Study The Protective Effect And Mechanism Of Scutellaria Baicalensis Stem-leaf Total Flavonoid Against The Human Umbilical Vein Endothelial Cells Oxidative Injury Induced By High Concentration Of Glucose

Background:The incidence of diabetes mellitus(DM) within global scope has increased fastly as the improvement of living standard and the change of dietetic structure. Atherosclerosis(AS) is a serious and common complication of DM. Vascular endothelial cell injury is an initiating agent of AS. To heighten blood glucose is the main clinical characteristic of DM. The chief action of high glucose is to oxidative damage vascular endothelial cells through facilitating oxidative stress state of vascular endothelial cells. Oxidative stress is defined as the accumulation of reactive oxygen species(ROS) in vivo inducing the organism’s damage because of excessive ROS in vivo surpassing the organism’s capability of cleaning. There are many kinds of enzymes in vivo which participate in the formation of ROS, and nicotinamide adenine dinucleotide phosphate oxidase(NADPH oxidase) is now considered as the main enzyme of the endovascular ROS generation. Vascular endothelial cells mainly express NADPH oxidase4(NOX4) and NADPH oxidase2(NOX2) in NOX family, and the expression of NOX4is about20times higher than that of NOX2.Scutellaria baicalensis stem-leaf total flavonoid(SSTF) which main ingredients are flavonoids compounds is the extract of scutellaria baicalensis stem-leaf. The pharmacology study has already showed that SSTF has extensive functions in cardiovascular system, such as antioxidant, reduce blood pressure, adjust blood-lipid, improve cardiovascular blood etc. Our studies have showed that SSTF had protection from Hydrogen peroxide and hypertriglyceridemia-serum revulsive HUVECs oxidative damage. So whether SSTF has protection on the vascular endothelial cells oxidative injury induced by HTG and its mechanism concerns inhibiting the expression of NOX? This is another research task of the project, its research results which can provide experiment basis are conducive to finding new targets of anti-AS drug.Objectives:1. To establish oxidative damage model of human umbilical vein endothelial cells (HUVECs) by using high concentration of glucose to injure HUVEC.2. To observe the protective effects of scutellaria baicalensis stem-leaf total flavonoid (SSTF) on HUVEC oxidative injury induced by high concentration of glucose, and to discuss its possible mechanisms at the molecular level.Methods:1. The foundation of oxidative injury model of HUVEC:HUVEC were cultured in vitro and were treated with different concentrations of were treated with different concentrations of glucose (5.5mmol/L,10mmol/L,20mmol/L,30mmol/L,40mmol/L,50mmol/L) for different time course (24hours,48hours,72hours,96hours). Then cell vitality (OD value) was measured by tetrazolo colorimetric method (MTT), superoxide dismutase (SOD) activity of cell was examined by spectrophotometric assay,malondialdehyde (MDA) content was determined by thibabituric acid (TBA) method and intracellular ROS level was detected by flow cytometry (FCM). And we obtained an optimum concentration and most appropriate time for endothelial cells oxidative indury induced by high concentration of glucose.2. The protective effects and mechanism of SSTF on HUVEC oxidative injury induced by high concentration of glucose:Different concentrations of SSTF (100,200,400mg/L) and positive control drug Vitc (20mg/L) were cultured in vitro together with HUVEC oxidative injury induced by high concentration of glucose for48hours. We could obsere the protective effects of SSTF through measuring cell vitality, SOD activity, MDA content and intracellular ROS level, then compare with which of VitC.We could investigate the changes of HUVEC NOX4expression after treated with SSTF and VitC through detecting mRNA expression by RT-PCR. Results:1. The foundation of oxidative injury model of HUVEC:1.1Results from the MTT assays showed that compared with control (5.5mmol/L) group, the the cell vitality (OD value) decreased (P<0.05) of HUVEC oxidative injury induced by glucose (10mmol/L) group for72h, glucose (20mmol/L) group for48h and glucose (30,40mmol/L) group for24h, decreased significantly (P<0.01) by glucose (10mmol/L) group for over96h, glucose (20mmol/L) group for over72h, glucose (30,40mmol/L) guoup for over48h and glucose (50mmol/L) guoup for over24h in a concentration-dependent manner.1.2Results from the activity of SOD and the content of MDA showed that compared with control (5.5mmol/L) group, the activity of SOD decreased (P<0.05) of HUVEC oxidative injury induced by glucose (20mmol/L) group for48h and glucose (30mmol/L) group for24h, and the content of MDA decreased (P<0.05) of HUVEC oxidative injury induced by glucose (20mmol/L) group for48h and72h, glucose (30mmol/L) group for24h; the activity of SOD decreased significantly and the content of MDA increased significantly (P<0.01) by glucose (30mmol/L) group for over48h, glucose (40,50mmol/L) group for over24h, in a concentration-dependent manner.1.3Results from the ROS level showed that intracellular ROS had a baseline production in cultured HUVEC and were increased significantly after high concentration of glucose treatment. Compared with control (5.5mmol/L) group, the ROS level decreased (P<0.05or P<0.01) of HUVEC oxidative injury induced by glucose (10,20,30,40,50mmol/L) group for48h; the ROS level increased significantly (P<0.01) of HUVEC oxidative injury induced by glucose (30mmol/L) group for24h,48h,72h,96h, in a concentration-dependent manner.2. The protective effects and mechanisms of SSTF on HUVEC oxidative injury induced by high concentration of glucose.2.1Results from the MTT assays showed that compared with the high concentration of glucose oxidative injury group, the cell vitality (OD value) increased in every SSTF group and VitC group, increased significantly (P<0.01) in SSTF (400mg/L) group and VitC group.2.2Results from the activity of SOD and the content of MDA showed that compared with the high concentration of glucose oxidative injury group, the activity of SOD increased (P<0.05or P<0.01) and the content of MDA decreased (P<0.05or P<0.01) in every SSTF group and VitC group.2.3Results from the ROS level showed that compared with the high concentration of glucose oxidative injury group, the intracellular ROS level decreased significantly (P<0.01) in every SSTF group and VitC group.2.4Results from the RT-PCR showed that compared with the high concentration of glucose oxidative injury group, NOX4mRNA expression decreased (P<0.05or P<0.01) in every SSTF group and VitC group.Conclusion:1. High concentration of glucose can induce human umbilical vein endothelial cells oxidative injury, and the oxidative injury degree correted with the concentrations of high concentration of glucose. The study choosed that HUVEC oxidative injury induced by glucose (30mmol/L) group for48hours was the optimum condition.2. SSTF showed obvious protective effects against the vascular endothelial cells oxidative injury induced by high concentration of glucose, which can promote vascular endothelial cells antioxidant capacity, inhibit lipid peroxidation effect, reduce intracellular ROS generation. The probable mechanisms of SSTF protective effects on HUVEC oxidative injury induced by high concentration of glucose may be associated with its down-regulating NOX4mRNA and protein expression, through which can reduce intracellular ROS generation.