Study On The Mechanism Of Triptolide-induced Liver Injury

Tripterygium wilfordii,a trandition Chinese medicine from the roots of Tripterygium wilfordii Hook.f.,is widely used in clinic for the treatment of rheumatoid arthritis,nephrotic syndrome,lupus erythematosus and other systermic immune diseases.It has an excellent curative effect,but its toxic effect is also very severe.The components of Tripterygium wilfordii are complex and it is extremely difficult to study the mechanism of its toxicity.Triptolide is one of the major active and toxic compounents extracted from tripterygium wilfordii.Triptolide has many pharmaceutical activities,such as immunosuppression,anti-inflammation,anti-tumor and anti-fertility.However,the incidences of triptolide-induced organ toxicity always occur during use and it has a narrow therapeutic window.The study of the mechanism of triptolide-induced liver injury could objectively reflect the toxicity of Tripterygium wilfordii,and it is also of great significance in avoiding its toxicity in clinical applications.Although there are many studies on studying the meachanism of Triptolide-indeuced liver injury,the precise mechanism remains unclear,and further researches are in need.In this study,the effect of endoplasmic reticulum stress?ER stress?directly mediated apoptosis and pyroptosis by activation of NLRP3 inflammasome in triplide-induced liver injury was studied by animal expriments in vivo and cell exprements in vitro.In addition,combined with metabolomics and transcriptomics techniques,the mechanism of triptolide-induced liver injury was further studied,which provided strategies to find new targets in triptolide-induced liver injury.Part one Studies on Role of Endoplasmic Reticulum Stress-mediated CellDeath in Triptolide-induced Liver InjuryObjective:To investigate the role of ER stress mediated apoptosis directly and pyroptosis by activation of NLRP3 inflammsome in triplolide-induced liver injury,histopathology and oxidative stress were used as observational indicators and molecular biology and cell biology techniques were applied.Methods:1 Dosage-toxicity and time-toxicity relationships in triptolide-induced liver injury:Adult male C57BL/6N mice were intragastrically administered with different doses of triptolide?0.2 mg/kg,0.4 mg/kg,0.6 mg/kg and 0.8mg/kg?for 24 h or treated with triptolide?0.8 mg/kg?for 2 h,6 h,12 h,24 h,48 h and 72 h to evaluate triptolide-induced liver injury.The degree of hepatic injury was evaluated by detection of the pathological changes of hepatic tissues using serum alanine aminotransferase?ALT?and glutamate aminotransferase?AST?levels and hematoxylin-eosin?HE?staining.2 Effect of RIPK3-mediated necroptosis in triptolide-induced liver injury:Adult male RIPK3 knockout mice and wild-type mice with the background of adult C57BL/6N were used as study subjects.Mice in both groups were treated with triptolide for 24 h to observe the difference of liver injury between the two groups of mice.3 The Role of Endoplasmic Reticulum stress directly mediated apoptosis and pyroptosis by activation of NLRP3 inflammasome in triptolide-induced liver injury:according to the results of studies on dosage-toxicity and time-toxicity relationships in triptolide-induced liver injury,liver tissues and liver sections from mice treated with 0.8 mg/kg triptolide for 24 h were selected to detect the cell death in triptolide-induced liver injury with TUNEL kit and western bolt technology.ER stress and NLRP3 inflammasome pathway-associated genes?Atf4,Ddit3,Ero1a,Gadd34,Dr5,Trailr2,Bim,Bax,Nlrp3,Pycard,Panx1 and Casp1?and protein expressions?p-Eif2?,p-IRE1?and CHOP?were detected.Adult male C57BL/6N mice were selected as study subjects,ER stress inhibitors TUDCA and Caspase-1inhibition Ac-YVAD-cmk were used to block the ER stress pathway and the NLRP3 inflammasome pathway to observe triptolide-induced liver injury.Primary liver cells from C57BL/6N mice,the study objects,were treated with different doses of triptolide to evaluate the effect of triptolide on the expression of oxidative stress-related genes?Gstm1,Gstm2,Gstm3,Gstm6,Gstp1,Gstp2,Gstt2,Mgst1,Nrf2,Hmox1,Gclm,Sod1,Sod2,Cat,Prdx1,Txnrd2 and Txnrd3?,what caused ER stress.Results:1 There was no significant diffecence on the serum ALT and AST levels and no severe liver injure was observed in histological examination at low-dose triptolide?0.2 mg/kg and 0.4 mg/kg?treatment.The serum ALT and AST levels and histological examination showed that there was a dose response relationship between triptolide and liver injury.Within 12 h after triptolide treatment,serum ALT and AST and liver tissue sections did not change significantly and ALT and AST levels rose to the maximum at 48 h after triptolide treatment.2 Compared with the wild-type mice group,serum ALT and AST levels in the RIPK3-null group were not significantly different.Liver tissue sections showed that the degree of liver injury did not improve.It suggested that triptolide-induced liver injury was not directly involved to RIPK3-meidated necroptosis.3 Compared with the control group,the apoptosis protein CASP3 and pyroptosis protein CASP1?p20,p22?were significantly increased.ER stress and NLRP3 inflammasome pathway-related genes and proteins were also increased in triptolide treatment group.The contents of the drug group were all increased.TUDCA and Ac-YVAD-cmk could significantly relieve the degree of liver damage caused by triptolide.Besides,Triptolide coud deplete GSH content and inhibit the expressions of GSH synthase and ROS scavenging enzyme synthesis related genes.Conclusions:The molecular mechanism of triptolide-induced liver injury is that it can consume GSH and inhibit the synthesis of GSH synthase and ROS scavenger enzyme,which changes the redox homeostasis in vivo and leads to ER stress.The aspect directly mediates apoptosis and,on the other hand,mediates cell death via the NLRP3 inflammatory body pathway.Part two Metabonomics-Based Study on Triptolide-induced Liver InjuryObjective:To establish a metabolomics analysis method for mice serum and liver tissues based on UPLC-Q-TOFMS,and to further investigate the mechanism of triptolide-induced