Protective Role Of GKT137831 Against Atorvastatin-induced Impaired Glucose Uptake By Suppressing ROS Level

Background:Dysis-China study indicated that the compliance rate of low-density lipoprotein(LDL-C)in dysis-China patients with dyslipidemia was only 61.5%,especially in the extremely high-risk and high-risk patients,the compliance rate of LDL-C was only 39.7%and 54.8%,respectively.According to the results of epidemiological investigation,it is predicted that the increase of serum cholesterol level will lead to an increase of about 9.2 million cardiovascular events in China from 2010 to 2030[1].Dyslipidemia characterized by elevated LDL-C or total cholesterol(TC)is a significant risk factor for Atherosclerotic cardiovascular disease(ASCVD).Lower levels of LDL-C can significantly reduce the risk of ASCVD morbidity and mortality[2].Statins are specific competition inhibitors of hydroxy methylglutaryl coenzyme A reductase inhibitor(HMG-CoA reductase).The HMG-CoA reductase is a microsomal enzyme that catalyzes the conversion of HMG-CoA to mevalonic acid.This catalytic reaction is a rate-limiting step in cholesterol metabolism,which can directly reduce the synthesis of cholesterol.In the 1980s?statins began to be used for clinical treatment.A number of studies have reported that statins can effectively reduce the level of LDL-C[3-4],which is currently the best drug for reducing the incidence of adverse cardiovascular events.However,recent clinical data suggest that prolonged statin use is associated with an increased risk of new diabetes mellitus(NODM).The incidence of Type 2 Diabetes Mellitus Type 2(T2DM)increased by 25%,after 20mg/day after 1.9 years with rosuvastatin[5].Subsequent analyses of RCTs and observational studies found similar results,but with varying degrees of increased NODM risk.Therefore,the Food and Drug Administration(FDA)has updated the safety instructions for the use of statins,indicating that such drugs can increase the level of hemoglobin(HbA1C)and fasting blood glucose[6].Currently,there is no effective intervention for this side effct of statins.The study on the possible mechanism of NODM induced by statins has important clinical significance for the intervention of this opposite effect.NADPH Oxidase(Nicotnamide Adenine Dinucleotidephosphate Oxidase,NOXs)is one of the me1bers of the family of superoxide and hydrogen peroxide enzyme[7]in skeletal muscle,there are three kinds of NADPH Oxidase,respectively Noxl,Nox2 and Nox4[40-44],and Nox4 in mice show high expression in skeletal muscle C2C12 cells among Nox1,Nox2 and Nox4[41-43].Interestingly,the N-terminal of Nox4 is connected to the mitochondrial intima[45],so the change of Nox4 expression may affect mitochondrial functionGKT137831 is the first-generation Noxl Nox4 inhibitor developed by Genkyotex company.GKT137831 can inhibit the fibrosis and apoptosis of liver cell[8],inhibit the NADPH oxidase activity from the renal cortex of diabetic mice,inhibit the production of superoxide and hydrogen peroxide,but does not affect the expression of Noxl or Nox4 protein[7].Currently,GKT137831 protection in the kidney is in three different kinds of animal models of diabetic nephropathy are confirmed.GKT137831 can significantly reduce the ROS levels in the rat model of diabetic nephropathy,diabetes renal vascular lesions[9-11],because the drug with good antioxidant function,has now entered the second phase of clinical trial,wich are used in the study of prevention and control of diabetes complications.However,whether GKT137831 can exert its antioxidant functions in skeletal muscle,which reduce the level of ROS in the skeletal muscle,protect the sugar metabolism of skeletal muscle,it is unclear[7].Therefore,this study aims to explore the role of GKT137831 in the intervention of atorvastatin in skeletal muscle cells,and to provide new ideas for the clinical application of GKT137831.Objective:The mechanism of statin-induced NODM is still unclear,and there is no effective intervention at present.This study aims to explore the effect of GKT137831 on atorvastatin-induced NODM,and to provide new ideas for clinical intervention of statin adverse reactions.Methods:Skeletal muscle C2C12 was used to investigate the effect of GKT137831 on atorvastatin-induced NODM.(1)in vitro,culturing of skeletal muscle C2C12 cells,,then C2C12 will be induced differentiation and maturation;(2)the effect of atorvastatin on C2C12 cells after intervention with CCK8 kit was tested.(3)flow cytometry was used to detect the glucose uptake and ROS production of skeletal muscle cells under the treatment of atorvastatin;(4)the effect of atorvastatin on the expression of Nox4 protein in skeletal muscle cells by Western blot(5)the effect of atorvastatin on the mitochondrial biosynthesis of skeletal muscle cells by real-time fluorescence quantitative PCR(RT-PCR);(6)the same method was used to observe the effect of GKT137831 on skeletal muscle glucose uptake,ROS level and mitochondrial biosynthesis function.Results:(1)atorvastatin interfered in skeletal muscle C2C12 cells,but had no effect on the cell viability.(2)atorvastatin can reduce the proportion of cells in C2C12 cells that absorb sugar and increase the level of ROS.(3)atorvastatin can increase the expression of Nox4 protein in C2C12 cells.(4)atorvastatin can reduce the function of mitochondrial biosynthesis;(5)GKT137831 can partially alleviate the decline in the proportion of C2C12 cells with glucose uptake caused by atorvastatin,reduce the ROS level,and partially alleviate the impaired mitochondrial function.Conclusion:(1)the decrease of glucose uptake in C2C12 cells induced by atorvastatin is not related to cell viability,but related to the increase of ROS level;(2)atorvastatin can reduce the biosynthesis ability of mitochondria;(3)GKT137831 can alleviate the reduced glucose uptake capacity of skeletal muscle cells caused by atorvastatin,reduce ROS level and increase the biosynthesis capacity of mitochondria.