Triptolide-induced Hepatotoxicity Research Articles

Novel detoxifier of spironolactone against triptolide-induced hepatotoxicity through inhibition of RPB1 degradation

Ethnopharmacological relevanceTriptolide is a major bioactive and toxic ingredient isolated from the traditional Chinese herb Tripterygium wilfordii (T. wilfordii) Hook F. It exhibits potent antitumor, immunosuppressive, and anti-inflammatory biological activities; however, its clinical application is hindered by severe systemic toxicity. Two preparations of T. wilfordii, including T. wilfordii glycoside tablets and T. wilfordii tablets, containing triptolide, are commonly used in clinical practice. However, their adverse side effects, particularly hepatotoxicity, limit their safe use. Therefore, it is crucial to discover potent and specific detoxification medicines for triptolide. Aim of the studyThis study aimed to investigate the detoxification effects and potential mechanism of action of spironolactone on triptolide-induced hepatotoxicity to provide a potential detoxifying strategy for triptolide, thereby promoting the safe applications of T. wilfordii preparations in clinical settings. Materials and methodsCell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and crystal violet staining. Nuclear fragmentation was visualized using 4′,6-diamidino-2-phenylindole (DAPI) staining, and protein expression was analyzed by Western blotting. The inhibitory effect of spironolactone on triptolide-induced hepatotoxicity was evaluated by examining the effects of spironolactone on serum alanine aminotransferase and aspartate aminotransferase levels, as well as liver pathology in a mouse model of triptolide-induced acute hepatotoxicity. Furthermore, a survival assay was performed to investigate the effects of spironolactone on the survival rate of mice exposed to a lethal dose of triptolide. The effect of spironolactone on triptolide-induced global transcriptional repression was assessed through 5-ethynyl uridine staining. ResultsTriptolide treatment decreased the cell viability, increased the nuclear fragmentation and the cleaved caspase-3 levels in both hepatoma cells and hepatocytes. It also increased the alanine aminotransferase and aspartate aminotransferase levels, induced the hepatocyte swelling and necrosis, and led to seven deaths out of 11 mice. The above effects could be mitigated by pretreatment with spironolactone. Additionally, molecular mechanism exploration unveiled that spironolactone inhibited triptolide-induced DNA-directed RNA polymerase II subunit RPB1 degradation, consequently increased the fluorescence intensity of 5-ethynyl uridine staining for nascent RNA. ConclusionsThis study shows that spironolactone exhibits a potent detoxification role against triptolide hepatotoxicity, through inhibition of RPB1 degradation induced by triptolide and, in turn, retardation of global transcriptional inhibition in affected cells. These findings suggest a potential detoxification strategy for triptolide that may contribute to the safe use of T. wilfordii preparations.

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

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.

Variable p53/Nrf2 crosstalk contributes to triptolide-induced hepatotoxic process

This study was to investigate the potential mechanism of triptolide-induced hepatotoxicity. We found a novel and variable role of p53/Nrf2 crosstalk in triptolide-induced hepatotoxic process. Low doses of triptolide led to adaptive stress response without obvious toxicity, while high levels of triptolide caused severe adversity. Correspondingly, at the lower levels of triptolide treatment, nuclear translocation of Nrf2 as well as its downstream efflux transporters multidrug resistance proteins and bile salt export pump expressions were significantly enhanced, so did p53 pathways that also increased; at a toxic concentration, total and nuclear accumulations of Nrf2 decreased, while p53 showed an obvious nuclear translocation. Further studies showed the cross-regulation between p53 and Nrf2 after different concentrations of triptolide treatment. Under mild stress conditions, Nrf2 induced p53 highly expression to maintain the pro-survival outcome, while p53 showed no obvious effect on Nrf2 expression and transcriptional activity. Under high stress conditions, the remaining Nrf2 as well as the largely induced p53 mutually inhibited each other, leading to a hepatotoxic result. Nrf2 and p53 could physically and dynamically interact. Low levels of triptolide enhanced the interaction between Nrf2 and p53. Reversely, p53/Nrf2 complex dissociated at high levels of triptolide treatment. Altogether, variable p53/Nrf2 crosstalk contributes to triptolide-induced self-protection and hepatotoxicity, modulation of which may be a potential strategy for triptolide-induced hepatotoxicity intervention.

