吸烟COPD模型大鼠肺组织内质网相关凋亡蛋白CHOP的表达

Expression of endoplasmic reticulum-associated apoptosis protein CHOP in lung tissues of COPD model rats

目的:研究吸烟所致慢性阻塞性肺疾病(COPD)模型大鼠肺组织CCAAT/增强子结合蛋白同源蛋白(CHOP)表达的情况。方法:40只成年雄性Wistar大鼠随机分为对照组、吸烟2个月组、吸烟4个月组及戒烟组。采用单纯被动吸烟法复制大鼠COPD模型,测各组大鼠0.3秒用力呼气容积与用力肺活量比(FEV0.3/FVC)和最高峰值流速(PEF);采用TUNEL法检测肺结构细胞凋亡情况;采用原位杂交和RT-PCR检测肺组织CHOP的mRNA表达水平;免疫组化和Western blot检测其蛋白质水平;同时采用Western blot检测蛋白激酶R样内质网激酶(PERK)、p-PERK、真核生物起始因子(e IF)2α和p-e IF2α的蛋白水平。结果:大鼠吸烟2个月后,肺功能较对照组明显下降(P<0.05),肺结构细胞凋亡明显增加,凋亡细胞主要是肺泡上皮细胞、血管内皮细胞和支气管上皮细胞,肺结构出现破坏;吸烟4个月后,FEV0.3/FVC显著下降(P<0.05),肺结构凋亡细胞进一步增加,肺结构破坏明显;戒烟组肺功能较4个月组稍好转,肺结构破坏仍明显。与对照组相比,p-PERK、p-e IF2α和CHOP表达在吸烟2个月大鼠中升高(P<0.05),在吸烟4个月大鼠中进一步升高(P<0.05);戒烟组大鼠CHOP较吸烟4个月大鼠稍下降但差异无统计学意义;PERK和e IF2α在各组大鼠中表达的差异无统计学显著性。肺结构细胞凋亡与CHOP表达呈正相关;结论:吸烟可通过PERK/e IF2α/CHOP信号通路促进CHOP表达,从而促进COPD的发生与发展。

AIM： To investigate whether cigarette smoke（ CS） promotes the expression of endoplasmic reticulum-associated apoptosis protein CCAAT/enhancer-binding protein homologous protein（ CHOP） in rat lung tissues.METHODS： Adult male Wistar rats（ n = 40） were randomly divided into 4 groups with 10 rats in each group： control group,CS-2 group（ exposed to CS for 2 months）,CS-4 group（ exposed to CS for 4 months） and ex-smoking（ Ex-S） group（ exposed to CS for 4 months and then quit smoking for 1 month）. The percentage of forced expiratory volume in 0. 3 second to forced vital capacity（ FEV0. 3/FVC） and peak expiratory flow（ PEF） were measured. TUNEL assay was used to detect the apoptotic cells. In situ hybridization and RT-PCR were used to determine the mRNA expression of CHOP. The methods of immunohistochemistry and Western blot were used to determine the protein expression of CHOP. Western blot was also used to determine the protein levels of protein kinase R-like endoplasmic reticulum kinase（ PERK）,p-PERK,eukaryotic initiation factor（ e IF） 2α and p-e IF2α. RESULTS： The pulmonary function greatly decreased in the rats exposed to CS for2 months in comparison with control group（ P 〈 0. 05）,markedly decreased in the rats exposed to CS for 4 months as compared with the rats after exposure to CS for 2 months（ P 〈 0. 05）,and was improved little in ex-smoking rats（ P 〉 0. 05）.The structural destruction of the lung was observed in the rats exposed to CS for 2 months,and more obvious changes were found in the rats exposed to CS for 4 months. However,the structural destruction of the lung remained obvious in ex-smoking rats. The apoptotic cells were markedly increased in the rats exposed to CS for 2 months and were even more in the rats exposed to CS for 4 months. The apoptotic cells were alveolar epithelial cell I（ ACE I）,ACE II,vascular endothelial cells and bronchial epithelial cells. The protein levels of p-PERK,p-e IF2α and CHOP were remarkably increased in the rats after exposure to CS for 2 months compared with the control rats（ P 〈 0. 05）,significantly elevated in the rats exposed to CS for 4 months compared with the rats exposed to CS for 2 months（ P 〈 0. 05）,and slightly decreased in ex-smoking rats in comparison with the rats after exposure to CS for 4 months（ P 〉 0. 05）. The total protein levels of PERK and e IF2α did not change between the control rats and those exposed to CS. CONCLUSION： CS promotes the development of chronic obstructive pulmonary disease（ COPD） by inducing the expression of endoplasmic reticulum-associated apoptosis protein CHOP via PERK/e IF2α/CHOP signaling pathway.