p38丝裂原活化蛋白激酶在不可分型流感嗜血杆菌致人单核细胞炎症反应中的作用

The role of p38 mitogen activated protein kinase in the inflammatory response of human monocytes infected with nontypeable haemophilus influenzae

目的 通过不可分型流感嗜血杆菌（NTHi）与单核细胞相互作用，研究丝裂原活化蛋白激酶（MAPK）信号传导通路在NTHi致人体免疫细胞炎症反应中的作用。方法NTHi为临床分离株，经血清学方法和16SrRNA测序证实。外周血单核细胞来自健康成年人静脉血，分为4组：培养基组、NTHi刺激组、SB203580（p38MAPK抑制剂）干预组和U0126（p44／42MAPK抑制剂）干预组。NTHi与单核细胞共培养1h、4h后收集细胞，用Western blot法检测p38、p44／42 MAPK的磷酸化程度；16h后用流式细胞仪检测细胞表面Toll样受体（TLR）4的表达。预先用SB203580或U0126与单核细胞共孵育1h，然后加入NTHi（感染复数为200），分别在4h、16h后收集上清，用酶联免疫吸附法检测肿瘤坏死因子α（TNF-α）的水平。采用SPSS11．5统计软件，组间比较用t检验，单核细胞TNF-俚的表达用单因素方差分析，组间比较用LSD检验。结果NTHi可迅速诱导p38、p44／42 MAPK通路的磷酸化，并至少持续到刺激后4h。与培养基组比较，NTHi刺激16h后单核细胞表面TLR4的表达明显增加（11．8±1．6，4．8±0．6），差异具有统计学意义（t=4．08，P〈0．05）。NTHi刺激4h和16h后上清液中的TNF-α（16．4±5．3，30．2±10．7）较培养基组（0．6±0．6，1．4±1．1）显著增加，差异具有统计学意义（4h时I-J值为15．78，16h时I-J值为28．82，P均〈0．01）。与细菌组比较，SB203580干预组单核细胞TNF-α水平显著降低（4h时I—J值为11．26，16h时I—J值为21．32，P均〈0．05），而U0126干预组TNF-α水平无明显变化（4h时I-J值为6．32，16h时I-J值为12．57，P均〉0．05）。结论 TLR4可能参与了NTHi诱导的单核细胞反应，p38 MAPK是该反应的关键信号分子。

Objective To investigate the role of mitogan activated protein kinase （MAPK） pathway in the inflammatory response induced by nontypeable haemophilus influenzae （NTHi）. Methods NTHi was a clinical isolate identified by serum agglutination test and 16S rRNA gene sequencing. The peripheral monocytes isolated from adult healthy donors were divided into medium-treated group, NTHi-stimulated group, SB203580 （ p38 MAPK inhibitor ） -pretreated group and UO126 （ p44/42 MAPK inhibitor ） -pretreated group. Monocytes were co-cultured with NTHi and harvested 1 h and 4 h after stimulation. The phosphorylation of p38 and p44/42 MAPKs was detected by Western blot. The expression of toll-like receptor 4 （TLR4） was examined by flow cytometry 16 h after bacterial stimulation. Monocytes were preincubated with SB203580 or UO126 for I h and then stimulated with NTHi for4 h and 16 h, respectively. The level of TNF-α in the supernatants was determined by Enzyme-linked immunosorbent assay （ ELISA ）. Results The phosphorylation of p38 MAPK and p44/42 MAPK was rapidly induced by NTHi and continued for at least 4 h after stimulation. The expression of TLR4 on monocytes after NTHi stimulation was siguificanfly up-regulated compared with the control group （ 11.8 ±1.6 vs 4. 8 ± 0. 6, P 〈 0. 05 ）. The level of TNF-α in the supornatants was increased 4 h and 16 h after bacterial stimulation compared with the control group （4h：16.4±5.3 vs 0.6±0. 6,P〈0.01;24 h：30.2±10.7 vs 1.4±1.1,P〈0.01）. SB203580 pretreatment decreased remarkably the TNF-α secretion from monecytes （P 〈 0. 05 ） whereas U0126 had no significant effect on TNF-α level （P 〉 0.05）. Conclusion TLR4 is probably involved in the inflammatory response of monocytes induced by NTHi whereas p38 MAPK is the key signal molecule in this inflammatory reaction.