Isoquercitrin Ameliorates Insulin Resistance Via Regulating MiR-29a And PI3K/AKT/Gsk3β/GLUT4 Pathway In HepG2 Cells

Objective:At present,diabetes has become a global health problem,and its prevalence has increased year by year.Type 2 diabetes（T2DM）accounts for more than 90%,mainly caused by insulin resistance（IR）and insufficient insulin secretion,while insulin resistance is dominant in type 2 diabetes.Insulin resistance is closely associated with type 2 diabetes,and it is consistent throughout its occurrence and development.Therefore,insulin resistance has become the key to the treatment of type 2 diabetes and has become a hot spot in diabetes research.However,the mechanism of insulin resistance is still unclear.miRNA is a non-coding small RNA consisting of 18 to 23 nucleotides which can targeted regulate one or more mRNAs,and inhibit or degrade the mRNA of the target gene through translational level to regulate gene expression.In recent years,a large number of reseraches have shown that miRNAs play an important role in the development and progression of insulin resistance and diabetes.Among them,it has been reported that miRNA-29a is overexpressed in diabetes and causes diabetes or blood glucose increased in fasting mice.Isoquercitrin is a biologically active monomer widely found in plants and Chinese medicinal materials,and produces antioxidant,anti-inflammatory,anti-cancer and decreases blood glucose.Our previous studies have shown that the flavonoids of Bidens bipinnata L.Flavonoids（BBLF）regulated the PI3K/AKT/GSK3β/GLUT4 signaling pathway and improved insulin resistance.The role and mechanism of isoquercitrin in insulin resistance is unclear.This study will clarify whether isoquercitrin has an effect of ameliorating insulin resistance and the underlying mechanisms at the cellular and molecular levels.Methods:Firstly,we constructed insulin resistance HepG2 cells model by treating HepG2 cells with 1×10-7 mol/L insulin and 125 μmol/L palmitic acid for 24 h according to the previous study.Then,insulin resistance HepG2 cells were identified by glucose oxidase method;Quantitative Real Time-PCR was used to measure the expression of miRNA-29a in insulin-resistant HepG2 cells.Then bioinformatics analysis was used to predict the target gene of miRNA-29a,and the candidate target gene PIK3R1 related to insulin signaling pathway was selected for further experiments.Dual luciferase assay was used to verify whether miRNA-29a can targeted inhibit the activity of PIK3R1 and then verify whether miRNA-29a can inhibit mRNA expression and protein expression of PIK3R1 at the cellular level;then CCK8 cell proliferation assay and glucose oxidase method were used to determine the optimal concentration of isoquercitrin.Finally,miRNA-29a mimic was transiently transfectedand into insulin-resistant HepG2 cells and isoquercitrin was used to treat insulin-resistant HepG2 cells at the same time.The effects and mechanism of isoquercitrin on insulin-resistant HepG2 cells were studied by Western blot and Quantitative Real Time-PCR.Results:The expression of miRNA-29a was high in insulin-resistant HepG2 cells;the optimal concentration of isoquercitrin was 20μm;PIK3R1 was the target gene of miRNA-29a;isoquercitrin increased the glucose consumption of insulin-resistant HepG2 cells;isoquercitrin repressed the expression of miRNA-29a in insulin-resistant HepG2 cells and increased the expression of various genes and proteins in PI3K/AKT/GSK3β/GLUT4.Conclusions:Isoquercitrin significantly inhibits miRNA-29a and increases mRNA expression and protein expression of PIK3R1,p-AKT,p-GSK3β and GLUT4 in insulin-resistant HepG2 cells;isoquercitrin ameliorates insulin resistance by suppressing miRNA-29 expression to regulate PI3K/AKTGSK3β/GLUT4 pathway.