The Involvement Of Ferroptosis In Valproic Acid-Induced Hepatotoxicity Via The ACSL4/GPX4 Pathway

Objective:Valproic acid(VPA)is a broad-spectrum antiepileptic drug used to therapy epilepsy of numerous types in clinic.However,hepatotoxicity is one of the most common and serious adverse drug reactions of VPA,mostly in children,with pathological manifestations of hepatic lipid accumulation and microvesicular steatosis,and enhanced prevalance to nonalcoholic fatty liver disease(NAFLD)in patients.There are plenty studies on the mechanisms of VPA hepatotoxicity,including disturbance of lipid metabolism,mitochondrial toxicity,and toxic metabolites,which leads to hepatic oxidative stress with production of lipid peroxides.Ferroptosis is an iron-dependent programmed cell death driven by lipid peroxidation processes.Currently,ferroptosis is widely studied in the field of liver,including hepatocellular carcinoma and liver fibrosis.However,the relationship between ferroptosis in NAFLD and VPA-induced liver injury has not been fully elucidated.In this study,we used VPA-induced hepatotoxicity in mice and cells as models to explore the occurrence of ferroptosis and related mechanisms in a VPA-induced hepatotoxicity model,and to preliminarily investigate the effect of ferroptosis and acyl-Co A synthase long chain family member 4(ACSL4),the critical enzyme of lipid peroxidation process,in VPA-induced hepatotoxicity.Methods:1.8-week-old C57BL/6J male mice were allocated to control(CON)and valproic acid(VPA)groups randomly,and the mice of VPA group were given VPA by gavage for 4 weeks at 500 mg/kg daily to obtain a VPA hepatotoxic mouse model,and the control group was treated with an equal volume of normal saline;human hepatoma cells(HepG2)were cultured in vitro and treated with graded concentrations of VPA(0,1,2 and5 m M)for 24 h to obtain a VPA hepatotoxic HepG2 cell model.Mouse serum and cell supernatant were used to determine biochemical indices of liver function,and mouse livers were used for histopathological staining to evaluate the establishment of in vivo and in vitro models of VPA-induced hepatotoxicity.2.The alterations of total and ferrous iron levels in the mouse and cellular hepatotoxicity models of VPA induced hepatotoxicity were determined by iron staining and colorimetry.3.Fluorescence probe and microscopic method were used to determine the alterations of lipid peroxidation indicators such as malondialdehyde(MDA),glutathione(GSH)and reactive oxygen species(ROS)in the VPA cellular hepatotoxicity model.4.The effects of pretreatment with Ferrostatin-1(Fer-1)on cell viability and glutathione peroxidase 4(GPX4)protein expression were measured by CCK-8 and Western Blot.Then,Western Blot was used to determine the alterations in the expression levels of ferroptosis suppressor protein 1(FSP1),light chain subunit of system Xc~-(xCT),cyclooxygenase 2(COX2),lysophosphatidylcholine acyltransferase 3(LPCAT3),ACSL4 and GPX4 to determine the mechanism of ferroptosis in VPA-induced hepatotoxicity.5.Small interfering RNA(siRNA)interfered with ACSL4 expression,and its effect on lipid peroxidation level and GPX4 protein expression was investigated by fluorescent probe method and Western Blot method to further investigate the mechanism of VPA-induced hepatic ferroptosis.Results:1.The levels of biochemical indices of liver function were increased in mice treated with VPA in oral,and the results of lipid level detection indicated that liver total cholesterol(TC)and triglyceride(TG)levels were remarkably elevated,and the hepatotoxicity of VPA was manifested as steatosis and lipid accumulation;the biochemical indices of liver function,TG and TC levels were significantly increased in VPA-treated HepG2 cells,which means that VPA caused lipid accumulation in HepG2 cells.2.In vitro and in vivo study demonstrated that VPA induced hepatic iron accumulation,as evidenced by increased levels of total and ferrous iron.3.In vitro studies showed that VPA induced hepatic lipid peroxidation,manifested by elevated levels of MDA and ROS and decreased levels of GSH,and that administration of exogenous iron or GSH could exacerbate or reduce the level of VPA-induced lipid peroxidation,respectively.4.Fer-1 pretreatment improved cell viability and GPX4 protein levels down-regulated by VPA in a VPA hepatotoxicity cell model.In vitro and in vivo studies showed that VPA caused alterations in ferroptosis-related proteins:GPX4 and FSP1 expression levels decreased and xCT,COX2,LPCAT3 and ACSL4 expression levels increased.5.After siRNA transfection with ACSL4,lipid peroxidation caused by VPA was alleviated and the level of VPA-induced GPX4 decrease was improved and siACSL4 attenuated VPA-induced ferroptosis.Conclusion:This study demonstrates that ferroptosis occurs in VPA-induced hepatotoxicity,that inhibition of ACSL4 expression ameliorates VPA-induced liver injury,and that ACSL4 may be a new target for the potential treatment of VPA-induced liver injury.