Studies On The Effects And Mechanisms Of Neferine Using As A Protective Agent Against The Damages Of Vascular Endothelial Cell And Sympathetic Ganglion In Diabetes

Introduction:Diabetes is one of four desease threatened to human health. The incidence of diabetic disease in China is highest in the world. Diabetes is seriously harm to human health and the social development. Prevention and herapy to diabetes have been thought as an important task for medical researchers. The main harm for diabetic is the chronic complicantions. The complicantions of sympathetic nerve and cardiovascular injury induced by diabetes are the most popular and seriously threatening factors to the living quality of diabetic patients. The complicantions of diabetic also are the major causes of disablity and death. Vascular endothelial cells (VEC) as the critical organ are possessed with vascular regulation and endocrine function. The postganglionic fibers of superior cervical ganglion (SCG) are distributed to cardiac plexus and regulate the activity of heart and vessel. It is reported that the seeds of Nelumbo micifera Gaertn have hypoglycemic activity. Neferine is a bisbenzylisoquinline alkaloid isolated from a Chinese medicinal herb (Nelumbo micifera Gaertn). The aim of this study was to investigate effects of neferine on the diabetes and its associated complications of sympathetic injury and the dysfunctional vascular endothelium, then provide experimental data for prevention and herapy to diabetes.Objective:1. The aim of the studies was to investigate the protective effect and its'mechanism of neferine against damage of VEC induced by chronic high glucose (HG) by ovserving the change of gene expression in VEC induced by HG and the effect of nefferin on the change of gene expression.2. Almost all human genes known to be associated with disease have orthologues in the rat genome. The studies observed the effects of neferine on the blood glucose, blood lipid, blood insuline, blood pressure of model rat for 2 type diabeteic and the expressions of CCL5 and CCR5 in SCG of diabetic rats. The mechanisms of neferine acting to the pathogenesy of diabetes and its complications were investigated.3. The change of relavant protein in SCG of diabetic rats was investigated by differential-disply proteomics. Drug target to reveal the effective mechamism of neferine in prevention and cure diabetic complications was screened. The studies provide experimental basis for clinical application.Methods:1. The primary cultured human umbilical vein endothelial cells (HUVECs) were treated with glucose (44 mM) for 5 d to simulate the model of endothelial cells injury. The content of nitric oxide (NO) in the cultured medium was measured by nitrate reductase method. The level of intracellular reactive oxygen species (ROS) was detected by fluorospectrophotometer analysis. Multiple genes analysis was undertaken by Superarray RT2 ProfilerTM PCR. array. The expressions of CCL5 and CCR5 screened from genetic expression and associated with diabetic complications were detected by immunohistochemistry, in situ hybridization, RT-PCR and western blot.2. Diabetic model of SD male rat was established with high calorie diet and intrapcritoneal injection of low dose streptozotocin. Methods of multiple studies were used as including biochemistry, radiommunoassay. two-dimensional electrophoresis, immunohistochemistry, in situ hybridization, RT-PCR and western blot. The studies were used to observe the effect of neferine on diabetic rat body weight, fasting blood glucose, insulin, lipid, blood pressure, the expression of CCL5 and CCR5 mRNA and protein in SCG and myocardium.3. The change of relavant protein in SCG of diabetic rats was identified by using differential-disply proteomics. The studies reveal the effect of neferine on diabetic complications. The studies provide experimental basis for clinical application. The expression of the dimethylarginine dimethylaminohydrolase 1 (DDAH1) and ubiquitin carboxy terminal hydrolase L1(UCHL1) in SCG was tesed by western blot. Effect of of neferine on the expression of the proteins (DDAH1 and UCHL1) associated with diabetic complications was confirmed..Results:1. Compared with control group, the content of NO in the medium was significantly decreased when exposure of HUVECs to high glucose for 5 days and the level of intracellular ROS was increased. Neferine significantly enhanced the decreased level of NO and decreased the increased level of intracellular ROS. The result of PCR Array showed that there were 16 genes upregulation and 10 genes downregulation in HUVECs following chronic culture with high glucose. Of these, CCL5 was the most significantly upregulation. The expressions of CCL5 and CCR5 mRNA and protein were upregulation in HUVECs treated with high glucose. The elevated expressions of CCL5 and CCR5 mRNA and protein were inhibited by neferine.2. Blood pressure,heart rate, fasting blood glucose, insulin, total cholesterol and triglyceride were decreased and body weight, high density lipoprotein were increased in 2 type diabetic model rats treated with neferine. The results of RT-PCR and in situ hybridization indicated that the expressions of CCL5 and CCR5 mRNA were enhanced in SCG and myocardium of diabetic rats. The expressions of CCL5 and CCR5 protein detected by immunohistochemistry and western blot were enhanced in SCG and myocardium of diabetic rats. The elevated expressions of CCL5 and CCR5 were decreased in SCG and myocardium of diabetes treated with neferine.3. The results tested by differential-disply proteomics in SCG of diabetic rats showed that 23 differential protein spots were identified and its were classified into 17 categories of proteins. Except NADH dehydrogenase was upregulation. others of proteins were downregulation in SCG of diabetic rats. The expression changes of most proteins were reverted in diabetic rats treated with neferine. Proteins identified were involved in cytoskeleton. energy metabolism, signal transduction, regulation of NO generation, oxidative stress and imflammatory reaction. DDAH1 was involved in the regulation of NO generation. UCHL1 was associated with oxidative stress. The expressions of DDAH1 and UCHL1 protein were decreased in SCG and myocardium of diabetic rats. The downregulated expressions of DDAH1 and UCHL1 protein in diabetic rats were elevated by neferine.Conclusions:High glucose can significantly affect the expressions of mRNA and protein in VEC and SCG. Neferine can decrease the upregulated expressions of CCL5 and CCR5 in endothelial cells, SCG and myocardium of diabetic rats. Neferine can also increase the downregulate expressions of DDAH1 and UCHL1 in SCG and myocardium of diabetic rats. Consequently, neferine increased the generation of NO and decreased the production of ROS. These results may suggest that neferine has preventive and therapeutic effect on endothelial cells and SCG injury induced by high glucose.