The Inhibitory Effect Of Herb Pair Salvia Miltiorrhiza Bunge-radix Puerariae On Diabetic Aorta In Rats

Objective: This study was designed to investigate the inhibitory effect of water extracts of herb pair Salvia miltiorrhiza Bunge(Danshen)-Radix Puerariae(Gegen)(DG) on aorta in diabetic rats.Methods: The DG was prepared by water extract at the ratio of 1∶1. The rat diabetic model was established by intraperitoneal injection of streptozotocin(STZ)(50 mg/kg/day) for two consecutive days. The rats with blood glucose level ≥11.1 mmol/L after 72 h of STZ injection were considered as diabetic rats. These rats were random Ly divided into four groups: control group, model group, low dosage DG(50 mg/kg/day) treated group, and high dosage DG(200 mg/kg/day) treated group. Rats in the control group and model group were administrated with same volume of saline by intragastric administration once a day, kept for 5 to 6 weeks. The blood glucose levels and body weights were measured once a week. The aorta rings were prepared and the vascular reactivity to acetylcholine(Ach) was determined; The plasma was collected and the levels of malondialdehyde(MDA), superoxyde dismutase(SOD), insulin(INS), hydrogen peroxide(H2O2), nitric oxide(NO), intercellular cell adhesion molecule-1(ICAM-1), and vascular cell adhesion molecule-1(VCAM-1) were determined by commercial kits. The aortas were stained with HE staining for observing the pathological alterations. The protein expression of ICAM-1, VCAM-1, NADPH oxidase 4(NOX4), NADPH oxidase 2(NOX2), and protein kinase C-β(PKC-β) in the aorta tissues were measured by Western blot.Results: STZ treatment significantly increased blood glucose levels in rats with an average level at 13.99 mmol/L. Compared with normal rats, the body weights in STZ treated rats were decreased significantly. DG administration for 5-6 weeks showed no effect on fasting blood glucose levels but significantly decreased the plasma levels of INS,MDA, H2O2, NO, ICAM-1, and VCAM-1, with increasing the plasma levels of SOD. The relaxation to ACh of aortic rings of the model group was much stronger than the normal group(P < 0.01), the DG significantly reduced the relaxation to ACh of diabetic aortic rings(P < 0.01). HE staining showed that DG improved the pathological alterations in diabetic rats, such as aortic wall thickening, edema, disordered arrangement of nucleus and the increase of nucleus. DG significantly inhibited the protein expression of ICAM-1, VCAM-1, NOX2, NOX4 and PKC-β in aorta of diabetic rats.Conclusion: DG demonstrated protective effect on aorta in STZ-induced diabetic rats. The mechanisms may include the inhibition of PKC-β, NOX2 and NOX4 activation, oxidative stress, and the expression of cell adhesion molecules such as ICAM-1 and VCAM-1.Objective: This present research used cultured human umbilical vein endothelial cells(HUVECs) as the tool, intends to explore the effect of water extracts of herb pair Salvia miltiorrhiza Bunge(Danshen)-Radix Puerariae(Gegen)(DG) on high glucose induced adhesion molecule expression in human umbilical vein endothelial cells.Methods: Cultured human umbilical vein endothelial cells were treated with different concentrations of DG for 24 h, then used microscope to watch the morphology change; The cytotoxic effect of DG on HUVECs was determined by assessment of cell viability using the MTT assay. Cells were divided into 5 groups: control group(Con)(5.5 mmol/L glucose), model group(Mod)(30 mmol/L glucose), DG low-dose group(DG-L)(10μg/m L DG + 30 mmol/L glucose), DG medium-dose group(DG-M)(25 μg/m L DG + 30 mmol/L glucose), DG high-dose group(DG-H)(50 μg/m L DG +30 mmol/L glucose), the intervention was kept for 12 h. The formation of intracellular ROS was determined with fluorescence probe DCFH2-DA. The human monocyte THP-1 cells, labeled with DAPI,were co-cultured with the HUVECs for 30 min, then used fluoroscence microscope to observe the influence of DG on the mononuclear-endothelial adhesion induced by high glucose. Western blotting was used to detect the protein expression of ICAM-1、VCAM-1、NOX 2、NOX 4 and PKC-β.Results: Treated with DG, the morphology of endothelial cells had no obvious change. According to the results of MTT assay, DG mildly promoted endothelial cell proliferation. As NAC can suppresse high glucose-induced ROS formation, DG significantly inhibited high glucose-induced ROS generation in endothelial cells. DG significantly suppressed the mononuclear cell-endothelial cell adhesion effect induced by high glucose. Western blotresults showed that the high glucose significantly induced protein expression of ICAM-1, VCAM-1, NOX2, NOX4 and PKC-β in endothelial cells, DG significantly reversed the high glucose-induced protein expression of ICAM-1、VCAM-1、NOX2、NOX4 and PKC-β.Conclusion:DG gentlely promoted endothelial cell proliferation, significantly inhibited high glucose-induced endothelial cell adhesion damage. The mechanism may include the inhibition of protein expression of PKC-β, NOX2 and NOX4 and the inhibition of ROS activation.