Effect Of Oxymatrine On Insulin Resistance And Lipid Metabolism In HepG2 Cells Exposed To Palmitic Acid

Insulin resistance(IR)is a common pathophysiological state associated with obesity,type 2 diabetes mellitus(T2DM),hyperlipidemia,non-alcoholic fatty liver disease(NFALD)and other metabolic diseases.Oxymatrine,a monomer component extracted from Sophora flavescens Ait,is reported to have many pharmacological effects,such as anti-inflammation,anti-tumor,anti-fibrosis and immune regulation.Recent studies have reported that oxymatrine combined with metformin was an effective and safe remedy for the treatment of NAFLD,which can improve liver function,dramatically attenuate hepatic lipid accumulation and enhance insulin sensitivity in patients with NAFLD.However,it remains unclear about the underlying mechanism of these effects.In this study,we established a model of insulin resistance in HepG2 cells which were exposed to palmitic acid for 24 hours.After 48-hour treatment of oxymatrine,we investigate the effect of oxymatrine on IR and lipid metabolism.An additional goal was to elucidate the molecular mechanism of oxymatrine in improving insulin resistance and fat metabolism,thus to provide evidence on clinical practice.Objective: To explore the effect of oxymatrine on insulin resistance and fat accumulation in HepG2 cells exposed to palmitic acid(Pa)and to elucidate the underlying molecular mechanisms through the evaluation of insulin signaling pathway,hepatic lipid metabolism and oxygen stress.Methods: HepG2 cells were cultured in medium containing 0.25 mmol/L palmitic acid to induce insulin resistance.HepG2 cells were randomly divided into two groups,normal-medium group and palmitic acid-medium group,and cultured for 48 hours respectively.The concentration of glucose in the medium was measured by glucose oxidase method.Glucose consumption rate was calculated to identify whether the model is established successfully.After successful modeling,the palmitic acid-medium group were randomly subdivided into 5 groups: Pa group(Pa)(0.25 mmol/L),metformin-medium group(Met)(0.2 mg/mL),low,middle and high dose of oxymatrine-medium group(Omt-L,Omt-M,Omt-H)(0.04 mg/mL,0.08 mg/mL,0.16 mg/mL).The normal-medium group(N)was established at the same time.After 48 hours of drug intervention,the cells were collected respectively.Triglyceride content and the related markers of oxygen stress were measured by kits,including the activity of glutathione peroxidase(GSH-Px)and total superoxide dismutase(T-SOD)and the content of malondialdehyde(MDA).Reactive oxygen radicals(ROS)were detected by 2,7-dichlorofuorescin diacetate(DCFH-DA)staining.The expressions of the major molecules that regulate insulin signal transduction(including insulin receptor(INSR),insulin receptor substrate-1(IRS-1),protein kinase beta(Akt),and glucose transporter 4(GLUT4))and lipid metabolism pathway(including SREBP-1c?FAS,ACC,SCD-1,CPT-1 and CD36)were examined by real time PCR.The expression of GLUT4,CD36,FAS,ACC,SCD-1,IRS,phospho-IRS(p-IRS),Akt and phospho-Akt(p-Akt)were determined by western blot.Results:1 Model of insulin resistance in HepG2 cells in vitroAfter 24 hours intervention with palmitic acid,there were increased level of glucose concentration and decreased insulin sensitivity in Pa group compared with N group,and the difference was statistically significant(P<0.05).It showed that the in vitro model of IR in HepG2 cells induced by Palmitic acid was successfully established.2 The effect of drug intervention on proliferation in HepG2 cells observed by MTT assayCompared with N group,the inhibitory rate of cell proliferation was obviously decreased in HepG2 cells exposed to palmatic acid for 24 hours(P<0.05).Compared with Pa group,HepG2 cells cultured in Met,Omt-L,Omt-M,Omt-H group showed lower cell inhibitory rate(P<0.05),but failed to recover to normal level.However,the inhibitory rate in HepG2 cells cultured in Omt-H2 group were increased dramatically than