Evaluation And Mechanism Study For Idiosyncratic Liver Injury Of Cis-SG And Trans-SG Based On The NLRP3 Inflammasome

Objectives:A composite cell model for evaluation of idiosyncratic drug-induced liver injury(IDILI)was established in vitro from the perspective of NLRP3 inflammasome,and this model was used to evaluate the risk of IDILI for cis-stilbene glucoside(Cis-SG)and trans-stilbene glucoside(Trans-SG).To investigate the possible mechanism of Cis-SG-induced immunological idiosyncratic hepatoxicity from the perspectives of inflammation,which provides an important basis for predicting and solving the idiosyncratic hepatoxicity of drugs.Methods:This experimental study is divided into three chapters.Chapter 1.To determine the low,medium,and high dosage of Cis-SG and Trans-SG,CellTiter-Glo(?)3D Cell Viability Assay was used to detect the effect of Cis-SG and Trans-SG on cell viability of HepG2 cells in three dimensional(3D)culture,and MTT assay was used to detect the effects of Cis-SG and Trans-SG on cell viability of THP-1 derived macrophages.THP-1 derived macrophages were incubated by Cis-SG and Trans-SG directly or supernatants from HepG2 cells incubated with them.Enzyme linked immunosorbent assay(ELISA)was used to detect the levels of interleukin-1β(IL-1β)in the supernatants of the THP-1 derived macrophages.Western blot and reverse transcription-polymerase chain reaction(RT-PCR)were used to determine the expression of apoptosis-associated speck-like protein(ASC),Nod-like receptor protein 3(NLRP3),cysteinyl aspartate specific proteinase-1(caspase-1),and IL-1β in THP-1 derived macrophages.ELISA was used to detect the effect of Cis-SG and Trans-SG on the levels of HMGB1 secreted by HepG2 cells in 3D culture.Chapter 2.The HepG2 cell suspension and the same volume of medium containing Cis-SG or Trans-SG were applied to a 96U plate with ultra-low adsorption on the bottom surface.After incubation for 7 days,the supernatant of HepG2 cells in each group was lyophilized.Non-target metabolomics analysis was performed on the supernatant of HepG2 cells in each group by GC-MS technique to find the different metabolites between the control group and the administration group.The effects of Cis-SG and Trans-SG on the metabolism of 3D cultured HepG2 cells were explored through the enrichment analysis of metabolic pathways.Chapter 3.The rats were randomly divided into five groups as follow:Control group,Cis-SG group(50 mg/kg),LPS group(2.8 mg/kg),LPS+Cis-SG group(2.8 mg/kg,50 mg/kg)and LPS+Cis-SG+DG group(2.8 mg/kg,50 mg/kg,60 mg/kg).After three days adaptive feeding,the rats were given DG or the same amount of saline by intraperitoneal injection at a dose of 60 mg/kg for five days.On the sixth day,the rats were gavaged 50 mg/kg Cis-SG or the same of amount of saline.Three hours later,2.8 mg/kg LPS or its saline vehicle was injected through a tail vein.After injected LPS seven hours,all rats were anesthetized by intraperitoneal injection of 1%pentobarbital sodium,and blood were collected from the abdominal aorta together with liver samples.The levels of alanine transaminase(ALT)and aspartate aminotransferase(AST)in the plasma were determined by microplate method.The levels of interleukin-1β(IL-1β),interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)in the plasma were measured by ELISA.HE staining was used to examine the pathological changes of liver tissue sections.Immunohistochemical staining was used to detect the expressions of HMGB1 and NLRP3 in liver tissue sections.Western blot and RT-PCR were used to detect the expressions of HMGB1,TLR4,MYD88,NF-κB and NLRP3 inflammasome activation-related proteins and mRNA.Results:Chapter 1.The results showed that there was no effect on the secretion of IL-1βin THP-1 derived macrophages incubated with 1,5,and 25 μM Cis-SG or Trans-SG directly.However,the secretion of IL-1β,the protein and mRNA expression of ASC,NLRP3,caspase-1,and IL-1β significantly increased in THP-1 derived macrophages incubated by supernatants from HepG2 cells incubated with 1,5,and 25 μM Cis-SG or 25 μM Trans-SG(P<0.01).There was no effect on the secretion of HMGB1 in 3D-cultured HepG2 cells with 1,5,and 25μM Cis-SG or Trans-SG directly.Chapter 2.The results showed that a total of 99 metabolites were identified from the supernatant samples of HepG2 cells in each group,and there were differences in metabolites from the supernatant of cells between the administration group and the control group.A total of 47 significantly different metabolites such as phenylalanine,proline,pyruvic acid,serine,stearic acid were identified between the Cis-SG group and the control group,which mainly involved 7 possible metabolic pathways including aminoacyl-tRNA biosynthesis,tricarboxylic acid cycle,amino acid metabolism and biosynthesis.A total of 16 significantly different metabolites such as isoleucine,nicotinic acid,lysine,malic acid,lactulose were identified between the Trans-SG group and the control group,but no eligible metabolic pathways were screened.Chapter 3.The results showed that neither Cis-SG nor LPS alone induced any apparent liver injury.LPS and Cis-SG co-treatment significantly increased the levels of ALT and AST in plasma(P<0.01),induced severe liver injury.Compared with LPS group,LPS and Cis-SG co-treatment significantly increased the levels of IL-1β,IL-6 and TNF-α in plasma(P<0.01),increased the expressions of HMGB1,TLR4,MYD88,NF-κB and activiation of NLRP3 inflammasome(P<0.01).Compared with LPS+Cis-SG group,DG pretreatment significantly alleviated Cis-SG-induced immunological idiosyncratic liver injury,decreased the levels of ALT,AST,IL-1β,IL-6 and TNF-α in plasma(P<0.01)and suppressed the expressions of HMGB1,TLR4,MYD88,NF-κB and activiation of NLRP3 inflammasome(P<0.01).Conclusions:1.The composite cell model for evaluation of IDILI established in vitro has been successfully applied in testing Cis-SG and Trans-SG.At the same dose gradient,Cis-SG has a stronger effect on activating the NLRP3 inflammasome in THP-1 derived macrophages than Trans-SG after being incubated by hepatocytes.This composite cell model is helpful to evaluate and screen drugs with IDILI risk in vitro preliminarily.2.The results of non-target metabolomics showed that there were significant differences in the metabolites from the supernatant of Cis-SG and Trans-SG after hepatocyte incubation.The pro-inflammatory effect of CIS-SG after hepatocyte incubation might be related to its interference with amino acid metabolism and glucose metabolism of hepatocytes.3.Cis-SG could significantly induce immunological idiosyncratic hepatoxicity in LPS-treated rats through the activation of HMGB1/TLR4/NF-κB signaling pathway and up-regulation of NLRP3 inflammasome,which may be a novel mechanism of Cis-SG-induced immunological idiosyncratic hepatoxicity.Furthermore,DG could effectively exert a protective effect against immunological idiosyncratic liver injury induced by Cis-SG and its protective mechanism may be related to suppress the activiation of HMGB1/TLR4/NF-κB signaling and NLRP3 inflammasome.