Involvement Of LKB1/AMPK/ACC Pathway In The Protective Effect Of Metformin Hydrochloride Against Alcoholic Fatty Liver

Alcoholic liver disease(ALD)is the general term of a series of chronic alcoholic liver diseases caused by excessive alcohol consumption for a long time.Alcoholic fatty liver(AFL)is the earliest stage of ALD,and its typical feature is that a large amount of fat(mainly triacylglycerol)accumulates in hepatocytes.If AFL is not treated in time,it is likely to further develop into hepatitis,fibrosis,cirrhosis and even hepatocellular carcinoma.AMP-activated protein kinase(AMPK)is a conserved Ser/Thr protein kinase in the evolution of eukaryotes.Because it can sensitively sense energy changes in cells and perform metabolic regulation,it is often referred to as cellular "Energy Metabolism Switch".Numerous studies have confirmed that AMPK activity is often inhibited in fatty liver,which results in a decrease in the activity of the downstream transcription factor PPAR-α and an increase in the activity of SREBP1 c.At the same time,the rate-limiting enzyme ACC involved in fatty acid synthesis is also activated due to dephosphorylation.All of the above ultimately leads to increased fatty acid synthesis and weaker oxidative degradation in hepatocytes,which leads to lipid deposition in the liver.Metformin is a first-line drug widely used in the treatment of type 2 diabetes,and its diabetes treatment history has been more than half a century.In addition to lowering blood sugar,metformin has also been reported to have many effects such as weight loss,anti-cancer and anti-aging.A large number of studies have shown that metformin can effectively alleviate non-alcoholic fatty liver by improving liver lipid metabolism.The activation of AMPK-mediated lipid metabolism pathways by metformin is one of the critical mechanisms for exerting its hepatoprotective effect.However,the role of metformin in AFL is currently rarely reported.In view of the currently known pathogenesis of AFL,we speculate that metformin may effectively alleviate AFL by improving liver lipid metabolism.Next,we tested the two upstream regulatory pathways of AMPK and clarified the upstream regulatory mechanism of metformin activation of AMPK.Together,we also evaluated the safety of long-term administration of metformin.Objective: To explore the effect of metformin hydrochloride on AFL and its possible mechanism and evaluate the safety of metformin hydrochloride during the administration.Methods: In order to verify the above hypothesis,we successively explored the effect and mechanism of metformin hydrochloride on AFL through in vitro and in vivo experiments.In the in vitro experiment,we set up control group,model group,medication group,LKB1 interference group and Ca MKK2 interference group.In the AML12 cell line,we used 200 m M absolute ethanol to induce fat deposition model,2 m M metformin hydrochloride was used to establish the administration group,and LKB1 and Ca MKK2 si RNA were transfected into cells to establish the interference group.After modeling,we used Nile Red staining to detect the fat accumulation of each group of cells,real-time fluorescent quantitative PCR was used to detect the m RNA expression of SREBP1 c and PPAR-α in each group of cells,and the kit was used to detect intracellular TG(triacylglycerol)content,protein expression of LKB1,Ca MKK2,AMPK and ACC was detected by Western blotting.In the in vivo experiment,we set up control group,model group,administration group,interference negative control group,LKB1 interference group and Ca MKK2 interference group.The AFL model was established in C57BL/6mice with reference to the Gao-binge model.Simultaneously,the mice were treated with metformin hydrochloride at a dose of 200 mg/kg by oral gavage to obtain the administration group,the sh RNA packed in the adeno-associated virus was injected through the tail vein to establish the corresponding interference groups.After modeling,mouse serum and liver were collected.Then,we performed liver pathology detection by tissue sectioning,oil red O staining and HE staining,and detected serum ALT(Alanine aminotransferase)and AST(Aspartate aminotransferase),TG and TC(total cholesterol)content,the protein expression of LKB1,Ca MKK2,AMPK and ACC in the liver was detected by Western blotting.Finally,we evaluated the safety of long-term administration of the drug through the detection of relevant biochemical and pathological sections.Results:(1)In vitro experiment: The results of Nile Red staining showed that compared with the control group,after AML12 cells were stimulated with 200 m M absolute ethanol for 24 hours,cell fat deposition increased significantly,indicating that the model was successfully constructed.At the same time,the TG content and the m RNA level of SREBP1 c in the model group increased significantly,while the m RNA level of PPAR-α decreased significantly.In addition,compared with the control group,the phosphorylation levels of LKB1,AMPK and ACC proteins in the model group were significantly reduced.However,compared with the model group,fat deposition was significantly improved after metformin hydrochloride administration,TG content and SREBP1 c m RNA level were significantly decreased,PPAR-α m RNA level was significantly increased,and the phosphorylation level of LKB1,AMPK and ACC protein rebounded significantly.Then,after knocking down LKB1 in the cell,it was found that the effects of metformin hydrochloride administration were reversed,but knocking down Ca MKK2 did not have a significant effect.(2)In vivo experiments: HE staining and Oil Red O staining showed that compared with the control group,ethanol exposure caused the formation of obvious fatty vacuoles in the liver of mice,the accumulation of liver fat was serious,the TG content in the serum increased,and both ALT and AST were significant increased,all above indicate that the AFL model was successfully constructed.At the same time,ethanol significantly inhibits the activity of AMPK in the liver of mice to activate ACC.The administration of metformin hydrochloride can effectively reverse the above changes.However,after knocking down LKB1 in the liver,the alcoholic liver injury was once again intensified,the AMPK level was significantly down-regulated,and the p-ACC level also decreased.(3)Safety assessment: Compared with the control group,continuous administration of metformin hydrochloride for two months can increase the serum ALT content and decrease the TG content in mice,but the heart,liver,spleen,lung and kidney have no obvious pathological changes in the two groups.Conclusions:(1)Metformin hydrochloride can effectively improve the steatosis of AML12 cells caused by alcohol by activating the LKB1/AMPK/ACC pathway;(2)Metformin hydrochloride can effectively alleviate AFL in mice by activating the LKB1/AMPK/ACC pathway;(3)Metformin hydrochloride administered at a dose of 200mg/kg per day for two months will not cause significant organ damage to mice.