that in Pa group.Then the drug level of Omt-L,Omt-M,and Omt-H group were chosen as the intervention concentration.3 The effect of drug intervention on insulin sensitivity in HepG2 cells3.1 Gene expression of key markers in insulin signal pathwayCompared with N group,the Pa group exhibited obviously decreased mRNA level of INSR,IRS-1,Akt and GLUT4.The differences were statistically significant(P<0.05).Compared with Pa group,Met,Omt-M and Omt-H treatment resulted in a significant up-regulation of GLUT4 which achieved the normal level.The mRNA levels of IRS-1,Akt and INSR increased significantly in Met,Omt-M,Omt-H group(P<0.05),but failed to recover to the normal level(P<0.05).Low-dose oxymatrine treatment could increase the mRNA expression of INSR,Akt and GLUT4,and the differences were statistically significant.Increased mRNA expression of IRS-1 in Omt-L were observed,although the changes were not statistically significant(P>0.05).3.2 The protein expression of key molecules in insulin signal PathwayCompared with N group,the ratio of p-IRS-1/IRS-1 in Pa group increased significantly,while the ratio of p-Akt/t-Akt and GLUT4/?-actin decreased significantly(P<0.05).Compared with Pa group,the ratio of p-IRS-1/IRS-1 declined significantly after metformin and oxymatrine administration,while the ratio of p-Akt/t-Akt,GLUT4/?-actin increased obviously(P<0.05).However,the ratio of p-Akt/Akt in drugs intervention groups failed to recover to the normal level.4 The effect of drug intervention on lipid metabolism in HepG2 cells4.1 The effect of drugs administration on lipid accumulationRed Oil O staining and measurement of TG showed that there was lipid accumulation in HepG2 cells exposed to palmitic acid for 24 hours,while the lipid accumulation ameliorated obviously after metformin and oxymatrine administration.4.2 The effect of palmitic acid on the expression of key elements involved in lipid metabolismCompared with N group,the mRNA level of SREBP-1c,FAS,ACC,SCD-1,and CPT-1 in Pa group decreased significantly,while CD36 increased significantly.Compared with N group,the protein levels of SREBP-1c,FAS,ACC,and SCD-1 in Pa group decreased significantly,while CD36 increased significantly.All the differences were statistically significant(P<0.05).4.3 The effect of drugs administration on the expression of key elements involved in lipid transportation and oxydation.Compared with Pa group,the mRNA level and protein expression of CD36 decreased significantly after metformin and different concentrations of oxymatrine administration,while the mRNA level of CPT-1 increased significantly.All the differences were statistically significant(P<0.05).5 The effect of drugs intervention on oxygen stress in HepG2 cells.5.1 Compared with N group,the ROS generation of HepG2 cells were significantly increased in Pa group.After different concentrations of oxymatrine and metformin administration,the ROS generation decreased dramatically.5.2 Compared with N group,the activities of anti-oxidative enzymes such as GSH-Px and T-SOD were significantly decreased in Pa group,while the content of MDA increased.After the treatment with oxymatrine and metformin,the generation of MDA reduced significantly,while the activities of GSH-Px and T-SOD increased when compared with Pa group.All the differences were statistically significant(P<0.05).While there were no significant differences between the intervention groups(P>0.05).Conclusions:1 Oxymatrine could play a role in insulin signaling pathway and ameliorate insulin resistance in HepG2 cells exposed to high-fat intervention.2 Oxymatrine could improve lipid accumulation in Pa-treated HepG2 cells through reducing the long-chain fatty acid uptake and increasing the ?-oxydation.3 Oxymatrine treatment could improve the oxidative stress,reduce ROS production and MDA content,and recover the activity of GSH-Px and SOD in HepG2 cells induced by high-fat treatment,thus improving glucose and lipid metabolism in HepG2 cells.