【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1d、3d、7d、14d、28d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 protein78,GRP78)、钙网蛋白(calreticulin,CRT)和CHOPmRNA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7d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CRTmRNA表达于术后1d即发生显著上调,较对照组增加136%(P<0.01),而蛋白在术后7d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7d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GRP78mRNA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(14d)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14d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。更多还原

【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 ERS-associated 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.更多还原