Activation of cDCs and iNKT cells contributes to triptolide-induced hepatotoxicity via STING signaling pathway and endoplasmic reticulum stress.

Triptolide (TP) exhibits therapeutic potential against multiple diseases. However, its application in clinics is limited by TP-induced hepatoxicity. TP can activate invariant natural killer T (iNKT) cells in the liver, shifting Th1 cytokine bias to Th2 cytokine bias. The damaging role of iNKT cells in TP-induced hepatoxicity has been established, and iNKT cell deficiency can mitigate hepatotoxicity. However, the activation of iNKT cells in vitro by TP requires the presence of antigen-presenting cells. Therefore, we hypothesized that TP could induce dendritic cells (DCs) to activate iNKT cells, thereby leading to hepatotoxicity. The hepatic conventional DCs (cDCs) exhibited immunogenic activities after TP administration, upregulating the expression of CD1d, co-stimulatory molecules, and IL-12. Neutralization with IL-12p40 antibody extenuated TP-induced hepatotoxicity and reduced iNKT cell activation, suggesting that IL-12 could cause liver injury by activating iNKT cells. TP triggered the activation and upregulation of STING signaling pathway and increased endoplasmic reticulum (ER) stress. Downregulation of STING reduced cDC immunogenicity, inhibiting the activation of iNKT cells and hepatic damage. These indicated the regulatory effects of STING pathway on cDCs and iNKT cells, and the important roles it plays in hepatoxicity. ER stress inhibitor, 4-phenylbutyrate (4-PBA), also suppressed iNKT cell activation and liver injury, which might be regulated by the STING signaling pathway. Our results demonstrated the possible mechanisms underlying TP-induced hepatoxicity, where the activation of cDCs and iNKT cells was stimulated by upregulated STING signaling and increased ER stress as a result of TP administration.

Inflammation aggravated the hepatotoxicity of triptolide by oxidative stress, lipid metabolism disorder, autophagy, and apoptosis in zebrafish.

Triptolide is a major compound isolated from the Tripterygium wilfordii Hook that is mainly used for the treatment of autoimmune disorders and inflammatory diseases. Though triptolide-induced hepatotoxicity has been widely reported, the hepatic effects when the patients are in an inflammatory state are not clear. In this study, we used low-dose Lipopolysaccharides (LPS) to disrupt the inflammation homeostasis in the liver of zebrafish and explored the hepatotoxicity of triptolide under an inflammatory state. Compared with the Triptolide group, LPS-Triptolide cotreatment exacerbate the liver injury with a remarkable decrease of liver size and liver-specific fluorescence intensity, accompanied by significant elevation of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities. Liver cell damages were further demonstrated by histological staining and scanning electron microscopy observation. Lipid metabolism was severely impaired as indicated by delayed yolk sac absorption, accumulated triglycerides in the liver, and dysregulation of the related genes, such as ppar-α, cpt-1, mgst, srebf1/2, and fasn. Oxidative stress could be involved in the molecular mechanism as the Nrf2/keap1 antioxidant pathways were down-regulated when the zebrafish in an inflammatory state. Moreover, the expression of autophagy-related genes such as beclin, atg5, map1lc3b, and atg3 was also dysregulated. Finally, apoptosis was significantly induced in responses to LPS-Triptolide co-treatment. We speculate that triptolide could exacerbate the immune response and impair lipid metabolism, resulting in enhanced sensitivity of the zebrafish liver to triptolide-induced toxic effects through disruption of the antioxidant system and induction of apoptosis.

The molecular pathogenesis of triptolide-induced hepatotoxicity.

Triptolide (TP) is the major pharmacologically active ingredient and toxic component of Tripterygium wilfordii Hook. f. However, its clinical potential is limited by a narrow therapeutic window and multiple organ toxicity, especially hepatotoxicity. Furthermore, TP-induced hepatotoxicity shows significant inter-individual variability. Over the past few decades, research has been devoted to the study of TP-induced hepatotoxicity and its mechanism. In this review, we summarized the mechanism of TP-induced hepatotoxicity. Studies have demonstrated that TP-induced hepatotoxicity is associated with CYP450s, P-glycoprotein (P-gp), oxidative stress, excessive autophagy, apoptosis, metabolic disorders, immunity, and the gut microbiota. These new findings provide a comprehensive understanding of TP-induced hepatotoxicity and detoxification.

