Study On The Mechanism Of Liraglutide In Improving Nonalcoholic Fatty Liver Disease Through MiR-124a/ATGL/Sirt1 Signaling Pathway

Objectives:Due to the prevalence of obesity and type 2 diabetes(T2DM),the incidence of nonalcoholic fatty liver disease(NAFLD)has increased year by year and NAFLD has become a major cause of chronic liver disease around the world.Effective treatment of NAFLD can not only reduce the incidence of end-stage liver disease,but also decrease T2 DM macrovascular and microvascular complications and cardiovascular mortality.Therefore,the prevention and treatment of NAFLD is urgent.In recent years,a large number of clinical and basic studies have shown that glucagon-like peptide-1 receptor agonist(GLP-1Ra)liraglutide can safely and effectively improve NAFLD,but its underlying mechanism is still unclear.Therefore,this study used high-fat diet(HFD)-induced NAFLD mice and palmitic acid(PA)-induced steatosis normal human liver cell line(HL-7702)as research subjects,through animal and cell experiments to explore the key gene adipose triglyceride lipase(ATGL),which affects liver lipid metabolism,and detect its expression;Using gene chip detection technology and searching bio-information database,we screened out the ATGL upstream regulatory signal miR-124 a,and verified whether miR-124 a affects lipid accumulation and inflammation in hepatocytes by inhibiting ATGL/Sirt1 signal through cell transfection technology;To determine whether liraglutide improves NAFLD via miR-124a/ATGL/Sirt1 signaling pathway by establishing in vitro and in vivo study.Contents and methods:1.The HFD-induced NAFLD mice were randomly divided into two groups,normal diet group(chow)and HFD group.After 12 weeks of HFD intervention,HE staining and oil red O staining were used to detect the deposition of triglyceride(TG)in liver tissue to evaluate the effect of HFD on liver lipid deposition in mice;Immunohistochemistry staining and q RT-PCR were used to detect the expression of inflammatory cytokines in liver tissue to evaluate the effect of HFD on inflammatory infiltration of liver.The expression of ATGL m RNA,a key gene of lipid metabolism in mouse liver tissue,was screened by q RT-PCR.Western blot and immunofluorescence staining were performed to further detecte the protein expression of ATGL;Gene chip technology was used to detect the expression of miRNAs in the liver tissues of the two groups of mice.2.PA-induced HL-7702 cells were divided into normal control group(NC),PA intervention group(PA),oil red O staining and OD520 was performed to detect intracellular lipid deposition.Western blot was used to detect the expression of ATGL protein in adipogenic hepatocytes;Bioinformatics database and q RT-PCR were used to detect miRNAs regulating ATGL in PA-induced hepatocytes;Dual luciferase reporter assay further verified the relationship between miRNA and ATGL;Transfection of miRNA inhibitors and agonists for functional gain and loss of function studies to explore the role and related mechanisms of miRNAs in NAFLD.3.After 12 weeks of HFD intervention,HFD-induced NAFLD mice were randomly divided into 3 groups: chow group,HFD group,and liraglutide intervention group(HFD+Lira group).The liver weight of the three groups of mice was weighed,the general metabolic index,intrahepatic lipid and inflammation index were measured,and the effect of liraglutide on lipid deposition in the liver was evaluated.Immunofluorescence staining and western blot were used to detect the effect of liraglutide on the liver.The expression of ATGL/Sirt1 signaling protein and inflammatory factors in tissues was detected.q RT-PCR was used to detect the effect of liraglutide on liver miR-124 a,and whether liraglutide could regulate the expression of ATGL by regulating the action of miR-124 a.4.Cellular immunofluorescence staining and western blot were used to detect the effect of liraglutide on the expression of ATGL/Sirt1 signaling protein and inflammatory factors in PA-induced HL-7702 cells;After treatment with liraglutide,fatty hepatocytes were transfected with miRNA agonists,and western blot was used to detect the expression of signaling proteins ATGL,Sirt1 and inflammatory markers,and to explore whether miR-124 a mediates liraglutide to ATGL./Sirt1 signaling pathway regulation.Results:Effect of HFD on metabolic index and liver lipid deposition and inflammatory infiltration in mice1.Compared with the chow group,the body weight,blood glucose and blood lipids in the HFD group were significantly increased,and the TG in the liver was significantly increased.The expression of macrophage marker CD68 was significantly increased in the HFD group,and the q RT-PCR showed the expression levels of inflammatory factors such as IL-1?,NF-?B p65 and TNF-?in the liver of mice in the HFD group increased significantly;2.Compared with the chow group,the expression of liver fatty acid synthesis related genes,TG synthesis related genes and lipid uptake-related genes increased in the HFD group,while the expression of lipid-degrading related genes ATGL decreased.Effect and mechanism of miR-124 a on hepatocyte lipid deposition and inflammatory response1.HL-7702 cells were treated with 0.05 m M,0.1 m M PA for 24 hours,and the intracellular lipid deposition increased in a concentration-dependent manner;ATGL decreased in the fatty liver cells;2.Compared with the chow group,the expression of miR-124 a,miR-34 a and miR-28a-5p was significantly increased in the liver tissues of mice in the HFD group;miR-124 a,miR-148 a,miR-148 b,miR-152 and miR-485 could bind to the3' non-coding region(UTR)of ATGL m RNA,and the expression of miR-148 a,miR-148 b and miR-152 is decreased,while the expression level of miR-124 a increased significantly in PA-induced hepatocytes;3.Mi R-124 a and ATGL 3' UTR have a conserved binding site,and miR-124 a could directly inhibit ATGL transcription and translation;inhibition of miR-124 a expression could promote ATGL/Sirt1 protein expression,inhibit lipid deposition and inflammatory factors in hepatocytes under high fat condition.Effect and mechanism of liraglutide intervention on HFD-induced NAFLD mice1.Liraglutide intervention can significantly improve liver weight,liver function,blood glucose and insulin sensitivity in HFD group mice; 2.Liraglutide promoted the expression of ATGL/Sirt1 proteins in livers of mice in HFD group,and improved lipid deposition in liver tissue of mice in HFD group,as well as inhibited the expression of inflammatory factors;3.Liraglutide inhibited the expression of miR-124 a in the livers of mice in the HFD group.Effect and mechanism of liraglutide intervention on PA-induced hepatocyte steatosis1.With the treatment of liraglutide,the expression of ATGL and Sirt1 protein increased,and the transferction of NF-?B p65 into the nucleus and the expression of inflammatory factors inhibited;2.Overexpression of miR-124 a could inhibit the effect of liraglutide on upreglating ATGL/Sirt1 protein expression,and block the effect of liraglutide on lipid deposition and inflammatory infiltration in PA-induced hepatocytes;3.Mi R-124 a agonists could inhibit the effect of liraglutide on inhibiting the transferction of NF-?B p65 into the nucleus.Conclusions:1.As the expression of ATGL protein in the liver of HFD group mice decreased,metabolic disorders and liver lipid deposition and inflammatory infiltration occurred.2.Mi R-124 a promotes lipid deposition and inflammatory response in hepatocytes by directly inhibiting the expression of ATGL m RNA.3.Liraglutide could delay and prevent the development of NAFLD through upregulating the expression of ATGL/Sirt1 signaling pathway in liver.4.Liraglutide can decrease the lipid deposition and inflammatory reaction in PA-induced hepatocytes by inhibiting the level of miR-124 a and upregulating the expression of ATGL/Sirt1,thereby improving NAFLD.