Effects Of Salvianolic Acid A On Diabetic Vascular Complications And The Related Mechanisms

Diabetes is a chronic disease.It happens when the pancreatic islets can not produce enough insulin or when the body resists the physiological effects of the insulin. Hyperglycaemia and other related disorders in the body metabolism can lead to serious damage of many tissues consequently.The pathological character of diabetic complications is vascular disease.The Study of the drugs' controlling of diabetic vascular disease is a major task as social-demanded in the field of pharmaceutical research.Salvianolic acid A(SalA) is the major water-soluble active constituent of the Chinese herb Salvia.,and was shown of many pharmacological effects,such as strong anti-oxidation,scavenging oxygen free radicals,and protecting the brain and the heart,also some therapeutic effects on hepatitis,hepatic fibrosis,and anti-tumor.Based on these knowledge,we predicted that SalA maybe have protecting effect on diabetic vascular complications.In this project,we investigated the effect of SalA on diabetes and diabetic vascular complications with the aim to find a new drug for the treatment of diabetic complications.Part1:Effect of SalA on blood glucose level and diabetic complications in diabetic rats with high fat dietMale Sprague-Dawley rats weighting 180-220g were feeded of high fat/high sucrose diet for 4 weeks,then were injected intra-peritoneally of Streptozotocin(STZ) 30mg/kg, once or twice,to induce the high blood glucose level.The rats with fasting blood glucose higher than 10mmol/L were selected as the diabetic rats.The rats were divided into 7 groups:the normal rats,the diabetic model rats,the diabetic rats treated with metformine(150mg/kg),the diabetic rats treated with Captopril(20mg/kg),the diabetic rats treated with SalA(10mg/kg,3mg/kg,lmg/kg).In each group,gastric infusion was performed continuously after grouping,once per day for 10 weeks,with high fat diet.After drug adminitration for 4 weeks,blood samples from rat orbit were collected and the blood TG,CHO,HDL,LDL,glucose,insulin level were assayed.Planter microcirculation was also investigated.In the 7th weeks,the inclined plate was used to test the rat staying time and test pain threshold of the rat planter.After drug adminitration for 10 weeks,we determined the rat nerve-intramuscular conduction velocity,planter microcirculation,blood pressure,intraventricular pressure,and electrocardiogram(ECG).We measured also blood TG,CHO,HDL,LDL,AGEs,NO,MDA,SOD,vWF,glucose,glycosylated hemoglobin(GHB),creatinine,urea nitrogen,GOT,GPT,and lactic acid level.Urine was collected from the bladder,and the uric NAG,and creatinine level,were determined.In addition,we separated the thoracic aorta,tested the aorta contraction and relaxaion reactivity by aortic rings,and investigated the change of AGEs,MDA level,SOD,NOS activity,and eNOS,VCAM-1 protein expression.Aorta,kidney and liver tissues were investigated by pathological section with HE stained.The results show that:(1) SalA at 1-3mg/kg reduced blood glucose,GHB,AGEs level, and improved glucose metabolism disorder and oxidative stress situation of the diabetic rats. (2)SalA at 1-3mg/kg reduced the CHO,TG,LDL,and increased HDL level.(3)SalA at 1-3mg/kg decreased the blood vWF level,suggesting that SalA can protect the diabetes related endothelium injury and meliorate the hypercoagulable state.(4)SalA at 1-3mg/kg meliorated the thoracic aorta contraction and relaxation reactivity disorder,and downregulated aortic eNOS,VCAM-1,AGEs level.(5)SalA at 1-10mg/kg improved planter blood perfusion of the diabetic rats,and elevated the ending vascular relaxation reactivity,suggesting that SalA has the effect of endothelium protection.(6) SalA at 1mg/kg decreased blood creatine level,uric NAG/Creatine ratio,suggesting the drug has protection effect on diabetic nephropathy.(7)SalA at 1-10mg/kg decreased the pain threshold time of diabetic rats,increased the inclined plate staying time,and decreased the nerve-intramuscular conduction velocity,which suggests that SalA can improve diabetic rats peripheral neuropathy.Part2:Effect of SalA on blood glucose and lipid level and the related mechanism1.Effect of SalA on the blood glucose levelNormal mice and normal diet diabetic rats were used to study the role of SalA on blood glucose.The human hepatoma cell line Bel-7401 was also used to study the role of SalA on cell glucose assumption.Our results showed that SalA reduced the normal mice blood glucose level,and the effect lasted for more than 15h.SalA can reduce the mice body weight.SalA can also reduce fasting and postprandial blood glucose of the diabetic rats. 1mg/kg SalA has the strongest effect