目的:研究不可分型流感嗜血杆菌(NTHi)诱导肺组织炎症反应的关键信号通路.方法:肺组织与NTHi(1010 CFU/L)共孵育4 h和24 h.Western blotting检测肺组织的磷酸化p38丝裂原活化蛋白激酶(p38 MAPK),电泳迁移率法检测核因子κB(NF-κB)核转位,实时定量RT-PCR检测Toll样受体(TLR)2 mRNA,酶联免疫吸附试验检测上清的白细胞介素(IL)-8水平.另外,肺组织预先与抗TLR2单抗(anti-TLR2:5 mg/L)、p38 MAPK抑制剂(SB203580:20 μmol/L)或NF-κB抑制剂(PDTC:25 μmol/L)孵育2 h,再加入NTHi(1010 CFU/L)刺激24 h,收集组织上清,测定IL-8.结果:肺组织感染NTHi 4 h后,肺组织TLR2-p38 MAPK-NF-κB信号通路迅速被激活.感染24 h后,肺组织IL-8表达较未感染组显著增加 (P<0.05).抗TLR2单抗、特异性p38 MAPK和NF-κB分子阻断剂可以明显抑制NTHi诱导的IL-8表达.结论:NTHi通过TLR2-p38 MAPK-NF-κB信号通路诱导肺组织分泌炎症因子.人体外肺组织感染模型为研究病原体和宿主相互作用提供了新的平台.%AIM: To investigate the key signal pathways of inflammatory responses in lung tissues induced by the infection of nontypeable Haemophilus influenzae( NTHi ).METHODS: Human lung tissues were co - incubated with NTHi ( 10 CFU/L ) for 4 h and 24 h, respectively.The phosphorylation of p38 mitogen - activated protein kinase ( MAPK ) was detected by Western blotting.The nuclear translocation of nuclear factor ( NF ) - κB was examined by electrophoretic mobility shift assay ( EMSA ).The expression of Toll - like receptor ( TLR ) 2 was measured by real - time quantitative RT - PCR, and the level of interleukin ( IL ) - 8 was detected by enzyme - linked immunosorbent assay ( ELISA ).Furthermore, lung tissues were incubated with anti - TLR2 monoclonal antibody ( 5 mg/L ), p38 MAPK inhibitor SB203580 ( 20 μmol/L ), or NF - κB inhibitor PDTC ( 25 μmol/L ) for 2 h, then stimulated with NTHi ( 1010 CFU/L ) for another 24 h.The supernatants were collected for IL - 8 detection.RESULTS: The TLR2 - p38 MAPK - NF - κB signaling pathway in lung tissues was rapidly activated 4 h after NTHi stimulation.IL - 8 secreted from lung tissues infected with NTHi was significantly increased compared with uninfected lungs ( P ＜0.05 ).The pre - incubation with anti - TLR2 antibody, p38 MAPK inhibitor or NF - κB inhibitor markedly decreased IL - 8 production induced by NTHi.CONCLUSION: NTHi induces inflammatory responses in lung tissues by activation of TLR2 - p38 MAPK - NF - κB signaling pathway.Human lung infection model provides a new research tool for the study of interaction between pathogens and hosts.
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