liver injury,the changes of metabolites and metabolic pathways at different triptolide dose were analyzed.Methods:Forty male C57BL/6N mice were randomly divided into five groups with eight mice per group as control group and different doses of triptolide groups.After triptolide exprosure for 24 h,the mice were killed and serum and liver tissues were collected.Mice serum and liver tissues samples were analyized by UPLC-Q-TOFMS equipped with Waters Acquity BEH Amide colume?2.1 mm×50 mm,1.7?m?at ESI positive and negative ion modes.The raw data was pre-treated and analyzed with multivariate statistical analysis.The diferential metabolites were screened out and comfirmed by matching their m/z and secondary spectrum with online database?Metlin,HMDB and Pubmed?.The metabolic pathways of potential biomarkers were obtained by using online Metabo Analyst 4.0 software and KEGG database.Results:In the metabolomics study of serum and liver tissues,raw data were processed by data processing and multivariate analysis.And 77diferential metabolites were screened out and confirmed,including lipids,choline,fatty acids,ribose and nucleic acids.These diferential metabolites mainly involved in gluthathione metabolism,purine metabolism,pantothenic acid and coenzyme A,glycerophospholipid metabolism,citric acid cycle,pyrimidine metabolism,niacin and niacinamide metabolism,taurine and hypotaurine metabolism and amino acid metabolism.Conclusions:In this study,a non-targeted metabolomics technique based on systems biology was established to analyze serum and liver tissues form mice treated with different doses of triptolide.The analysis of metabolic pathway of metabolites showed that triptolide could cause changes of multiple metabolic pathways,including oxidative stress,lipid metabolism and amino acid metabolic disorder.All of them played important roles in triptolide-induced liver injury.It provided guidances for the final discovery of biomarkers in triptolide-induced liver injury and further study of the mechanism of liver injury.Part three Transcriptomics-Based Study on Triptolide-induced Liver InjuryObjective:To investigate the mechanism of triptolide induced-liver injury,RNA-seq sequencing technology was used to dectect the gene expression profiles of mice liver tissues under different doses of triptolide treatment,and the differentially expressed genes and toxic patheays were anlayized.Methods:Forty male C57BL/6N mice were randomly divided into five groups with eight mice per group as control group and different doses of triptolide groups.After triptolide exprosure for 24 h,the mice were killed and liver tissues were collected.Total RNA was extracted from liver tissues,dectected and purified,and then subjected to sequencing.IPA software was used to analyze differentially expressed genes for toxic pathways and toxicity function.Then further in-depth bioinformatics anslysis was performed.Results:The gene expression profiles of mice treated with different doses of triptolide were detected and analyzed in depth,and 133,1234,5839and 7312 differentially expressed genes were found,respectively.The number of differentially expressed genes showed a dose-dependent manner.These genes enriched in several pathways,including AHR signaling,Nrf2-mediated oxidative stress response,SAPK/JNK signaling,death receptor signaling,apoptotic signal macrophage production of NO and ROS,LPS/1L-mediated inhibition of RXR function,hepatic fibrosis/activation of hepatic stellate cells,IL-8 signaling,NK-?B signaling,PPAR signaling and hepatic cholestasis.Conclusions:In this study,RNA-seq technology was used to study liver gene expression profiles of mice treated with different doses of triptolide for toxic pathway and functions by IPA software.The results indicated that triptolide-induced liver injury was mainly related to hepatocyte death and inflammatory react and metabolic disorders.This study provided a new research strategy for studies on mechabism of traditional Chinese medicine induced liver injury.Part four Integrative Analysis of Metabolomic and TranscriptomicsProfiling on Triptolide-induced Liver InjuryObjective:To investigate the mechanism of triptolide induced-liver injury,the changed metabolites and differentially expressed genes form mice treated with triptolide were integrated and analyzed with Sytoscape and IPA softeware.Combined with animal expreiments in vivo,the results of multiomic analysis were validated.Methods:1 Connected network analysis of changed metabolites and differentially expressed genes were analysis with Cytoscape and IPA software.Then,these metabolites and genes and their log₂?Fold change?values were import to IPA for the pathway analysis with“Core analysis”.2 Twenty male C57BL/6N mice were randomly divided into two groups with ten mice per group as macrophage depletion group and control group.In macrophage depletion group,mice were given clodronate liposomes and mice in control group were given empty liposomes by vein injection.2 days later,all mice were given triptolide by gavage.Serum ALT and AST were determined.HE staining was used to detect the pathological changes of liver tissues.Results:1 Through the integrative analysis of changed metabolites and differentially expression genes,we found that the production of nitric oxide and reactive oxygen species in macrophages changed significantly.2 The results of macrophage depletion expreiments showed that the levels of serum ALT and AST in macrophages depletion group were significantly decreased and the liver tissue sections showed normal structure after triptolide treatment.These results showed that macrophages played an important role in triptolide-induced liver injury.Conclusions:In this study,metabolic data and transcriptomic data were initially integrated with Cytoscape and IPA software to construct an interaction network and to performe metabolic pathway enrichment analysis.The results indicated that macrophage played a vital role in triptolide-induced liver injury.Meanwhile,combined with macrophage depletion experiments in vivo,the multi-omics analysis results were verified to further prove the role of macrophage in triptolide-induced liver injury.