甲苯二异氰酸酯诱导人支气管上皮细胞炎症反应及自噬

Toluene diisocyanate induces inflammatory response and autophagy in human bronchial epithelial cells

目的探讨甲苯二异氰酸酯(TDI)对人支气管上皮细胞(16HBE)自噬的活化及炎性因子人白细胞介素(IL)-4、IL-6表达的影响。方法①制备TDI-人血清白蛋白(HSA),测定TDI-HAS中TDI的质量浓度。②分别以剂量为0.00～400.00 mg/L的TDI-HSA和HSA处理细胞12 h后,以CCK-8实验测定细胞存活率,选择后续实验的TDI-HSA和HSA处理剂量。③分别以剂量为0.00～120.00 mg/L的TDI-HSA和HSA处理细胞12 h后,以流式细胞仪检测细胞中活性氧(ROS)水平,以酶联免疫吸附法检测细胞上清液中IL-4和IL-6的水平。④以剂量为0.00～120.00 mg/L的TDI-HSA处理细胞12 h后,在透射电子显微镜下观察细胞自噬活动,以蛋白免疫印迹法测定酵母自噬相关基因6 (Beclin1)、微管相关蛋白1轻链3(LC3β)和泛素结合蛋白P62的蛋白相对表达水平。结果①40.00、80.00、120.00 mg/L TDI-HSA组中TDI的质量浓度分别为0.44、0.89、1.33 mg/L。②CCK-8实验结果显示,剂量为120.00 mg/L以下的TDI-HSA和HSA不影响细胞存活率,选择0.00～120.00 mg/L为后续实验的TDI-HSA和HAS处理剂量。③40.00、80.00、120.00 mg/L TDI-HSA组16HBE细胞中ROS水平和细胞上清液中IL-4、IL-6的水平均高于同剂量HSA组(P<0.01);16HBE细胞中ROS水平和细胞上清液中IL-4、IL-6的水平均随着TDI-HSA剂量的增加而增加(P<0.01)。④透射电镜观察显示,与0.00 mg/L组比较,40.00、80.00、120.00 mg/L TDI-HSA组16HBE细胞内自噬溶酶体数量明显增多,线粒体空泡化数量增加。随着TDI-HSA剂量的增加,16HBE细胞上清液中Beclin1蛋白相对表达水平及LC3β-Ⅱ/Ⅰ比值逐渐升高(P<0.05),P62蛋白相对表达水平逐渐下降(P<0.01)。结论 TDI-HSA可诱导16HBE细胞ROS及炎性因子表达水平升高并诱导自噬的活化;细胞自噬可能是TDI诱导职业性哮喘发生中气道炎症反应的重要因素。

Objective To investigate the effect of toluene diisocyanate(TDI) on the activation of autophagy and expression of inflammatory cytokines interleukin(IL)-4 and IL-6 in normal human bronchial epithelial cells(16 HBE). Methods i) We prepared TDI-human serum albumin(HSA) and determined the mass concentration of TDI in TDI-HSA. ii) The cells were treated with TDI-HSA and HSA at concentrations of 0.00-400.00 mg/L for 12 hours. CCK-8 assay was used to determinate the cell viability, and TDI-HSA and HSA doses were selected for subsequent experiments. iii) The cells were treated with TDI-HSA and HSA at doses of 0.00-120.00 mg/L for 12 hours, and the levels of reactive oxygen species(ROS) in the cells were detected by flow cytometry. The levels of IL-4 and IL-6 in the cell supernatant were measured by enzyme-linked immunosorbent assay. iv) The cells were treated with TDI-HSA at doses of 0.00-120.00 mg/L for 12 hours, and the autophagy activity was observed under transmission electron microscope. Western blot was utilized to detect the expression of Beclin1, microtubule-associated protein 1 light chain(LC3β) and P62. Results i) The mass concentrations of TDI in 40.00, 80.00 and 120.00 mg/L TDI-HSA groups were 0.44, 0.89 and 1.33 mg/L respectively. ii) The results of CCK-8 showed that TDI-HSA and HSA at doses below 120.00 mg/L did not affect cell viability, and 0.00-120.00 mg/L was selected as the TDI-HSA and HSA treatment doses for subsequent experiments. iii) The level of ROS in cells and the levels of IL-4 and IL-6 in the supernatant of 16 HBE cells in the TDI-HSA group at 40.00, 80.00, and 120.00 mg/L were higher than that in HSA group at the same dose(P<0.01). The level of ROS in cells and the levels of IL-4 and IL-6 in the supernatant of 16 HBE cells increased with the increase of TDI-HSA doses(P<0.01). iv) Transmission electron microscopy showed that the number of autophagic lysosomes in 16 HBE cells increased significantly, and the number of mitochondrial vacuoles increased in 40.00, 80.00, 120.00 mg/L TDI-HSA group compared with 0.00 mg/L group. With the increase of TDI-HSA dose, the relative expression of Beclin1 protein and LC3β-Ⅱ/Ⅰ ratio in 16 HBE cell supernatant increased(P<0.05), and the relative expression of P62 protein decreased(P<0.05). Conclusion TDI-HSA induces increased expression of ROS and inflammatory factors and induces autophagy activation in 16 HBE cells. Autophagy may be an important factor for the development of airway inflammation in TDI-induced occupational asthma.