testicular injury.We also found increased reactive oxygen species(ROS)signaling and down-regulated pathways associated with spermatid development in somatic cells,especially Leydig and Sertoli cells,in triptolide-treated mice,indicating that dysregulation of these signaling pathways may contribute to triptolide-induced testicular toxicity.Overall,our high-resolution single-cell landscape offers comprehensive information regarding triptolide-associated gene expression profiles in major cell types of mouse testes at single cell resolution,providing an invaluable resource for understanding the underlying mechanism of triptolide-associated testicular injury and additional discoveries of therapeutic targets of triptolide-induced male reproductive toxicity.

Analysis of the synergistic effect of triptolide combined with gefitinib on EGFR-mutant NSCLC cells / 中国药房

OBJECTIVE To investigate the synergistic effect of triptolide （TPL） combined with epidermal growth factor receptor tyrosine kinase inhibitor （EGFR-TKI） gefitinib on EGFR-mutated non-small cell lung cancer （NSCLC） cells and its potential mechanism. METHODS Human NSCLC cell lines H1975 （EGFR T790M/L858R mutated drug-resistant cell lines） and H1299 （EGFR wild-type non-drug-resistant cell lines） were cultured in vitro. MTT method was used to detect cell activity， and the effect of combined medication was evaluated by the combination index （CI）. The H1975 cells were divided into blank group， low- concentration and high-concentration groups of TPL （5 nmol/L or 15 nmol/L）， gefitinib group （2 μmol/L）， low-concentration and high-concentration groups of TPL+gefitinib （5 nmol/L TPL+2 μmol/L gefitinib， 15 nmol/L TPL+2 μmol/L gefitinib）. Flow cytometry was used to detect the apoptosis of H1975 cells and the distribution of the cell cycle. Molecular docking studies were used to predict the binding ability of TPL to EGFR. The expressions of phosphatidylinositol 3 kinase （PI3K）/protein kinase B （Akt）/mammalian target of rapamycin （mTOR） pathway and autophagy-related proteins [microtubule-associated protein 1 light chain 3α （MAP1LC3A）， MAP1LC3B] in H1975 cells were detected by flow cytometry. RESULTS TPL had a strong inhibitory effect on the proliferation of H1975 and H1299 cells in a time-dependent and dose-dependent manner. Forty-eight hours treatment of 5 or 15 nmol/L TPL combined with gefitinib had a synergistic inhibitory effect on the proliferation of H1975 cells （CI＜1）， while there was no synergistic inhibitory effect on H1299 cells （CI＞1）. Compared with the blank group， the apoptosis rate and the proportion of H1975 cells at G0/G1 phase were increased significantly in administration groups， while the proportions of cells at S phase and G2/M phase （except for several TPL groups） were decreased significantly， and the combination group had better effects （P＜0.05）. Molecular docking studies showed that the hydroxyl radical of TPL could form hydrogen bonds with the Thr854 residue of the product encoded by EGFR T790M/L858R mutation. Compared with the blank group， the expressions of pathway-related proteins were down-regulated significantly in administration groups， while those of autophagy-related proteins were up-regulated significantly， and the combination group had better effects （P＜0.05）. CONCLUSIONS TPL combined with gefitinib can synergically inhibit the proliferation activity of EGFR-mutated NSCLC cells， the mechanism of which may be related to the down-regulation of PI3K/Akt/ mTOR pathway and induction of autophagy.