on blood glucose regulation.SalA can improve the glucose absorption of human hepatoma cell line Bel-7401,and have no significant influence on cell proliferation.2 Effect of SalA on blood lipid level of hyperlipemia ratsIn order to clarify the effect of SalA on animal blood lipid level,we used the high fat diet to induce the hyperlipemia rat model.Rats were given of SalA(0.5mg/kg,1.5mg/kg, 5mg/kg) by intraperitoneal injection,while Simvastation(3.5mg/kg) and XueZhikang (200mg/kg) by gastric infusion were taken as positive control.The results showed that SalA decreased blood CHO,LDL,TG,and increased the HDL level of the hyperlipaemia rats,which suggests that SalA can improve the lipid metabolic decompensation.3 Effect of SalA on cell ATP productionIn this study,endothelial cells cells,neonatal rats myocardiocytes,rat vascular smooth muscle cells(VSMC),and rat vascular fibroblasts,were treated with SalA under incubation for different time.Intracellular ATP content was measured by CellTiter-GloTM Luminescent Cell Viability Assay.And we detected the lysosome uptake ability by Neutral Red test,detected the ATP production ability of mitochondria and the open of PT well induced by Ca²⁺.Results showed that SalA can improve the ATP product ability of the cells for at list 6h,and can improve the mitochondria ATP production function.Enhanced lysome Neutral Red uptaking,and inhibited the Ca²⁺ induced rnitochondrial PT well opening,were found.These results suggest that SalA can inhibit mitochondria dysfunction induced apoptosis.Part3:Effect of SalA on myocardial ischemic/reperfusion injury and neonatal rat myocardial cell1.Effect of SalA on myocardial ischemic/reperfusion injuryIn this study,we observed the effect of SalA on myocardial ischemic/reperfusion injury in rats in vivo.SalA was given by intravenous injection at 10 min before ischemia. Comparing with the sham group,ECG of the model group had significant elevation of the ST-segment,and had arrhythmia after ischemia and reperfusion.SalA at 1.5mg/kg can decrease the ischemic/reperfusion arrhythmia and fibrillation,and reduce the blood CK and CK-MB levels.SalA at 5mg/kg had the trend of enhancing the myocardial activity of ATPase,Na-K ATPase,Ca-MG ATPase.The results suggested that SalA can protect the myocardial ischemia/reperfusion injury in rats in vivo.2.Effect of SalA on neonatal rat myocardiocyteIn this experiment,we observed the protective effect of SalA on hypoxia/reoxygenation injury on SD rats neonatal primary cultured myocardiocytes.The intracelluar Ca2+,mitochondrial membrane potential,ATP production,cell proliferation, and beat rhythm were measured.The results showed that SalA at 10^-7-10^-5 M had the protective effect on myocardiocyte hypoxia/reoxygenation.SalA 10^-5 M can decrease the KCl-induced intracelluar Ca²⁺ elevation.SalA at 10^-7M-10^-5M can increase the intracelluar ATP production.10^-5M salA can inhibit the neonatal rat myocardiocyte proliferation and myocardiocyte beat rhythmically.Part 4:Effect of SalA on endothelial cells injuryIn order to understand the action of SalA on diabetic vascular complications,we explored the influence of SalA on endothelial cells injury induced by a few factors.1.AGEs-induced endothelial cells injuryIn this study,the results showed that 10^-6 M-10^-4M SalA can inhibit the AGEs formation,and break the AGES too.AGEs can injure the endothelial cell EA.hy926 directly, and 10^-6M-10^-5M SaLA can protect the AGEs-induced endothelial cells injury.2.Endothelial cells injury induced by other factorsPrevious studies disclosed that ox-LDL,angiotensionlI,free fat acid,homocysteine, and hypoxia/reoxygenation can injure endothelial cells directly.The above results had been validated in our studies.We had investigated the effects of SalA on these injury factors,and found that 10^-9M-10^-6M SalA can protect ox-LDL,10^-7M angiotensionâ…¡,50μM free fat acid,10/20mM homocysteine and hypoxia/reoxygenation on endothelial cell EA.hy926 injury. 3.The methods of cellular viability testIn the cell-based studies,a very important criterion is cell viability.The cell viability study can help us to know the mechanism of a drug better.In this section,we summarized the methods of celluar viablilty test that had used in our studies.The methods include MTT, Neutral Red uptake test,Fluorescein Diacetate(FDA) test,ATP content test,and Sulforhodamine B(SRB) test.In summary,SalA can improve the animal's blood glucose metabolism and vascular complications.Shown by our study,the mechanism is that Sal A can improve the glucose absorption and metabolic rate,thus alleviate the diabetic vascular disease.SalA have a bright prospection on the prevention of human diabetic vascular complications.