首页|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

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

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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r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(f=-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±0.214、1.101±0.496、1.697±0.959、1.697±0.959,4-PBA+I/R 组蛋白表达(0.716±0.305、0.416±0.207、0.687±0.214)均低于 I/R 组分别为:(0.308±0.043、0.748±0.274、1.101±0.496,t=-13.485,P<0.01),TM+I/R 组蛋白表达(1.589±0.349、1.323±0.298、1.697±0.959)均高于 I/R 组(0.308±0.043、0.748±0.274、1.101±0.496,t=62.011,P<0.01);GRP78 与 iNOS蛋白表达具有正相关性[相关系数(r1)=0.998,P<0.01];p65、GRP78与iNOS蛋白表达具有正相关性[相关系数(r2)=0.989,(r3)=0.978,P<0.01].结论 在MIRI中,ERS和M1型巨噬细胞,两者具有正相关性,ERS是巨噬细胞极化重要调节因子;心肌ERS可能是通过NF-κB p65来调节巨噬细胞极化.
Correlation between endoplasmic reticulum stress and macrophage polarization in myocardial ische-mia-reperfusion injury
Objective 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 clin-damycin+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 ante-rior 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 anteri-or 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-KB(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.Results The results of flow cytometry,the number of macrophages recruited was higher in the 4-PBA+I/R group(0.192±0.019),the 1/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 po-larization marker protein iNOS showed weak positive cytoplasmic staining in myocardial tissue staining in the Sham group[(9.3±3.2)%,(7.6±3.9)%,P<0.05;and(18.4±2.4)%,(22.3±2.1)%,P<0.05]in the 4-PBA+I/R group.Weak positive cytoplasmic staining;positive cytoplasmic staining ap-peared in the I/R group[(46.0±1.3)%,(33.6±4.9)%,P<0.05],and strong positive cytoplasmic staining appeared in the TM+I/R group[(76.2±9.3)%,(79.3±5.2)%,P<0.05].The results of Western blotting showed that the 4-PBA+I/R group,I/R and TM+1/R groups had higher GRP78,iNOS,and p65 protein expression than the Sham group(t=1.298,3.934,and 6.128,P<0.01),and the GRP78 protein expression in the 4 groups was 0.716±0.305,0.308±0.043,1.589±0.349,0.518±0.141,4 groups of iNOS protein expression were 0.416±0.207,0.748±0.274,1.323±0.298,0.223±0.132,and 4 groups of p65 protein expression were 0.687±0.214,1.101±0.496,1.697±0.959,1.697±0.959.4-PBA+I/R group protein expression(0.716±0.305,0.416±0.207,0.687±0.214)was lower than that of the I/R group(0.308±0.043,0.748±0.274,1.101±0.496,t=-13.485,P<0.01),TM+I/R group protein expression(1.589±0.349,1.323±0.298,1.697±0.959)were higher than that of the I/R group(0.308±0.043,0.748±0.274,1.101±0.496,t=62.011,P<0.01);and GRP78 had a positive correlation with iNOS protein expression(correlation coeffi-cient(r1)=0.998,P<0.01);p65,GRP78 had positive correlation with iNOS protein expression[corre-lation coefficient(r2)=0.989,(r3)=0.978,P<0.01].Conclusion In MIRI,ERS,and M1-type macrophages,which have a positive correlation,ERS is an important regulator of macrophage polarization;myocardial ERS may regulate macrophage polarization through NF-κB p65.

Myocardial ischemia-reperfusion injuryEndoplasmic reticulum stressMacro-phage Ml polarizationGlucose regulated protein 78Nuclear factor-KB

刘新磊、冯旭、罗程、黎玉贵、蔡雄伟、谢晓勇

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广西医科大学第一附属医院心胸外科,南宁 530021

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

2024

中华实验外科杂志
中华医学会

中华实验外科杂志

CSTPCD
影响因子:0.759
ISSN:1001-9030
年,卷(期):2024.41(11)