ACE2基因缺失对止血带休克后主动脉收缩反应性的影响  

Effect of ACE2 deletion on vasoconstriction reactivity of aortic segments in mice with tourniquet shock

作　　者：方芳 王丽君 杨羚 张文丽 张晓伏 杨秀红 FANG Fang;WANG Lijun;YANG Ling;ZHANG Wenli;ZHANG Xiaofu;YANG Xiuhong(Hebei Key Laboratory for Chronic Diseases,School of Basic Medical Sciences,North China University of Science and Tech-nology,Tangshan 063210,China;Department of Pathology,Dongzhimen Hospital Beijing University of Chinese Medi-cine,Beijing 100007,China;School of Public Health,North China University of Science and Technology,Tangshan 063210,China)

机构地区：[1]华北理工大学基础医学院,河北省慢性疾病重点实验室,河北唐山063210 [2]北京中医药大学东直门医院病理科,北京100007 [3]华北理工大学公共卫生学院,河北唐山063210

出　　处：《中国病理生理杂志》2023年第5期802-810,共9页Chinese Journal of Pathophysiology

基　　金：Supported by the National Natural Science Foundation of China(No.81970359;No.81372029);the Natural Science Foundation of Hebei Province(No.H2022209081);the Foundation of Key R&D Program of Hebei Province(No.20277718D)。

摘　　要：目的:观察血管紧张素转化酶2(angiotensin-converting enzyme 2,ACE2)基因缺失对止血带休克(tourniquet shock,TS)小鼠主动脉收缩反应性的影响。方法:8月龄C57BL/6雄性小鼠,分为野生型(WT)对照组、WT-TS组、基因敲除(knockout,KO)组、KO-TS组,每组10只小鼠,其中5只小鼠血管用于测定血管反应性,另外5只用于其他检测。WT-TS组和KO-TS组小鼠用止血带造成双后肢缺血2 h再灌注4 h后处死。WT组和KO组不进行套扎与再灌注,其余操作同模型组。应用离体血管张力测定仪测定主动脉收缩反应性,光学显微镜结合透射电子显微镜评价血管形态学变化,蛋白免疫印迹法检测AT1、MAS、ACE和ACE2蛋白的表达。采用酶联免疫吸附法检测血清中血管紧张素(angiotensin,Ang) Ⅱ和Ang-(1-7)含量。结果:与WT相比,WT-TS组小鼠主动脉对去甲肾上腺素(norepinephrine,NE)反应性降低,损伤明显;ACE蛋白表达水平升高(P<0.01),ACE2蛋白表达水平降低(P<0.05);AT1蛋白表达水平降低,MAS蛋白表达水平升高,ATI/MAS比值降低(P<0.01);血清Ang Ⅱ生成增多,Ang-(1-7)水平降低,Ang Ⅱ/Ang-(1-7)比值升高(P<0.05)。与WT组相比,KO组小鼠主动脉在NE低浓度(<10^(-7)mol/L)时的收缩反应性升高,在NE高浓度(>10^(-7)mol/L)时的收缩反应性反而下降,但血管损伤不明显;主动脉AT1、MAS和ACE蛋白表达均增高(P<0.05),AT1/MAS比值降低;血清Ang Ⅱ水平升高(P<0.05),Ang-(1-7)水平无明显变化。与KO和WT-TS组小鼠相比,KO-TS组小鼠主动脉收缩曲线明显右移,对NE的反应性明显下降(P<0.05);血管损伤程度轻度增加(P<0.01);主动脉AT1和ACE表达水平轻度升高(P<0.05),但MAS表达水平显著升高(P<0.01);血清Ang Ⅱ和Ang-(1-7)水平分别升高和降低,因此Ang Ⅱ/Ang-(1-7)比值增大(P<0.01)。结论:TS后ACE2基因敲除小鼠体内缺失的ACE2蛋白可引起主动脉更严重的收缩低反应性,其机制可能与肾素-血管紧张素系统失衡程度增加有关。AIM:To observe the effect of angiotensin-converting enzyme 2(ACE2)deletion on vasoconstric⁃tion reactivity of aortic segments in ACE2 knockout(KO)mice with tourniquet shock(TS).METHODS:The 8-monthold male mice with C57BL/6 background were divided into wild-type(WT)control group,WT-TS group,KO group and KO-TS group,with 10 mice in each group,of which five were used for determination of vascular reactivity,and the other five for the other assays.The hindlimbs of the mice in WT-TS group and KO-TS group were ligated with tourniquet for 2 h and loosened for 4 h.The mice in WT group and KO group were subjected to the same treatment except for tourniquet liga⁃tion.The vasoconstriction reactivity of the aorta was measured on tensiometer.The morphological damage of the aorta was evaluated by vascular histopathology.Western blot was used to detect the expression of AT1,MAS,ACE and ACE2 pro⁃teins in aorta.The serum levels of angiotensin(Ang)Ⅱ and Ang-(1-7)were determined by enzyme-linked immunosorbent assay.RESULTS:Compared with WT group,the mice in WT-TS group had lower vascular reactivity to norepinephrine(NE)and obvious vascular lesions.The expression of ACE protein increased significantly(P<0.01),while the expres⁃sion of ACE2 decreased(P<0.05).The expression of AT1 protein in aorta decreased significantly,the expression of MAS protein increased significantly,and the AT1/MAS ratio decreased(P<0.01).Serum Ang II level increased,serum Ang-(1-7)level decreased,and Ang Ⅱ/Ang-(1-7)ratio increased(P<0.05).Compared with WT group,vascular reactivity in KO group increased at low concentration of NE(<10^(-7) mol/L),and decreased at high concentration(>10^(-7) mol/L)without vascular lesion.The expression levels of aortic AT1,MAS and ACE were all elevated(P<0.05).The serum level of Ang Ⅱ increased(P<0.05),but the level of Ang-(1-7)had no obvious change.Compared with KO and WT-TS groups,the aortic reactivity in KO-TS group subtracted apparently(P<0.05),representing its curve shifting to the right obviously.The morpholog

关 键 词：血管紧张素转换酶2 止血带休克 血管低反应性 基因敲除 

分 类 号：R363.2[医药卫生—病理学] R543.1[医药卫生—基础医学]