目的 探讨内质网应激(endoplasmic reticulum stress,ERS)在PM2.5诱导人气道上皮细胞炎症和凋亡中的作用及机制.方法 采用免疫组织化学法观察人体手术切除肺组织标本中内质网标志蛋白葡萄糖调节蛋白78(Glucose response protein 78,GRP78)和蛋白激酶R样内质网激酶(PKR-like ER kinase,PERK)蛋白的表达.构建PM2.5与人气道上皮细胞HBE16体外细胞模型,予以不同浓度的PM2.5溶液染毒,观察细胞染毒前后的生长和增殖情况,以CCK-8法测定各组细胞存活率,并选取合理的染毒浓度与时间.以不加处理的气道上皮细胞HBE16为空白组对照,设立PM2.5组和PM2.5+选择性PERK抑制剂GSK2606414组.先予选择性PERK抑制剂GSK2606414预处理细胞,再以PM2.5染毒.采用Western blot法检测干预前后 ERS 相关蛋白 GRP78、PERK、C/EBP 同源蛋白(C/EBP homologous protein,CHOP)、磷酸化 PERK(Phos-phorylation of PKR-like ER kinase,p-PERK)、真核生物起始因子 2α(Eukaryotic translation initiation factor 2 alpha,eIF2α)和磷酸化 eIF2α(Phosphorylation of eukaryotic translation initiation factor 2,p-eIF2α)表达量;以 ELISA 法检测干预前后炎性因子肿瘤坏死因子(Tumor necrosis factor,TNF)-α、白介素(Interleukin,IL)-6及黏蛋白(Mucin,MUC)5AC相对含量;以免疫荧光化学法观察干预前后GRP78蛋白的表达情况;以流式细胞仪检测干预前后各组细胞凋亡情况.结果 GRP78、PERK蛋白在正常组和慢性阻塞性肺疾病(COPD)组的支气管上皮黏膜中均有表达,其中在COPD组的表达较正常组明显升高,差异有统计学意义(P＜0.05).细胞实验中,选取100 μg/ml的PM2.5染毒24 h为最适作用浓度及时间,分不同组处理细胞.检测到PM2.5染毒24h后,细胞活性氧含量、细胞凋亡率及细胞上清中TNF-α、IL-6和MUC5AC蛋白相对含量较空白组均有明显增加,差异有统计学意义(P＜0.05);免疫荧光检测到GRP78蛋白表达较空白组增强;ERS相关蛋白GRP78、CHOP、p-PERK、p-eIF2α的表达也较空白组明显增加,差异有统计学意义(P＜0.05).使用抑制剂GSK干预后均使上述ERS相关蛋白表达明显降低,活性氧含量明显减少,细胞凋亡率明显降低,细胞上清中炎症因子、MUC5AC表达明显下降,GRP78蛋白荧光表达明显减弱,差异有统计学意义(P＜0.05).结论 PM2.5可通过ERS促进气道上皮的氧化应激、凋亡、炎症和黏液高分泌.此外,ERS相关信号通路PERK/eIF2α可能在调节气道上皮氧化应激、凋亡、炎症和黏液高分泌等方面发挥重要作用.
Role of endoplasmic reticulum stress in PM2.5 induced airway inflammation and apoptosis
Objective To understand the role and mechanism of endoplasmic reticulum(ER)stress in inflammation and apoptosis of human airway epithelial cells induced by fine particulate matter(PM2.5).Methods The expression of endoplasmic reticulum marker protein-glucoregulatory protein 78(GRP78)and protein kinase R-like endoplasmic reticulum kinase(PERK)protein in human surgical resection lung tissue specimens was observed by immunohistochemistry.Human airway epithelial cells,HBE16,were cultured in vitro.In order to select a reasonable intervention concentration and time,the CCK8 assay was performed to evaluate the cell viability of each group before and after stimulation with PM2.5 at different times.The cells HBE16were divided into blank group,PM2.5 group,and PM2.5+selective PERK inhibitor GSK2606414 group.Western blot was used to detect endoplasmic reticulum stress-related proteins GRP78,PERK,C/EBP homologous protein(CHOP),phosphorylation of PKR-like ER kinase(p-PERK),and eukaryotic initiation factor 2α(Eukaryotic translation)before and after intervention Expression levels of initiation factor 2 alpha(eIF2α)and phosphorylation of eukaryotic translation initiation factor 2(p-eIF2α);The relative levels of tumor necrosis factor(TNF)-α,interleukin(IL)-6 and mucin(MUC)5AC were detected by ELISA before and after intervention.Immunofluorescence chemistry was used to observe the expression of GRP78 protein before and after intervention.Flow cytometry was used to detect the apoptosis of each group before and after the intervention.Result GRP78 and PERK proteins were expressed in bronchial epithelium and mucosa of normal people and patients with chronic obstructive pulmonary disease(COPD),and the expression of GRP78 and PERK proteins in COPD patients was signifi-cantly higher than that in normal people(P＜0.05).In the cell experiment,100 μg/ml PM2.5 exposure for 24 hours was selected as the optimal concentration and time,and cells were treated in different groups.The content of reactive oxygen species,apopto-sis rate and the relative contents of TNF-α,IL-6 and MUC5AC protein in cell supernatant were significantly increased 24 hours after PM2.5 stimulation compared with blank group(P＜0.05).The expression of GRP78 protein was higher than that of blank group by immunofluorescence detection.The expressions of ER stress-related proteins GRP78,CHOP,p-PERK and p-eIF2α were also significantly increased compared with the blank group(P＜0.05).After the intervention of inhibitor GSK,the expression of the above ER stress-related proteins,the content of reactive oxygen species,the apoptosis rate,the expression of inflammatory factors and MUC5AC in the cell supernatant,and the fluorescence expression of GRP78 protein were significantly decreased(P＜0.05).Conclusion PM2.5 can promote airway epithelial oxidative stress,apoptosis,inflammation and mucus hyper-secretion through ER stress.In addition,PERK/eIF2α,the ER stress-related signaling pathway,may play an important role in regulating oxidative stress,apoptosis,inflammation and mucus hypersecretion of airway epithelial cells.
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