Effect Of Jinlida On Insulin Resistance And Oxidative Stress In HepG2 Cells

Insulin resistance plays a central role in the development of type 2 diabetes mellitus(T2DM)and other metabolic diseases. Oxidative stress is proposed to be a primary factor in the etiology of insulin resistance. Jinlida(JLD), composed of seventeen Chinese medical herbs, is a widely used Chinese herbal prescription for treating T2 DM. JLD plays a role in improving the symptoms of diabetes, lowering blood glucose, protecting of beta cell of islet, increasing insulin secretion, and reducing insulin resistance. However, to our knowledge, the mechanism underlying this effect remains unclear.In the present study, we estabolished a model of insulin resistance in Hep G2 cells by exploring to palmitic acid for 24 hours. After 48-hour treatment of JLD, we measured the oxidative stress indicators, detected the gene expression and protein of insulin signaling pathway and stress-sensitive signaling pathways. We aimed to elucidate the molecular mechanism of JLD on insulin resistance, thus to provide evidence on clinical practice.Objective: To explore the effect of JLD on insulin resistance and oxidative stress in Hep G2 cells exposed to palmitic acid(PA) through insulin signaling pathway and to elucidate the molecular mechanism of JLD on ameliorating insulin resistance.Methods: Insulin resistance in Hep G2 cells was induced by culturing with medium containing 0.25 mmol/L PA. Hep G2 cells were randomly divided into two groups, control-medium group and PA-medium group, and were cultrued for 24 hours, respectively, the concentration of glucose in the medium was measured by glucose oxidase method, glucose uptake rate was calculated. After successful modeling, the PA-medium group were randomly divided into 5 groups: PA-control-medium group(PA)(0.25 mmol/L), metformin-medium group(Met)(0.2 mg/m L), low, middle and high dose of JLD-medium group(JLD-L, JLD-M, JLD-H)(0.75 mg/m L, 1.5 mg/m L, 3.0 mg/m L). The normal-control-medium group(Con) was established at the same time, and metformin was the positive control drug. After 48 hours intervention, the cells were collected respectively, liver peroxidation(LPO), reduced glutathione(GSH), antioxidant enzymes including total superoxide dismutase(T-SOD), glutathione peroxidase(GSH-Px) and catalase(CAT) were measured; reactive oxygen radicals(ROS) was detected by dihydroethidium(DHE) staining. The expression of the major signaling pathway molecules that regulate glucose uptake were examined by real time PCR(RT-PCR), including, insulin receptor(INSR), insulin receptor substrate-1(IRS-1), phosphoinositide-3-kinase(PI3K), protein kinase beta(AKT), and glucose transporter 2(GLUT2). The expression of AKT and phospho-AKT(p-AKT) proteins as well as its downstream PI3 K, IRS, phospho-IRS(p-IRS) and c-Jun N-terminal kinase(JNK), phospho-JNK(p-JNK), p38 mitogen-activated protein kinases(p38MAPK), phospho-p38MAPK(p-p38MAPK) were determined by western blot.Results:1 In vitro model of insulin resistance in Hep G2 cellsHep G2 cells were cultrued for 24 hours, respectively, compared with Con group, there were increased level of glucose concentration and decreased insulin sensitivity in the PA group, the difference was statistically significant(P<0.05), shows that the in vitro model of insulin resistance in Hep G2 cells induced by palmitic acid were successfully established.2 The effect of drugs intervention on proliferation in Hep G2 cells by MTT assayCell inhibitory rate was obviously increased in PA group as compared with control group, and the cell proliferation rate was significantly decreased(P<0.05) in Hep G2 cells culcured for both 24 hours and 48 hours. Compared with PA group, Hep G2 cells culcured in Met、JLD-L、JLD-M、JLD-H group showed higher cell proliferation rate(P<0.05),but failed to recover to the Con group level.3 Insulin sensitivity in Hep G2 cells(1) The gene expression of key mediators in insulin signal pathwayCompared with Con group, the PA group exhibited significantly decreased m RNA expression of INSR, IRS-1, PI3 K, AKT and GLUT2. The differences were statistically significant(P<0.05). Compared with PA group, JLD-L、JLD-M treatment resulted in a modest up-regulation of IRS-1, AKT and GLUT2(P<0.05), but failed to show any change in INSR, PI3 K gene expression(P>0.05). While metformin and high dose of Jinlida treatment could up-regulation the m RNA expression of INSR, IRS-1, AKT, PI3 K and GLUT2, but failed to recover to the Con group level.(2) The protein expression of key mediators in insulin signal pathwayCompared with Con group, the ratio of p-IRS-1/IRS-1 was significantly increased in PA group, while the ratio of p-Akt/t-Akt, PI3K/β-actin was significantly decreased(P<0.05). Compared with PA group, the ratio of p-IRS-1/IRS-1 was significantly decreased in Met、JLD-M、JLD-H groups, while the ratio of p-Akt/t-Akt, PI3K/β-actin was significantly increased(P<0.05). Although treatment with JLD-L could decrease the rate of p-IRS-1/IRS-1, but failed to increase the rate of p-AKT/AKT, PI3K/β-actin.4 Oxidative stress of Hep G2 cells.Compared with Con group, the ROS generation of Hep G2 cells and LPO were significantly increased in PA group, while the activities of anti-oxidative enzymes such as T-SOD, CAT, GSH-Px and the content of GSH were significantly decreased(P<0.05). Treatment with JLD and metformin significantly reduced ROS and LPO generation, while increased T-SOD, GSH-Px, CAT activity and GSH level when compared with PA group(P<0.05). While there were no significant differences between the intervention groups(P>0.05).5 JNK and p38 MAPK signaling pathways.Compared with Con group, the ratios of p-JNK/JNK and p-p38MAPK/p38 MAPK were significantly increased in PA group(P<0.05). Compared with PA group, the ratios of p-JNK/JNK and p-p38MAPK/p38 MAPK were significantly decreased in metformin and JLD groups(P<0.05). It is worth mentioning that treatment of metformin could restore the above ratio to normal levels(P>0.05).Conclusions:1 Jinlida treatment can play a role in the insulin signaling pathway, and reduce insulin resistance in Hep G2 cells.2 JLD treatment can reduce oxidative stress in Hep G2 cells, decrease ROS and LPO generation, while increase anti-oxidatant such as SOD, GSH-Px, CAT activity and GSH level.3 Jinlida treatment can inhibit JNK and p38 MAPK signaling pathway, restore Hep G2 cells insulin signal transduction.In summary, Jinlida treatment improved insulin sensitivity in Hep G2 cells. The effect may be attributed to the antioxidant properties of JLD.