Objective: To explore the role of autophagy in PM2.5-induced inflammation in human nasal epithelial cells and related mechanism. Methods: Human nasal epithelial cells were exposed to different concentration of PM2.5 for different times, and the expression levels of microtubule-associated protein-1 light chain-3 Ⅱ (LC3 Ⅱ) and Beclin1 proteins were measured by Western blot. The typical autophagosome and autolysosome were observed by using transmission electron microscopy (TEM). To observe autophagic flux, mRFP-GFP-LC3 plasmid was transfected to nasal epithelial cells and the punctate staining of mRFP-GFP-LC3 were determined by confocal laser scanning microscope. The expression of inflammatory cytokines interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α) in cell culture supernatant were assessed by enzyme-linked immunosorbent assay (ELISA). To assess the role of autophagy in PM2.5-mediated inflammation, autophagy related gene Atg5 and Beclin-1 were silenced by siRNA knockdown, and inflammatory cytokines were analyzed.GraphPad Prism 6.0 was used for statistical analysis. Results: PM2.5 exposure increased the expression of LC3 Ⅱ and Beclin-1 proteins in a dose- (in PM2.5 group with concentration of 0, 15, 30, 60, 120 μg/ml, the expression of LC3 Ⅱ was 0.021±0.001(x±s), 0.037±0.002, 0.058±0.005, 0.075±0.006, 0.085±0.004, respectively, F=126.8, P<0.05; the expression of Beclin-1 was 0.002±0.000, 0.003±0.000, 0.005±0.000, 0.007±0.001, 0.008±0.001, respectively, F=137.3, P<0.05) and time-dependent manner (in PM2.5 group with exposure time of 0, 3, 6, 12, 24 h, the expression of LC3Ⅱ was 0.160±0.007, 0.222±0.003, 0.251±0.015, 0.483±0.029, 0.585±0.035, respectively, F=215.3, P<0.05; the expression of Beclin-1 was 0.059±0.002, 0.080±0.002, 0.087±0.002, 0.183±0.007, 0.228±0.005, respectively, F=137.3, P<0.05) in human nasal epithelial cells. TEM analysis showed typical autophagosome and autolysosome in cells after PM2.5 exposure for 24 h. PM2.5 significantly increased the number of yellow and red dots representing autophagosomes and autolysosomes respectively, indicating autophagic flux was elevated. Moreover, PM2.5 enhanced the secretion of inflammatory cytokines such as IL-6 and TNF-α, which was dramatically prevented by Atg5-siRNA and Beclin-1-siRNA. Conclusion: Autophagy plays an important role in PM2.5-caused inflammation response in nasal epithelial cells, which can induce release of inflammatory factors such as IL-6 and TNF-α and advance the inflammatory reaction.
目的： 研究细胞自噬在PM2.5暴露致鼻黏膜上皮细胞炎性反应中的作用及其作用机制。 方法： 将鼻黏膜上皮细胞暴露于不同浓度PM2.5，蛋白印记法检测不同浓度PM2.5和不同暴露时间下鼻黏膜上皮细胞微管相关蛋白1轻链3Ⅱ（microtubule-associated protein-1 light chain-3Ⅱ，LC3Ⅱ）及Beclin-1蛋白的表达。透射电镜下观察PM2.5暴露后鼻黏膜上皮细胞自噬体形成情况；采用腺病毒mRFP-GFP-LC3转染鼻黏膜上皮细胞，激光共聚焦显微镜下观察PM2.5暴露后鼻黏膜上皮细胞自噬流变化情况。采用酶联免疫吸附测定法（ELISA）观察PM2.5对鼻黏膜上皮细胞炎性因子白细胞介素6（interleukin 6，IL-6）及肿瘤坏死因子α（tumor necrosis factorα，TNF-α）表达的影响。利用自噬相关基因5（autophagy-related 5，Atg5）-siRNA及Beclin-1-siRNA特异性下调自噬水平，观察阻断细胞自噬是否可缓解PM2.5引发的鼻黏膜上皮细胞炎性反应。采用GraphPad Prism 6.0软件进行统计学分析。 结果： PM2.5暴露可引起鼻黏膜上皮细胞LC3Ⅱ及Beclin-1蛋白表达明显升高，随着PM2.5暴露浓度升高，LC3Ⅱ及Beclin-1蛋白表达量逐渐升高，呈明显的剂量依赖效应[浓度为0、15、30、60、120 μg/ml的PM2.5暴露组的LC3Ⅱ表达量分别为0.021±0.001（x±s，下同）、0.037±0.002、0.058±0.005、0.075±0.006和0.085±0.004，F=126.8，P<0.05；Beclin-1表达量分别为0.002±0.000、0.003±0.000、0.005±0.000、0.007±0.001和0.008±0.001，F=137.3，P<0.05]。随着PM2.5暴露时间延长，LC3Ⅱ及Beclin-1蛋白表达量也逐渐升高，呈明显的时间依赖效应(干预时间为0、3、6、12、24 h的PM2.5暴露组的LC3Ⅱ表达量分别为0.160±0.007、0.222±0.003、0.251±0.015、0.483±0.029和0.585±0.035，F=215.3，P<0.05；Beclin-1表达量分别为0.059±0.002、0.080±0.002、0.087±0.002、0.183±0.007和0.228±0.005，F=137.3，P<0.05)。PM2.5暴露24 h后鼻黏膜上皮细胞内可见大量特征性自噬体结构，对照组未见明显自噬体形成。将mRFP-GFP-LC3质粒转入细胞后，共聚焦显微镜下可观察到PM2.5暴露组细胞内多个明亮的黄色及红色荧光斑点，分别代表自噬体及自噬溶酶体，提示PM2.5引起了鼻黏膜上皮细胞发生自噬，自噬流顺畅。同时，PM2.5暴露组的鼻黏膜上皮细胞IL-6及TNF-α炎性因子表达上调，采用Atg5-siRNA和Beclin-1-siRNA下调细胞自噬水平可显著缓解IL-6及TNF-α炎性因子的表达。 结论： 细胞自噬在PM2.5暴露致鼻黏膜上皮细胞炎性反应中发挥重要作用，可诱发鼻黏膜上皮细胞IL-6及TNF-α炎性因子释放，促进鼻黏膜上皮细胞炎性反应。.
Keywords: Autophagy; Inflammation; Nasal mucosa; PM2.5.