PM诱导支气管上皮细胞炎症反应及内质网应激机制研究  

Regulatory mechanism of particular matter-induced inflammatory response and endoplasmic reticulum stress in bronchial epithelial cells

作　　者：施强强 董年 刘莉[1] 王强[1] 陈俊杰[1] 陈成水[1] SHI Qiangqiang;DONG Nian;LIU Li;WANG Qiang;CHEN Junjie;CHEN Chengshui(Department of Respiratory and Critical Care Medicine,the First Affiliated Hospital of Wenzhou Medical University,Key Laboratory of Interventional Pulmonology of Zhejiang Province,Wenzhou 325015,China)

机构地区：[1]温州医科大学附属第一医院呼吸与危重症医学科、浙江省介入肺脏病学重点实验室,325015

出　　处：《浙江医学》2022年第17期1825-1832,共8页Zhejiang Medical Journal

基　　金：浙江省自然科学基金资助项目(LQ20H010002);温州市基础性科研项目(Y20190522);浙江省医药卫生科技计划项目(2022RC203)。

摘　　要：目的探讨细颗粒物(PM)诱导气道炎症中支气管上皮细胞内质网应激(ERS)现象和氧化应激的调控机制。方法雄性C57BL/6小鼠用4 mg/kg PM连续气道滴注2 d,构建动物模型。获取空白组和PM组小鼠肺组织进行病理分级评分;采用Western blot法检测肺组织葡萄糖调节蛋白前体78(GRP78)和CCAAT增强子结合蛋白(CHOP)的相对表达量,采用免疫组化法检测GRP78和CHOP的累计光密度值,采用硫代巴比妥酸(TBA)法测定肺组织中丙二醛(MDA)质量摩尔浓度。获取肺泡灌洗液,ELISA法检测IL-6、IL-8和前列腺素E(PGE)的分泌水平。用100、200、400 mg/L PM和2.5 mmol/L N-乙酰半胱氨酸(NAC)、200 mg/L PM与支气管上皮细胞BEAS-2B共培养,构建细胞模型。检测各组细胞活性氧(ROS)变化。Western blot法检测NAC干预下的BEAS-2B中GRP78和CHOP表达变化,ELISA法检测炎症介质分泌水平。结果气道滴注PM诱导C57BL/6小鼠支气管病理形态学改变,肺组织GRP78、CHOP表达升高(P<0.05),肺组织MDA质量摩尔浓度升高(P<0.05),伴随肺泡灌洗液中IL-6、IL-8和PGE分泌增多(P<0.05)。细胞模型中,PM作用下各组细胞ROS表达水平均升高(均P<0.05)。NAC干预后细胞ROS表达水平降低(P<0.05),CHOP/GRP78相对表达量降低(P<0.05),IL-6、IL-8和PGE分泌水平降低(P<0.05)。结论PM诱导的支气管上皮细胞炎症中存在内质网应激现象,其调控机制为氧化应激。Objective To investigate the effects of endoplasmic reticulum stress(ERS)in particulate matter(PM)-induced airway inflammation and the regulatory role of oxidative stress in bronchial epithelial cells.Methods C57BL/6 mice were randomly divided into blank group and PM group.The PM group was exposed to 4 mg/kg PM by intratracheal instillation for 2 consecutive days.The lung tissue samples were harvested and bronchoalveolar lavage fluid(BALF)were collected after the animals were sacrificed,The expression of glucose-regulated protein 78(GRP78)and CCAAT/enhancer binding protein(CHOP)in lung tissue was detected by Western blot and immunohistochemistry,the lung MDA content was determined with TBA method;the concentration of IL-6,IL-8 and prostaglandin E(PGE)in BALF were measured with ELISA.Human bronchial epithelial BEAS-2B cells were treated by 200 mg/L PM to establish airway inflammation cell model.BEAS-2B cells were pretreated or not treated with N-Acety-L-Cysteine(NAC)before challenged with PM.The level of reactive oxygen species(ROS)in BEAS-2B exposed to PM with or without NAC were detected.The protein levels of GRP78/CHOP from cell lysate as well as the secretion of inflammatory mediators IL-6,IL-8 and PGEwere quantified.Results In vivo,PM exposure resulted in airway inflammation with up-regulated expression of GRP78/CHOP(P<0.05),elevated MDA content(P<0.05)and up-regulated secretion of inflammatory mediators IL-6,IL-8 and PGE(P<0.05).Consistently,the NAC exerted a reversal effect on the up-regulated expression of GRP78/CHOP and secretion of inflammatory mediators IL-6,IL-8 and PGE(P<0.05).Conclusion The ERS exists in PM-induced airway inflammation,and oxidative stress is involved in the regulatory mechanism.

关 键 词：细颗粒物 支气管上皮细胞 内质网应激 氧化应激 

分 类 号：R122[医药卫生—环境卫生学] X513[医药卫生—公共卫生与预防医学]