Triptolide regulates the balance of Tfr/Tfh in lupus mice

Abstract Introduction/objectives Systemic lupus erythematosus (SLE) is a classic prototype of the multisystem autoimmune disease and follows a relapsing and remitting course. Triptolide is a diterpene triepoxide extracted from Chinese medicine Tripterygium wilfordii Hook F, with potent immunosuppressive and anti-inflammatory properties. Our previous work observed that triptolide alleviated lupus in MRL/lpr lupus mice with the upregulation of regulatory T cells (Treg) proportion in previous study. In this study, we explored the proportion of follicular T regulatory (Tfr), follicular T helper (Tfh) and germinal center (GC) B cells in lupus mice and evaluated the efficacy of triptolide for lupus treatment in vivo. Methods 20 female MRL/lpr mice were randomly divided into 2 treatment groups and treated orally with vehicle or triptolide. C3H mice were all housed as controlled group and treated orally with vehicle. The percentage of Tfr cells, Tfh cells and GC B cells in spleen of mice were detected by Flow cytometric analysis and immunohistochemistry after 13 weeks of treatment. Results We found that the percentage of Tfr cells decreased in MRL/lpr mice compared with controlled mice. The percentage of Tfh cells in MRL/lpr mice was significantly higher compared with that in controlled mice. The ratio of Tfr/Tfh is also decreased in lupus mice. After treated with triptolide in MRL/Lpr mice in vivo, the percentage of Tfr cells and ratio of Tfr/Tfh increased. The proportion of GC B cells also decreased in mice treated with triptolide by FACS and immunohistochemistry. Conclusions Our results demonstrate that the effect of triptolide in alleviating lupus is partly by reversing immune imbalance with increased percentage of Tfr cells and ratio of Tfr/Tfh. Triptolide might also has effect on immune response through inhibiting proliferating GC B cells.

Triptolide inhibits inflammatory response and migration of fibroblast like synovial cells in rheumatoid arthritis through the circRNA 0003353/JAK2/STAT3 signaling pathway / 南方医科大学学报

OBJECTIVE@#To investigate the effect of triptolide (TPL) on inflammatory response and migration of fibroblast like synovial cells (FLS) in rheumatoid arthritis (RA-FLS) and the mechanism of circular noncoding RNA (circRNA) 0003353 for mediating this effect.@*METHODS@#We collected peripheral blood mononuclear cells (PBMCs) and serum samples from 50 hospitalized RA patients and 30 healthy individuals for detecting the expression of circRNA 0003353, immune and inflammatory indexes (ESR, CRP, RF, anti-CCP, IgA, IgG, IgM, C3, and C4) and DAS28 score. Cultured RA-FLS was treated with 10 ng/mL TPL and transfected with a circRNA 0003353 overexpression plasmid, and cell counting kit-8 (CCK-8) assay and Transwell assay were used to detect the changes in the viability and migration of the cells. Enzyme-linked immunosorbent assay (ELISA) was used to examine the cytokines IL-4, IL-6, and IL-17, and real-time fluorescence quantitative PCR (RT-qPCR) was performed to detect the expression of circRNA 003353; Western blotting was used to detect the expressions of p-JAK2, pSTAT3, JAK2 and STAT3 proteins in the treated cells.@*RESULTS@#The expression of circRNA 0003353 was significantly increased in PBMCs from RA patients and showed a good performance in assisting the diagnosis of RA (AUC=90.5%, P < 0.001, 95% CI: 0.83-0.98). CircRNA 0003353 expression was positively correlated with ESR, RF and DAS28 (P < 0.05). Treatment with TPL significantly decreased the expression of circRNA 0003353, suppressed the viability and migration ability, decreased the expressions of IL-6 and IL-17, and increased the expression IL-4 in cultured RA-FLS in a time-dependent manner (P < 0.01). TNF-α stimulation of RA-FLS significantly increased the ratios of p-JAK2/JAK2 and p-STAT3/STAT3, which were obviously lowered by TPL treatment (P < 0.01). TPL-treated RA-FLS overexpressing circRNA 0003353 showed significantly increased cell viability and migration ability with decreased IL-4 expression and increased IL-6 and IL-17 expressions and ratios of p-JAK2/ JAK2 and p-STAT3/STAT3 (P < 0.01).@*CONCLUSION@#The expression of circRNA 0003353 is increased in PBMCs in RA patients and in RA-FLS. TPL treatment can regulate JAK2/STAT3 signal pathway and inhibit the inflammatory response and migration of RA-FLS through circRNA 0003353.

