基于Nrf2/ARE信号通路探讨少腹逐瘀汤对寒凝血瘀证大鼠血管内皮损伤的保护作用及机制  

Effect and Mechanism of Shaofu Zhuyutang on Endothelial Injury of Rats with Cold Coagulation and Blood Stasis Syndrome Based on Nrf2/ARE Signaling Pathway

作　　者：李凤金 牛雯颖[2] 刘欣欣[2] 肖洪彬 王伟明[1] LI Feng-jin;NIU Wen-ying;LIU Xin-xin;XIAO Hong-bin;WANG Wei-ming(Institute of Chinese Medicine,Heilongjiang Academy of Traditional Chinese Medicine,Harbin 150047,China;Institute of Chinese Medicine,Heilongjiang Academy of Traditional Chinese Medicine,Harbin 150040,China)

机构地区：[1]黑龙江省中医药科学院中药研究所,哈尔滨150047 [2]黑龙江中医药大学基础医学院,哈尔滨150040

出　　处：《中国实验方剂学杂志》2021年第1期31-37,共7页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(81703981)。

摘　　要：目的:通过制备寒凝血瘀证大鼠模型,观察少腹逐瘀汤对寒凝血瘀证大鼠胸主动脉血管组织核转录因子E2相关因子2(Nrf2)/抗氧化反应元件(ARE)信号通路的影响,探讨少腹逐瘀汤对血管内皮损伤的保护作用及机制。方法:将50只SPF级大鼠随机分为少腹逐瘀汤高、中、低剂量组(4.8,2.4,1.2 g·kg^(-1)),模型组和空白组,每组10只。采用冰水浴联合皮下注射盐酸肾上腺素的方法建立寒凝血瘀证大鼠模型。造模的同时开始灌胃给药,连续给药28 d后,采用全自动血液流变仪检测血液流变学指标;酶联免疫吸附测定(ELISA)检测血清中一氧化氮(NO),内皮素-1(ET-1),超氧化物歧化酶(SOD),谷胱甘肽过氧化物酶(GSH-Px),细胞间黏附分子-1(ICAM-1),血管细胞黏附分子-1(VCAM-1),血管性血友病因子(vWF)水平;苏木素-伊红(HE)染色观察胸主动脉血管损伤情况;蛋白免疫印迹法(Western blot)检测胸主动脉血管组织Nrf2,血红素加氧酶-1(HO-1)蛋白表达;实时荧光定量聚合酶链式反应(Real-time PCR)检测胸主动脉血管组织Nrf2,HO-1 mRNA的表达。结果:与空白组比较,模型组大鼠全血黏度及血浆黏度均明显升高(P<0.05,P<0.01),vWF,ICAM-1,VCAM-1含量显著升高(P<0.01),NO,SOD,GSH-Px含量显著降低(P<0.01),ET-1的含量显著升高(P<0.01),胸主动脉血管组织Nrf2,HO-1 mRNA表达显著升高(P<0.01),细胞核内的Nrf2蛋白表达明显升高(P<0.05),细胞浆中Nrf2的蛋白表达水平明显降低(P<0.05),HO-1的蛋白表达水平显著升高(P<0.01)。与模型组比较,少腹逐瘀汤高、中剂量组全血黏度(高、中切),血浆黏度均明显降低(P<0.05);vWF,ICAM-1,VCAM-1,ET-1含量明显降低(P<0.05,P<0.01);NO,SOD,GSH-Px含量明显升高(P<0.05,P<0.01);胸主动脉血管内皮细胞增生、肿胀、脱落,内弹力膜断裂、平滑肌排列紊乱等病理变化有所改善;Nrf2,HO-1蛋白及mRNA表达水平显著升高(P<0.01)。结论:少腹逐瘀汤对寒凝血瘀证大鼠胸主动脉血�Objective:To observe the effect of Shaofu Zhuyutang on nuclear factor erythroid-2-related factor 2(Nrf2)/antioxidant response element(ARE)signaling pathway in blood vessels by establishing the model of rats with cold coagulation and blood stasis syndrome, and to explore the protective effect and mechanism of Shaofu Zhuyutang on vascular endothelial injury. Method: The 50 SPF rats were randomly divided into high dose group(4.8 g·kg^(-1)),middle dose group(2.4 g·kg^(-1)),low dose group(1.2 g·kg^(-1)),model group and normal group(ten of each group). The rat model of cold coagulation and blood stasis syndrome was established by subcutaneous injection of epinephrine hydrochloride combined with ice bath. At the same time of modeling,the drug was administered by gavage. After 28 days of continuous administration,the hemorheology indexes were detected by automatic hemorheology instrument. Levels of nitric oxide(NO),endothelin(ET)-1,superoxide dismutase(SOD),glutathione(GSH-Px),intercellular adhesion molecule-1(ICAM-1),vascular cell adhesion molecule-1(VCAM-1),von Willebrand factor(v WF)in serum were determined by ELISA.Hematoxylin and eosin(HE)staining was used to observe the endothelial injury of vascular tissue of thoracic aorta. The protein expression of Nrf2 and HO-1 in vascular tissue of thoracic aorta was detected by Western blot.Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR)was used to observe the expression of Nrf2 and heme oxygenase-1(HO-1) m RNA in vascular tissue of thoracic aorta. Result:Compared with the blank group,model group rats whole blood viscosity and plasma viscosity were significantly increased(P<0.05,P<0.01),v WF,ICAM 1,VCAM 1 content increased significantly(P<0.01),NO,SOD,gsh-px levels decreased significantly(P<0.01),significantly increased the content of ET-1(P<0.01),thoracic aorta vascular tissue Nrf2, HO-1 m RNA expression was significantly increased(P<0.01), Nrf2 protein expression in the cell nucleus increased significantly(P<0.05), The protein expression leve

关 键 词：少腹逐瘀汤 内皮细胞损伤 核转录因子E2相关因子2(Nrf2)/抗氧化反应元件(ARE) 寒凝血瘀证 

分 类 号：R2-0[医药卫生—中医学] R289[医药卫生—方剂学]