Melatonin Protects Against Ferroptosis During Acetaminophen-induced Acute Liver Injury

Acetaminophen(APAP)is a kind of widely used analgesic-antipyretic nonsteroidal anti-inflammatory drugs(NSAIDs).Under the influence of unreasonable medication and some occasional factors,APAP administration could lead to the liver injury,even acute liver failure.APAP-induced hepatocyte death is associated with the overproduction of lipid peroxides due to disorders in the cellular antioxidant system.Ferroptosis is a mode of cell death dependent on iron and oxidative stress,which is different from classical cell death,such as apoptosis and necrosis.It has unique morphological characteristics.The accumulation of lipid peroxides in cells is the direct cause of ferroptosis.Studies have shown that iron chelators inhibit ferroptosis.The fenton reaction mediated by intracellular iron ions and reactive oxygen species is the key link leading to the accumulation of lipid peroxides in cells,and the genes and proteins associated with antioxidant activities in cells may participate in inhibiting this process.APAP-induced liver injury is associated with accumulation of lipid peroxides and intracellular accumulation of iron,including ferroptosis.Melatonin,as an active amine hormone produced by the pineal body,is a highly effective antioxidant.Melatonin has direct and indirect antioxidant and free radical scavenging biological activities.Studies have shown that melatonin can improve the survival ability of mice with liver injury induced by APAP and inhibit the oxidative stress state of hepatocytes,but the specific mechanism is not clear.The purpose of this study was to investigate the effect of melatonin on ferroptosis during APAP-induced hepatocyte death.1.This study firstly examined the protective effect of melatonin on APAP-induced liver injury in mice.To investigate the protective effect of melatonin from the liver function and pathological damage index,and then to discuss whether melatonin can play the protective effect of liver through ferroptosis pathway,APAP-induced liver injury model was established using C57BL/6J mice.The ferroptosis inhibitor Ferrostatin-1(FER-1)was used as a positive drug in the positive control group,the normal control group,the APAP model group,the Fer-1(5 mg/kg)group,and the high-dose melatonin(100 mg/kg)and low-dose melatonin(25 mg/kg)groups.The administration methods of melatonin and Fer-1 were gavage and intraperitoneal injection respectively.1)Serum transaminases(ALT and AST)were used as liver function indexes to detect the degree of liver injury.The results showed that ALT and AST levels increased significantly in the APAP group compared with the normal group(P<0.01,P<0.01).ALT and AST levels in the melatonin groups were significantly lower than those in the APAP group(P<0.01,P<0.05;P<0.01,P<0.05).ALT and AST levels in Fer-1 group were significantly lower than those in APAP group(P<0.05,P<0.05).The results showed that melatonin had a significant protective effect on APAP-induced ALI in mice.2)The pathological features of liver injury were detected by H&E and TUNEL staining.The results showed that the liver tissues of ALI model mice induced by APAP showed obvious necrosis and hemorrhage,and high and low doses of melatonin could well alleviate these pathological injuries.Fer-1 also alleviated these pathological injuries.3)The content of malondialdehyde(MDA)and GSH in the liver were measured by microplate method to evaluate the antioxidant system of the liver.The results showed that MDA level in the liver tissues of ALI mice induced by APAP was significantly increased(P<0.01),and GSH in the liver was significantly depleted(P<0.01).High and low doses of melatonin not only significantly reduced MDA level,but also significantly increased liver GSH level(P<0.05,P<0.05;P<0.05,P<0.05).Compared to the APAP group,Fer-1 also significantly reduced MDA level and significantly increased liver GSH level(P<0.05,P<0.05).4)The mRNA levels of prostaglandin peroxidase 2(Ptgs2),a representative biomarker of ferroptosis,were detected by RT-PCR.The results showed that Ptgs2 mRNA expression in APAP-induced ALI mouse liver tissues increased