Antibiotic pretreatment promotes orally-administered triptolide absorption and aggravates hepatotoxicity and intestinal injury in mice

Ethnopharmacological relevanceTriptolide (TP) exhibits extensive pharmacological activity, but its hepatotoxicity and intestinal injury are significant and limit its clinical use. Aim of the studyTo investigate the effect of gut microbiota disturbance after antibiotic pretreatment on TP-induced hepatotoxicity, intestinal injury and their mechanism. Materials and methodsWe compared the characteristics of TP-induced hepatotoxicity and intestinal injury in mice with or without antibiotic pretreatment. The levels of cytokines in the serum, immunohistochemistry, and the pharmacokinetics of TP were determined. ResultAntibiotic pretreatment aggravates TP-induced hepatotoxicity and ileum/colon injury. TP induces hepatotoxicity in a dose-dependent manner after antibiotic pretreatment. Serum IL-1β and IL-6 levels were increased in mice given oral TP after antibiotic pretreatment. TP can increase the expression of NLRP3 inflammasome in hepatocytes, and Oral TP after antibiotic pretreatment can significantly enhance its expression, but NLRP3 inflammasome no significant change in colon and ileum. The pharmacokinetic characteristics of TP are altered significantly by antibiotic pretreatment, as shown by a 145.87% increase in Cmax, a 155.11% increase in AUC0–t, a 155.1% increase in relative bioavailability, and a 15.44% delay in MRT. Moreover, TP causes hepatotoxicity in a time-dependent manner. ConclusionsAntibiotic pretreatment aggravates triptolide-induced hepatotoxicity and intestinal injury through elevated inflammatory response and promoted triptolide absorption.

Triptolide Shows High Sensitivity and Low Toxicity Against Acute Myeloid Leukemia Cell Lines Through Inhibiting WSTF-RNAPII Complex.

Triptolide exhibits superior and broad-spectrum antitumor activity. However, the narrow safety window caused by the toxicity of triptolide limits its clinical applications. Although several characterized targets for triptolide are reported, the association between triptolide and its targets in cancer therapy is not fully understood. Here, we show that acute myeloid leukemia (AML) cell lines are sensitive to triptolide by constructing an in vitro cell and in vivo xenograft models. Meanwhile, the triptolide-induced hepatotoxicity increases with increasing dosages within the xenograft models. Additionally, the expression levels of WSTF-RPB1 are strongly associated with the sensitivity to triptolide in hematological cancer cells and can be downregulated in a dose and time-dependent manner. Finally, we show that optimizing dosing regimens can achieve the same pharmaceutical effect and reduce toxicity. In summary, this study aims to search for triptolide-sensitive cell lines as well as the underlying molecular mechanisms in order to broaden the safety window of triptolide; thus, increasing its clinical utility.

Protection of catalpol against triptolide-induced hepatotoxicity by inhibiting excessive autophagy via the PERK-ATF4-CHOP pathway.

Catalpol significantly reduces triptolide-induced hepatotoxicity, which is closely related to autophagy. The aim of this study was to explore the unclear protective mechanism of catalpol against triptolide. The detoxification effect of catalpol on triptolide was investigated in HepaRG cell line. The detoxification effects were assessed by measuring cell viability, autophagy, and apoptosis, as well as the endoplasmic reticulum stress protein and mRNA expression levels. We found that 5–20 µg/L triptolide treatments increased the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH), as well as the expression of autophagy proteins including LC3 and Beclin1. The expression of P62 was downregulated and the production of autophagosomes was increased, as determined by transmission electron microscope and monodansylcadaverine staining. In contrast, 40 µg/L catalpol reversed these triptolide-induced changes in the liver function index, autophagy level, and apoptotic protein expression, including Cleaved-caspase3 and Cleaved-caspase9 by inhibiting excessive autophagy. Simultaneously, catalpol reversed endoplasmic reticulum stress, including the expression of PERK, which regulates autophagy. Moreover, we used the PERK inhibitor GSK2656157 to prove that the PERK-ATF4-CHOP pathway of the unfolded protein response is an important pathway that could induce autophagy. Catalpol inhibited excessive autophagy by suppressing the PERK pathway. Altogether, catalpol protects against triptolide-induced hepatotoxicity by inhibiting excessive autophagy via the PERK-ATF4-CHOP pathway. The results of this study are beneficial to clarify the detoxification mechanism of catalpol against triptolide-induced hepatotoxicity and to promote the application of triptolide.

