心肌缺血再灌注损伤中内质网应激与巨噬细胞极化的相关性研究  

Correlation between endoplasmic reticulum stress and macrophage polarization in myocardial ischemia-reperfusion injury

作　　者：刘新磊 冯旭[1] 罗程[1] 黎玉贵 蔡雄伟[1] 谢晓勇[1] Liu Xinlei;Feng Xu;Luo Cheng;Li Yugui;Cai Xiongwei;Xie Xiaoyong(Department of Cardiothoracic Surgery,the First Affiliated Hospital of Guangxi Medical University,Nanning 530021,China)

机构地区：[1]广西医科大学第一附属医院心胸外科,南宁530021

出　　处：《中华实验外科杂志》2024年第11期2551-2555,共5页Chinese Journal of Experimental Surgery

基　　金：国家临床重点专科建设项目;广西医疗卫生重点学科建设项目;广西壮族自治区临床重点专科建设项目。

摘　　要：目的:探究在心肌缺血再灌注损伤(MIRI)中内质网应激(ERS)与巨噬细胞极化的相关性。方法:广西医科大学实验动物中心提供的C57BL/6小鼠,20~40 g,20只(雌雄各半),使用随机数表法分为4组,每组5只,假手术组(Sham组)、心肌缺血再灌注组(I/R组)、衣霉素+I/R组(TM+I/R组)、4-苯基丁酸+I/R组(4-PBA+I/R组)。Sham组单纯开胸后垫高冠状动脉;I/R组结扎左冠状动脉前降支30 min,再灌注2.5 h;TM+I/R组及4-PBA+I/R组,分别腹腔注射TM及4-PBA,结扎左前降支30 min,再灌注2.5 h。流式细胞术检测巨噬细胞募集数量;苏木精-伊红(HE)染色观察心肌组织、免疫组织化学法观察心肌细胞形态;采用蛋白质印迹法(Western blot)检测心肌ERS标记蛋白葡萄糖调节蛋白78(GRP78)、巨噬细胞极化标记蛋白诱导型一氧化氮合酶(iNOS)及核因子-κB(NF-κB)通路标记蛋白p65的蛋白表达量。组间比较采用完全随机设计双样本t检验。结果:流式细胞术结果,4-PBA+I/R组(0.192±0.019)、I/R组(0.308±0.043)和TM+I/R组(13.112±0.462)巨噬细胞募集数目均高于Sham组(0.116±0.03,t=8.835、9.928、62.941,P均<0.05),4-PBA+I/R组低于I/R组(t=-13.485,P<0.05),而TM+I/R组则高于I/R组(t=62.011,P<0.05)。心肌ERS标记蛋白GRP78、巨噬细胞极化标记蛋白iNOS免疫组织化学,Sham组为[(9.3±3.2)%、(7.6±3.9)%,P<0.05]心肌组织染色出现弱阳性细胞质染色;4-PBA+I/R组为[(18.4±2.4)%、(22.3±2.1)%,P<0.05]出现弱阳性细胞质染色;I/R组为[(46.0±1.3)%、(33.6±4.9)%,P<0.05]出现阳性的细胞质染色,TM+I/R组为[(76.2±9.3)%、(79.3±5.2)%,P<0.05]出现强阳性细胞质染色。Western blot结果显示4-PBA+I/R组、I/R组和TM+I/R组GRP78、iNOS、p65蛋白表达量高于Sham组(t=1.298、3.934、6.128,P<0.01),4组GRP78蛋白表达量分别为0.716±0.305、0.308±0.043、1.589±0.349、0.518±0.141、4组iNOS蛋白表达量分别为0.416±0.207、0.748±0.274、1.323±0.298、0.223±0.132、4组p65蛋白表达量分别为0.687±0.2Objective To investigate the correlation between endoplasmic reticulum stress(ERS)and macrophage polarization in myocardial ischemia-reperfusion injury(MIRI).Methods C57BL/6 mice,20-40 g,20(half male and half female),provided by the Experimental Animal Center of Guangxi Medical University,were divided into four groups of 5 mice each using the random number table method,the sham operation group(Sham group),the myocardial ischemia-reperfusion group(I/R group),the clindamycin+I/R group(TM+I/R group),and the 4-phenyl-butyric acid+I/R group(4-PBA+I/R group).In the Sham group,the coronary artery was padded after simple chest opening;in the I/R group,the anterior descending branch of the left coronary artery was ligated for 30 min and reperfused for 2.5 h.In TM+I/R and 4-PBA+I/R groups,TM and 4-PBA were injected intraperitoneally,respectively,and the anterior descending branch of the left anterior descending branch was ligated for 30 min and then reperfused for 2.5 h.The number of macrophages recruited was detected by flow cytometry;the myocardial tissues were stained by hematoxylin and eosin(HE)staining and immunohistochemistry Myocardial cell morphology was observed;protein expression of myocardial ERS marker protein glucose regulated protein 78(GRP78),macrophage polarization marker protein inducible nitric oxide synthase(iNOS)and nuclear factor-κB(NF-κB)pathway marker protein p65 were detected by Western blotting.comparisons between groups were made using a t-test for two-sample comparisons in a completely randomized design.ResultsThe results of flow cytometry,the number of macrophages recruited was higher in the 4-PBA+I/R group(0.192±0.019),the I/R group(0.308±0.043)and the TM+I/R group(13.112±0.462)than in the Sham group(0.116±0.03,t=8.835,9.928,62.941,P<0.05),and the 4-PBA+I/R group was lower than the I/R group(t=-13.485,P<0.05),while the TM+I/R group was higher than the I/R group(t=62.011,P<0.05).Immunohistochemistry of myocardial ERS marker protein GRP78 and macrophage polarization marker protein iNOS show

关 键 词：心肌缺血再灌注损伤 内质网应激 巨噬细胞M1极化 葡萄糖调节蛋白78 核因子-ΚB 

分 类 号：R54[医药卫生—心血管疾病]