Protective Role And Mechanisms Of MitoQ In Acetaminophen-induced Hepatocyte Ferroptosis

Background & Aim: Acetaminophen(APAP)is one of the most widely used antipyretic and analgesic drugs for clinical treatment.Although therapeutic dosage is relatively safe,APAP overdose can lead to acute liver injury(ALI)and even acute liver failure.APAP has been the leading cause of acute liver failure worldwide.Currently,the mechanism of APAP-induced liver injury has not been fully understood.Ferroptosis is a new type of programmed cell death mode.Recent studies have reported that ferroptosis is involved in APAP-induced liver injury,but the specific mechanism is unclear.Therefore,the objective of the present study was to investigate the role of mt ROS in APAP-induced hepatocyte ferroptosis.Moreover,we aimed to evaluate the protective effect of MitoQ on APAP-induced hepatocyte ferroptosis and ALI.Methods Animal experiments: Experiment 1,to determine APAP-induced ALI,forty overnight fasted mice except controls were intraperitoneally injected with APAP(300mg/kg).Mice were euthanized at 2,6,and 24 h after APAP.The levels of GSH and two secondary products of lipid peroxidation,MDA and 4-HNE,in liver tissue were detected.The levels of ferroptosis marker ACSL4 protein and ptgs2 m RNA in liver tissues were detected.Hepatocyte death was detected by TUNEL assay.Ultrastructure of hepatocytes was observed by transmission electron microscopy.ALT and AST levels in serum were detected.Histopathological changes of liver were observed.Experiment2,to explore the role of ferroptosis in APAP-induced ALI,sixty mice were randomly divided into four groups: Ctrl,APAP,Fer-1 and APAP+Fer-1groups.In the APAP and APAP+Fer-1 groups,mice were intraperitoneally injected with APAP(300 mg/kg).In the Fer-1 and APAP+Fer-1 groups,mice received an intraperitoneal injection of Fer-1(5 mg/kg).All mice were euthanized either 2 or 6 h after APAP.Blood and liver tissues were collected for subsequent analysis.Experiment3,to explore the protective effect of MitoQ on APAP-induced ferroptosis,sixty mice were randomly divided into four groups: Ctrl,APAP,MitoQ,APAP+MitoQ groups.In the APAP and APAP+MitoQ groups,mice were intraperitoneally injected with APAP(300 mg/kg).In the MitoQ and APAP+MitoQ groups,mice received an intraperitoneal injection of MitoQ(4 mg/kg).All mice were euthanized either 2 or 6 h after APAP.Blood and liver tissues were collected for detection of the indicators mentioned previously.Experiment 4,to evaluate the effects of MitoQ on animal survival,twenty mice were randomly divided into two groups: APAP and APAP+MitoQ groups.In the APAP group,mice were intraperitoneally injected with APAP(500 mg/kg).In the APAP+MitoQ group,mice received an intraperitoneal injection of MitoQ(4 mg/kg)1 h before APAP(500 mg/kg).Animal survival was observed for 48 h.Cell experiments: Experiment 1,for time courses of APAP-induced lipid peroxidation and mitochondrial dysfunction,AML-12 cells were treated with APAP(25 m M)and harvested at different time points(3,6,or 12 h).Oxidative lipid levels were detected.Mitochondrial disfunctions,assessed by mitochondrial membrane potentials(MMPs),oxidative phosphorylation complex protein levels,ATP content and mt ROS,were detected.Experiment 2,to explore the role of ferroptosis in APAP-induced hepatocyte ferroptosis,AML-12 cells were pretreated with Fer-1,DFO(iron chelator)and z VADfmk(apoptosis inhibitor)for 1h,and then co-cultured with APAP(25m M)for 24 h to observe the effects of different inhibitors on hepatocyte death.Experiment 3,to assess the effect of MitoQ on APAP-induced ferroptosis,AML-12 cells were cultured with APAP(25 m M)in the presence or absence of MitoQ(180 n M).Cells were harvested 12 or 24 h after APAP.The changes of mitochondrial function,mt ROS,oxidized lipids and hepatocyte death were