Integrating systematic pharmacology-based strategy and experimental validation to explore mechanism of Tripterygium glycoside on cholangiocyte-related liver injury / 中草药·英文版

Objective: Tripterygium glycoside (TG) is widely used in clinical practice for its multiple bioactivities including anti-inflammatory and immunosuppressive effects. However, emerging studies have frequently reported TG-induced adverse reactions to multiple organs, especially liver. Here, this study aimed to investigate the mechanism of liver damage induced by TG and explore representative components to reflect TG hepatotoxicity. Methods: Network pharmacology was used to determine the potential targets of bile duct injury caused by TG. Next, the hepatotoxic effects of TG, triptolide (TP) and celastrol (CEL) were investigated and compared in vivo and in vitro. Liver function was determined by measuring serum transaminase and histopathology staining. The cell proliferation and apoptosis were determined by cell viability assay, scratch assay and flow cytometry. The expression of gene of interest was determined by qPCR and Western blot. Results: Based on the network pharmacological analysis of 12 bioactive ingredients found in TG, a total of 35 targets and 15 pathways related to bile duct injury were obtained. Both TG and TP resulted in cholangiocyte damage and liver injury, as illustrated by increased levels of serum transaminase and oxidative stress, stimulated portal edema and lymphocytic infiltration and decreased expression of cholangiocyte marker, cytoskeletal 19. In addition, TG and TP inhibited cell proliferation and migration, arrested cell cycle and promoted Caspase-dependent apoptosis of cholangiocytes via suppressing the phosphorylation of extracellular regulated protein kinases 1/2 (ERK1/2) and protein kinase B (AKT). While, CEL at equivalent dosage had no obvious hepatotoxicity. Conclusion: We revealed that TG-stimulated liver injury was specifically characterized by cholangiocyte damage and TP might be the decisive ingredient to reflect TG hepatotoxicity. Our results not only provide novel insights into the mechanism underlying the hepatotoxicity effects of TG but also offer reference for clinical rational use of TG.

Chronic Toxicity of Triptolide and Ferulic Acid Ethosomes Gel for Percutaneous Administration / 中国实验方剂学杂志

ObjectiveTo investigate the long-term safety of triptolide ferulic acid ethosome gel in percutaneous administration. MethodWe mixed triptolide with ferulic acid to make liposomes gel in different doses and then administrated the gel to SD rats of both sexes with intact skin and damaged skin for 12 weeks. The daily dosages calculated based on triptolide for the low-， middle-， and high-dose groups were 63.75， 127.50， 255.00 μg·kg-1， respectively. The body weight of each rat was measured weekly. The rats were sacrificed in the last week for the determination of serum biochemical parameters and organ indexes as well as the observation of histopathology. The toxicity was assessed based on the body weight and all the parameters and indexes. ResultAfter long-term administration， the body weight and serum biochemical parameters did not show significant difference between the gel-treated groups and the blank group with intact skin， which indicated that the percutaneous administration of triptolide and ferulic acid ethosomes gel was relatively safe. However， the rats in the high-dose group showed sparse hair and were easy to die in the case of unhairing with chloral hydrate at the late stage of the study. Comprared with the female rats with intact skin in the blank control group， the female rats with damaged skin in the middle-dose group showed decreased heart index （P<0.05）， which indicated certain cardiotoxicity. Moreover， damage appeared in skin and lung， which may be influeneced by dosage， sex， and skin state. ConclusionFerulic acid in combination with triptolide is relatively safe for percutaneous administration， whereas there are some risks of skin and lung damage in the case of long-term administration. Individualized administration scheme should be developed according to liver and kidney function and skin conditons to ensure the safety of clinical medication.

Qualitative and quantitative analyses of Tripterygium hypoglaucum in Yinning Tablets, a compound traditional Chinese herbal preparation / 南方医科大学学报

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.

Improvement of the quality standard for Kunxian capsules / 药学实践杂志

Objective To improve the quality standards of Kunxian capsules (KC) and effectively control the product quality. Methods Triptolide, icariin and hypericin were used as the indicator components, to increase or improve the thin layer chromatography (TLC) identification methods of Kunming begonia, epimedium and dodder. Agilent ZORBA SB-C18 (4.6 mm×250 mm, 5 μm) as a chromatographic column, the HPLC method for the determination of triptolide was improved with acetonitrile-0.1% formic acid solution as the mobile phase and 220 nm as the detection wavelength. Results The spots in the TLC method of Kunming begonia, epimedium and dodder has strong specificity, good and clear separation of characteristic spots, negative and no interference. The quantitative analysis of the content of triptolide in KC showed that there is a good linear relationship (r=0.9995) between the mass concentration of triptolide and the peak area in the range of 40.16-502.00 μg/ml, the average recovery was 98.12%, RSD was 8.25%, and the accuracy was good. Conclusion The TLC identification method and HPLC method established in this experiment have strong specificity and good reproducibility, and can effectively control the quality of KC.

