β_1-肾上腺素受体自身抗体通过激活内质网应激诱导心肌细胞凋亡  

Endoplasmic reticulum stress is involved in cardiomyocyte apoptosis induced by β_1-adrenoceptor autoantibody

作　　者：侯晓鸿 宁娜 李杨[2] 何金玲[2] 燕子[2] 原媛 王丽[1] 王晓晖[1] HOU Xiao-hong;NING Na;LI Yang;HE Jin-ling;YAN Zi;YUAN Yuan;WANG Li;WANG Xiao-hui(Department of Pathology,School of Basic Medical Sciences,Shanxi Medical University,Taiyuan 030001,China;Department of Physiology,School of Basic Medical Sciences,Shanxi Medical University,Taiyuan 030001,China;Laboratory of Morphology,School of Basic Medical Sciences,Shanxi Medical University,Taiyuan 030001,China)

机构地区：[1]山西医科大学基础医学院病理教研室,山西太原030001 [2]山西医科大学基础医学院生理学系,山西太原030001 [3]山西医科大学基础医学院形态学实验室,山西太原030001

出　　处：《中国病理生理杂志》2018年第11期1921-1927,共7页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.31401006);山西省应用基础研究项目(No.201601D021146);山西医科大学博士启动研究基金资助项目(No.03201407);代谢紊乱相关心血管疾病北京市重点实验室提供部分资助

摘　　要：目的:探讨内质网应激在β_1-肾上腺素受体自身抗体(β_1-AA)引起心肌细胞凋亡中的作用。方法:采用β_1-肾上腺素受体细胞外第二环抗原肽段主动免疫大鼠,应用SA-ELISA法检测大鼠血清中β_1-AA的水平,TUNEL染色检测心肌组织的凋亡水平,Western blot法和免疫组化法检测心肌组织中葡萄糖调节蛋白78(GRP78)、C/EBP同源蛋白(CHOP)及caspase-12的蛋白表达。利用亲和层析法纯化的β_1-AA处理H9c2心肌细胞,CCK-8法检测细胞活力,Annexin V-FITC/PI双染流式细胞术检测心肌细胞凋亡;采用内质网应激抑制剂4-苯基丁酸(4-PBA)预处理H9c2心肌细胞,再给予β_1-AA干预,CCK-8法和流式细胞术分别检测细胞活力及凋亡的变化情况。结果:与对照组相比,主动免疫大鼠血清中β_1-AA水平在免疫2周时显著增加,进一步增加至8周,并且主动免疫2周大鼠心肌组织凋亡率明显升高,持续升高至8周。与对照组相比,主动免疫大鼠心肌组织中GRP78、CHOP及caspase-12的蛋白表达在免疫4周和8周时均明显增加。β_1-AA引起H9c2心肌细胞活力持续降低,凋亡明显增加。与β_1-AA单独处理组相比,内质网应激抑制剂4-PBA预处理H9c2心肌细胞可以有效逆转β_1-AA诱导的细胞凋亡增加和活力下降。结论:β_1-AA可以通过激活内质网应激诱导心肌细胞凋亡。AIM:To investigate the role of endoplasmic reticulum(ES)stress in cardiomyocyte apoptosis induced byβ1-adrenoceptor autoantibody(β1-AA).METHODS:The rat model of active immunization with the second extracellular loop ofβ1-adrenoceptor was established,and SA-ELISA was applied to detect the level ofβ1-AA in serum of actively immunized rats.The apoptosis of cardiomyocytes was detected by TUNEL staining,and the protein expression levels of glucose-regulated protein 78(GRP78),C/EBP homologous protein(CHOP)and caspase-12 in rat heart tissues were determined by Western blot and immunohistochemistry.After purifiedβ1-AA obtained by affinity chromatography was used to treat H9c2 myocardial cells,the cell viability was measured by CCK-8 assay and the apoptosis was analyzed by flow cytometry with Annexin V-FITC/PI double staining.The H9c2 cells were treated with ER stress inhibitor 4-phenoxybutyric acid(4-PBA)before interfered withβ1-AA,and the changes of cell viability and apoptosis were determined by CCK-8 assay and flow cytometry.RESULTS:Compared with vehicle group,the level ofβ1-AA in the serum of rats was significantly increased after active immunization for 2 weeks and further rised in 8 weeks,and increased apoptosis was observed in cardiomyocytes after active immunization for 2 weeks,lasting till 8 weeks.Compared with vehicle group,the protein expression of GRP78,CHOP and caspase-12 increased after active immunization for 4 weeks and 8 weeks.Continuous reduction of cell viability and increased apoptosis of H9c2 cells were induced byβ1-AA.ER stress inhibitor 4-PBA pretreatment in H9c2 cells reversed the increased apoptosis and decreased cell viability induced byβ1-AA,indicating that suppression of ER stress effectively reduced cardiomyocyte apoptosis.CONCLUSION:β1-AA induces increased apoptosis in cardiomyocytes by activating ER stress.

关 键 词：β1-肾上腺素受体自身抗体 内质网应激 细胞凋亡 葡萄糖调节蛋白78 C/EBP同源蛋白 CASPASE-12 

分 类 号：R363.2[医药卫生—病理学] R541[医药卫生—基础医学]