益气活血方调控cAMP/Epac1/Rap1信号改善冠心病气虚血瘀证大鼠的验证  

Effect of Yiqi Huoxue Prescription on cAMP/Epac1/Rap1 Signaling Pathway in Coronary Heart Disease Rats with Qi Deficiency and Blood Stasis Syndrome

作　　者：吴华英 邓凯 李静 毛一之 李亮 彭清华 WU Huaying;DENG Kai;LI Jing;MAO Yizhi;LI Liang;PENG Qinghua(College of Medicine,Hunan Normal University,Changsha 410013,China;School of Traditional Chinese Medicine(TCM),Hunan University of Chinese Medicine,Changsha 410208,China;Changsha Hospital of TCM,Changsha 410002,China)

机构地区：[1]湖南师范大学医学院,长沙410013 [2]湖南中医药大学中医学院,长沙410208 [3]长沙市中医医院,长沙410002

出　　处：《中国实验方剂学杂志》2024年第18期107-116,共10页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：湖南省自然科学基金青年基金项目(2022JJ40287);湖南省教育厅优秀青年项目(21B0081);湖南省中医药管理局项目(D2022027);长沙市自然科学基金项目(kq2202255);湖南师范大学大学生创新创业训练计划校级项目(2022115)。

摘　　要：目的:基于环磷酸腺苷(cAMP)/环磷酸腺苷调节鸟嘌呤核苷酸交换因子1(Epac1)/Ras-同源蛋白1(Rap1)信号通路探讨益气活血方对冠心病气虚血瘀证大鼠的心肌保护机制。方法:88只SPF级雄性SD大鼠,按随机数字表分为假手术组(n=12)和实验组(n=76),实验组大鼠采用限制饮食及力竭性游泳复合冠状动脉左前降支(LAD)结扎术构建冠心病气虚血瘀证模型,采集大鼠心电图,将造模成功后的实验组大鼠随机分为模型组、益气活血方低、中、高剂量组(4.28、8.55、17.1 g·kg^(-1))、西药组(单硝酸异山梨酯片,3.6 mg·kg^(-1)),连续给药4周;假手术组及模型组灌胃等体积双蒸水。末次给药后24 h取材,行心脏超声检测左心室舒张末期内径(LVEDD)、左心室收缩末期内径(LVESD),左室短轴缩短率(FS)、射血分数(EF),腹主动脉采血行血液流变学测定,酶联免疫吸附测定法(ELISA)检测血浆cAMP水平;留取心肌组织进行苏木素-伊红(HE)染色、马松(Masson)染色观察心肌病理损伤情况,透射电镜观察心肌组织超微结构改变;实时荧光定量聚合酶链式反应(Real-time PCR)检测心肌Epac1、Rap1GTP酶激活蛋白(Rap1GAP)及Rap1 mRNA表达,蛋白免疫印迹法(Western blot)检测心肌Epac1、Rap1GAP及Rap1蛋白表达。结果:与假手术组比较,模型组大鼠的LVEDD、LVESD显著增加(P<0.01),EF及FS比率显著下降(P<0.01),表现出心功能减退的“心气虚”症状;全血黏度及血浆黏度显著升高(P<0.01),cAMP含量显著升高(P<0.01),心肌组织胶原纤维显著增生(P<0.01),心肌超微结构受损严重,表现出“血瘀”的病理改变;Real-time PCR结果显示模型组大鼠Epac1及Rap1 mRNA显著降低(P<0.01),Rap1GAP mRNA显著增加(P<0.01);Western blot显示Epac1蛋白表达显著降低(P<0.01),Rap1GAP蛋白表达明显增加(P<0.05);与模型组比较,益气活血方能改善冠心病气虚血瘀证大鼠心功能、降低血液黏度、降低血浆cAMP含量、减少胶原纤维�Objective Based on cyclic adenosine monophosphate(cAMP)/cAMP-regulated guanine nucleotide exchange factor 1(Epac1)/Ras-homologous protein 1(Rap1)signaling pathway to explore the myocardial protective mechanism of Yiqi Huoxue prescription on coronary heart disease(CHD)rats with Qi deficiency and blood stasis syndrome.Method Eighty-eight specific-pathogen-free(SPF)grade male Sprague-Dawley(SD)rats were divided into a sham operation group(n=12)and an experimental group(n=76)according to the random number table.The experimental group underwent a restricted diet and exhaustive swimming combined with left anterior descending(LAD)coronary artery ligation to construct a model of CHD with Qi deficiency and blood stasis syndrome,and electrocardiograms(ECGs)of rats before and after the LAD operation were collected.After the model was successfully established,the rats were randomly divided into model group,Yiqi Huoxue prescription low-dose group(4.28 g·kg^(-1)),medium-dose group(8.55 g·kg^(-1)),high-dose group(17.1 g·kg^(-1)),and Western medicine group(isosorbide mononitrate tablets,3.6 mg·kg^(-1)).Rats were intragastrically administered assigned drugs for 4 weeks consecutively,while the sham operation group and the model group were administered with equal volumes of double distilled water.Twenty-four hours after the final administration,the rats were anesthetized with isoflurane to detect the left ventricular end-diastolic diameter(LVEDD),left ventricular end-systolic diameter(LVESD),left ventricular fractional shortening(FS,%),and ejection fraction(EF,%)by echocardiography.Blood samples were collected from the abdominal aorta for hemorheological measurements,and plasma cAMP levels were determined using enzyme-linked immunosorbent assay(ELISA).Myocardial tissue was collected for hematoxylin-eosin(HE)staining and Masson staining to observe myocardial pathological damage,and a transmission electron microscope was used to observe ultrastructural changes of myocardial tissue,fluorescent quantitative real-time polymerase cha

关 键 词：益气活血方 补阳还五汤 冠心病 气虚血瘀证 环磷酸腺苷(cAMP)/环磷酸腺苷调节鸟嘌呤核苷酸交换因子1(Epac1)/Ras-同源蛋白1(Rap1)信号通路 

分 类 号：R284.2[医药卫生—中药学] R285[医药卫生—中医学] R289R287R22R2-031R33R24