Preparation and characterization of biomimetic liposomes coated with erythrocyte membrane co-loading triptolide and celastrol / 药学学报

Drug combination can effectively enhance the anti-tumor effect, reduce the drug dose, and improve medication safety. The use of nano-carrier for drug co-delivery can effectively avoid the differences in drug delivery behavior in vivo. Triptolide and celastrol are the main anti-tumor active components of Tripterygium wilfordii Hook f. Modern studies have shown that the combination of triptolide and celastrol can significantly enhance the antitumor effect, but they are limited by poor water solubility and low tumor tissue delivery rate. In this study, a biomimetic erythrocyte membrane liposome co-loaded with triptolide and celastrol was prepared to characterize the morphology, particle size, potential, drug release, serum stability, and other properties. The immunogenicity, uptake behavior, and anti-cell proliferation ability of the biomimetic liposome was compared. All the animal experiments were carried out in accordance with protocol evaluated and approved by the Ethics Committee of Chengdu University of Traditional Chinese Medicine (Chengdu, China). The results showed that the biomimetic erythrocyte membrane liposome co-loaded with triptolide and celastrol (C+T/RBCm@Lip) in this study had an average particle size of 119.12 ± 2.78 nm and a spherical "core-shell" structure. The zeta potential value was -16.9 ± 1.2 mV, and the drug release behavior in vitro was slow. In addition, the process of coating the cell membrane maintained the characteristics of erythrocyte membrane protein, had good stability in serum, and could effectively avoid the recognition and clearance of macrophages, without causing immunogenicity in vivo. The uptake effect of co-loaded biomimetic liposomes on HepG2 hepatocellular carcinoma cells was enhanced compared with that of uncoated cell membrane liposomes, and the inhibitory effect on proliferation of HepG2 cells was enhanced. In conclusion, the biomimetic liposomes coated with erythrocyte membrane prepared in this study is beneficial to the anti-tumor delivery of triptolide and celastrol, and could enhance the inhibitory effect on the growth of HepG2 liver cancer cells, providing a new idea for the anti-tumor application of Tripterygium wilfordii Hook f.

In vitro and in vivo evaluation of folate-mediated triptolide exosome targeted delivery system against malignant melanoma / 药学学报

In this study, exosomes were extracted from human malignant melanoma cell A375. Folic acid (FA) receptor was used as target and triptolide (TPL) was used as model drug to prepare exosome targeted drug delivery system, FA-Exo/TPL. The physicochemical properties and antitumor effect were evaluated in vivo and in vitro. Gradient centrifugation method was applied to collect exosomes. Then, exosome was modified with FA for loading TPL. The particle sizes of the FA-Exo/TPL were about 100 nm with a double-layer membrane structure like a tray. It is characteristic of high encapsulation efficiency and drug loading. In vitro experiments showed that FA-Exo/TPL could be effectively uptaken by A375 cells, thus significantly inhibiting proliferation and promoting apoptosis the cells. In vivo experiment results showed that FA-Exo/TPL could effectively inhibit the growth of tumor tissue, prolong the model mice life cycle, and significantly reduce the systemic toxicity of the free drug, playing a synergistic and toxic role. Animal welfare and experimental procedures follow the regulations of the Animal Ethics Committee of Fudan University Shanghai Cancer Center. This study provides a new strategies and methods for the preparation of TPL against malignant melanoma.

