PM2.5通过自噬活化NLRP3炎症小体诱导16HBE细胞炎症反应

Autophagy activates NLRP3 inflammasome in 16HBE cells with PM2.5-induced inflammatory response

目的:探讨PM2.5诱导的细胞自噬调控支气管上皮细胞炎症反应的潜在分子机制。方法:PM2.5体外染毒支气管上皮细胞(16HBE),采用MTT法检测细胞存活率,Westernblot法检测微管相关蛋白1轻链3(LC3)和自噬相关蛋白Beclin1的表达水平;通过转染自噬相关蛋白7(ATG7)小干扰RNA(siATG7)来抑制细胞自噬,利用ELISA法检测自噬抑制前后PM2.5诱导的细胞上清液中IL-1β和IL-18的含量,Westernblot法检测自噬抑制前后Nod样受体蛋白3(NLRP3)和依赖半胱氨酸天冬氨酸酶-1(caspase-1)的蛋白表达水平。结果:随着PM2.5浓度的增加和刺激时间的延长,细胞存活率降低;PM2.5导致细胞中LC3-Ⅱ和Beclin1蛋白表达水平增高而LC3-I蛋白表达水平降低;siATG7下调16HBE细胞中ATG7的表达水平后,PM2.5诱导的细胞自噬水平降低,细胞培养液中的炎症因子IL-1β和IL-18含量显著减少,NLRP3和caspase-1蛋白表达水平降低。结论:PM2.5可诱导支气管上皮细胞发生自噬,过度的细胞自噬可参与活化NLRP3炎症小体,促进IL-1β和IL-18的分泌,导致机体炎症反应。

Objective: To explore the molecular mechanism of autophagy on the inflammatory response in bronchial epithelial cells induced by PM 2.5 . Methods: The bronchial epithelial cells (16HBE) were exposed to PM 2.5 in vitro,the cell viability of 16HBE cells exposed to different concentrations of PM 2.5 was detected by MTT assay and the protein expression of microtubule-associated protein 1 light chain 3 and Beclin1 was detected by Western blot.The autophagy was inhibited by transfection of siATG7,the levels of interleukin-1β(IL-1β) and IL-18 in the supernatant were tested by ELISA and the expression of Nod-like receptor protein 3 (NLRP3) and caspase-1 protein were measured by Western blot before and after autophagy inhibition. Results: The survival rate of 16HBE cells decreased with the increase of PM 2.5 concentration and prolongation of exposure time,and there was a time-dose dependent effect.After treated with different concentrations of PM 2.5 for 24 h,the expression levels of LC3-Ⅱ and Beclin1 were increased and the expression of LC3-I protein was decreased,which induced autophagy.After siATG7 down-regulated the expression level of ATG7 in 16HBE cells,the level of autophagy induced by PM 2.5 decreased,the contents of IL-1β and IL-18 in cell culture medium were significantly decreased,and the expression levels of NLRP3 and caspase-1 were decreased. Conclusion: PM 2.5 could induce autophagy in bronchial epithelial cells.Excessive autophagy may partially participate in the activation of NLRP3 inflammatory bodies and promote the secretion of IL-1β and IL-18,leading to inflammation.