红景天苷对PM_(2.5)诱导大鼠肺泡Ⅱ型上皮细胞损伤及凋亡的影响  

Effects of salidroside on damage and apoptosis of alveolar type II epithelial cells induced by PM_(2.5)in rats

作　　者：柯鸿阳 赵佳丽 马璇 欧阳钏 刘玉梅 张丽雯[1,2,3] 李晓红 谭金峰[4] 于丽 徐东群 王秦 李万伟[1,2] KE Hong-yang;ZHAO Jia-li;MA Xuan;OU YANG-Chuan;LIU Yu-mei;ZHANG Li-wen;LI Xiao-hong;TAN Jin-feng;Yu Li;XU Dong-qun;WANG Qin;LI Wan-wei(School of Public Health,Weifang Medical University,Weifang,Shandong 261053,China;Weifang Key Laboratory of Health Inspection and Quarantine,Weifang,Shandong 261053,China;Experimental Center of Public Health and Management,Weifang Medical University,Weifang,Shandong 261053,China;Weifang Environmental Monitoring Center station,Weifang,Shandong 261044,China;School of Basic Medicine,Weifang Medical University,Weifang,Shandong 261053,China;National Institute of Environment Health,China CDC,Beijing 100021,China)

机构地区：[1]潍坊医学院公共卫生学院,山东潍坊261053 [2]潍坊市卫生检验与检疫重点实验室,山东潍坊261053 [3]潍坊医学院公共卫生与管理实验中心,山东潍坊261053 [4]潍坊市环境监测中心站,山东潍坊261044 [5]潍坊医学院基础医学院,山东潍坊261053 [6]中国疾病预防控制中心环境与健康产品安全所,北京100021

出　　处：《毒理学杂志》2023年第4期303-311,322,共10页Journal of Toxicology

基　　金：山东省自然基金项目(ZR2020MH336);中华医学会医学教育分会和中国高等教育学会医学教育专业委员会2020年医学教育研究立项课题项目(2020B⁃N08142);潍坊市科学技术发展计划项目(2022GX010,2022GX015)。

摘　　要：目的探讨红景天苷(salidroside,SAL)对大气细颗粒物(fine particulate matter,PM_(2.5))导致大鼠肺泡Ⅱ型上皮细胞(RLE⁃6TN)损伤及凋亡的保护机制。方法梯度浓度SAL预处理细胞24 h,加入PM_(2.5)染毒24 h,实验分为6组:0组(无血清培养基),SAL组(160μmol/L SAL),PM_(2.5)组(50μg/ml的PM_(2.5)溶液),40μmol/L+PM_(2.5)组(40μmol/L SAL+50μg/ml PM_(2.5)),80μmol/L+PM_(2.5)组(80μmol/L SAL+50μg/ml PM_(2.5)),160μmol/L+PM_(2.5)组(160μmol/L SAL+50μg/ml PM_(2.5))。倒置显微镜观察细胞形态变化,噻唑蓝法测定细胞活力;试剂盒结合酶标仪测定细胞上清液中乳酸脱氢酶(lactate dehydrogenase,LDH)水平,细胞内总超氧化物歧化酶(total superoxide dismutase,T⁃SOD)、谷胱甘肽(glutathione,GSH)和丙二醛(malondialdehyde,MDA)水平;采用DCFH⁃DA、Annexin V⁃FITC/PI和JC⁃1探针法结合激光共聚焦检测荧光强度以确定细胞活性氧(reactive oxygen species,ROS)、细胞凋亡和线粒体膜电位水平;Western blot检测细胞内Bcl⁃2、p53、BAX、Fas、FADD、Cytochrome c、Caspase⁃3和Caspase⁃9蛋白表达。结果PM_(2.5)组细胞形态变圆,细胞间隙变大,各SAL+PM_(2.5)组形态有所好转,与0组相比,细胞活力降低(P<0.05),LDH和MDA水平升高(P<0.05),T⁃SOD和GSH活力下降(P<0.05),ROS、凋亡水平升高(P<0.05),线粒体膜电位下降(P<0.05),胞内p53、BAX、Fas、FADD、Cytochrome c、Caspase⁃3和Caspase⁃9表达提高,Bcl⁃2下降(P<0.05)。与PM_(2.5)组相比,160μmol/L+PM_(2.5)组细胞活力好转(P<0.05),80~160μmol/L+PM_(2.5)组LDH水平下降,GSH活力提高(P<0.05),160μmol/L+PM_(2.5)组MDA水平下降,SOD活力提高(P<0.05),160μmol/L+PM_(2.5)组ROS、凋亡水平均明显下降(P<0.05),线粒体膜电位上升(P<0.05),80~160μmol/L+PM_(2.5)组p53、BAX、Fas、FADD、Cytochrome c和Caspase⁃3蛋白表达均下降(P<0.05),160μmol/L+PM_(2.5)组Caspase⁃9表达下降(P<0.05),160μmol/L+PM_(2.5)组Bcl⁃2表达明显提升(P<0.05)。结论50μg/ml PM_(2.5)暴露可�Objective To investigate the protective mechanism of Salidroside(SAL)on the damage and apoptosis of rat alveolar type II epithelial cells(RLE⁃6TN)induced by fine particulate matter(PM_(2.5)).Methods Cells were pretreated with gradient concentration of SAL for 24 h and exposed to PM_(2.5)for 24 h.The experiment was divided into six groups:Group 0(serum⁃free medium),SAL group(160μmol/L SAL),PM_(2.5)group(50μg/ml PM_(2.5)solution),40μm+PM_(2.5)group(40μmol/L SAL+50μg/ml PM_(2.5)),80μm+PM_(2.5)group(80μmol/L SAL+50μg/ml PM_(2.5)),160μm+PM_(2.5)group(160μmol/L SAL+50μg/ml PM_(2.5)).The morphological changes of the cells were observed by inverted microscope,and the cell viability was determined by thiazolyl blue method.The level of lactate dehydrogenase(LDH)in cell supernatant was determined by using the kit combined with a microplate reader.The levels of total superoxide dismutase(T⁃SOD),glutathione(GSH),and malondialdehyde(MDA)in cells were measured with the kit combined with a microplate plate.DCFH⁃DA,Annexin V⁃FITC/PI and JC⁃1 probes combined with laser confocal fluorescence intensity were used to determine the levels of reactive oxygen species(ROS),apoptosis and mitochondrial membrane potential.The protein expressions of Bcl⁃2,p53,BAX,Fas,FADD,Cytochrome C,Caspase⁃3 and Caspase⁃9 were detected by Western blot.Results The morphology of the cells in the PM_(2.5)group became round and the intercellular space became larger,and the morphology of each SAL+PM_(2.5)group was improved.Compared with group 0,the cell viability was decreased(P<0.05),the levels of LDH and MDA were increased(P<0.05),and the activities of T⁃SOD and GSH were decreased(P<0.05).The levels of reactive oxygen species and apoptosis were increased(P<0.05),the mitochondrial membrane potential was decreased(P<0.05),the intracellular expressions of p53,BAX,Fas,FADD,Cytochrome C,Caspase⁃3 and Caspase⁃9 were increased,and the expression of Bcl⁃2 was decreased(P<0.05).Compared with PM_(2.5)group,the cell viability of 160�

关 键 词：细颗粒物 氧化应激 细胞凋亡 红景天苷 

分 类 号：R114[医药卫生—卫生毒理学] R99[医药卫生—公共卫生与预防医学]