The Effects Of Glucagon-like Peptide-1on High Glucose Induced Rat Glomerular Mesangial Cells Oxidative Stress

[Background]The prevalence of diabetes rockets nowadays in our country along with the fast economic developing. the survey held by CDS in2008revealed that there were already9percent of the adult Chinese population who had got diabetes, not to mention the rate of15percent on the IGT(Impaired Glucose Tolerance) situation.The chronic complications of DM are the most threatening things to the patients,which also has become great burden of the whole country.Along with the extension in the course of Diabetes Mellitus, more and more chronic complications come out in while the Diabetic Nephropathy (DN), the main cause of death in T1DM, definitely plays a principal role. DN is now worldwide the leading cause of End-stage renal disease (ESRD), and comes the second place causing ESRD in our country next to chronic nephritis.Until now, the pathogenesis of DN is not quite clear, but the basic pathological change of DN includes thickening of glomerular basement membrane (GBM), hyperplasia of mesenteric cells and accumulation of extracellular stroma. The kidney injury could be caused by hyperglycemia through a number of ways, and it is believed by most of the researchers that DN is resulted from the combined effect of multiple factors. Many researches revealed that the accumulation of reactive oxygen species (ROS),such as superoxide anion, hydroxy radical,H2O2and NO destroys the balance of oxidation-reduction, which finally mediates the development of DN through oxidative-stress in the renal tissue.There are definitely oxidative-stress reactions in the state of hyperglycemia, but what causes the ROS accumulation in such state is remaining unclear. Previous points show that it may related with the autoxidation of the glucose, nonenzymatic glycosylation of the protein, activation of polylol pathways and protein kinase C (PKC) and the decresing ability of oxydate cleaning-up. Hyperglycemia-reduced ROS is recently reported of coming from the mitochondria, firstly hyperglycemia enhances the ROS in the mitochondria which brings disturbance to the mitochondria itself, then the oxidative-stress and downstream pathways are activated, which finally get the tissue damaged.Mitochondria plays an important part in intracellular energy synthesis, about80%～90%ATP is generated during the process of oxidative phosphorylation in the mitochondrial respiratory chain. As the main place of aerobic respiration in cell, Mitochondria synthetizes ATP with ADP and inorganic phosphate through oxidative phosphorylation reaction which is coupled with electron transport chain, and the ROS is generated in the process of respiration in which the electron is transferred to molecular oxygen by NADH or FADH2. As the terminal receptor of electrons and proton hydrogen in the electron transport chain, molecular oxygen can trigger the process of chemical oxidation, change into superoxide anion after accepting a leaked electron, and subsequently generate hydroxyl free radicals. Because the mitochondrial DNA is close to the respiratory chain, oxidative damage is inevitable, then the vicious circle forms and oxidative damage is enlarged. ROS can also be generated during the process of oxidative phosphorylation in mitochondria. The mitochondria is not only the main place that ROS is generated, but also where ROS attacks. Therefore, the state of mitochondrial function has a direct impact on cell function. Too much ROS generated in mitochondrial might be the common mechanism of the chronic complications in patients with diabetes mellitus. Therefore, to protect the mitochondrial structure integrity and vitality and to avoid the mitochondrial oxidative damage could become a potential new direction of prevention and treatment for chronic complications in patients with diabetes mellitus and an important breakthrough for the research and development of related drugs. Because the ROS has high reactivity, can quickly react with surrounding material and is difficult for detection, the quantitative description of oxidative stress in the body is mainly based on the determination of antioxidant enzymes and oxidative metabolites from ROS reacted with lipid, DNA or protein. The degree of oxidative stress damage could be indirectly determined by testing the quantity of MDA, GSH and SOD.A large number of clinical and animal experiments at home and abroad indicated that a variety of antioxidants can effectively protect against oxidative damage in diabetes, such as glutathione (GSH), vitamin, DL-thioctic acid, coenzyme Q10and biological flavonoids. Besides, some clinical commonly used drugs, such as TZDS, repaglinide, statins, angiotensin converting enzyme inhibitors, angiotensin Ⅱ receptor antagonists and calcium channel blockers, were found to have strong antioxidant activity, which opened up a new direction for the treatment of DN. Since alone or combined treatment with the above drugs could also reduce glucose, lower blood pressure, regulate blood lipid and reduce inflammation, they have broad