摘要
目的探讨高迁移率族蛋白1(HMGB1)在甲苯二异氰酸酯(TDI)诱导人支气管上皮细胞(HBECs)中核苷酸结合寡聚化结构域样受体家族含热蛋白结构域蛋白3(NLRP3)炎症小体活化中的作用。方法 (1)TDI-人血清白蛋白(HSA)刺激实验:将对数生长期HBECs随机分为对照组和低、中、高剂量组,分别予剂量为0.00、40.00、80.00、120.00 mg/L的TDI-HSA偶联物处理12 h。(2)HMGB1表达抑制实验:将对数生长期HBECs随机分为对照组、TDI-HSA处理组、TDI-HSA+阴性-siRNA组和TDI-HSA+HMGB1-siRNA组,TDI-HSA+阴性-siRNA组和TDI-HSA+HMGB1-siRNA组细胞分别利用携带阴性-siRNA慢病毒和携带HMGB1-siRNA慢病毒感染后,与TDI-HSA处理组细胞均予剂量为120.00 mg/L的TDI-HSA处理12 h,对照组细胞不予TDI-HSA处理。(3)各组细胞采用蛋白质印迹法检测HMGB1、NLRP3、凋亡相关斑点样蛋白(ASC)、半胱氨酸天冬氨酸蛋白酶(caspase-1)前体(pro-caspase-1)和caspase-1 p20蛋白表达,以免疫荧光法观察TDI-HSA刺激实验中NLRP3和caspase-1炎症小体数量。结果 (1)TDI-HSA刺激实验:中、高剂量组HBECs中HMGB1和ASC蛋白相对表达水平均高于对照组(P值均<0.01);3个剂量组HBECs中NLPR3-casepase-1 p20蛋白相对表达水平和NLRP3-caspase-1炎症小体计数均高于对照组(P值均<0.01);随着TDI-HSA染毒剂量的升高,HBECs中NLRP3-caspase-1炎症小体计数增加(P值均<0.01)。(2)HMGB1表达抑制实验:TDI-HSA处理组和TDI-HSA+阴性-siRNA组HBECs中HMGB1、NLRP3、ASC、pro-caspase-1、caspase-1 p20的蛋白相对表达水平均高于对照组(P值均<0.01);TDI-HSA+HMGB1-siRNA组HBECs中上述指标均低于TDI-HSA处理组和TDI-HSA+阴性-siRNA组(P值均<0.01)。结论 TDI处理HBECs可诱导HMGB1蛋白表达增加,活化NLPR3炎症小体;抑制HMGB1表达可下调NLPR3及其相关蛋白的表达。
Objective To investigate the role of high mobility group protein 1(HMGB1) in toluene diisocyanate(TDI) induced nucleotide-binding oligomerization domain like receptor family pyrin domain-containing 3(NLRP3) inflammasome activation in human bronchial epithelial cells(HBECs). Methods i) The TDI-human serum albumin(HSA) stimulation experiment: the HBECs in logarithmic growth phase were randomly divided into control group, low-, medium-and high-dose groups that were pretreated with TDI-HSA with the final concentration of 0.00, 40.00, 80.00 and 120.00 mg/L for 12 hours. ii) The HMGB1 expression inhibition experiment: the HBECs in logarithmic growth phase were divided into control group, TDI-HSA group, TDI-HAS+negative-siRNA group, and TDI-HAS+HMGB1-siRNA group. The cells in TDI-HAS+negative-siRNA group and TDI-HAS+HMGB1-siRNA group were infected with HBECs with negative-siRNA lentivirus and HMGB1-siRNA lentivirus, respectively. Cells in these two groups and the TDI-HSA group were treated with 120.00 mg/L of TDI-HSA for 12 hours. The cells in the control group were not treated with TDI-HAS. iii) The expression of HMGB1, NLRP3, apoptosis-associated speck-like protein containing CARD(ASC), pro-caspase-1 and caspase-1 p20 proteins in all groups were detected by Western blot. The number of NLRP3 and caspase-1 inflammasome in TDI-HSA stimulation experiment was observed by immunofluorescence method. Results i) TDI-HSA stimulation experiment: the relative protein expression of HMGB1 and ASC was higher in HBECs of medium-and high-dose groups than that of control group(all P values were <0.01). The relative protein expression of NLRP3 and casepase-1 p20 and the number of NLRP3-caspase-1 inflammasome were higher in HBECs of 3 dose groups than that of control group(all P values were <0.01). The number of NLRP3-caspase-1 inflammasome in HBECs increased obviously in low-, medium-and high-dose groups as compared to the control group(all P values were <0.05). The number of NLRP3-caspase-1 inflammasome in HBECs increased with the increase of TDI-HSA dose(all P values were <0.01). ii) The HMGB1 expression inhibition experiment: the relative protein expression of HMGB1, NLRP3, ASC, pro caspase-1 and caspase-1 p20 in HBECs were higher in the TDI-HSA group and TDI-HSA + negative-siRNA group than those of the control group(all P values were <0.01). The above indexes of HBECs were lower in the TDI-HAS + HMGB1-siRNA group than those in the TDI-HSA group and TDI-HSA + negative-siRNA group(all P values were <0.01).Conclusion TDI treatment in HBECS can induce the increase of HMGB1 protein expression and activate NLPR3 inflammasome. Inhibition of HMGB1 expression can down-regulate the expression of NLPR3 and its related proteins.
作者
焦博
杨晓涵
张晓帆
厉铭
李超
张玉
薄存香
赛林霖
贾强
JIAO Bo;YANG Xiaohan;ZHANG Xiaofan;LI Ming;LI Chao;ZHANG Yu;BO Cunxiang;SAI Linlin;JIA Qiang(Shandong Academy of Occupational Health and Occupational Medicine,Shandong First Medical University&Shandong Academy of Medical Sciences,Jinan,Shandong 250062,China)
出处
《中国职业医学》
CAS
北大核心
2020年第5期526-532,共7页
China Occupational Medicine
基金
国家自然科学基金(81872603)
山东省自然科学基金(ZR2016YL015,ZR2017YL001)。
