摘 要: 目的 探讨线粒体过度分裂对甲苯二异氰酸酯(TDI)诱导人支气管上皮细胞(HBECs)NLRP3炎性小体激活的影响。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(0.00、40.00、80.00和120.00 mg/L)处理HBECs 12 h,采用荧光探针法检测细胞中活性氧(ROS)水平,酶联免疫吸附法(ELISA)检测细胞上清液中白细胞介素-18(interleukin-18,IL-18)和白细胞介素-1β(interleukin-1β,IL-1β)的水平,蛋白质印迹法(Western blot)检测NLRP3炎性小体相关蛋白(ASC、NLRP3、Caspase-1)的表达水平以及线粒体分裂蛋白DRP1、融合相关蛋白MFN2和OPA1的表达水平。(3)采用线粒体分裂抑制剂Mdivi-1(10 μmol/L)预处理HBECs 2 h后,再用TDI-HSA(120.00 mg/L)处理12 h,检测细胞中ROS及IL-18、IL-1β的水平,测定NLRP3炎性小体相关蛋白ASC、NLRP3、Caspase-1,线粒体分裂蛋白DRP1及融合相关蛋白MFN2、OPA1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22.80、2 120.00 μg/L,摩尔比为4.19。(2)与对照组相比,随着TDI-HSA染毒剂量的增加,HBECs细胞中R0S和IL-18、IL-1β水平明显增加,差异有统计学意义(P<0.05);与对照组相比,TDI-HSA 80.00、120.00 mg/L剂量染毒组NLRP3炎性小体相关蛋白ASC、NLRP3、Cleaved Caspase-1的表达水平增加(P<0.05);线粒体分裂蛋白DRP1表达水平增加,融合相关蛋白MFN2、OPA1表达降低(P<0.05)。(3)与TDI-HSA 120.00 mg/L染毒组相比,TDI-HSA+Mdivi-1组HBECs细胞中ROS和IL-18、IL-1β水平明显降低,差异具有统计学意义(P<0.05);NLRP3炎性小体相关蛋白ASC、NLRP3、Cleaved Caspase-1的表达明显降低,差异均有统计学意义(P<0.05);线粒体分裂蛋白DRP1表达明显降低,融合相关蛋白MFN2、OPA1表达升高,差异有统计学意义(P<0.05)。结论 TDI可以剂量依赖性方式促进HBECs线粒体过度分裂及融合抑制,促进NLRP3炎性小体活化。采用线粒体分裂抑制剂Mdivi-1处理HBECs后,可显著抑制NLRP3炎性小体的活化,表明TDI诱导的线粒体过度分裂是NLRP3炎性小体活化的重要原因。 |
关键词: 甲苯二异氰酸酯(TDI) 人支气管上皮细胞(HBECs) 线粒体 线粒体分裂抑制剂 炎性小体 |
中图分类号: R994.3
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 mitochondrial division on activation of inflammatory bodies NLRP3 of human bronchial epithelial cell induced by toluene diisocyanate |
ZHANG Xiaoxia,YANG Xiaohan,DONG Shuangyan,LI Ming,LI Chao,MENG Xiangjing,JIA Qiang
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Shandong First Medical University/Shandong Provincial Academy of Medical Sciences/Shandong Provincial Academy of Occupational Health and Occupational Medicine,Jinan,Shandong 250062,China
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Abstract: Objective To investigate the effect of mitochondrial over-division on activation of NLRP3 inflammasomes in human bronchial epithelial cells(HBECs)induced by toluene diisocyanate(TDI). Methods (1)TDI human serum albumin(HSA)conjugate was prepared,and the levels of TDI and HSA in the conjugate were determined by Gutmann method and BCA protein quantitative method,respectively.(2)HBECs were treated with different concentrations of TDI-HSA(0.00,40.00,80.00,and 120.00 mg/L)for 12 h,the levels of reactive oxygen species(ROS)in cells were detected by fluorescence probe method,the levels of IL-18 and IL-1β in cell supernatant were detected by enzyme-linked immunosorbent assay(ELISA)method,furthermore,the expression levels of NLRP3 inflammasome-related proteins ASC,NLRP3,Caspase-1 as well as the expression levels of mitochondrial mitogen DRP1,fusion-related proteins MFN2 and OPA1 were detected by Western blot.(3)After two hours pretreatment with Mdivi-1(10 μmol/L),the HBECs were poisoned with TDI-HSA(120.00 mg/L)for 12 h,then detect the levels of ROS,IL-18,IL-1β and the expressions of NLRP3 inflamnasome-related proteins ASC,NLRP3 and Caspase-1 as well as the mitochondrial mitogen DRP1,fusion-related proteins MFN2 and OPA1 in cells. Results The results showed that(1)the mass concentrations of TDI and HSA in synthesized TDI-HSA conjugate were 22.80 μg/L and 2 120.00 μg/L,respectively,and the molar ratio was 4.19.(2)Compared with control group,with the increase of TDI-HSA exposed dose,the levels of ROS,IL-18 and IL-1β in HBECs cells were significantly increased(P<0.05),the expression levels of NLRP3 inflammasome-related proteins ASC,NLRP3 and Cleaved Caspase-1 in 80.00 mg/L and 120.00 mg/L TDI-HSA groups also increased significantly(P<0.05),the expression level of mitochondrial mitogen DRP1 was significantly increased,the expressions of fusion-related proteins MFN2 and OPA1 were decreased significantly(P<0.05).(3)Compared with 120.00 mg/L TDI-HSA group,the levels of ROS,IL-18 and IL-1β in HBECs of TDI-HSA+Mdivi-1 group were significantly reduced(P<0.05),meanwhile,the expressions of NLRP3 inflammasome-related proteins ASC,NLRP3,Cleaved Caspase-1 and mitochondrial mitogen DRP1 were all decreased significantly(P<0.05),the expressions of fusion-related proteins MFN2 and OPA1 were significantly increased(P<0.05). Conclusion The results suggested that TDI could promote mitochondrial excessive division,fusion inhibition of HBECs and NLRP3 inflammasomes activation in a dose-dependent manner. After treatment of mitochondrial division inhibitor Mdivi-1,the activation of NLRP3 inflammasomes of HBECs could be significantly inhibited,which suggested TDI-induced mitochondrial excessive division was an important reason for the activation of NLRP3 inflammasomes. |
Keywords: toluene diisocyanate(TDI) human bronchial epithelial cells(HBECs) mitochondria mitochondrial mitotic inhibitor inflammasome |