detected.Experiment 4,to further explore the role of FSP1 and GPX4 on MitoQ-mediated protection,AML-12 cells were transfected with either FSP1 or GPX4 si RNA and then cultured with APAP(25 m M)in the presence of or absence of MitoQ(90 n M).Cells were harvested 12 or 24 h after APAP.The changes of oxidized lipids and hepatocyte death were detected.Results 1.Hepatocyte death and ferroptosis-characterized mitochondrial ultrastructural alterations at early stage of APAP-induced ALI.Hepatocyte death was elevated as early as 2 h and continued increased 24 h after APAP.Ferroptosis-characterized mitochondrial ultrastructural alteration,mainly mitochondrial shrinkage,was observed in APAP-exposed mice.Quantitative analysis showed that hepatic mitochondrial area was reduced as early as 2 h and continued decreased 24 h after APAP.2.APAP rapidly induces lipid peroxidation in mouse livers and hepatocytes.Hepatic GSH content was reduced dramatically,with the lowest GSH levels at 2 h after APAP.Hepatic MDA and4-HNE,two secondary products of lipid peroxidation were markedly increased as early as 2 and 6 h after APAP.Hepatic ACSL4 was elevated as early as 2 h and remained increased 6 and 24 h after APAP.Hepatic ptgs2 m RNA as upregulated,beginning 6 h and remaining increased 24 h after APAP.APAP-induced oxidized lipids were elevated as early as 3 h and remained increased 12 h in AML-12 cells.3.Ferroptosis occurs in early stage of APAP-induced ALI.In vitro results indicated that APAP-induced hepatocyte death in vitro was partly alleviated by Fer-1 and DFO,but not z VAD-fmk.In vivo results showed Fer-1 pretreatment partially restored the hepatic GSH levels,significantly reduced the MDA and 4-HNE,downregulated the ptgs2 m RNA,improved hepatocyte death and mitochondrial ultrastructural damage,and finally significantly alleviated APAP-induced ALI in mice.4.Mitochondrial ROS are rapidly increased in APAP-exposed hepatocytes.MMPs were reduced as early as 3 h and remained decreased 12 h after APAP.Five subunits of OXPHOS were downregulated as early as3 h and continued decreased 12 h after APAP.Correspondingly,cellular ATP content was reduced as early as 3 h and continued decreased 12 h after APAP.Mt ROS were increased as early as 3 h and continued increased 12 h after APAP.5.MitoQ attenuates APAP-evoked oxidized lipids and ferroptosis in AML-12 cells.MitoQ pretreatment partially improved the decreased MMPs,partially restored intracellular ATP content,partially scavenged mt ROS,but almost reversed APAP-induced lipid peroxidation,and finally significantly alleviated ferroptosis in AML-12 cells.6.MitoQ protects against APAP-induced ferroptosis and ALI in mice.MitoQ pretreatment partially restored the hepatic GSH levels,significantly reduced the MDA and 4-HNE,downregulated the ptgs2 m RNA,improved hepatocyte death and mitochondrial ultrastructural damage,significantly alleviated APAP-induced ALI and improved animal survival in APAPexposed mice.7.FSP1 knockdown weakens protective effect of MitoQ on APAPinduced lipid peroxidation and hepatocyte ferroptosis.Knockdown of FSP1 had little effect on APAP-induced lipid oxidation but significantly increased APAP-induced hepatocyte ferroptosis.Besides,knockdown of FSP1 partially weakened the protection of MitoQ on APAP-induced lipid peroxidation and hepatocyte ferroptosis.8.GPX4 knockdown does not affect protective effect of MitoQ on APAP-induced ferroptosis in hepatocytes.Knockdown of GPX4 exacerbated APAP-induced oxidized lipids and hepatocyte ferroptosis but did not influence the protective effect of MitoQ on APAPinduced lipid peroxidation and hepatocyte ferroptosis.Conclusion Ferroptosis occurs in early stage of APAP-induced ALI.Mt ROS may be an initiator of APAP-induced hepatocyte ferroptosis.MitoQ protects against APAPinduced hepatocyte ferroptosis partially in an FSP1-dependent manner.