摘要
目的通过体外实验探讨高迁移率族蛋白B1(high mobility group box 1,HMGB1)在甲苯二异氰酸酯(toluene diisocyanate,TDI)所致的职业性哮喘中趋化中性粒细胞的作用机制。方法制备TDI-人血清白蛋白(human serum albumin,HSA)偶联物(TDI-HSA),并通过Gutmann法与BCA法分别测定偶联物中TDI及HSA含量。分别以0、40、80、120 mg/L TDI-HSA染毒人支气管上皮细胞(human bronchial epithelial cells,HBECs)12 h,ELISA法检测细胞培养上清中白细胞介素-8(interleukin-8,IL-8)、趋化因子12(C-X-C motif chemokine 12,CXCL12)的水平;Western blot法检测细胞中HMGB1、CXCL12及核因子κB(nuclear factor kappa-B,NF-κB)相关蛋白的表达;活细胞荧光探针法检测活性氧(reactive oxygen species,ROS)的水平;免疫荧光法观察HMGB1的核转位情况。使用不同浓度(100、200 mmol/L)甘草素(glycyrrhizin,GL)预处理HBECs细胞24 h抑制HMGB1后,继续用TDI-HSA染毒12 h,检测细胞培养上清中IL-8水平,HMGB1、CXCL12及NF-κB相关蛋白表达,ROS释放水平及HMGB1的核转位情况。结果不同浓度TDI-HSA染毒HBECs 12 h后,随着染毒浓度的升高,HBECs细胞内HMGB1发生明显核转位,细胞中ROS和细胞上清中IL-8水平明显增加,差异有统计学意义(P<0.05);与对照组相比,120 mg/L染毒组HBECs细胞HMGB1、磷酸化P65蛋白表达显著增加,差异有统计学意义(P<0.05)。以含有不同浓度GL的培养基联合120 mg/L TDI-HSA处理HBECs细胞12 h后,与对照组相比,GL作用于HBECs细胞可显著抑制TDI-HSA导致的HMGB1的核转位,抑制HBECs细胞中的ROS和上清液中的IL-8水平的升高,显著减少细胞中HMGB1、磷酸化P65蛋白的表达,差异有统计学意义(P<0.05)。结论TDI可通过增加HBECs细胞HMGB1蛋白表达和核转位,激活NF-κB,促进IL-8的释放,不影响CXCL12的表达。GL可通过降低HMGB1表达,抑制NF-κB活化,减少IL-8释放水平。
Objective:To investigate the mechanism of high mobility group box 1(HMGB1)in chemotaxis of neutrophils in occupational asthma induced by toluene diisocyanate(TDI)in vitro.Methods:TDI and human serum albumin(TDI-HSA)conjugate were prepared,and the contents of TDI and HSA in the conjugate were determined by Gutmann method and BCA method,respectively.On 0,40,80,120 mg/L TDI-HSA infected human bronchial epithelial cells(human bronchial epithelial cells,HBECs)12 h,ELISA method to detect cell culture supernatant of interleukin 8(interleukin-8,IL-8)and C-X-C motif chemokine 12(CXCL12)levels;Western blot was used to detect the expression of HMGB1,CXCL12 and nuclear factor kappa-B(NF-κB)related proteins in cells.The level of reactive oxygen species(ROS)was detected by live cell fluorescent probe method.The nuclear translocation of HMGB1 was observed by immunofluorescence.HBE cells were pretreated with different concentrations(100 and 200 mmol/L)of glycyrrhizin(GL)for 24 h to inhibit HMGB1,and then treated with TDI-HSA for 12 h.The level of IL-8 in cell culture supernatant was detected.The expression of HMGB1,CXCL12 and NF-κB related proteins,the level of ROS release and the nuclear translocation of HMGB1 were observed.Results:HBE cells were exposed to different concentrations of TDI-HSA(0,40,80,120 mg/L)for 12 h.The results showed that nuclear translocation of HMGB1 occurred in HBECs with the increase of TDI-HSA concentration.ROS in the cells and the cell supernatant of IL-8 levels increased significantly(P<0.05);Compared with the control group,the 120 mg/L exposure group had significant increases in the protein expression of HMGB1 and phosphorylated P65 in HBECs cells(P<0.05).Not found HBE cells CXCL12 protein expression in cells with TDI-HSA infected concentration increase is on the rise,in cell supernatant CXCL12 levels are not checked out.In order to further verify the regulatory role of HMGB1,useing different concentrations(100,200 mmol/L)GL medium and 120 mg/L TDI-HSA on cells after 12 h,the results showed that compared with the control group,100 and 200 mmol/L GL effected on HBE cells can inhibit the nuclear transfer caused by TDI-HSA,HMGB1 can significantly inhibit suppression ROS in the HBE cells and rise the level of IL-8 in the supernatant.The expression of HMGB1 and phosphorylated P65 protein in the cells was significantly reduced(P<0.05).Conclusions:TDI can increase the expression and nuclear translocation of HMGB1,activate NF-κB and promote the release of IL-8 in HBE cells,but does not affect the expression of CXCL12.GL can reduce the expression of HMGB1,inhibit the activation of NF-κB,and reduce the release of IL-8.
作者
翟美玲
厉铭
孟祥敬
李超
杨晓涵
马洁
潘志峰
贾强
ZHAI Meiling;LI Ming;MENG Xiangjing;LI Chao;YANG Xiaohan;MA Jie;PAN Zhifeng;JIA Qiang(Shandong Academy of Occupational Health and Occupational Medicine,Toxicology laboratory,Jinan 250062,China;Shandong First Medical University&Shandong Academy of Medical Sciences,Jinan 250117,China)
出处
《山东第一医科大学（山东省医学科学院）学报》
CAS
2024年第9期519-527,共9页
Journal of Shandong First Medical University ＆ Shandong Academy of Medical Sciences
基金
国家自然科学基金(81872603)。
