The Effects And Mechanisms Of Zhimu-Huangbai Herb Pair And Its Main Effective Components Mangiferin On Glucolipid Metabolism In Insulin Resistance HepG2 Cells

Objective:To establish the insulin resistance model of HepG2 cells,and observe the effect of Zhimu-Huangbai herb pair and its effective components mangiferin on insulin resistance with glucose and lipid metabolism disorder.Then to further explore its intervention effect on APN,AdipoR2,APPL1,AMPK in APN transduction AMPK signaling pathway on the upstream of AMPK signaling pathway,and IRS-1,AKT,GLUT4 in insulin signal transduction pathway on the downstream of AMPK signaling pathway,to illuminate the mechanism of the Zhimu-Huangbai herb pair and mangiferin on improving insulin resistance.It will provide theoretical foundation and clinical development value in metabolic diseases such as type 2 diabetes mellitus.Methods:1.The HepG2 cells were treated with different concentrations of oleic acid,palmitic acid and their mixture for 24 h to induce insulin resistance,Cell viability and glucose consumption were measured.The cell status was observed by oil red 0 staining to test whether the model was successfully induced.2.The antiproliferation and toxicity efficiency of Zhimu-Huangbai herb pair and mangiferin on HepG2 cell was assessed with MTT assasy to screen the non-toxic concentration rangeand.The HepG2 cells were treated with 2mM oleic acid and 1mM palmitic acid for 24 h to induce an insulin resistance model and were divided into model group,positive control group(metformin),Zhimu-Huangbai herb pair treatment group(384,192,96 mg·L-1),mangiferin treatment group(500,250,125 ?M)and solvent group(0.2%DMS0),and another normal control group.After 24 h of administration in each group,the supernatant and cell samples were collected to detect the change of glucose consumption,triglyceride,cholesterol,to observe their hypoglycemic and lipid-lowering effect.3.After treated with Zhimu-Huangbai herb pair and mangiferin in HepG2 cells with insulin resistance for 24 h,the cell samples were collected to extract total RNA,in order to measure the mRNA expression of APPL1,AdipoR2,APN,AMPK,IRS1,AKT,GLUT4 factor by Quantitative Real-time PCR.4.After 24 hours of Zhimu-Huangbai herb pair and mangiferin treatment in HepG2 cells with insulin resistance,the cell samples were collected to extract total protein for detecting the protein expression of p-AMPK and AMPK factor by Western blotting.Results:1.Compared with the normal group,oleic acid,palmitic acid and their mixture at different concentrations can reduce the glucose consumption of HepG2 cells.Among them,the mixture of oleic acid and palmitic acid has a better effect,especially the combination of 1mM palmitic acid and 2mM oleic acid has the best induction effect(P<0.01),and did not affect the cell viability.While palmitic acid group emerges obvious cytotoxicity.After oil red 0 staining,a large number of oil droplets in HepG2-IR cells which induced by 1mM palmitic acid and 2mM oleic acid were observed and stained red under the microscope.The result show that HepG2 insulin resistance cells model was successfully established.2.After treated with different concentrations of Zhimu-Huangbai herb pair and mangiferin in HepG2 cells with insulin resistance for 24 h,Zhimu-Huangbai herb pair and mangiferin can significantly increased glucose consumption(P<0.01),and decreased triglyceride and cholesterol content(P<0.05)compared with the model group;the improving effect of Zhimu-Huangbai herb pair was the most obvious in 192 mg·L-1,and the effect on hypoglycemic and lipid-lowering was equivalent to metformin;the effect of mangiferin on lipid-lowering was the most obvious in 125?M,and its triglyceride-lowering effect was better than metformin,while the cholesterol-lowering effect was equivalent to metformin.3.After treated with different concentrations of Zhimu-Huangbai herb pair and mangiferin for 24 h in insulin-resistant HepG2 cells,compared with the model group,both the 192 mg·L-1 of Zhimu-Huangbai herb pair group and the 125 ?M of mangiferin group could significantly upregulate the mRNA expression of APN,AdipoR2,APPL1 and AMPK in APN transduction AMPK signaling pathway on the upstream of AMPK signaling pathway(P<0.05),and significantly upregulate the mRNA expression of IRS-1,AKT,and GLUT4 in insulin signal transduction pathway on the downstream of AMPK signaling pathway(P<0.05).4.After treated with different concentrations of Zhimu-Huangbai herb pair and mangiferin in HepG2 cells with insulin resistance for 24 h,compared with the model group,administration of Zhimu-Huangbai herb pair and mangiferin at each dose group could significantly increasing the phosphorylation of AMPK(P<0.05),among which had a better effect on the 192 mg·L-1 of Zhimu-Huangbai herb pair group and the 125 ?M of mangiferin group.Conclusion:This study demonstrated that Zhimu-Huangbai herb pair and its effective components mangiferin can improve the disorder of glucose and lipid metabolism in HepG2 cells with insulin resistance by up-regulating the mRNA expression of APN,AdipoR2 and APPL1 in APN transduction AMPK signaling pathway on the upstream of AMPK signaling pathway,thereby activating AMPK signaling pathway,increasing the phosphorylation of AMPK and promoting the expression of AMPK protein,thus further up-regulating the mRNA expression of IRS1,AKT and GLUT4 in insulin signal transduction pathway on the downstream of AMPK signaling pathway,promoting glucose and lipid metabolism,reducing the level of glucose and lipid in vivo and ultimately improving insulin resistance.