Targeting tryptophan metabolism reveals Clematichinenoside AR alleviates triptolide-induced hepatotoxicity

Liver toxicity induced by Triptolide (TP) has limited its clinical application on rheumatoid arthritis (RA). Saponins have been proved as an efficacious remedy to mitigate hepatotoxicity. However, the mechanism of reducing hepatotoxicity by saponins intervention remains incompletely characterized. Tryptophan (Trp) metabolites activate transcriptional regulators to mediate host detoxification responses. Our study aimed to investigate whether Clematichinenoside AR (C-AR) could attenuate TP-induced liver damage by regulating Trp metabolism. We used targeted metabolomics to quantify Trp metabolites in the serum and liver samples of collagen-induced arthritis rats treated by TP. Multiple comparison analyses helped the evaluation of promising biomarkers. The pronounced changed levels of Trp, indole acetic acid, and indole-3-carboxaldehyde in the serum and indole acetic acid, indole, and tryptamine in the liver are relevant to TP-induced liver injury. Intervention with C-AR could relieve TP-induced hepatotoxicity evidenced by ameliorative serum parameters and hepatic histology. In addition, C-AR regulated the levels of these indoles biomarker candidates to normal. Therapeutic modulation with natural compounds might be a useful clinical strategy to ameliorate toxicity induced by TP. Deciphering Trp metabolism will facilitate a better understanding of the pathogenesis of diseases and drug responding.

Role of MicroRNA-155 in Triptolide-induced hepatotoxicity via the Nrf2-Dependent pathway

Ethnopharmacological relevanceTriptolide (TP), the main bioactive and toxic ingredient of Tripterygium wilfordii Hook F, causes severe toxicity, particularly for hepatotoxicity. However, the underlying mechanisms for its hepatotoxicity are not entirely clear. Aim of the studyThe purpose of the study was to explore the role of miR-155, a microRNA closely related to various liver injuries and a regulator of the nuclear factor erythroid 2-related factor 2 (Nrf2) antioxidant pathway, in TP-induced liver injury in vitro and in vivo. Materials and methodsFirst, in vitro L02 cells were treated with different concentrations of TP. The protein levels of Nrf2 and its downstream genes Heme oxygenase1 (HO-1) were determined by Western blot. The mRNA expression of miR-155, Nrf2, NAD(P)H: quinone oxidoreductase 1 (NQO1) and HO-1 were measured using qRT-PCR. And we transfected miR-155 inhibitor and miminc before TP treatment to determine the mRNA and/or protein levels of miR-155, Nrf2 and HO-1. Then, we further confirmed the interaction between miR-155 and Nrf2 pathway in TP-induced hepatic injury in BALB/C mice. The degree of liver injury was determined by HE staining and serum biochemical. The mRNA expression of miR-155 was examined with qRT-PCR and Nrf2 and HO-1 gene expression in liver were evaluated by immunohistochemistry and/or Western blot. ResultsThe results showed that TP significantly induced the expression of miR-155 both in L02 cells and in rodents liver tissue, and the inhibition of miR-155 could mitigate the hepatic damages caused by TP. Further experiments demonstrated that the inhibition of miR-155 reversed the down-regulation of Nrf2 and HO-1 by TP, while the miR-155 mimic enhanced the effects of TP. Animal experiments also showed that the inhibition of miR-155 by miR-155 antagomir reversed the decrease of Nrf2 induced by TP administration. ConclusionsThese results indicated that miR-155 played an important role in TP-induced hepatotoxicity by regulating the Nrf2 signaling pathway.

Catalpol coordinately regulates phase I and II detoxification enzymes of Triptolide through CAR and NRF2 pathways to reduce Triptolide-induced hepatotoxicity

