摘要
为了验证PM_(2.5)进入肺部后,在肺巨噬细胞清除PM_(2.5)组分中是否发生细胞焦亡效应,本研究以THP-1细胞作为人巨噬细胞模型,以不同浓度PM_(2.5)暴露于THP-1细胞,采用CCK-8法检测细胞存活率,试剂盒检测乳酸脱氢酶(LDH)释放水平,荧光显微镜下观察细胞PI染色情况,流式细胞仪检测Annexin V/PI染色,蛋白免疫印迹(Western blotting)检测焦亡相关蛋白NLRP3、ASC、caspase-1、GSDMD的表达,以及ELISA法检测IL-1β、IL-18的分泌等来检测PM_(2.5)暴露诱导THP-1细胞的焦亡发生和炎症因子释放水平.结果显示:THP-1细胞暴露于PM_(2.5)后,细胞存活率的降低与PM_(2.5)暴露浓度的增加呈正相关;THP-1暴露PM_(2.5)48 h后,细胞胀大并呈气泡状;与空白对照(PBS)组相比,PM_(2.5)暴露组与阳性对照组(1.0μg·mL^(-1)LPS+5.0 mmol·L^(-1)ATP)的细胞上清中LDH水平显著提高;流式细胞仪检测显示Annexin V/PI染色双阳区的细胞比例显著提高;Western blotting结果显示,与对照组相比,PM_(2.5)暴露组炎症小体蛋白NLRP3、ASC、caspase-1表达水平显著提高,而且caspase-1和焦亡执行蛋白GSDMD发生了切割;ELISA结果显示,与对照组相比,PM_(2.5)暴露组IL-1β与IL-18分泌显著提高.研究表明,PM_(2.5)暴露可诱导THP-1细胞炎症性细胞焦亡效应.
To explore the pyroptosis of macrophage cells induced by PM_(2.5)exposure,in this study,THP-1 cells were used as human macrophage cell model to investigate PM_(2.5)-induced pyroptosis.THP-1 cells were exposed to different comcentration of PM_(2.5),the viability of THP-1 cells was detected using a Cell Counting Kit-8 assay.Lactate dehydrogenase(LDH)in the culture medium of THP-1 cells were measured using a colorimetric assay.In the THP-1 cells,the levels of IL-1β and IL-18 were detected using ELISA.The pyroptosis of THP-1 cells was detected using Annexin V/PI double-positive staining.Expression of proteins associated with the nucleotide-binding oligomerization domain,leucine rich repeat and pyrin domain-containing protein3 inflammasome(NLRP3),including NLRP3,apoptosis associated speck-like protein(ASC),stimulated the cleavage of caspase-1 and the pyroptosis executive protein Gasdermin D(GSDMD),were measured by western blotting.PM_(2.5)significantly inhibited cell viability,induced cell swelling and cell membrane rupture,and increased LDH activity in THP-1 cells.In addition,PM_(2.5)markedly promoted the production of IL-1β and IL-18 by THP-1 cells.Treatment with PM_(2.5)significantly increased the protein of NLRP3、ASC、pro-caspase-1,Caspase-1,GSDMD,and cleaved gasdermin D(GSDMD-N)in THP-1 cells.The results from the present study suggest that PM_(2.5)can induce the pyroptosis in THP-1 cells.
作者
洪启浩
王国镇
冯董董
刘紫佳
唐铎
薛姗
赵子杰
王谦
姚孟菲
侯潇楠
任阵海
周志祥
HONG Qihao;WANG Guozhen;FENG Dongdong;LIU Zijia;TANG Duo;XUE Shan;ZHAO Zijie;WANG Qian;YAO Mengfei;HOU Xiaonan;REN Zhenhai;ZHOU Zhixiang(Faculty of Environment and Life,Beijing University of Technology,Beijing 100124;Department of Laboratory Medicine,China-Japan Friendship Hospital,Beijing 100029)
出处
《环境科学学报》
CAS
CSCD
北大核心
2022年第11期475-481,共7页
Acta Scientiae Circumstantiae
基金
国家自然科学基金(No.42077399)。
