黄芪苷Ⅳ对微囊泡损伤的大鼠胸主动脉环舒张功能的保护作用

摘要

目的:以缺氧/复氧诱导人脐静脉内皮细胞(HUVECs)释放的微囊泡(H/R-EMVs)处理大鼠胸主动脉环,造成其舒张功能损伤,探究黄芪苷Ⅳ(AST)对大鼠胸主动脉环舒张功能的影响及相关机制.方法:采用缺氧12 h/复氧4h的方法诱导体外培养的HUVECs产生MVs,H/R-EMVs保存于D-Hank's液中备用雄性Wistar大鼠开胸取出胸主动脉,制备3～4 mm宽、内皮完整的胸主动脉环 实验分为6组:H/R-EMVs组,在孵育胸主动脉环的培养基中加入H/R-EMVs,使其终浓度为10μg/ml;不同剂量AST组分别采用10、20、40、60 mg/L AST与10 μg/ml H/R-EMVs共同孵育胸主动脉环;对照组给予等体积的D-Hank's溶液孵育时间为4h,每组各测定5个血管环观察AST对舒张功能的影响,检测一氧化氮(N0)含量及t-eNOS、p-eNOS、t-Akt、p-Akt、ERK1/2和p-ERK1/2蛋白质水平. 结果:H/R-EMVs对大鼠胸主动脉环舒张功能有明显的抑制作用(P＜0.01).与H/R-EMVs组相比,AST 20、40和60 mg/L组剂量依赖性地提高大鼠胸主动脉环的舒张率(P＜0.01),使NO含量增加(P＜0.05,P＜0.01);t-eNOS、t-Akt和ERK1/2蛋白质水平不变,p-eNOS、p-Akt和p-ERK1/2蛋白质水平增高(P＜0.01). 结论:AST可显著改善H/R-EMVs损伤的大鼠胸主动脉环的舒张功能,其机制与提高NO含量及增加p-eNOS、p-Akt和p-ERK1/2蛋白质水平有关.%Objective:To investigate the effects of Astragaloside Ⅳ (AST) on diastolic function of rat thoracic aorta rings which was injured by microvesicles derived from hypoxia/reoxygenation (H/R)-treated human umbilical vein endothelial cells (HUVECs),and the mechanism of AST.Methods:H/R-induced endothelial microvesicles (H/R-EMVs) were generated from cultured HUVECs in vitro under the condition of hypoxia for 12 hour/Reoxygenation for4 hour,H/R-EMVs were stored in D-Hank's solution.Male Wistar rats were underwent thoracotomy,the thoracic aorta with intact endothelium were carefully removed and cut into 3 ～ 4 mm rings.The experiment was divided into six groups.H/R-EMVs group:thoracic aortic rings of rats were incubated in culture medium and treated with H/R-EMVs in a final concentration of 10μg/ml;different doses of A.ST groups:thoracic aortic rings of rats were treated with 10,20,40,60 mg/L AST co-incubated with 10 μg/ml H/R-EMVs respectively;control group were treated with the same volume of D-Hank's solution.Duration of incubation was 4 h,each group was tested in five replicate aortic rings.Effects of AST on endothelium-dependent relaxation were detected.The production of nitric oxide (NO) and the level of endothelial NO synthase (eNOS),phosphorylated eNOS (p-cNOS,Ser-1177),serine/threonine kinase (Akt),phosphorylated Akt (p-Akt,Ser-473),extracellular regulated protein kinases (ERK1/2) and phosphorylated ERK1/2 (p-ERK1/2,Thr202/Tyr204) of rat thoracic aortic rings were detected.Results:Ten μg/ml H/R-EMVs could impaire the relaxation of rat thoracic aortic rings significantly (P ＜ 0.01).Compared with H/R-EMVs group,relaxation of rat thoracic aortic rings was increased by 20,40 and 60 mg/L AST in a concentration-dependent manner (P ＜ 0.01),the level of NO production was also enhanced (P ＜ 0.05,P ＜ 0.01).The level of t-eNOS,t-Akt and ERK1/2 was not changed,but the level of p-eNOS,p-Akt and p-ERK1/2 increased by the treatment with AST (P ＜ 0.01).Conclusion:AST could effectively ameliorate endothelium-dependent relaxation of rat thoracic aortic rings impaired by H/R-EMVs in a concentration-dependent manner,the mechanism might involve the increase in production of NO,and the protein level of p-eNOS,p-Akt and p-ERK1/2.