Study On The Mechanism Of Metformin On Sepsis-induced Liver Injury By AMPK/FOXO3a Pathway

Objective:Metformin（Met）is widely used in the treatment of type 2 diabetes.It can play anti-inflammatory and antioxidant effects by activating adenylate-activated protein kinase（AMPK）.In this study,lipopolysaccharide（LPS）was used to establish a septic liver injury model to verify the protective role of Met in septic liver injury induced by LPS,and to explore the relationship between its anti-injury effect and AMPK/FOXO3a signal pathway,so as to provide new ideas for clinical treatment.Methods:1.Forty-two healthy male C57BL/6J mice aged 6-8 weeks weighing 25±5 g were randomly divided into 6 groups with 7 mice in each group:normal control group,LPS group,LPS+Met group,LPS+Met+Dor group,Met+Dor group and Met group.After 1 week of adaptive feeding,the model was established by intraperitoneal injection of LPS（12.5mg/kg,dissolved in dd H₂O）.Met（400mg/kg,soluble in dd H₂O）was injected 6 hours before LPS was given.AMPK inhibitor dorsomorphin（15mg/kg,soluble in DMSO）was injected 1 hour before Met.Other groups of mice were given the same amount of normal saline at the time of injection.After LPS stimulation for16 hours,the body weight of mice was recorded,and the mice were anesthetized with10%chloral hydrate（1g/kg,dissolved in dd H₂O）.Blood and complete liver tissue samples were collected.2.The liver tissue was measured with a uniform scale after the excess fluid was absorbed by filter paper.The changes of liver surface of mice in each group were observed and the ratio of liver weight to body weight was calculated.The pathological changes of mouse liver were observed after hematoxylin/eosin staining.Serum levels of TNF-α,IL-6 and IL-10,alanine aminotransferase（ALT）and aspartate aminotransferferase（AST）were detected after blood samples were collected.The levels of IL-1,IL-6 and IL-10 in liver tissue were detected by RT-q PCR.The expression levels of phosphorylated AMPK,phosphorylated FOXO3a,thioredoxin-1（Trx-1）and apoptotic protein cleaved caspase-3 in liver tissue were detected by western blot technique.3.Human normal hepatocytes（LO2 cells）were cultured and set up the same group as the mouse model.The septic environment was simulated by LPS（3μg/m L）stimulation.Met（450μM）was added 6 hours before LPS stimulation.Dorsomorphin was added 1 hour before Met（5μM）.After 16 hours of LPS stimulation,the total proteins were collected and the expression levels of phosphorylated AMPK,phosphorylated FOXO3a and Trx-1 were detected by western blot technique.4.One-way ANOVA and t-test were used for compare the differences of sample means,and P<0.05 indicated statistical differences between groups.Results:1.Compared with control group,there were inflammatory punctate changes on the surface of liver tissue in LPS group.The ratio of liver weight to body weight increased.The structure of hepatic lobules was destroyed,accompanied by balloon-like transformation,and inflammatory cells infiltrated the hepatic sinusoid under microscope.The levels of serum transaminase（ALT,AST）,systemic and liver inflammatory factors（TNF-α,IL-1,IL-6,IL-10）were significantly increased.The level of cleaved caspase-3 protein increased.The inflammatory exudation on the surface of liver tissue of mice pretreated with metformin decreased.Metformin also reduced the ratio of liver weight to body weight,and reduced liver structural damage.The levels of serum transaminase and inflammatory factors decreased significantly.The protein level of cleavedcaspase-3 was also decreased.2.Compared with the control group,AMPK phosphorylation and Trx-1 level decreased,and FOXO3a phosphorylation level increased after LPS stimulation.Metformin could stimulate the phosphorylation of AMPK and increase the level of Trx-1,but decrease the level of FOXO3a phosphorylation.After adding AMPK inhibitor dorsomorphin,the changes of protein levels were consistent with those of LPS group.3.Both LPS and AMPK inhibitor dorsomorphin could increase the level of serum ALT and the amount of cleaved caspase-3 protein in mice.These decreased after the intervention of metformin.Conclusion:1.Metformin attenuates LPS-induced liver injury in sepsis.2.Metformin attenuates septic liver injury by regulating AMPK/FOXO3a signal pathway.