Mechanistic Study Onrole Of Melatonin In APAP-induced Liver Injury Via The Regulation Of FOXO3a-mediated Mitochondrial Quality Control

Objective:Acetaminophen(APAP)overdose is one of the major causes of hepatotoxicity and acute liver failure.A growing body of research evidence suggests that hepatocyte necrosis plays a key role in APAP-induced liver injury.However,the mechanisms of APAPinduced necrosis and liver injury are not fully understood.In this study,the mechanism of APAP-induced liver injury was investigated by constructing an APAP acute liver injury model in Foxo3a-KO and Foxo3a-WT mice,which were given melatonin pretreatment.The results showed that Foxo3a-KO could inhibit APAP-induced mitochondrial autophagy and mitochondrial biosynthesis and other mechanisms,while melatonin not only had strong antioxidant effects,but also exerted protective effects on APAP-induced hepatocyte necrosis and liver injury by inducing FOXO3 a.Methods:1.FVB wild-type mice were randomly divided into 4 groups: control group(saline),APAP model group(i.g.400 mg/kg APAP),melatonin-protective drug group(i.p.10mg/kg Mel+i.g.400 mg/kg APAP),melatonin alone group(i.p.10 mg/kg Mel).2.Foxo3 a knockout mouse model construction: Paired breeding of stable offspring,genotype identification,RT-q PCR,Western blot and other means to verify the success of the model construction.3.In vivo experiments: serum glutamic oxalyl transaminase and glutamic alanine transaminase levels were measured using kits;malondialdehyde(MDA),SOD and ATP contents,GSH/GSSG ratio in liver were measured;H&E staining and immunohistochemical staining were performed;total RNA from liver tissues was extracted,and the relative copy number of mt DNA was examined for oxidative stress The total protein of liver tissues was extracted for the detection of Foxo3a-regulated oxidative stress pathway proteins and mitochondrial quality-controlled signaling pathway-related proteins,etc.The fresh tissues were subjected to frozen sections and immunohistochemical staining,and the tissue damage was observed by transmission electron microscopy in ultrastructure.Results:1.melatonin plays an important role in antioxidant levels in mouse liver by activating FOXO3 a.Melatonin reduced APAP-induced hepatic lipid peroxidation and ROS levels in Foxo3a-WT mice,increased SOD,GSH/GSSG ratio,and ATP content,and alleviated hepatic peroxidation;while its antioxidant effects disappeared after Foxo3 a knockdown in mice.2.Compared with the control group,liver SIRT3,AMPKα,PGC-1α,NRF1,TFAM and other mitochondrial biosynthetic pathway protein expressions were significantly down-regulated in APAP model group mice,and mitochondrial autophagy pathway protein expressions such as PINK1,Parkin,Beclin1,Atg3,ULK1,LC3-II were significantly down-regulated.The expression of mitochondrial biosynthetic pathway proteins in the liver of Foxo3a-KO mice was down-regulated in the melatonin-protected drug group compared with Foxo3a-WT mice.The above results indicate that melatonin has significantly promoted mitochondrial biosynthetic pathway protein expression,involved in inducing the patency of autophagic flow pathway and promoting mitochondrial autophagy.the pro-expression effect disappeared after Foxo3 a knockdown,indicating that melatonin has a protective effect against liver injury in APAP mice through regulating FOXO3 a involved in mitochondrial quality control.Conclusions.(1)Melatonin activates FOXO3 a,regulates oxidative stress,and protects against APAP hepatotoxicity.(2)FOXO3a deficiency is more sensitive to APAP-induced acute hepatotoxicity with higher levels of oxidative stress.(3)Melatonin protects against APAP liver injury in mice by promoting mitochondrial biosynthesis and mitochondrial autophagy to modulate mitochondrial quality control.