Triptolide (TP), as the main component of Tripterygium Wilfordii (TW), can induce obvious liver injury when exerting the therapeutic effect. However, in our previous study, Catalpol (CAT), the main active ingredient of Rehmannia Glutinosa (RG), was shown to increase the drug clearance rate of TP and to attenuate TP-induced hepatotoxicity. Thus the present study aims to address the roles of phase I and II metabolic enzymes and the nuclear receptors in the detoxification process of TP, to analyze the mechanism of CAT reducing hepatotoxicity. For this purpose, SD rats and human liver cell line L-02 and HepG2 cells were selected, and treated with TP or the combination of TP and CAT in our study. Then the effect of CAT on detoxification of TP was analyzed, and the roles of phase I metabolic enzymes cytochrome P450 3A2/4 (CYP3A2/4) and phase II metabolic enzyme UDP-glucuronosyltransferase 1A6 (UGT1A6) and their related nuclear receptor regulations were evaluated. It was found that TP inhibited the transcription of CYP3A2/4. And through the constitutive androstane receptor (CAR) pathway, CAT not only significantly changed this inhibition and increased the expression of CYP3A2/4 but also increased the expression of CYP2C9, both of which are phase I detoxification enzymes of TP. And with the gene-silenced experiment, it was confirmed that this regulation was CAR-dependent. We also found that CAT could continue to exert a certain protective effect after CAR was silenced, with UGT1A6, the phase II detoxification enzyme of TP, significantly induced. And this was closely related to the enhanced transcriptional regulation of the nuclear factor erythroid 2-related factor 2 (NRF2) pathway. In conclusion, our results reveal that CAT can induce TP’s phase I detoxification enzymes CYP3A2/4 and CYP2C9 through the CAR pathway, and induce TP’s phase II detoxification enzyme UGT1A6 via the NRF2 pathway when CAR is strongly inhibited. And this coordinate regulation of CAT may be an important source of the effect for CAT to increase TP metabolic conversion and reduce TP hepatotoxicity.

Gut microbiota protects from triptolide-induced hepatotoxicity: Key role of propionate and its downstream signalling events

As a potential drug for treating inflammatory, autoimmune diseases and cancers, triptolide (TP) is greatly limited in clinical practice due to its severe toxicity, particularly for liver injury. Recently, metabolic homeostasis was vitally linked to drug-induced liver injury and gut microbiota was established to play an important role. In this study, we aimed to investigate the functions of gut microbiota on TP-induced hepatotoxicity using metabolomics in mice. Here, predepletion of gut microbiota by antibiotic treatment strikingly aggravated liver injury and caused mortality after treated with a relatively safe dosage of TP at 0.5 mg/kg, which could be reversed by gut microbial transplantation. The loss of gut microbiota prior to TP treatment dramatically elevated long chain fatty acids and bile acids in plasma and liver. Further study suggested that gut microbiota-derived propionate contributed to the protective effect of gut microbiota against TP evidenced by ameliorative inflammatory level (Tnfa, Il6 and Cox2), ATP, malondialdehyde and hepatic histology. Supplementing with propionate significantly decreased the mRNA levels of genes involved in fatty acid biosynthesis (Srebp1c, Fasn and Elovl6), resulting in the decreased long chain fatty acids in liver. Moreover, TP restricted the growth of Firmicutes and led to the deficiency of short chain fatty acids in cecum content. In conclusion, our study warns the risk for TP and its preparations when antibiotics are co-administrated. Intervening by foods, prebiotics and probiotics toward gut microbiota or supplementing with propionate may be a clinical strategy to improve toxicity induced by TP.

Mitochondria-dependent apoptosis in triptolide-induced hepatotoxicity is associated with the Drp1 activation

How triptolide is associated with mitochondrial dysfunction and apoptosis in connection with its hepatotoxicity remains unclear. The objective of our study was to find out the link between mitochondrial dynamics and cell death in triptolide induced hepatotoxicity. We treated L02 cells with 25 nM concentration of triptolide. The results demonstrated that triptolide treatment caused an increase in apoptotic cell death, mitochondrial depolarization, ROS overproduction, a decrease in ATP production, and mitochondrial fragmentation which in turn is associated with the activation of Drp1 fission protein. Triptolide treatment led to the translocation of Drp1 from the cytosol into outer mitochondrial membrane where it started mitochondrial fission. This fission event is coupled with the mitochondrial release of cytochrome c into the cytosol and subsequently caspase-3 activation. TEM analysis of rat liver tissues revealed the distortion of mitochondrial morphology in triptolide-treated group. Western blot analysis explained that disruption in mitochondrial morphology was attached with the recruitment of Drp1 to mitochondria, cytochrome c release, and caspase-3 activation. However, Mdivi-1 co-treatment inhibited the activation of Drp1 and caspase-3 and blocked the release of cytochrome c into the cytosol. In short, inhibiting Drp1 protein activation may provide a new potential target for curing Drp1-associated apoptosis in triptolide-induced hepatotoxicity.

