Monoclonal antibody against Toll-like receptor 4 attenuates ventilator-induced lung injury in rats by inhibiting MyD88-and NF-κB-dependent signaling
Monoclonal antibody against Toll-like receptor 4 attenuates ventilator-induced lung injury in rats by inhibiting MyD88-and NF-κB-dependent signaling
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DOI：
10.3892/ijmm.2017.2873
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发表时间：
2017-03-01
影响因子：
5.4
通讯作者：
Fu, Ruili
中科院分区：
文献类型：
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作者：
Huang, Cuiyuan;Pan, Linghui;Fu, Ruili
机械通气引起非感染性炎症性疾病和肺损伤的机制尚不清楚。这种类型损伤的动物模型表明，它涉及toll样受体（TLR）4和9介导的信号传导。在本研究中，为了进一步了解TLR4在这类损伤中的作用，我们进行了体内和体外实验，以确定TLR4触发炎症的机制。我们还研究了使用TLR4单克隆抗体（mAb）是否可以减轻这种类型的损伤。为此，将大鼠切开气管，气管内注射抗TLR4单抗或生理盐水，然后以40 ml/kg的高潮气量（HTV）通气4小时或在相同时间内允许自主呼吸（对照组）。从大鼠支气管肺泡灌洗液（BALF）中分离肺泡巨噬细胞（AMs），在抗tlr4单抗存在或不存在的情况下，用肿瘤坏死因子(TNF)- α刺激16小时。通过检测肺组织病理学、肺干湿比、BALF总蛋白和BALF及血浆中细胞因子水平评估肺损伤。测定培养巨噬细胞中TLR4、TLR9、髓样分化因子88 （Myd88）、核因子- κ B mRNA和蛋白表达水平。与对照组（自发呼吸）相比，通气大鼠表现出更大的肺通透性，更严重的炎症细胞浸润/肺水肿，以及BALF和血浆中更高水平的白细胞介素(IL)-1 β、IL-6和tnf - α。通气大鼠AMs中TLR4、TLR9、Myd88和NF-kappa B mRNA和蛋白表达水平高于自然呼吸大鼠。经抗TLR4单抗预处理的通气大鼠表现出明显减弱的通气损伤迹象，如肺部炎症和肺水肿减轻，BALF细胞减少，BALF和血浆中il和tnf - α水平降低。同样，当tnf - α与抗TLR4单抗联合使用时，am中TLR4、Myd88和nf - κ B mRNA和蛋白表达的tnf - α依赖性增加比单独使用tnf - α时减弱。同时给予抗tlr4单抗也降低了tnf - α依赖性il的分泌。总的来说，我们的数据表明，TLR4通过激活Myd88/NF-kappa B通路，对通气诱导的肺损伤有重要作用，并且通过抑制Myd88/NF-kappa B信号传导，用抗TLR4单抗预处理大鼠，部分保护它们免受这种类型的损伤。
The mechanisms through which mechanical ventilation causes non-infectious inflammatory diseases and lung injury are poorly understood. Animals models of this type of injury suggest that it involves signaling mediated by Toll-like receptor (TLR)4 and 9. In this study, in order to gain further insight into the involvement of TLR4 in this type of injury, we performed in vivo and in vitro experiments to determine the mechanisms through which TLR4 triggers inflammation. We also examined whether the use of TLR4 monoclonal antibody (mAb) can alleviate this type of injury. For this purpose, rats were tracheotomized and administered intratracheal injections of anti TLR4 mAb or saline, and then ventilated for 4 h at a high tidal volume (HTV) of 40 ml/kg or allowed to breathe spontaneously for the same period of time (controls). Alveolar macrophages (AMs) were isolated from the bronchoalveolar lavage fluid (BALF) of the rats and stimulated for 16 h with tumor necrosis factor (TNF)-alpha in the presence or absence of anti-TLR4 mAb. Lung injury was assessed by examining lung histopathology, lung wet/dry weight ratio, BALF total protein and cytokine levels in BALF and plasma. The mRNA and protein expression levels of TLR4, TLR9, myeloid differentiation factor 88 (Myd88) and nuclear factor (NF)-kappa B were measured in cultured macrophages. Compared to the controls (spontaneous breathing), the ventilated rats exhibited greater pulmonary permeability, more severe inflammatory cell infiltration/lung edema, and higher levels of interleukin (IL)-1 beta, IL-6 and TNF-alpha in BALF and plasma. The AMs from the ventilated rats expressed higher mRNA and protein levels of TLR4, TLR9, Myd88 and NF-kappa B compared with the macrophages from the spontaneously breathing rats. The ventilated rats pre-treated with anti TLR4 mAb exhibited markedly attenuated signs of ventilation-induced injury, such as less lung inflammation and pulmonary edema, fewer cells in BALF, and lower levels of ILs and TNF-alpha in BALF and plasma. Similarly, the TNF-alpha-dependent increases in the mRNA and protein expression of TLR4, Myd88 and NF-kappa B in AMs were attenuated when TNF-alpha was co-administered with anti TLR4 mAb than when TNF-alpha was administered alone. Co-administering anti-TLR4 mAb also reduced the TNF-alpha-dependent secretion of ILs. On the whole, our data demonstrate that TLR4 contributes significantly to ventilation-induced lung injury by activating the Myd88/NF-kappa B pathway, and pre-treating rats with anti-TLR4 mAb partially protects them against this type of injury by inhibiting Myd88/NF-kappa B signaling.