摘 要: 目的 探讨N-乙酰半胱氨酸(NAC)对于甲苯二异氰酸酯(TDI)诱导人支气管上皮细胞(HBECs)自噬激活的影响。方法 (1)制备TDI-人血清白蛋白(HSA)偶联物,分别采用Gutmann法和BCA蛋白定量法测定偶联物中TDI和HSA的水平。(2)以不同浓度TDI-HSA(0、40.00、80.00和120.00 mg/L)处理HBECs 12 h,采用活细胞成像技术检测细胞中活性氧(ROS)水平,酶联免疫吸附法(ELISA)检测细胞上清液中IL-4和IL-6的水平,蛋白质印迹法(Western blot)检测自噬相关蛋白腺苷酸活化蛋白激酶(AMPK)、磷酸化AMPK(p-AMPK)、哺乳动物雷帕霉素靶蛋白(mTOR)、磷酸化mTOR(p-mTOR)、酵母自噬相关基因6(Beclin1)、微管相关蛋白1A/1B-轻链3(LC3)和泛素结合蛋白p62的表达水平。(3)采用NAC(5 mmol/L)预处理HBECs 4 h后,再用TDI-HSA(120.00 mg/L)染毒12 h,检测细胞中ROS及IL-4、IL-6的水平,测定自噬相关蛋白AMPK、p-AMPK、mTOR、p-mTOR、Beclin1、LC3、p62的表达水平。结果 (1)合成的TDI-HSA偶联物中TDI和HSA质量浓度分别为21.90 mg/L、1 955.00 mg/L,摩尔比为4.41。(2)随着TDI-HSA染毒剂量的增加,HBECs细胞中ROS和IL-4、IL-6水平明显增加,差异具有统计学意义(P<0.05)。TDI-HSA中(80.00 mg/L)、高(120.00 mg/L)剂量染毒组自噬相关蛋白p-AMPK/AMPK比值、LC3Ⅱ/LC3Ⅰ比值和Beclin1表达明显增加,p-mTOR/mTOR比值和p62表达明显减少,差异均有统计学意义(P<0.05)。(3)与TDI-HSA高剂量染毒组相比,TDI-HSA+NAC组HBECs细胞中ROS和IL-4、IL-6水平明显降低,差异具有统计学意义(P<0.05);自噬相关蛋白p-AMPK/AMPK比值、LC3Ⅱ/LC3Ⅰ比值和Beclin1表达明显降低,p-mTOR/mTOR比值和p62表达明显升高,差异均有统计学意义(P<0.05)。结论 通过抑制TDI-HSA诱导的HBECs中ROS水平,可显著抑制HBECs自噬激活,并降低细胞上清液中IL-4、IL-6的水平。 |
关键词: 甲苯二异氰酸酯(TDI) 人支气管上皮细胞(HBECs) N-乙酰半胱氨酸(NAC) 活性氧(ROS) 自噬 炎性因子 |
中图分类号: R994.6
文献标识码: A
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基金项目: 国家自然科学基金面上项目(81872603),山东第一医科大学学术提升计划(2019QL001) |
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Effect of N-acetylcysteine on toluene diisocyanate-induced activation of autophagy in human bronchial epithelial cells |
GUO Su-mei,JIAO Bo,YANG Xiao-han,SUN Lei,ZHANG Xiao-xia,DONG Shuang-yan,LI Chao,JIA Qiang
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Shandong First Medical University/Shandong Provicial Academy of Medical Sciences/Shandong Provicial Institute of Occupational Health and Occupational Disease Prevention,Jinan 250062,China
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Abstract: Objective To investigate the effect of N-acetylcysteine(NAC)on autophagy activation of human bronchial epithelial cells(HBECs)induced by toluene diisocyanate(TDI). Methods (1)Prepare TDI-human serum albumin(HSA)conjugate,the levels of TDI and HSA in conjugate were determined by Gutmann method and BCA protein quantitative method.(2)HBECs were treated with different concentrations of TDI-HSA(0,40.00,80.00 and 120.00 mg/L,respectively)for 12 h,then,detect the reactive oxygen species(ROS)level with living cell imaging technology,determine the levels of IL-4 and IL-6 in cell supernatant by enzyme-linked immunosorbent assay(ELISA)and measure the expression levels of autophagy-related proteins including AMPK,p-AMPK,mTOR,p-mTOR,Beclin1,LC3 and p62,etc.using Western blot method.(3)HBECs were pretreated with NAC(5 mmol/L)for 4 h and exposed to TDI-HSA(120.00 mg/L)for 12 h,then,detect the levels of ROS,IL-4 and IL-6 of cells,and the expression levels of autophagy related proteins such as AMPK,p-AMPK,mTOR,p-mTOR,Beclin1,LC3 and p62. Results The results showed that:(1)The concentration of TDI and HSA was 21.90 mg/L and 1 955.00 mg/L,respectively in synthesized TDI-HSA conjugate,the molar ratio was 4.41.(2)With the increase of the TDI-HSA exposure dose,the levels of ROS,IL-4 and IL-6 in HBECs increased significantly,the difference was significant(P<0.05). Additionally,some ratios of autophagy-related proteins p-AMPK/AMPK,LC3Ⅱ/LC3Ⅰand the expression of Beclinl all increased significantly in the 80.00 and 120.00 mg/L dose groups,while the ratio of p-mTOR/mTOR and the expression of p62 were significantly reduced(P<0.05).(3)Compared with 120.00 mg/L dose group,the levels of ROS,IL-4 and IL-6 in TDI-HSA+NAC group were significantly reduced(P<0.05),meanwhile,the ratios of autophagy-related protein mentioned above such as p-AMPK/AMPK,LC3Ⅱ/LC3Ⅰand the expression of Beclin1 were significantly reduced,the ratio of p-mTOR/mTOR and the expression of p62 were increased significantly(P<0.05). Conclusion The results suggested that by inhibiting ROS level of HBECs induced by TDI-HSA,the autophagy of HBECs can be significantly inhibited,and the levels of IL-4 and IL-6 in cell supernatant can be reduced as well. |
Keywords: toluene diisocyanate(TDI) human bronchial epithelial cells(HBECs) N-acetylcysteine(NAC) reactive oxygen species(ROS) autophagy inflammatory factors |