NCOA4-mediated Ferritinophagy Contribute To APAP-induced Hepatocyte Ferroptosis

Background and Purpose Overdose of APAP(acetaminophen)is a common cause of drug-induced liver injury.N-acetylcysteine(N-acetylcysteine,NAC)is the only FDAapproved drug for the treatment of APAP overdose,but its time-limited action limits its application.There is an urgent need to explore effective therapeutic targets.Ferroptosis is a newly discovered cell death mode in recent years,and its main features are GSH depletion and excessive free iron overload.Studies have shown that ferroptosis is autophagy-dependent,and excess free iron can be released through the ferritinophagy,leading to cell ferroptosis.However,whether ferritinophagy plays a role in APAPinduced acute liver injury remains unknown.Therefore,the purpose of this study was to investigate whether ferroptosis-mediated by ferritinophagy causes APAP-induced acute liver injury.Methods This study is mainly divided into two aspects: in vivo experiment and in vitro experiment.Experiments in vivo: Experiment 1: Establishing a model of acute liver injury induced by APAP in mice.24 ICR mice(male)were randomly divided into 4groups.After fasting for 12 hours,all mice were killed at 0,2,6,and 24 hours after APAP administration,and liver blood samples were collected for detecting the indicators of liver function,autophagy and iron ferroptosis.Experiment 2: Using ferroptosis inhibitor Fer-1 to observe whether it can protect APAP-induced acute liver injury by inhibiting ferroptosis.24 mice were randomly divided into control group,Fer-1 group,APAP group,and APAP+Fer-1 group.Fer-1(5 mg/kg)was administered 1hour through pre-administration,after APAP(300 mg/kg)was administered 6 hours,mice were killed and collected the livers.Experiment 3: To observe the effect of iron chelator DFO on the survival rate of mice after exposure to APAP,20 ICR mice(male)were randomly divided into APAP group and APAP+DFO group,and the mice were intraperitoneally injected with APAP(500 mg/kg),and DFO(200 mg/kg)was injected intraperitoneally 1 h before APAP,the survival of the mice was observed until 48 h.In vitro experiment: Experiment 1: Establish the time-effect relationship of APAP(25m M)inducing free iron,lipid peroxide and ferritinophagy-related proteins in AML-12 cells: use Ferroorange to detect intracellular free ferrous ions,and C11-BODIPY to detect lipid,WB was used to detect ferritinophagy-related proteins,and PI staining was used to detect cell death.Experiment 2: Observing the role of iron overload in APAPinduced ferroptosis in hepatocytes: DFO(400m M)was pretreated 1 hour before APAP(25m M)exposure,and changes in free iron,lipid peroxides,and hepatocyte death were detected.Experiment 3: Observing the effect of autophagy inhibitor CQ on ferroptosis:CQ(5u M)pretreatment was given before APAP(25m M)exposure to detect protein NCOA4 and FTH,and flow cytometry was used to detect changes in free iron.Experiment 4: Exploring the role of ferritinophagy in APAP-induced ferroptosis in hepatocytes: RNA silencing technology was used to knock down NCOA4 in AML-12 hepatocytes,and its effect on free iron and hepatocyte death was observed.Results The study showed that ferroptosis accelerated acute liver injury caused by APAP,and that ferroptosis occurred mainly in the early stages of acute liver injury,at 2and 6 hours after APAP administration,and Fer-1 intervention could protect APAPinduced acute liver injury.Further research showed that at 2h and 6h after administration,autophagy had been activated,and the m RNA level of NCOA4 was at an elevated level,but the protein was on a downward trend,indicating that ferroptosis at this period was mediated by ferritinophagy.Iron ion chelator DFO can improve the survival rate of mice.In vitro experiments showed that APAP stimulation could cause lipid peroxide and free iron overload in AML-12 hepatocytes.The autophagy inhibitor CQ can reduce the iron ion induced by APAP.After NCOA4 knockdown and subsequent APAP stimulation,the percentage of free iron and PI-positive decreased.Conclusions Ferroptosis is involved in APAP-induced acute liver injury;iron overload may be the initiating factor of APAP-induced ferroptosis in hepatocytes;iron overload mediated by ferritinophagy promotes APAP-induced hepatocyte ferroptosis.