问号钩端螺旋体溶血素Sph2经HMGB1放大炎症反应的研究  

Hemolysin Sph2 from Leptospira interrogans amplified inflammatory response in murine macrophages J774A.1 through HMGB1

作　　者：陈叙宏 高帅 孙爱华 严杰[1] 林旭瑷[1,3] Chen Xuhong;Gao Shuai;Sun Aihua;Yan Jie;Lin Xu′ai(Department of Medical Microbiology and Parasitology,Zhejiang University,School of Medicine,Hangzhou 310058,China;Faculty of Basic and Forensic Medicine,Hangzhou Medical College,Hangzhou 310053,China;National Clinical Research Center for Child Health,Children′s Hospital,Zhejiang University,School of Medicine,Hangzhou 310052,China)

机构地区：[1]浙江大学医学院病原生物学系,杭州310058 [2]杭州医学院基础医学与法医学院,310053 [3]浙江大学医学院附属儿童医院国家儿童健康与疾病临床医学研究中心,杭州310052

出　　处：《中华微生物学和免疫学杂志》2021年第8期592-597,共6页Chinese Journal of Microbiology and Immunology

基　　金：浙江省自然科学基金(LY17H190002)。

摘　　要：目的探讨问号钩端螺旋体溶血素Sph2经高迁移率组蛋白B1(high mobility group box-1,HMGB1)放大炎症反应的可能机制。方法一定浓度的rSph2与小鼠J774A.1巨噬细胞共孵育一定时间,乳酸脱氢酶(LDH)量的变化检测细胞的膜结构损伤情况;冷冻电镜观察细胞结构的改变;ELISA方法检测细胞上清液中HMGB1的含量变化。商品化的HMGB1与小鼠J774A.1巨噬细胞共孵育一定时间,Western blot检测NF-κB、p38-MAPK及JNK炎症信号通路关键分子的磷酸化水平;ELISA检测IL-1β、IL-6、KC(IL-8)等促炎细胞因子的表达情况。结果重组问号钩端螺旋体溶血素rSph2可导致J774A.1细胞明显的细胞核消失、细胞膜结构损伤、细胞裂解、膜泡胀;LDH释放量及HMGB1的含量明显增加;HMGB1增加NF-κB、p38-MAPK及JNK信号通路关键蛋白质的磷酸化水平;HMGB1上调J774A.1细胞IL-1β、IL-6、KC的表达且该上调作用可被3条通路抑制剂所抑制。结论重组问号钩端螺旋体溶血素rSph2诱导小鼠J774A.1巨噬细胞产生损伤并释放大量的HMGB1,HMGB1经NF-κB、p38-MAPK及JNK信号通路调控下游促炎细胞因子的表达,从而放大rSph2造成的炎症效应。Objective To investigate the possible mechanism of high mobility group box-1(HMGB1)in amplifing inflammatory responses in Leptospira interrogans hemolysin Sph2-treated J774A.1 macrophages.Methods Recombinant Sph2 was incubated with J774A.1 macrophages.The damage of cell membrane was detected by lactate dehydrogenase(LDH)determination;the changes of cell structure were observed by cryo-electron microscope;ELISA was used to determine the expression of HMGB1.After the commercial recombinant HMGB1 was incubated with mouse J774A.1 macrophages,the phosphorylation of NF-κB,p38-MAPK and JNK signaling pathway wsa detected by Western blot,and the expression of IL-1β,IL-6,and KC(IL-8)was detected by ELISA.Results Recombinant hemolysin rSph2 induced significant changes in the structures of J774A.1 cells,including nucleus disappearance,cell membrane structure damage,cell lysis and membrane swelling.The yields of LDH and HMGB1 also increased significantly.Phosphorylated-NF-κB,-p38-MAPK and-JNK were increased by HMGB1.The expression of IL-1β,IL-6 and KC in J774A.1 cells was up-regulated by HMGB1 and inhibited via inhibitors of NF-κB,p38-MAPK and JNK signal pathways.Conclusions Hemolysin rSph2 damaged the membrane of J774A.1 cells,and induced the secretion of HMGB1.Secreted-HMGB1 might induce the expression of IL-1β,IL-6 and KC in J774A.1 cells via NF-κB,p38-MAPK and JNK signal pathways,thus amplifying the inflammatory responses caused by Sph2.

关 键 词：问号钩端螺旋体 溶血素Sph2 HMGB1 促炎细胞因子 炎症放大反应 

分 类 号：R514.4[医药卫生—内科学]