Ferrostatin-1 Protects Acetaminophen-induced Acute Liver Injury In Mice By Mitigating Ferroptosis

Background: Acetaminophen(APAP)is one of the most commonly antipyretic and analgesic drug in clinical practice,it is also the most common drug that causes acute drug-induced liver injury even liver failure.APAP overdose induces various forms of hepatocyte death with liver glutathione(GSH)depletion and iron overload that be considered to hallmarks of ferroptosis,a newly discovered cell death.Early vitro study found that the ferroptosis inhibitor ferrostatin-1(fer-1)reduced hepatocyte death in mice after APAP overdose,but the specific mechanism is not clear.At the same time,the role of ferroptosis in APAP-induced hepatotoxicity in mice and the therapeutic potential effect of ferroptosis inhibitor ferrostatin-1(fer-1)are also unclear.Objective: The purpose of this study was further to investigate the role of ferroptosis in APAP-induced acute liver injury in mice,and the potential therapeutic effect and possible mechanism of ferroptosis-specific inhibitor ferrostatin-1(fer-1).Methods: Our experiments were divided into two parts.In the first part: 6-8 weeks old60 female and 60 male CD-1 mice all randomly divide into 12 groups(n = 5 per group).In the control group,mice were i.p.injected with 16% Cremophor? EL.In fer-1 alone group,mice were administered with fer-1(5 mg/kg,i.p).In APAP alone group,mice injected with APAP(400mg/kg,i.p).In the APAP + fer-1 pretreatment groups,mice were administered with fer-1(5 mg/kg,i.p)at 0.5 h before APAP(400mg/kg,i.p).In the APAP + fer-1 posttreatment groups,mice were administered with fer-1(5 mg/kg,i.p)at0.5 h after APAP(400mg/kg,i.p).All mice were sacrificed at 0 to 24 h after APAP intoxication.In the APAP + NAC pretreatment group,mice were injected with NAC(300 mg/kg,i.p)at 0.5 h before APAP(400mg/kg,i.p),the mice were sacrificed at 24 h after APAP intoxication.Liver tissue and serum were collected for the measurement of biochemical parameters and molecular experiments.In the second experiment,thirty mice were randomly divided into three groups for survival assay.In the APAP alone group,mice were administered with APAP(500 mg/kg,i.p).In the APAP + fer-1pretreatment group,mice were administered with fer-1(5 mg/kg,i.p)at 0.5 h before APAP(500 mg/kg,i.p).In the APAP + NAC pretreatment group,mice were injected with NAC(300 mg/kg,i.p)at 0.5 h before APAP.Mice were continuously observed 7 d for survival studies after APAP injection.The data were recorded and the survival curve was drawn.Results: Our results showed that the weight of liver,serum ALT,AST and TBIL levels increased significantly at 1h to 24 h after APAP administration in mice.Histopathological observations also revealed significant intrahepatic hemorrhage and hepatocyte death,all of which indicated the serious liver damage with a decrease the survival rate of mice.In addition,compared with the control group,we found that APAP overdose increased the expression of liver inflammatory cytokines like MCP-1,KC,TNF-?,and IL-1? m RNA levels.Subsequently,our data suggested that APAP overdose cause the hepatic GSH depletion and reduction of GSH metabolic enzyme activities,as well as liver iron overload and the accumulation of lipid peroxide MDA.Furthermore,APAP treatment significantly up-regulated the length of acyl-Co A synthetase Chain family member 4(ACSL4)and prostaglandin endoperoxide synthase 2(PTGS2)m RNA levels,inhibited glutathione peroxidase 4(GPX4)m RNA level,these all the specific markers of ferroptosis.Pretreatment or posttreatment with fer-1 reduced APAP-induced acute liver injury and inflammation,improved the survival rate.Surprisely,fer-1 not significantly affected liver GSH depletion and the activity of GSH metabolic enzymes in APAP-induced acute liver damage.Then,we found that fer-1 pretreatment improved APAP-induced liver iron overload and inhibited the ferroptosis.In the present study,our results indicated that hepatic iron metabolism dysfunction induced by the abnormal expression of hepcidin,FPN,DMTI,HJV,HFE,TFR2,IRP1/2,and Ft-L in APAP-induced acute liver injury,and all of these change were markedly improved by fer-1.Conclusion: In summary,our study provided some new insights into the mechanisms of hepatic ferroptosis in APAP-induced acute liver injury,and suggested that the protective effect of fer-1 on APAP-induced hepatotoxicity may be not just the inhibition of ferroptosis,but also associated with its improvements of inflammatory responses and iron overload.