Protective Effects And Mechanisms Of Astragaloside Ⅳ On Experimental Vascular Complication In Diabetes Mellitus

Part Ⅰ Effects of Astragaloside Ⅳ on Aortic Complication in DM RatsAimTo explore the effects and mechanisms of AST Ⅳ on vascular complication in DM rats.MethodsMale rats were fed with high-glucose and fat diet for 4 weeks and intraperitoneal injected STZ 35mg/kg once to mimic DM model. Rats with blood glucose more than 16.7 mmol/L were identified as a DM working model. The model rats were randomly divided into model group, AST Ⅳ with low, medium and high dosages(20, 40, 80 mg/kg/d, ig, respectively) groups, and Ins(0.15 U/kg/d, ih) group. In addition, the control group was established. Weight and blood glucose of all rats were measured in specific time. The content of GHb in red cells, the level of PT and APTT in plasma, the contents of TG, LDL, CHOL and HDL in serum, the activity of SOD and the content of MDA in serum were measured by biochemical techniques. The level of AGEs in serum was detected by ELISA. The level of FISN in serum was measured by RIA. The pathologic histology of aorta in rats was observed by HE staining. Organelles of endothelial cells in aortic were inspected by scanning electron microscope. The positivecells expression of TGF-β1 in endothelial cells was detected by IHC. The expressions of Nox4, TGF-β1, Smad2, BCL-2, BAX and Caspase 3 m RNAs and proteins were measured by QPCR and WB respectively.Results(1) Compared with control group, rats in model group looked depressed, hair loss and weight lesson, the weight was dereased, the aortic weight index was increased. Compared with model group, AST Ⅳ(40, 80 mg/kg) and Ins groups improved mental state, increased weight, decreased aortic weight index.(2) The content of BG, TG, LDL and CHOL were significantly higher, the content of HDL was significantly decreased, the content of GHb and the level of FISN were significantly increased in model group. On the contrary, these results in AST Ⅳ(40, 80 mg/kg) and Ins groups were alleviated.(3) The level of AGEs was markly increased, PT and APTT were significantly shortened in model group. However, AST Ⅳ(40, 80 mg/kg) and Ins reduced the level of AGEs, lengthened PT and APTT.(4) HE staining result showed that the endothelial cell layer, middle smooth muscle layer and lining and outer membrane had no obvious changes in model group. Nevertheless, EM result showed that endothelial cells in aortic was separated with middle smooth muscle layer, mitochondria vacuoles was degenerated, rough endoplasmic reticulum was mild expanded, nuclei karyopyknosis was obvious, the aortic endothelial cells had obvious ultrastructure damage in model group. Compared with model group, AST Ⅳ and Ins could improve aortic endothelial cell ultrastructure damage.(5) The activity of SOD was significantly reduced, the content of MDA was significantly increased, the expressions of Nox4 m RNA and protein in aorta weresignificantly up-regulated in model group. Compared with model group, AST Ⅳ and Ins could significantly increase SOD activity, lower MDA content, down-regulate the expressions of Nox4 m RNA and protein.(6) The expressions of TGF-β1 and Smad2 m RNAs and proteins in aorta were significantly up-regulated in model group. IHC results showed that TGF-β1 positive expression cells in aortic endothelial cells were significantly increased in model group. Compared with model group, AST Ⅳ(40, 80 mg/kg) and Ins could down-regulate the expressions of TGF-β1 and Smad2 m RNAs and proteins, reduce TGF-β1 expression of positive in aortic endothelial cells.(7) The expressions of BAX and Caspase 3 m RNAs and proteins were significantly up-regulated, the expressions of BCL-2 m RNA and protein were significantly down-regulated in model group. However, AST Ⅳ(40, 80 mg/kg) and Ins could obviously down-regulate the expressions of BAX and Caspase 3 m RNAs and proteins, up-regulate the expressions of BCL- 2 m RNA and protein.Conclusion(1) Male rats which were fed with high glucose and fat diet for 4 weeks and intraperitoneal injected with STZ 35 mg/kg once could mimic a DM model successfully.(2) AST Ⅳ had a protective effect on aortic injury in DM rats.(3) The specific mechanisms of AST Ⅳ could be related with regulation of glucose and fat disorder, inhibition of oxidative stress, down-regulation the expressions of TGF-β1 and Smad2, and regulation of apoptosis related genes expressions.Part Ⅱ Effects of Astragaloside Ⅳ on H2O2-Induced Human Umbilical Vein Endothelial Cells ApoptosisAimTo explore the protective effects and mechanisms of AST Ⅳ on H2O2-induced human umbilical vein endothelial cells apoptosis.MethodsThe vascular injury of DM in vitro was mimicked by incubating H2O2 100 μmol/L for 18 h to induce HUVECs damage. Each drug was treated in the mediums pre-exposing to H2O2 1h. The EC50 of AST Ⅳ on H2O2-damage HUVECs was determined by methyl thiazolyl tetrazolium(MTT) assay. Annexin-V and PI staining was used to detect apoptosis rate in HUVECs with H2O2.Intracellular ROS production was detected by FCM. The expressions of Nox4, Smad2, TGF-β1, BCL-2, BAX and Caspase 3 m RNAs and proteins were measured by QPCR and WB respectively.Results(1) H2O2 up-regulated Nox4 expression, increased the level of ROS, up-regulated the expressions of TGF-β1, Smad2, BAX and Caspase 3, lowered the expression of BCL-2 and increased apoptosis rate in HUVECs.(2) Adiminitration with AST Ⅳ could down-regulate the expression of Nox4, reduce the level of ROS, down-regulate the expressions of TGF-β1 and Smad2, regulate the expressions of apoptosis-related genes and lower HUVECs apoptosis.(3) Treatment with DPI, a specific inhibitor of Nox4, could down-regulate the expression of Nox4, reduce the level of ROS, down-regulate the expressions of TGF-β1and Smad2, regulate the expressions of apoptosis-related genes and lower HUVECs apoptosis.(4) Exposing to LY2109761, a selective inhibitor of TGF-β1/Smad2 pathway, could down-regulate the expressions of TGF-β1/Smad2, regulate the expressions of apoptosis-related genes, and lower HUVECs apoptosis, but have no effect on the expression of Nox4 and the level of ROS.Conclusion(1) H2O2 up-regulated Nox4-dependent, induced ROS aggeration, up-regulated the expressions of TGF-β1 and Smad2, resulted in apoptosis of HUVECs.(2) AST Ⅳ had a protective effect on H2O2-induced HUVECs apoptosis. The mechanisms of which were related with down-regulating the expression of Nox4, decreasing the level of ROS, down-regulating the expressions of TGF-β1 and Smad2, regulating the expressions of apoptosis-related genes.