Acetaminophen Aggravates Fat Accumulation In NAFLD By Inhibiting Autophagy Via The AMPK/mTOR Pathway

Non-alcoholic fatty liver disease(NAFLD)is a clinical disease characterized by liver fat exceed 5% of the total liver weight,it can cause severe liver damage and metabolic comorbidities if not controlled.NAFLD includes four pathological processes: simple fatty liver,fatty hepatitis,fatty fibrosis and fatty cirrhosis,is a serious threat to the health of people the world over,with the morbidity rising every year and the age of sufferers getting younger.Patients with steatosis often do not feel that they have the disease for their mild symptoms,when they really realize they are sick,the condition is already very heavy.Acetaminophen(APAP),commonly known as paracetamol,is the active metabolite of the same kind drug-Phenacetin.APAP is one of the most frequently used analgesic and antipyretic drugs,because of its effectiveness on reducing fever and pain,the minor irritation to the gastrointestinal tract and the complete oral absorption.It is safe and effective at therapeutic doses,but an overdose can cause hepatotoxicity,acute liver failure(ALF)and even death.APAP overdose is the leading cause of drug-induced acute liver injury,In recent years,severe adverse reactions such as liver injury caused by overdose or misuse of APAP have been widely reported all over the world.Autophagy is a ubiquitous metabolic phenomenon in eukaryotic cells,also it is a self-renewal process of cells and a self-defense mechanism of body.Autophagy can be divided into the following three types: macroautophagy,microautophagy and chaperone-mediated autophagy.At present,studies on autophagy are mainly focused on macroautophagy,and the mechanism is relatively clear,which is often referred to as autophagy in a narrow sense.Autophagy plays an important role in coping with multiple forms of cellular stress,including nutrient or growth factor deprivation,hypoxia,or damaged organelles.Aims:This study investigated the effect of acetaminophen on nonalcoholic fatty liver disease through autophagy,and further explored the role of AMPK/mTOR signaling pathway in this process.Methods:Male C57 BL /6J mice at 6-8 weeks were randomly selected,dividing into 5 groups: normal group,model group,APAP(50,100,200 mg/kg)group.A nonalcoholic fatty liver model was established by feeding mice with high-fat and highsugar diet,and the normal group was fed with control diet,with a modeling period of 56 days(8 weeks).The mice were given APAP by gavage on the last day of modeling,and the mice were sacrificed after 24 h.The weight changes of mice in each group were recorded every week,the mice were weighted before death and mice liver were weighted after death to calculate the liver index.Serum ALT,AST and TG levels were determined.Pathological changes were observed by HE staining of liver tissue,and lipid deposition in liver was observed by Oil Red O staining.Immunohistochemistry(IHC)was used to determine the expression of AMPK and mTOR in liver tissues.Lipid droplets and autophagosomes in mice liver were detected by transmission electron microscopy(TEM).Western blot was used to detect the expressions of autophagy related proteins LC3,Beclin1,lipid metabolism related proteins SREBP-1c,AMPK and mTOR in mouse liver.In vitro,anhydrous ethanol combined with oleic acid were used to stimulate human normal hepatocytes(L02)to establish a lipid deposition model of hepatocytes,dividing into six groups: normal group,model group,APAP(2,4,8 mM)group,APAP 4 mM+ RAPA(1 μM)group.Oil Red O staining was used to detect the lipid accumulation in each group,and the intracellular TG level was determined,Western blot was used to detect the expressions of autophagy related proteins LC3,Beclin1,lipid metabolism related proteins SREBP-1c,AMPK and mTOR in L02 cells.Results:1.Compared with normal group,the body weight and liver index of mice in model group and APAP group were significantly increased,and the liver index of APAP group was significantly higher than that of model group.The levels of ALT,AST and TG in serum of model group were significantly higher than those of normal group,and the levels of ALT,AST and TG were further increased in APAP group.HE and Oil Red O staining showed that model group had more lipid accumulation than normal group,and APAP group had more lipid accumulation than model group.2.Immunohistochemical results showed that AMPK level in model group was lower than that in normal group,and further decreased after APAP administration,while mTOR level was the opposite compared to AMPK level.TEM results showed that the number of autophagosomes decreased in model group and decreased further in APAP group.Western blot results showed that the expressions of LC3,Beclin1 and AMPK were decreased compared with normal group,and the expressions of mTOR and SREBP-1c were increased after the APAP administration,and were further increased after the APAP administration.3.In model of anhydrous ethanol joint oleic acid stimulated L02 cells lipid accumulation,Oil Red O staining and semi-quantitative results show that compared with normal group,lipid accumulation of model group increased significantly,APAP administration further aggravating,while RAPA can reverse this effect,compared with normal group,intracellular TG was significantly increased in model group,further increased after APAP treatment,and significantly decreased after RAPA treatment.4.In vitro experiments,western blot results showed that compared with normal group,the expressions of LC3,Beclin1 and AMPK were decreased and further decreased after the APAP administration,while the expressions of mTOR and SREBP-1c were increased and further increased after the APAP administration,and the autophagy agonist RAPA could reverse the effect APAP administration.Conclusion:APAP aggravates hepatic fat deposition in NAFLD,which may be related to its inhibition of autophagy associated with the AMPK/mTOR pathway,and the patients with NAFLD should use a lower dose of APAP.