Molecular Mechanism Of MAFLD Risk Factors And Improvement Of Lipid Metabolism In Hepatocytes By Dagliflozin

Purpose:Clinical observations suggest that dagliflozin has an ameliorative effect in patients with metabolic associated fatty liver disease(MAFLD),but the mechanism of its ameliorative effect is not clear.In this study,we investigated the relevant differences between non-obese and obese MAFLD patients by observing clinical characteristics;we investigated the role of genetics on MAFLD by single nucleotide polymorphism(SNP);we investigated the molecular mechanism of dagliflozin to improve lipid metabolism in hepatocytes by biochemical analysis techniques and cellular experiments,so as to provide a theoretical basis for dagliflozin to improve MAFLD.Methods:Chapter I Influence of relevant factors on MAFLD1.Clinical characteristics of non-obese and obese MAFLD patientsInpatients with MAFLD who met the criteria(439 cases)were collected as study subjects and divided into two groups.Patients with BMI≤24 kg/m~2were classified as the non-obese MAFLD group(n=235),and patients with BMI>24 kg/m~2were classified as the obese MAFLD group(n=204).The differences in demographic and laboratory indices between the two groups were compared.2.Relationship between PNPLA3 and APOC3 gene polymorphisms and the risk of MAFLDWhole blood specimens were obtained from patients(n=839)and normal controls(n=357)who met the criteria,and genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism analysis(PCR-RFLP)to compare the risk of MAFLD due to different genotypes.Demographic and laboratory data of the study subjects were collected to compare the differences between the different genotypes of MAFLD patients.Chapter II Inhibition of lipid accumulation in hepatocytes by dagliflozin1.A human hepatocellular carcinoma cell line(Hep G2)of logarithmic growth phase was taken to construct a fatty liver cell model.The experiment was divided into 3 groups:control group(Control),model group(FFA),and dagliflozin dosing group(FFA+Da).2.CCK-8 was used to detect the effect of the drug on cell viability and to determine the concentration and duration of action of the drug;oil red O staining and triglyceride(TG)test kit were used to detect the lipid content in each group.Chapter III Molecular mechanism of dagliflozin inhibition of lipid accumulation and inflammatory response in hepatocytes1.Bioinformatics techniques to screen the action pathwayWe screened the GEO database to obtain the differentially expressed genes between MAFLD patients and normal subjects;used the DAVID database to perform GO enrichment analysis on the differential genes;performed PPI network analysis by STRING11.0 database;used Cyto Scape 3.8.0 software and Cyto Hubba plug-in to screen the top 10The key genes were screened by Cyto Scape 3.8.0 software and Cyto Hubba plug-in;finally,the molecular pathways were identified by KEGG enrichment analysis.2.Cellular experiments to verify the effect of dagliflozin in treating MAFLD through the screened molecular pathways(1)A human hepatocellular carcinoma cell line(Hep G2)was taken to construct a fatty liver cell model.There were 4 groups:control group(Control),model group(FFA),dagliflozin dosing group(FFA+Da),and dagliflozin+pathway inhibitor group(FFA+Da+AG-490).(2)Oil red O staining and TG content measurement to detect TG content in each group;EISA to detect IL-6 content;RT-PCR to detect JAK2 m RNA,STAT3 m RNA,SOCS3m RNA expression levels;immunoblotting to detect changes in JAK2,p-JAK2,STAT3,p-STAT3,SOCS3 protein expression in different groups The changes of JAK2,p-JAK2,STAT3,p-STAT3 and SOCS3 protein expression in different groups were detected by immunoblotting.Results:Chapter I.Influence of relevant factors on MAFLD1.Clinical characteristics of non-obese and obese MAFLD patients(1)ALT,AST and GGT were higher in obese MAFLD patients than in non-obese MAFLD patients,and ALP and HDL-C were lower in obese MAFLD patients than in non-obese patients(P<0.05).(2)In the univariate regression analysis of risk factors for obese MALFD,the risk factors were gender,weight,ALT,ALP,and GGT(P<0.05).(3)In the multifactorial regression analysis of risk factors for obese MALFD,the risk factors included gender,ALT,ALP,and GGT(P<0.05).Chapter II Inhibition of lipid accumulation in hepatocytes by dagliflozin(1)Hep G2 cells showed the best effect at a concentration of 1 m M in the modeling solution and an action time of 24 h.The cell viability was determined by CCK-8 and the drug concentration was chosen as 20μM.(2)The results of both oil red O staining and TG content assay reagents showed that the TG content was significantly lower in the dagliflozin plus group compared with the model group(0.79±0.34 vs.0.23±0.12,P<0.0001).Chapter III Molecular mechanism of dagliflozin inhibition of lipid accumulation and inflammatory response in hepatocytes1.Bioinformatics techniques to screen the pathways of actionA total of 553 differential genes were screened from the GEO database,and GO enrichment analyses were performed to obtain the biological,cellular and molecular functional pathways involved in the differential genes,and PPI network analysis was performed to obtain the key genes as IL-6,IL-1β,CXCL8,CCL2,IL-10,PTGS2,CXCR4,SOCS3,TNFRSF1A,CCL3;key pathways were obtained including cytokine and cytokine receptor pathway,JAK-STAT signaling pathway,TNF signaling pathway,etc.Finally,JAK2/STAT3/SOCS3 signaling pathway was selected as the experimental pathway.2.Cellular experiments verified that dagliflozin could produce therapeutic effects for MAFLD through the screened molecular pathways(1)Both oil red O staining and TG content assay showed that TG content was significantly lower in the dagliflozin group(FFA+Da)compared with the model group(FFA)(0.76±0.32 vs 0.24±0.11,P<0.001);AG-490 increased TG expression compared with the dosing group(0.24±0.11 vs 0.63±0.50,P=0.012).(2)Elisa results showed that dagliflozin reduced the expression of inflammatory factor IL-6 in cell supernatant;AG-490 increased the expression of inflammatory factor IL-6 in cell supernatant compared with the FFA+Da group(0.45±0.03 vs 0.79±0.30,P<0.05).(3)RT-q PCR results showed that dagliflozin increased JAK2 m RNA and STAT3 m RNA expression and decreased SOCS3 m RNA expression;AG-490 decreased JAK2 m RNA and STAT3 m RNA expression and increased SOCS3 m RNA expression(P<0.05),suggesting that the pathway inhibitor AG-490 could reverse the effect of dagliflozin effect.(4)WB results showed that dagliflozin decreased the protein expression level of SOCS3and increased the protein expression levels of JAK2,p-JAK2,STAT3 and p-STAT3,and AG-490 reversed the expression of each protein in dagliflozin plus group(P<0.05).Conclusion:(1)Men between 50 and 65 years of age are more likely to have MAFLD;men are risk factors for obese MAFLD patients;the higher the degree of obesity in patients,the more pronounced the inflammatory response.(2)Genetic polymorphisms were associated with the incidence of MAFLD;PNPLA3rs738409 allele G and APOC3 rs2854116 allele C were both risk factors for developing MAFLD.(3)Dagliflozin can effectively reduce triglyceride content in fatty liver cells.(4)The mechanism by which dagliflozin improves MAFLD may be related to the JAK2/STAT3/SOCS3 signaling pathway.