目的探讨脂磷壁酸(LTA)对人肺泡巨噬细胞(AM)凋亡及炎症因子释放的影响和莫西沙星(MXF)对其反应的抑制作用。方法收集、提纯及体外培养人AM,LTA刺激4h后,加或不加MXF与其共孵育,于各实验终点用MTr法计算细胞相对活力,光学显微镜观察细胞形态,流式细胞术检测细胞凋亡率,RT-PCR法检测TLR2、IL-1B、IL-8及TNF-α的mRNA水平,ELISA检测IL-8蛋白水平,验证RT-PCR。结果LTA对AM有细胞毒性,并呈浓度递增关系(P〈0.05),MXF对AM活力无影响(P〉0.05),且可抑制LTA的毒性作用(P〈0.05)。LTA促进AM凋亡(P〈0.05),此作用可被MXF抑制(P〈0.05)。TLA上调AM中TLR2、IL-1β、IL-8及TNF-α的mRNA表达(P〈0.05),各峰值时间及峰值分别为:12h(3.56±0.03)、6h(46.63±7.06)、12h(28.07±1.24)、3h(2.34±0.50),上调24h IL-8蛋白水平,上述效应可被MXF抑制。结论LTA对人AM有细胞毒性,促进AM凋亡,上调AM中TLR2及炎症因子IL-1β、IL-8及TNF-α的表达,MXF抑制LTA诱导的AM炎症及凋亡,可能在革兰氏阳性菌肺炎中发挥杀菌、抗炎、保护宿主AM免疫活性的作用。
Objective To investigate the lipoteichoic acid (LTA) induced apoptosis and the expression of inflammatory cytokines in human alveolar macrophage (AM) and the anti-apoptotic and anti-inflamatory effect of moxifloxacin (MXF). Methods Obtained human AM from bronchoalveolar lavage and used MTT assay to observe the effects of LTA and MXF on cell activity, optical microscope to investigate the change of the cell morphology, flow cytometry to assess cell apoptosis, RT-PCR to detect the mRNA levels of TLR2, IL-1β, IL-8 and TNF-α, ELISA for the production of IL-8 to exam RT-PCR. Results LTA showed cytotoxicity on AM in a dose-dependent manner ( P〈0.05 ) ; MXF inhibited the effect of LTA without cyto- toxicicy (P〈0.05). LTA promoted apoptosis ( P〈0.05 ) and the mRNA expressions of TRL2, IL-1 β, IL-8 and TNF-α significantly in AM ( P〈0.05 ), the peaks and peak time of the above factors were ( 3.56±0.03 ) at 12 h, (46.63±7.06) at6 h, (28.07±1.24) at 12 h and (2.34±0.50) at 3 h respectively and increased the release of IL-8 protein level at 24 h ( P〈0.05 ). MXF inhibited the cell apoptosis and the above mRNAexpression at 1 2 h ( P 〈 0.0 5 ) , and inhibited the IL-8 protein level at 24 h( P〈0.05 ). Conclusion LTA showed cytotoxicity on AM, induced AM apoptosis and increased the expression of TLR2, IL-1 β, IL-8 and TNF-α of AM ; MXF could protect AM through inhibiting of the above effects and may play a key role beside bactericidal effect in gram-positive bacteria pneumonia.