摘要
目的 研究细胞外高迁移率族蛋白B1(HMGB-1)诱导人正常支气管上皮细胞(NHBE)炎性反应的作用机制.方法 实验分组为空白对照组、HMGB-1组、HMGB-1+RAGE抗体(RAGE-Ab)组、HMGB-1+JNK抑制剂SP600125组、RAGE-Ab组.采用免疫荧光技术分析RAGE蛋白的表达;ELISA实验检测NHBE细胞上清液中的炎性细胞因子TNF-α、IL-8、IL-10和MCP-1的水平;采用蛋白免疫印迹分析RAGE、p-JNK和p-NF-κB p65蛋白的表达.结果 HMGB-1能剂量依赖性的诱导RAGE蛋白的表达.在浓度为5和10μg/ml时,与空白对照组相比HMGB-1能显著增加荧光的强度(P<0.05),HMGB-1+RAGE-Ab组RAGE表达显著下降(P<0.05),而HMGB-1+SP600125组RAGE蛋白的表达没有显著变化.与空白对照组相比,用HMGB-1处理后TNF-α、IL-8、IL-10和MCP-1的水平均显著增加(均P<0.05),HMGB-1+RAGE-Ab组和HMGB-1+SP600125组各炎性因子的释放均显著下降(均P<0.05).与空白对照组比较,用HMGB-1处理NHBE细胞后RAGE、p-JNK和p-NF-κB p65蛋白表达呈现剂量依赖性增加(P<0.05),HMGB-1+RAGE-Ab组中RAGE、p-JNK和p-NF-κB p65蛋白的表达显著下降(P<0.05),而HMGB-1+SP600125组中p-JNK和p-NF-κB p65蛋白的表达显著下降(P<0.05),但RAGE的表达没有显著变化.结论 NHBE细胞中HMGB-1诱导的炎性反应是通过RAGE/JNK/NF-κB信号通路介导的.
Objective To investigate the mechanism of extracellular high mobility group box 1 (HMGB-1)-induced inflammatory response in normal human bronchial epithelial cells(NHBE). Methods The subjects were divided into the control group,HMGB-1 group,HMGB-1+RAGE antibody(RAGE-Ab) group,HMGB-1+JNK inhibitor SP600125 group,and RAGE-Ab group. Immunofluorescence technique was used to analyze the expression of RAGE protein. ELISA assay was used to determine the levels of inflammatory cytokines TNF-α,IL-8,IL-10 and MCP-1 in NHBE cell supernatant. Western blotting was used to analyze the expression of RAGE,p-JNK and p-NF-κB p65 protein. Results HMGB-1 could dose-dependently induce RAGE protein expression. HMGB- 1 could significantly increase the intensity of fluorescence at the concentrations of 5 and 10μg/ml compared with that in the control group(P<0.05). The RAGE expression decreased significantly in the HMGB-1+RAGE-Ab group(P<0.05),whereas there was no significant change in RAGE protein expression in the HMGB-1+SP600125 group. Compared with the blank control group,the levels of TNF-α,IL-8,IL-10,and MCP-1 significantly increased after the treatment with HMGB-1(P<0.05). The release of inflammatory factors significantly decreased in the HMGB-1+RAGE-Ab group and HMGB-1+SP600125 group (P<0.05). Compared with the blank control group,the protein expression of RAGE,p-JNK and p-NF-κB p65 dose-dependently increased after the treatment of NHBE cells with HMGB-1(P<0.05). The protein expression of RAGE,p-JNK and p-NF-κB p65 in the HMGB-1+RAGE-Ab group significantly decreased(P<0.05). The protein expression of p-JNK and p-NF-κB p65 in the HMGB-1+SP600125 group significantly decreased (P<0.05),whereas there was no significant change in the expression of RAGE. Conclusion The inflammatory response induced by HMGB-1 in the NHBE cells is mediated through the RAGE/JNK/NF-κB signaling pathway.
出处
《中华生物医学工程杂志》
CAS
2018年第1期7-12,共6页
Chinese Journal of Biomedical Engineering
基金
江苏省卫生计生委基金项目(Z20627)%江苏省333高层次人才基金项目(BRA2017298)%徐州市医学青年后备人才基金 Foundation of Jiangsu Provincial Health and Family Planning Commission(Z20627)%Jiangsu 333 High Level Talent Foundation Project(BRA2017298)%Xuzhou Medical Youth Reserve Fund
