Study On The Mechanism Of Mangiferin In Improving Insulin Resistance

Object:To study the activity of mangiferin in vitro by HepG2 cells,C2C12 cells,3T3-L1 cells,and from its genes,proteins expression and inflammatory factors secretion level to explore the possible mechanism of mangiferin to improve the occurrence of insulin resistance.It lays a theoretical foundation for further study on mangiferin to improve the occurrence of type 2 diabetes.Methods:Insulin resistance model was established by using 10-6 mol/L insulin to stimulate HepG2 cells,0.5 mM palmitic acid to stimulate C2C12 cells,and 1 ?mol/L palmitic acid to stimulate 3T3-L1 cells.CCK-8 method was used to detect the effect of mangiferin on the activity of three cells in order to screen the appropriate drug concentration.The GOD-POD method to determine the effect of MGF on glucose consumption of three kinds of cells.RT-PCR detection was used to detect the expression of InsR,IRSI,PI3K(p85)and other genes mRNAs in PI3K/AKT signal pathway,and the expression of IKKa,IKK?mRNAs in inflammation pathway in HepG2 cells and C2C12 cells.Western Blot method was used to detect the expression of p-AKT(Thr308),p-GSK-3?(Ser9),AMPKa,IKKa and other related proteins in PI3K/AKT signal pathway and inflammation pathway in HepG2 cells and C2C12 cells.The effect of mangiferin on inflammatory factors such as TNF-?,IL-6,IL-1?in 3T3-L1 cells was detected by Enzyme-Linked ImmunoSorbent Assay.Results:1.The effect of mangiferin on HepG2 insulin-resistant cells:1)CCK-8 assay:Mangiferin inhibited the growth of HepG2 cells at a concentration greater than 125 ?g/mL.2)Determination of glucose content by GOD-POD method:Mangiferin in the high-dose and middle-dose groups can increase the glucose consumption to varying degrees(P<0.01).Although the low-dose group increased,it was not statistically significant.3)RT-PCR detection:MGF high-dose group can up-regulate IRS1,InsR,PDK1,AKT,AMPK,GLUT2,GLTUT4 mRNA expression,and down-regulate PCK1 mRNA expression(P<0.01 or P<0.05).Low-dose group can up-regulate GLUT2 mRNA expression(P<0.05),and other genes are different from the model group but not statistically significant.4)Western Blot assay:MGF high-dose group can up-regulate the expression of p-IRS1(Thr895),p-AKT(Thr308),p-GSK-3?(Ser9),InsR and other proteins,and down-regulate the protein expression levels of PCK1,IKKa and IKK?,and the difference were statistically significant compared with the model group(P<0.01 or P<0.05).The low-dose group could up-regulate the expression of GLUT2 protein and down-regulate the expression level of PCK1 protein(P<0.05).2.The effect of mangiferin on C2C12 insulin-resistant cells:1)CCK-8 assay:The mangiferin inhibited the growth of C2C12 cells at a concentration greater than 62.5 ?g/mL.2)Determination of glucose content by GOD-POD method:Mangiferin in the high-dose group can increase the glucose consumption of cells(P<0.05).Although the middle-dose and low-dose groups increased,there was no statistical significance.3)RT-PCR detection:MGF high-dose group can up-regulate the expression of IRS1,PI3K,PDK1,AKT,PDE3B,FoxO1 and other mRNA,and down-regulate the expression of IKKa and IKK? mRNA(P<0.01 or P<0.05).Low-dose group can increase IRS1,PI3K,AKT,AMPK,GLUT4 mRNA expression,the difference were statistically significant compared with the model group(P<0.01 or P<0.05).4)Western Blot assay:High-dose group of MGF can up-regulate p-PDK1(S241),P-AKT(Thr308),p-AmPK(Thr172),p-GSK-3?(Ser9),PI3K(p85)and InsR protein express-ion levels,down-regulate IKKa,IKK? Protein(P<0.01 or P<0.05).Low-dose group of MGF can increase InsR protein expression(P<0.05).There was no statistically significant difference in the m-TOR protein expression between normal group,model group and administration groups.3.The effect of mangiferin on 3T3-L1 insulin-resistant cells:1)CCK-8 assay:Compared with the control group,the mangiferin inhibited the growth of 3T3-L1 cells at a concentration greater than 62.5 ?g/mL.2)Determination of glucose content by GOD-POD method:The mangiferin in the high-dose and middle-dose groups can increase the glucose consumption of cells to varying degrees(P<0.01).Although the low-dose group increased,it was not statistically significant.3)Enzyme-Linked ImmunoSorbent Assay:Compared with the normal group,the TNF-?,IL-6,IL-1? inflammatory factors in the model group were significantly increased(P<0.01).Compared with the model group,the secretion of TNF-?,IL-6 and IL-1? inflammatory facto-rs in the high-dose group and the middle-dose group were significantly reduced(P<0.01),and the TNF-? inflammatory factor in the low-dose group was significantly reduced(P<0.05).Although the secretion of IL-6 and IL-1? in the low-dose group decreased,it was not statistically significant.Conclusion:1.In the HepG2 insulin resistance cell model,MGF can increase the level of cellular glucose consumption,and up-regulates the expression of key genes in PI3K/AKT signaling pathway,such as IRS1,InsR,AKT,GLUT2,AMPK,etc.And up-regulates the expression levels of InsR,GLUT2,AMPKa,and phosphorylated IRSI,AKT,GSK-3? and other proteins.Improves insulin resistance by affecting insulin dependence(AKT pathway).2.In the model cell of C2C12 insulin resistance,MGF can up-regulate the expression of key genes in PI3K/AKT signaling pathway such as PI3K,AKT,GSK-3? AMPK,etc.,and up-regulate the expression of InsR,PI3K,phosphorylated AKT,phosphorylated GSK-3?,phosphorylated AMPKa and other proteins to play a role in improving insulin resistance,and increase the glucose consumption level of cells.3.In the 3T3-L1 insulin resistance cell model,MGF can increase the glucose consu-mption level:of cells,and play a role in improving insulin resistance by inhibiting the secretion of inflammatory factors.