Protective Effect Of Salvianolic Acid B On Endothelial Function In Diabetic Rats With Fluctuating Blood Glucose And The Potential Mechanisms Implicated

Aim: To investigate the effect of Salvianolic acid B(Sal B)on endothelial function in blood glucose fluctuation state of diabetic rats and the possible mechanisms involved.Methods: 1.Animal experiment: Fifty male SD rats were randomly allocated to control group and diabetic model groups,control group were fed with standard laboratory chow while diabetic model groups kept high-fat diet.After 4 weeks on either diet,diabetic model groups rats were injected intraperitoneally(i.p.)with STZ(40mg/kg)dissolved in a 0.1 mmol/L citrate-phosphate buffer(pH 4.5)except controls.Rats with FBG levels ≥ 13.9 mmol/L were considered diabetic and randomly allocated to 4groups,namely intermittent high glucose(IHG)group,sustained high glucose(SHG)group,Sal B 160 group(Salvianolic acid B 160 mg/kg),Sal B 80 group(Salvianolic acid B 80 mg/kg).The models of blood glucose fluctuation of were established by streptozotocin-induced diabetic rats of which fed with high-fat diet combined with either subcutaneous injection with insulin or gavaging of glucose twice daily.Rats in Sal B groups were administered intragastrically with corresponding drug suspended in double distilled water for 6 weeks,while the rest groups were given double distilled water of the same volume.Body weight,fasting glucose(FBG),daily blood glucose levels of 5 time points and levels of MBG,SDBG,LAGE were collected to measure the stability of blood glucose during administration.The oral glucosetolerance test(OGTT)were performed and the concentrations of total cholesterol(TC),glycosylated hemoglobin(GHb)and fasting insulin(F-INS)were measured by commercially available kits after 6 weeks.Values of area under curve(AUC)and insulin sensitivity index(ISI)were calculated to evaluate insulin resistance(IR).The isolated aortic ring of rats was mounted on a bath system,and vascular reactivity experiments induced by acetylcholine and sodium nitroprusside.The primary histopathological changes of aorta were observed by HE staining.Protein expression of nuclear factor-κB(NF-κB)and vascular cell adhesion protein 1(VCAM-1)in aorta were detected by immunohistochemical staining.The aorta homogenates Caspase-3activity as well as total antioxidant capacity(T-AOC),total superoxide dismutase(T-SOD)activity and malondialdehyde(MDA)level in serum and aorta were measured by colorimetric assay.Endothelial cell apoptosis was evaluated by TUNEL assay.The protein expression of Bax,Bcl-2,NADPH oxidase 4(NOX4)and phosphorylation level of endothelial nitric oxide synthase(eNOS)in aorta were determined by western blot.2.HUVECs experiment: HUVECs were preincubated with Sal B(1×10-5,10-6,10-7mol/L)for 24 h,followed by incubation with intermittent high glucose(IHG,5.5mmol/L 12 h,33.3 mmol/L 12 h)for 72 h while glucose concentration in control group(Control,5.5 mmol/L)and sustained high glucose(SHG,33.3 mmol/L)group remained constant level.Cell viability and apoptosis were assayed by MTT assay and flow cytometry assay,respectively.Levels of T-AOC,MDA,nitric oxide(NO)and Caspase-3 activity were determined colorimetrically.Intracellular reactive oxygen species(ROS)was evaluated by a fluorescence microscope using a fluorescent probe DCFH-DA.Protein levels of NOX4,p-eNOS,Bax and Bcl-2 were determined by Western-blot analysis.Results: 1.Animal experiment:(1)After 6 weeks of blood glucose fluctuation,MBG,SDBG and LAGE of diabetic rats were all increased significantly than those in control group(P <0.01);The levels of SDBG and LAGE in blood glucose fluctuation state of diabetic rats were significant raised while MBG was reduced compared to the SHG group(P <0.01).(2)Sal B significantly reduced levels of FBG,TC and GHb(P<0.05 or P <0.01)while increased F-INS secretion in blood glucose fluctuation state of diabetic rats(P <0.05),accompanied by a marked amelioration of insulin resistance(P<0.05).(3)Sal B significantly elevated the expression of NF-κB and VCAM-1 and attenuated endothelium-dependent dysfunction to alleviate aortic lesions(P <0.05 or P<0.01).(4)Sal B markedly promoted activation of eNOS,with NO synthesis increased notably(P <0.05 or P <0.01).(5)Supplementation with Sal B significantly reduced endothelial cell apoptosis and Caspase-3 activity in diabetic rats,with protein expression of Bax down-regulated and Bcl-2 up-regulated markedly(P<0.05 or P<0.01).(6)In addition,Sal B effectively enhanced T-AOC and T-SOD activity in aortic,with protein expression of NOX4 and MDA content reduced notably(P<0.05 or P<0.01).2.HUVECs experiment:(1)Sal B evidently suppressed IHG-induced cell apoptosis,with the expression of Bax protein down-regulated and Bcl-2 protein up-regulated markedly(P<0.05 or P<0.01).The activity of Capase-3 was also significantly decreased in Sal B treated HUVECs(P<0.05 or P<0.01).(2)Pretreatment with Sal B significantly ameliorated IHG-induced cell injury as manifested by increased cell viability,eNOS activation and NO production in HUVECs(P<0.05 or P<0.01).(3)Preincubation with Sal B led to a significant enhancement of antioxidant capacity and reduction of NOX4 protein expression,accompanied by a marked decrease of intracellular ROS and MDA content(P<0.05 or P<0.01).Conclusion: Salvianolic acid B effectively ameliorates endothelial dysfunction in blood glucose fluctuation state of diabetic rats,which might be attributed to suppression of endothelial cell apoptosis and endothelium-dependent relaxing dysfunction.The beneficial effect of Salvianolic acid B on endothelial dysfunction might be derived fromits antioxidant capacity.