PM2.5激活内质网应激诱导肺组织细胞凋亡对慢性阻塞性肺疾病大鼠的影响

Effect of exposure to PM2.5 via endoplasmic reticulm strss to induced apoptosis of lung cells on rats with chronic obstractive pulmonary disease

目的：探讨PM2.5的暴露是否激活内质网应激诱导肺组织细胞凋亡及其对 COPD大鼠的影响。方法将40只健康的雄性 SD 大鼠[体质量为（200±20）g]按随机数字表法分为对照组（N组）、PM2.5组（P组）、COPD模型组（C组）、COPD＋PM2.5组（C＋P 组）。采取熏烟联合两次气管内注入脂多糖（LPS）（200μg/100μl）法建造 COPD大鼠模型,C＋P 组在构建 COPD模型基础上给予气管内注入PM2.5混悬液（5 mg/kg,体积0.25 ml/kg）,P 组大鼠给予气管内注入等量PM2.5混悬液,N组给予气管内滴注相同体积生理盐水。模型建立结束后,检测各组大鼠的肺功能；行 HE染色,观察各组肺组织病理学改变；TUNEL法测定肺结构细胞的凋亡程度；免疫组织化学检测肺组织中 GRP78、ATF6和CHOP蛋白的表达；Real-time RT-PCR检测CHOP mRNA表达水平。结果①肺功能：C＋P组 FEV0.3/FVC （%）、呼气峰流速均较其余3组显著降低（P〈0.05）,C组与 P 组及 N 组比较明显下降（P 〈0.05）,而 P 组与 N 组比较结果尚不能认为差异有统计学意义（P ＆gt;0.05）。②HE染色：N组大鼠肺组织结构正常；P 组肺组织结构基本比较完整,肺泡间隔破坏不明显,与 N组比较,支气管管壁周围有明显的炎性细胞浸润；C 组肺组织结构不完整,破坏明显,肺泡壁变薄、断裂,肺泡腔扩大,有的融合成肺大疱,支气管黏膜上皮细胞坏死、部分脱落,管壁周围有大量的炎性细胞浸润；C＋P 组肺组织结构破坏程度较 C 组更加严重。③肺组织细胞凋亡：P 组、C组、C＋P组细胞凋亡率均较 N 组升高（P 〈0.05）,其中 C 组凋亡率明显高于 P 组（P 〈0.05）, C＋P组较C组进一步升高（P〈0.05）。④免疫组织化学：GRP78、ATF6和CHOP均主要表达于支气管上皮细胞、肺泡上皮细胞及血管内皮细胞,而前两者主要表达于胞浆内,后者主要表达于胞核内。N组肺组织中 GRP78、ATF6和CHOP蛋白均无明显表达或极少量表达,而在 P 组、C组及 C＋P组这三种蛋白的表达均较 N组升高（P〈0.05）,其中 C组较 P 组均显著升高（P 〈0.05）,C＋P组较C组均进一步升高（P 〈0.05）。⑤Real-time RT-PCR 结果：P 组、C 组及 C＋P 组 CHOP mRNA表达含量均较 N组增高（P〈0.05）,其中C组较P组明显增高（P〈0.05）,C＋P组较C组则进一步增高（P〈0.05）。结论 PM2.5暴露可激活内质网应激途径诱导肺组织细胞凋亡,从而促进COPD加重和发展。

Objective To investigate whether exposure of PM2.5 activates the endoplasmic reticulum stress pathway to induce the apoptosis of lung structure and its effect on rats with COPD. Methods The 40 healthy male SD rats （body weight 200 ± 20 g） were randomly divided into control group（group N）,PM2.5 group（group P）,COPD model group（group C）,COPD＋PM2.5 group（C＋P）.The COPD rats model was built by the two times of smoke and the inj ection of lipopolysaccharide （200μg/100μl）.On the basis of constructing COPD model,the C＋P group was injected with PM2.5 suspension（5 mg/kg,volume 0.25 ml/kg）,the rats in group P were injected with the same amount of PM2.5 suspension and the same volume of normal saline was injected into the trachea of the rats in group N.After the model was established,the lung function of rats in each group was detected.HE staining was performed to observe the pathological changes of lung tissue in each group.TUNEL method was used to measure the degree of apoptosis of lung cells.The expression of GRP78,ATF6 and CHOP protein in lung tissue was detected by immunohistochemistry,and the expression level of mRNA CHOP was detected by real-time RT-PCR.Results ①Lung function：group C＋P FEV0.3/FVC（%）,peak expiratory flow（PEF） were significantly lower than the other three groups（P〈0.05）,compared with group P and group N,the group C was significantly decreased,group P and group N comparison results still can not believe that differences were statistically significant（P＆gt;0.05）.②HE staining：the normal structure of lung tissue in N group.P group of lung tissue structure integrity,alveolar septum damage is not obvious,compared with the N group,the bronchial wall around the obvious inflammatory cell infiltration.The lung tissue structure of C group was not complete,the damage was obvious,the alveolar wall became thin,and the alveolar space was enlarged,some of them formed into Bullaes,Bronchial epithelial cell necrosis and partial loss, there are a large number of inflammatory cell infiltration around the wall of the tube.The lung tissue structural damage in C＋P group was more serious than that in C group.③Lung tissue cell apoptosis：The apoptosis rate of P group,C group and C＋P group was higher than that of N group（P 〈0.05）.The apoptosis rate of C group was significantly higher than that of group P（P〈0.05）.Compared with group C＋P,the C group was further increased （P 〈0.05）.④Immunohistochemistry：GRP78,ATF6 and CHOP were mainly expressed in bronchial epithelial cells,alveolar epithelial cells and vascular endothelial cells.The former two are mainly expressed in the cytoplasm,the latter is mainly expressed in the cell nucleus.The expression of GRP78,ATF6 and CHOP protein in lung tissue of N group was little.The expression of these three proteins in P group,C group and C＋P group were higher than those in group N （P〈0.05）.The C group was significantly higher than that in the P group （P 〈0.05）.Compared with the C group,the C＋P group was further increased （P 〈0.05）.⑤Real-time RT-PCR results：The expression levels of mRNA CHOP in group P,group C and group C＋P were higher than those in group N （P〈0.05）.The C group was significantly higher （P 〈0.05） than that of the P group.C＋P group compared with the C group was further increased（P〈0.05）.Conclusions PM2.5 exposure can activate the endoplasmic reticulum stress pathway to induce the apoptosis of lung structure,and further promote the development and aggravation of COPD.