significantly(P<0.01).Compared with the APAP model group,the mRNA level of Ptgs2 in the high and low dose melatonin groups was significantly lower(P<0.01,P<0.01).Compared with APAP model group,FER-1 group also significantly reduced the mRNA level of Ptgs2(P<0.05).5)The characteristic structure of hepatocytes mitochondria was observed by transmission electron microscopy.The results showed that typical ferroptosis-related mitochondrial pathological changes occurred in the liver tissues of ALI mice induced by APAP,including significant enlargement of mitochondria and damage of mitochondrial cristae.The pathological changes of mitochondria were significantly improved in high and low dose melatonin groups.Fer-1 also alleviated the pathological changes of mitochondria.The above results indicated that there was ferroptosis in APAP-induced ALI mice,and melatonin,like Fer-1,a selective ferroptosis inhibitor,could inhibit ferroptosis in hepatocytes of APAP-induced ALI mice,thus playing a role of liver protection.2.The protective effect of melatonin on HepaRG hepatotoxicity model induced by APAP was further investigated.1)The optimal protective concentration of Fer-1 on ferroptosis in HepaRG caused by APAP was determined by cell toxicity analysis(Cell Counting Kit-8,CCK-8).The results showed that DFO(20 ?M),Fer-1(1?M)and melatonin(50 M)had significant protective effects on HepaRG hepatocyte injury by 10 mM APAP,respectively.2)On this basis,MDA was detected to investigate the accumulation of lipid peroxides in HepaRG cells to explore the protective effect of melatonin on hepatocytes and its effect on ferroptosis.The results showed that the lipid peroxides in HepaRG cells treated with 10 mM APAP were significantly increased(P<0.01),and melatonin significantly decreased(P<0.05).Fer-1 also significantly reduced lipid peroxides in HepaRG cells(P<0.05).These results indicated that melatonin had a significant inhibitory effect onAPAP-induced hepatocyte ferroptosis.3.APAP-induced ALI mice was used to explore melatonin inhibits iron signaling pathways involved in the death,in order to elucidate the oxidation of melatonin activation pathways involved in APAP-induced ferroptosis.1)RT-PCR was used to detect the mRNA levels of ferroptosis-related antioxidant factors,including E2-related nuclear factor 2(NF-erythroid2-related factor 2,Nrf2),Heme oxygenase-1(HO-1),and the reduced quinone oxidoreductase 1(NAD(P)H quinone oxidoreductase(NQO1)in mice liver.The results showed that Nrf2 and NQO1 in the APAP-induced ALI model group were not significantly changed compared with the normal group(P>0.05,P>0.05).Compared with the normal group,HO-1 in the APAP-induced ALI model group was significantly decreased(P<0.05).Melatonin significantly increased the expression of Nrf2,HO-1 and NQO1 mRNA in the liver of the APAP model group(P<0.05,P<0.05,P<0.05).DFO significantly increased Nrf2(P<0.05),HO-1(P<0.05)and NQO1 mRNA expression in liver of APAP-induced ALI model mice(P<0.05).2)Immunohistochemistry was used to detect the protein expressions of Nrf2 and HO-1 in the liver of mice in each group.The results showed that the protein expression of Nrf2 and HO-1 in the APAP model group was not significantly different(P>0.05,P>0.05),and melatonin could significantly improve the protein expression of Nrf2 and HO-1 in the APAP model group(P<0.05,P<0.05).DFO had the similar function.3)the ACSL4 pathway was detected by RT-PCR,and the results showed that there was no significant difference in ACSL4 mRNA between the APAP group and the normal group(P>0.05).Moreover,there was no significant difference in ACSL4 mRNA between the melatonin group and the DFO group(P>0.05,P>0.05),which indicate that ALI induced by 400 mg/kg APAP may be unrelated to lipid metabolism pathways.In conclusion,this study showed that melatonin inhibit the ALI induced by APAP by inhibiting ferroptosis.Furthermore,melatonin alleviates liver damage by activating Nrf2,NQO1 and HO-1 pathways to inhibit APAP-induced ferroptosis.The findings provide a deeper understanding of the mechanism of APAP-mediated liver injury and provide theoretical basis for the role of melatonin in liver protection through ferroptosis.