Effects of triptolide on radiosensitization of lung cancer A549 cells / 中国基层医药

Objective:To investigate the effects of triptolide on radiosensitization of lung cancer A549 cells and the underlying mechanism.Methods:During June-September 2019, lung cancer A549 cells were treated with different concentrations of triptolide for 24 and 48 hours in Animal Experiment Center, Zhejiang Chinese Medical University, China. The inhibitory effects of triptolide on the proliferation of lung cancer A549 cells were determined using MTT method. Appropriate concentrations of triptolide and double distilled water were added to the experimental and control groups, respectively. The effects of triptolide on radiosensitization of lung cancer A549 cells was determined by colony formation assay. Radiosensitization ratio was calculated. Lung cancer A549 cells were divided into blank control, triptolide, radiotherapy, and radiotherapy + triptolide groups. The effects of triptolide on apoptosis and cell cycle of lung cancer A549 cells were determined by flow cytometry.Results:The 10% inhibitory concentration (IC 10) and half maximal inhibitory concentration (IC 50) of triptolide for treating lung cancer A549 cells were 36.61 nmol/L and 259.38 nmol/L, respectively at 24 hours, and they were 9.05 nmol/L and 61.49 nmol/L, respectively at 48 hours. Triptolide had an radiosensitization effect on lung cancer A549 cells, with the radiosensitization ratio of 1.135. The apoptosis rate in the radiotherapy + triptolide group was significantly higher than that in the radiotherapy [(45.47 ± 8.29)% vs. (5.25 ± 0.59)%, t = 6.847, P = 0.002]. The proportion of lung cancer A549 cells at the G2/M phase in the radiotherapy group was significantly higher than that in the radiotherapy + triptolide group [(27.82 ± 0.96)% vs. (11.98 ± 0.55)%, t = 20.176, P < 0.05]. The proportion of lung cancer A549 cells at the G2/M phase in the black group was significantly higher than that in the triptolide group [(17.31 ± 3.42)% vs. (8.05 ± 0.71)%, t = 3.749, P = 0.02]. Conclusion:Triptolide has a radiosensitization effect on lung cancer A549 cells, and the underlying mechanism may be related to its participation in cell apoptosis and cycle regulation.

Altered integrity of hepatocyte tight junctions in rats with triptolide-induced cholestasis / 中国天然药物

Triptolide (TP), an active component of Tripterygium wilfordiiHook. f. (TWHF), has been widely used for centuries as a traditional Chinese medicine. However, the clinical application of TP has been restricted due to multitarget toxicity, such as hepatotoxicity. In this study, 28 days of oral TP administration (100, 200, or 400 μg·kg

Prediction of the pharmacological characteristics and therapeutic mechanism of Tripterygium wilfordii in treating oral lichen planus based on network pharmacology / 口腔疾病防治

Objective@#To explore the potential mechanism of the main active component Tripterygium wilfordii in the treatment of oral lichen planus based on network pharmacology.@*Methods@#The components of Tripterygium wilfordii and targets were searched through the Traditional Chinese Medicine system pharmacology database and analysis platform (TCMSP) and the Traditional Chinese Medicine integrated database (TCMID) databases. The related targets of oral lichen planus (OLP) were obtained through databases such as Gene Cards. The OLP targets were mapped by Venn analysis to the targets of Tripterygium wilfordii to screen out the common targets as the treatment of OLP targets of Tripterygium wilfordii. The Cytoscape software and STRING were used to construct a chemical component-target network and protein-protein interaction network, a network analyzer was used to compute the network topology properties, a cluster profiler software was used to analyze the GO classification enrichment analysis and KEGG signal path analysis, and a Tripterygium wilfordii chemical components-targets-pathway network diagram was constructed. @*Results@#Twenty-three components and 44 OLP treatmenttargets of Tripterygium wilfordii were obtained. The key active ingredients of Tripterygium wilfordii in the treatment of OLP are triptolide, kaempferol, and tangerine peel. The key targets include TNF and AKT1. The GO classification enrichment analysis obtained 63 GO terms, which are mainly involved in the leukocyte differentiation and reaction to lipopolysaccharides. The KEGG analysis identified 111 signaling pathways, which are mainly related to the TNF signaling pathway and IL17 signaling pathway. @*Conclusion@#Tripterygium wilfordii in the treatment of OLP. This study can provide a theoretical basis for further research to explore drugs with high activity and low toxicity to treat OLP from Tripterygium wilfordii.