As₂O₃ Induces Activation Of Hepatic Stellate Cells And Protective Effect Of Taurine Via PPAR?-autophagy Pathway

Objective:Liver fibrosis is a kind of pathological change based on chronic liver injury,which involves activation of multiple signaling pathways and self-repair.It will develop into cirrhosis or hepatocellular carcinoma eventually.In previous studies,we found that arsenic toxicity induced nonalcoholic fatty liver disease and lead to liver fibrosis in mice.A large number of studies have confirmed that activation of hepatic stellate cells is the key event of arsenic-induced hepatic fibrosis.Autophagy is an evolutionary conserved fundamental cellular process by which cells degrade and metabolize own constituents.Recent studies have demonstrated that autophagy stimulates the activation of HSCs by promoting the loss of lipid droplets.However,the toxicity of arsenic on hepatic stellate cells and the underlying mechanism of activation of hepatic stellate cells are still unclear.Taurine?Tau?is a sulfur-containing?-amino acid,which a major free intracellular amino acid present in many tissues of humans and animals.Taurine has been shown to protect against arsenic-induced non-alcoholic fatty liver disease.Our previous studies also have shown that taurine could reduce arsenic-induced autophagy and liver damage,but whether taurine protected arsenic-induced activation of hepatic stellate cells and the underlying mechanism is unclear.Therefore,the purpose of this study was exploring the protective effect of taurine on arsenic-induced hepatic stellate cell activation and the intrinsic mechanism.Methods:C57BL/6J male mice were exposed to arsenic trioxide at concentrations of 0,1,2,4 mg/L in drinking water.Taurine protection group was given 250 mg/kg taurine daily for 12 weeks on the basis of arsenic trioxide at concentrations of 4 mg/L in free drinking water.After 12 weeks,paraffin sections were taken from the liver of mice and the inflammation and edema of the liver were detected by hematoxylin staining;the pathological features of liver fibrosis were observed by Sirius red staining;the expression of hepatic stellate cell activation marker alpha smooth muscle actin?alpha-SMA?was detected by immunohistochemical staining;and the microtubules of autophagy-related protein were detected by Western blot.The expression of related protein light chain 3-II?LC3-II?,p62 and Collagen-1.In vitro experiments,human hepatic stellate cell line LX-2 was studied.The viability of LX-2 cells treated with arsenic trioxide at different concentrations was measured by MTT assay.LX-2 cells were treated with arsenic trioxide at 0,1,2,4?M for 24 hours.Autophagy-related proteins LC-3,p62,hepatic stellate cell activation-related proteins?-SMA,Collagen-1 and peroxisome proliferators-activated receptors?PPAR alpha?were detected by Western blot.And the changes of intracellular lipid droplets were observed by oil red staining.LX-2 cells were pretreated with autophagy inhibitor 3-methyladenine?3MA?,PPAR alpha inhibitor MK-886 and taurine,then treated with arsenic trioxide at a concentration of 4?M for 24 hours.Western blot was used to detect the level of autophagy,activation of hepatic stellate cells,and expression of PPAR alpha.Oil red was used to stain whether lipid droplets were lost in hepatic stellate cells.Results:After exposure to arsenic trioxide at concentrations of 0,1,2,4 mg/L,the expression of alpha-SMA in hepatic stellate cells of mice increased significantly,accompanied by inflammatory reaction and edema.Compared with the arsenic-treaded group,the inflammatory reaction and edema in liver and the expression levels of alpha-SMA protein in hepatic stellate cells of mice exposed to taurine were significantly improved;however,compared with the control group,sirius red staining showed no significant difference among groups.Western blot showed that the expression of autophagy-related protein LC3-II increased with the concentration of arsenic trioxide,while the expression of p62 decreased with the concentration of arsenic trioxide.In addition,the expression of LC3-II decreased and p62 increased in taurine group compared with the highest arsenic trioxide exposure group.In vitro experiments,LX-2 cell lines were treated with arsenic trioxide at different concentrations for 24 hours,and the cell survival rate decreased with the increase of arsenic trioxide concentration.After treated with arsenic trioxide at 0,1,2,4?M for 24hours,Western blot results showed that the expressions of PPAR alpha,alpha-SMA,Collagen-1 and LC3-II increased,while the expression of p62 protein decreased,and oil red staining results showed the lipid droplets loss in hepatic stellate cells increased with the increase of arsenic trioxide dosage.After pretreatment with 3MA and taurine,Western blot results showed that the expression of alpha-SMA,Collagen-1 and LC3-II was significantly lower than that of the highest arsenic trioxide exposure group?exposure to arsenic trioxide alone,concentration of 4?M?.Oil red staining showed that lipid droplets in cells of 3MA and taurine pretreatment groups increased significantly compared with those of the highest arsenic trioxide exposure group.After pretreatment with PPAR alpha inhibitor MK886 and taurine,Western blot results showed that the expression of autophagy-related proteins LC3-II,alpha-SMA,Collagen-1 decreased and the expression of p62 increased compared with the highest arsenic trioxide exposure group.Oil red staining showed that lipid droplets in hepatic stellate cells increased significantly in MK886 and taurine pretreatment groups compared with those in the most arsenic trioxide highly exposed group.Conclusion:Taurine protected As₂O₃-induced the activation of hepatic stellate cells through inhibiting PPAR?-autophagy pathway.