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C/EBP 同源蛋白介导的内质网应激相关凋亡途径参与腹主动脉狭窄致大鼠心肌肥厚

张振英, 刘秀华, 孙胜, 荣飞, 郭晓笋, 胡维诚

中国人民解放军总医院病理生理研究室，北京 100853；山东大学医学院病理生理教研室，济南 250012

摘要

内质网应激(endoplasmic reticulum stress, ERS)是细胞对环境改变的适应性反应，但过度ERS 可诱导细胞凋亡。 C/EBP 同源蛋白(C/EBP homologous protein, CHOP)是ERS 相关凋亡途径中重要的信号分子。本实验旨在探讨CHOP 介导的ERS 相关凋亡途径在大鼠腹主动脉狭窄致高血压心肌肥厚发生、发展中的作用。健康雄性Wistar 大鼠85 只，随机分为模型组(n=45) 和对照组(n=40)，模型组行腹主动脉狭窄术，对照组仅分离腹主动脉不行狭窄术，分别于术后1 d、3 d、7 d、14 d、 28 d 时观察各组血流动力学变化，测定全心重/ 体重比(HW/BW)和左心室重/ 体重比(LVW/BW)，RT-PCR 技术检测左心室心 肌组织ERS 相关分子葡萄糖调节蛋白78 (glucose-regulated protein 78, GRP78)、钙网蛋白(calreticulin, CRT)和CHOP mRNA表 达变化，Western blot 分析GRP78、CRT、CHOP，以及凋亡相关蛋白Bax 和Bcl-2 表达变化。结果显示，腹主动脉狭窄可诱 导大鼠心肌肥厚，与对照组比较，术后7 d 模型组大鼠血压升高，心功能代偿性增加，HW/BW和LVW/BW显著增加。模 型组内质网分子伴侣CRT mRNA 表达于术后1 d 即发生显著上调，较对照组增加136% (P<0.01)，而蛋白在术后7 d 开始出 现显著变化，较对照组升高69.2% (P<0.01)；GRP78 基因和蛋白表达均于术后7 d 显著增加，分别较对照组增加20% 和186% (均P<0.01)，在此后观察期间内CRT 和GRP78 mRNA 和蛋白均持续高水平表达。相关分析显示左心室内压最大上升速率 (+dp/dtmax)分别与CRT蛋白表达(r=0.780, P<0.01)和GRP78 蛋白表达(r=0.694, P<0.01)显著正相关。长期ERS (14 d)可触发CHOP 凋亡途径，模型组大鼠心肌组织CHOP mRNA和蛋白表达均于术后14 d 显著上调，分别较对照组增加22.2% 和76.0% (均P<0.01)， 同时促凋亡蛋白Bax 表达增加(较对照组增加41.1%，P<0.01)，而抗凋亡蛋白Bcl-2 表达降低(较对照组降低25.5%，P<0.01) ； 相关分析显示CHOP 蛋白表达与Bax 表达正相关(r=0.654, P<0.01)，而与Bcl-2 表达负相关(r = –0.671, P<0.01)。上述结果提示腹 主动脉狭窄早期即可诱导内质网分子伴侣表达变化，触发ERS，长期ERS 诱导心肌细胞凋亡，CHOP 介导的ERS 相关凋 亡途径可能参与了心肌肥厚过程，推测心肌细胞凋亡参与了心肌肥厚及失代偿的调节，决定肥厚心肌失代偿的进程。

关键词： 心肌肥厚; 内质网应激; 凋亡; C /EBP 同源蛋白

分类号：R363.2

[C/EBP homologous protein-mediated endoplasmic reticulum stress-related apoptosis pathway is involved in abdominal aortic constriction-induced myocardium hypertrophy in rats.] [Ariticle in Chinese]

ZHANG Zhen-Ying, LIU Xiu-Hua, SUN Sheng, RONG Fei, GUO Xiao-Sun, HU Wei-Cheng

Department of Pathophysiology, Chinese PLA General Hospital, Beijing 100853, China； Department of Pathophysiology, Medical School of Shandong University, Jinan 250012, China