prospects in the treatment of DN. as a newly found drug, glucagon-like peptide-1plays a significant role in the treatment of Diabetes. GLP-1is a kind of30-amino acids-polypeptide mainly secreted by the Langerhans cell in ileum and colon, and also minimal secreted by the L-cell in duodenum and jejunum, which is the product of proglucagon precursor gene. This gene encodes a peptide precursors composed of160amino acids. Until now, GLP-1is the strongest glucose-dependent secretagogue that promote the insulin secretion. GLP-1, first incretin be described, is secreted by digestive system during food-taking, which is able to promote the insulin-secreting function of glucose-dependent Beta-cells. Besides,It also reduces the level of glucagon and the speed of gastric emptying, increases satiety and stimulates the proliferation of Beta-cell. All these characters makes it a promising treatment for T2DM.GLP-1brings out its biological effects by combining with GLP-1receptor, which delivers the signals through cAMP in cells. It plays an important role in regulating the secretion of Insulin and glucagon, appetite and gastrointestinal functional homeostasis. Disturbance in GLP-1secretion may cause metabolism disorder, and it is also proved that T2DM is closely related to the reduction of GLP-1secretion. GLP-1receptors distribute widely in human body, not only in islets, but also in cardiac and pulmonary tissues. They dilate the vessels, lower blood pressure and protect endothelial cells. Some animal experiments indicated that GLP-1can stimulate the synthesis of glycogen in liver and skeletal muscle cell and that their receptors was also found in some parts of central nervous system, mainly in thalami. Food and water-taking could be obviously inhibited by injecting GLP-1into the encephalocoele. Besides, GLP-1have also been found in the glomerulus mensagial cells. Exendin-4is a GLP-1receptor agonist which is not easily degradated by DPP-IV. There are53%homology in amino acid sequence between GLP-1and exendin-4. It is reported that the progress of DN could be postponed by exendin-4, at the same time, the expression of glomerular mesangial cell transforming growth factorβ1and connective tissue growth factor could be lowered by using exendin-4 under the condition of high glucose. Also the GLP-1analogs Liraglutide could reduce the protein volume in urine of the rat with T1DM.At present, it is rarely reported about the mechanism how GLP-1relieve the diabetic nephropathy. In order to discuss the mechanism of the protection GLP-1offered to Glomerular mesangial cell in hyperglycemia environment, we arranged such experiments to observe the indices of oxidative stress after the intervention.[Objective]To investigate the impact of high glucose on the proliferation and oxidative stress of rat glomerular mesangial cells cultured in vitro, and to further explore the protective effect of glucagon-like peptide-1at different concentrations on ki dney.[Methods]1. GMC was incubated for24hours in the following medium respectively:5.5mM glucose (control group),25mM glucose(high glucose group),25mM glucose+GLP-1(3nmol/L),25mMglucose+GLP-1(10nmol/L),25mM glucose+GLP-1(30nmol/L),25mM glucose+GLP-1(100nmol/L).2. Cell proliferation was assessed by CCK-8.3. ROS was detected by flow cytometry method2’,7’-dichlorofluorescein diacetate(DCFH—DA)was used as a reactive oxygen species(ROS)capture agent,The fluorescent intensity of2’,7’-dichlorofluores cein (DCF), which was the product of cellular oxidation of DCFH—DA,was detected by flow cytometry, and the level of ROS was thus measured.4. The acitive of superxide dismutatse(SOD) and the levels of glutathione(GSH) in supernatant wered mesured by Enzyme standard instrument,and the levels of malo Ndialdechyde(MDA) were assayed using chromatometry. [Results]1、Compared with the control group, both the growth rate and the mean DCF fl uorescence fluorescence intensity of GGMC cultured in high-glucose medium were increased(P<0.05);2、Compared with the control group, reduced quantity of GSH,SOD and elevat ed quantity of MDA were observed in high-glucose medium group(P<0.05);3、Compared with the high glucose group, the GLP-1(3nmol/L) showed no sig nificant impact on the mean fluorescence intensity of intracellular DCF(P>0.05), while the GLP-1(10-100nmol/L) can reduce it significantly(P<0.05) with no difference between GLP-110and30nmol/l group.4、Compared with the high glucose group, the GLP-1(3nmol/L) showed no significant impact on the activity of SOD, the quantity of GSH and the level of MDA in supernatant (P>0.05), while the GLP-1(10-100nmol/L) can not only reduce the activity of SOD and the quantity of GSH significantly but also increase the level of MDA(P<0.05), with no difference between GLP-110and30nmol/L group.[Conclusion]Low concentration of GLP-1had no significant effect on proliferation and oxidative stress of glomerular mesangial cells cultured under high glucose, while high concentration of GLP-1can protect against the damage of high glucose. Therefore, the protective effect of GLP-1on glomerular mesangial cells is concentration dependent.