Autophagy in Triptolide-Mediated Cytotoxicity in Hepatic Cells.

Triptolide is a major active ingredient isolated from the traditional Chinese herb Tripterygium wilfordii Hook F. However, its use in clinical practice is limited due to its severe hepatotoxicity. Autophagy, a highly conserved intracellular process, is essential for maintaining cytoplasmic homeostasis. Considering that abnormalities in autophagy are closely associated with drug-mediated hepatotoxicity, we applied human normal liver HL7702 cells to elucidate the roles of autophagy in triptolide-induced hepatotoxicity. Our study revealed that triptolide was cytotoxic to HL7702 cells. It markedly increased autophagosome formation and expression of autophagy-related proteins, namely Beclin1 and microtubule-associated protein 1 light chain 3II, and induced oxidative stress. These proautophagic effects were counteracted by pretreatment with N-acetylcysteine, a reactive oxygen species scavenger. Moreover, the pharmacological suppression of autophagy further exacerbated triptolide-elicited decrease in cell viability, increase in lactate dehydrogenase leakage, and activation of apoptosis proteases (caspase 3 and caspase 9). Our findings suggest that triptolide-induced oxidative stress consequently enhances autophagic activity, and autophagy is a cytoprotective mechanism against triptolide-induced cytotoxicity in HL7702 cells.

Catalpol and panax notoginseng saponins synergistically alleviate triptolide-induced hepatotoxicity through Nrf2/ARE pathway

Triptolide (TP), as the main component of Tripterygium wilfordii (TW), could induce significant hepatic damage when exerting the therapeutic effect. However, The previous studies have shown that catalpol (CAT) and panax notoginseng saponins (PNS) have synergistic protecting effect against hepatotoxicity induced by TP. This study aims to address the role of the nuclear factor erythroid-2-related factor-2 (Nrf2)/antioxidant response element (ARE) pathway in their protecting effect, and to explore their synergistic mechanisms. For this purpose, the human hepatocyte cell line L-02 was selected, and the synergistic antioxidative effect of CAT and PNS was confirmed. Then the effects of CAT and PNS on different aspects of the Nrf2/ARE pathway were analyzed. The results showed that CAT significantly reduced TP-induced inhibition of Nrf2 transcription and made it increased, PNS significantly increased Nrf2 phosphorylation for relieving TP-induced potential inhibition of Nrf2/ARE binding activity, while the combination of the two further enhanced their activation by synergistically inducing Nrf2 transcriptional expression and phosphorylated Nrf2 (p-Nrf2) expression, followed with NAD(P)H:quinine oxidoreductase 1 (NQO1) expression and glutathione (GSH) activity increasing, which further enhanced their antioxidative effects. Subsequently, the gene silencing techniques were used, and that the Nrf2/ARE pathway is necessary in this effect was confirmed. In conclusion, the synergistic protecting effect of CAT and PNS is dependent on Nrf2/ARE pathway, while the natural mutual promotion between the regulatory links of CAT and PNS may be the main source of the synergistic effect.

Protective Effect of Vitamin C on Triptolide-induced Acute Hepatotoxicity in Mice through mitigation of oxidative stress.

Triptolide, a purified diterpenoid from the herb Tripterygium wilfordii Hook.f., was widely used to treat many diseases. However, the hepatotoxicity of triptolide limited its clinical use. Research showed oxidative stress played an important role in triptolide-induced liver injury. To investigate the effect of vitamin C, which was one of the most effective antioxidants, on triptolide-induced hepatotoxicity and its potential mechanism in mice. In the present study, acute liver injury was induced by intraperitoneal injection of triptolide and vitamin C was orally administered. The results showed treatment with vitamin C prevented the triptolide-induced liver injury by reducing the levels of aspartate transaminase from 286.86 to 192.48 U/mL and alanine aminotransferase from 746.75 to 203.36 U/mL. Histopathological changes of liver corresponded to the same trend. Furthermore, vitamin C also protected the liver against triptolide-induced oxidative stress by inhibiting the generation of malondialdehyde (2.22 to 1.49 nmol/mgprot) and hydrogen peroxide (14.74 to 7.19 mmol/gprot) and restoring the level of total superoxide dismutase (24.32 to 42.55 U/mgprot) and glutathione (7.69 to 13.03 μg/mgprot). These results indicated that vitamin C could protect against triptolide-induced liver injury via reducing oxidative stress, and vitamin C may pose a significant health protection in the clinical use of triptolide.