Abstract

Endoplasmic reticulum stress (ERS) is an adaptive process in response to circumstantial changes, but excessive and/or prolonged ERS can induce cell apoptosis. C/EBP homologous protein (CHOP) is a very important marker participating in ERSassociated cell apoptosis, while the role of the myocyte apoptosis induced by CHOP remains unclear in the development of hypertrophy. The present study aimed to investigate the effect of CHOP-mediated ERS-associated apoptosis on myocardial hypertrophy induced by abdominal aortic constriction in rats. Healthy male Wistar rats were randomly divided into model group (n=45) and control group (n=40). The rats in model group received abdominal aortic constriction. Hemodynamic changes, whole heart weight/body weight (HW/ BW) and left ventricular weight/body weight (LVW/BW) were measured on 1 d, 3 d, 7 d, 14 d and 28 d after surgery, respectively. The mRNA expression of glucose-regulated protein 78 (GRP78), calreticulin (CRT) and CHOP, which are important markers of ERS, were detected by RT-PCR, and Western blot was used to assess the protein level of GRP78, CRT, CHOP, and apoptosis-associated proteins, Bax and Bcl-2. The results obtained were as follows. Compared with control group, the blood pressure, LVW/BW, and HW/BW of rats in model group increased significantly and cardiac function enhanced compensatively on 7 d after surgery, and increased progressively during the experiment. As early as 1 d after surgery, the mRNA level of CRT in model group increased by 136% (P<0.01) compared with control, while the protein expression increased by 69.2% on 7 d after surgery (P<0.01). Both mRNA and protein expression of GRP78 increased by 20% and 186% (P<0.01) respectively on 7 d after surgery, and the expression sustained high level during the experiment afterwards. Correlation analysis indicated a positive correlation between +dp/dtmax and CRT protein expression (r=0.780, P<0.01) as well as GRP78 protein expression (r=0.694, P<0.01). Prolonged ERS triggered myocyte apoptosis, as both the mRNA and protein level of CHOP in model group increased by 22.2% (P<0.01) and 76.0% (P<0.01) respectively compared with control on 7 d after hypertrophy (14 d after surgery), and meanwhile, the protein expression of pro-apoptotic Bax increased by 41.1% (P<0.01) and anti-apoptotic Bcl-2 protein expression decreased by 25.5% (P<0.01). Correlation analysis indicated a positive correlation between CHOP and Bax expression (r=0.654, P<0.01), and a negative correlation between CHOP and Bcl-2 expression (r= –0.671, P<0.01). These results suggest that abdominal aortic constriction induces a significant up-regulation in ER molecular chaperones at early stage of post-surgery, indicating that ERS response is activated in the rat heart; while prolonged ERS could lead to myocyte apoptosis, and CHOP-mediated ERS-associated apoptosis may contribute to myocardial hypertrophy. We speculate that cell apoptosis may take part in the regulation of myocardial hypertrophy and heart failure, and determine the progression of decompensated hypertrophy.

Key words: hypertrophy; endoplasmic reticulum stress; apoptosis; C/EBP homologous protein

收稿日期：2008-07-22　　录用日期：2008-12-22

通讯作者：刘秀华　　E-mail: xiuhualiu98@yahoo.com.cn

引用本文：

张振英, 刘秀华, 孙胜, 荣飞, 郭晓笋, 胡维诚. C/EBP 同源蛋白介导的内质网应激相关凋亡途径参与腹主动脉狭窄致大鼠心肌肥厚[J]. 生理学报 2009; 61 (2): 161-168.

ZHANG Zhen-Ying, LIU Xiu-Hua, SUN Sheng, RONG Fei, GUO Xiao-Sun, HU Wei-Cheng. [C/EBP homologous protein-mediated endoplasmic reticulum stress-related apoptosis pathway is involved in abdominal aortic constriction-induced myocardium hypertrophy in rats.] [Ariticle in Chinese] . Acta Physiol Sin 2009; 61 (2): 161-168 (in Chinese with English abstract).