Protective Effects Of Ginkgo Biloba Extract On Diabetic Rats And ECV 304 Cells Exposed To High Glucose

Diabetes mellitus (DM) is a metabolic syndrome with chronic diabetes blood glucose level increasing as the main characteristics. Long-term high blood glucose level can cause a variety of complications; inducte multiple system damages, degrade life quality of patients, which result in a shorter life expectancy and increased mortality. With the improvement of people's living standards, the ageing of the population, lifestyles changing, Diabetes is becoming a prevalent disease in China. The incidence of type 2 diabetes develops into younger age. Thus, diabetes has become a serious threat to human health worldwide and a public health problem. The prevention and treatment of diabetes should be strengthened urgently.Recent researches suggest that the development and worsening of diabetes and its complications are closely related to oxidative stress. Lots of evidences showed that hyperglycemia induced the increasing of reactive oxygen species (ROS) which generated by the mitochondrial respiratory chain, resulted in oxidative stress in many organs and tissues, and insulin resistance.Ginkgo biloba extract (EGb) is extracted from natural plant. It has the effect of inhibiting oxidative damage by scavenging free radicals, increasing antioxidant enzyme activities and suppressing lipid peroxidation. It can also exert protective effects on the cardiovascular and nervous system by increasing blood flow, inhibiting platelet aggregation, suppressing the expression of pro-inflammatory cytokines, and reducing the concentration of amyloid protein.With a perfect antioxidant effect, EGb will be applied to diabetes prevention and treatment as a new research direction. The present research will study the protective effects of EGb on human umbilical vein endothelial cells ECV 304 against high level of glucose and diabetic rats, and the mechanism of its protective role.Aim: To explore the protective effects of EGb on diabetic rats and ECV 304 cells exposed to high level of glucose.Methods:Diabetic rats induced by streptozotocin (STZ) were divided into three groups: control group, diabetes mellitus (DM) group, and EGb protected group. EGb was given to diabetic rats 0.83 ml?kg-1 by intraperitoneal injection daily in 14 days. On day 1 and day 15 after injected, rats were sacrificed to determine levels of triglyceride (TG), high density lipoprotein (HDL-C), and low density lipoprotein (LDL-C) in serum. On day 15, insulin levels in serum were determined.To observe the effects of high level of glucose on ECV 304 ECV 304 were divided into different doses of glucose groups (glucose: 11.1, 15, 25, 35, 45 mmol?L-1) and mannitol group (glucose: 11.1mmol?L-1, mannitol: 25 mmol?L-1). After 24 h exposure, the intracellular Ca2+ concentration ([Ca2+]i) and mitochondrial membrane potential (?Ψm) of ECV 304 were determined by flow cytometry.To observe the effects of different doses of EGb on ECV 304 exposed to high level of glucose ECV 304 were divided into 5 groups: control group, high glucose group (glucose: 35 mmol?L-1), low EGb group (glucose: 35 mmol?L-1, EGb: 250μg?ml-1), high EGb group (glucose: 35 mmol?L-1, EGb: 500μg?ml-1), and mannitol group (10 mmol?L-1). After 24 h exposure, [Ca2+]i and ?Ψm of ECV 304 were determined by flow cytometry; the superoxide dismutase (SOD) activities, the ability of anti-superoxide anion, the suppression ability of hydroxyl radical and level of malondialdehyde (MDA) was measured by kit; the morphological changes of ECV 304 were observed by electronmicroscope.To observe the effects of EGb pretreatment on ECV 304 exposed to high level of glucose ECV 304 were divided into 4 groups: control group, high glucose group (glucose: 35 mmol?L-1), EGb group (glucose: 35 mmol?L-1, EGb: 500μg?ml-1), EGb pre-treatment group (glucose: 35 mmol?L-1, EGb: 500μg?ml-1, ECV 304 cells were pretreated by EGb 24 h in advance), and mannitol group (10 mmol?L-1). After 24 h exposure, [Ca2+]i and ?Ψm of ECV 304 were determined by flow cytometry; the cell membrane fluidity of ECV 304 cells were measured by electron spin resonance.Result:1. The level of TG, LDL-C in serum of diabetic rats were significantly higher than those in control group. The level of HDL-C and insulin in serum of diabetic rats was significantly decreased, compared with control group. EGb was able to decrease the level of TG, LDL-C, HDL-C and increased insulin significantly. 2. High level of glucose was able to increase the [Ca2+]i concentration and decrease ?Ψm in ECV-304. EGb reversed the effects of high of glucose significantly with dose-response relationship on ECV 304 cells. ECV 304 cells were pretreated by EGb could strengthen the protective effects of EGb. 3. The activities of SOD, the ability of anti-superoxide anion, and the suppression ability of hydroxyl radical was significantly decreased when ECV-304 were exposed to high level of glucose; while the level of MDA, production of lipid peroxidation, was significantly increased. EGb increased the activities of SOD, the ability of anti-superoxide anion and the suppression ability of hydroxyl radical of ECV 304 exposed to high level of glucose, and decreased the level of MDA. 4. EGb was able to reduce the damages of high level of glucose to ECV 304. 5. The cell membrane fluidity of ECV 304 was decreased by high level of glucose. EGb could raise the cell membrane fluidity, and ECV 304 cells were pretreated by EGb could strengthen the protective effects of EGb.Conclusion: 1. EGb was able to play its active role in the prevention of diabetes complication worsening through involving in the regulation of lipid metalism, reducing the level of TG, LDL-C and increasing the level of HDL-C. 2. High level of EGb and EGb pretreatment can reduce the damages to ECV 304 cells caused by high level of glucose. 3. EGb was able to maintain the normal function of ECV 304 cells through maintaining the intracellular calcium concentration, mitochondrial membrane potential, reducing the production of free radicals and damages to the cell membrane caused by free radicals.