Drp1-associated mitochondrial dysfunction and mitochondrial autophagy: a novel mechanism in triptolide-induced hepatotoxicity.

Triptolide being an active ingredient of Chinese herbal plant Tripterygium wilfordii Hook f. has severe hepatotoxicity. Previous studies from our lab reported triptolide-induced mitochondrial toxicity in hepatocytes. However, biomolecular mechanisms involved in triptolide-induced mitochondrial dysfunction are not yet entirely clear. We explored the connection between mitochondrial fragmentation and mitophagy in triptolide-induced hepatotoxicity. Triptolide caused an increase in ROS production, a decrease in mitochondrial depolarization, a diminution of ATP generation, a decline in mitochondrial DNA copy number, mitochondrial fragmentation, and disturbance in mitochondrial dynamics in a concentration-dependent manner in L02 cells. Disturbance in mitochondrial dynamics was due to an increased expression of Drp1 fission protein in vitro and in vivo. L02 cells exhibited an increase in the colocalization of lysosomes with mitochondria and autophagosomes with mitochondria in triptolide treated group as compared to control group which was inhibited by Mdivi-1. Transmission electron micrographs of rat liver tissues treated with triptolide (400μg/kg) revealed activation of mitophagy which was prevented by Mdivi-1 co-treatment. Taken together, our results showed that mitochondrial fission-associated mitophagy is a novel mechanism involved in triptolide-induced hepatotoxicity. For the alleviation of triptolide-induced hepatotoxicity, mitochondrial fission and mitochondrial autophagy signaling pathway can be targeted as a new therapeutic strategy. Graphical abstract ᅟ.

In vitro study of the cytotoxic effects of microcystin-LR, cylindrospermopsin and their mixture in the neuronal SH-SY5Y cell line

Hepatic macrophages are central players in the pathogenesis of some liver diseases, but few studies have examined the effect of triptolide (TP) on these cells. In this study, we investigated the possible role of hepatic macrophage recruitment and activation in triptolide-induced hepatotoxicity based on a non-hepatotoxic dose of lipopolysaccharides (LPS). The results showed that continuous administration of TP for two weeks and a single challenge of low-dose lipopolysaccharides increased the number of hepatic macrophages but inhibited their phagocytic function. TP induced the liver to recruit monocyte-derived macrophages (MoMFs) in response to a single challenge of low-dose LPS, resulting in acute inflammation and increased sensitivity to the endotoxin. Concurrent administration of TP with LPS resulted in obvious hepatotoxicity, but a single dose of LPS did not induce hepatotoxicity. These results indicate that TP could change the number and function of hepatic macrophages to reduce the ability of the liver to clear mild endotoxins, thus increasing blood endotoxin levels and increasing the sensitivity of the liver to low-dose LPS. By investigating the critical function of hepatic macrophages in TP-induced hepatotoxicity, this study elucidated the mechanism underlying TP-induced hepatotoxicity.

Possible role of hepatic macrophage recruitment and activation in triptolide-induced hepatotoxicity

Hepatic macrophages are central players in the pathogenesis of some liver diseases, but few studies have examined the effect of triptolide (TP) on these cells. In this study, we investigated the possible role of hepatic macrophage recruitment and activation in triptolide-induced hepatotoxicity based on a non-hepatotoxic dose of lipopolysaccharides (LPS). The results showed that continuous administration of TP for two weeks and a single challenge of low-dose lipopolysaccharides increased the number of hepatic macrophages but inhibited their phagocytic function. TP induced the liver to recruit monocyte-derived macrophages (MoMFs) in response to a single challenge of low-dose LPS, resulting in acute inflammation and increased sensitivity to the endotoxin. Concurrent administration of TP with LPS resulted in obvious hepatotoxicity, but a single dose of LPS did not induce hepatotoxicity. These results indicate that TP could change the number and function of hepatic macrophages to reduce the ability of the liver to clear mild endotoxins, thus increasing blood endotoxin levels and increasing the sensitivity of the liver to low-dose LPS. By investigating the critical function of hepatic macrophages in TP-induced hepatotoxicity, this study elucidated the mechanism underlying TP-induced hepatotoxicity.