Sun Xi Jia, Li Xiao Qian, Wang Xiao Long, Tan Wen Fei, Wang Jun Ke
Department of Anesthesiology, First Affiliated Hospital, China Medical University, Shenyang 110001, Liaoning, China.
PLoS One. 2015 Apr 14;10(4):e0122752. doi: 10.1371/journal.pone.0122752. eCollection 2015.
Infection is a common cause of acute lung injury (ALI). This study was aimed to explore whether Toll-like receptors 4 (TLR4) of airway smooth muscle cells (ASMCs) play a role in lipopolysaccharide (LPS)-induced airway hyperresponsiveness and potential mechanisms.
In vivo: A sensitizing dose of LPS (50 µg) was administered i.p. to female mice before anesthesia with either 3% sevoflurane or phenobarbital i.p. After stabilization, the mice were challenged with 5 µg of intratracheal LPS to mimic inflammatory attack. The effects of sevoflurane were assessed by measurement of airway responsiveness to methacholine, histological examination, and IL-1, IL-6, TNF-α levels in bronchoalveolar lavage fluid (BALF). Protein and gene expression of TLR4 and NF-κB were also assessed. In vitro: After pre-sensitization of ASMCs and ASM segments for 24h, levels of TLR4 and NF-κB proteins in cultured ASMCs were measured after continuous LPS exposure for 1, 3, 5, 12 and 24h in presence or absence of sevoflurane. Constrictor and relaxant responsiveness of ASM was measured 24 h afterwards.
The mRNA and protein levels of NF-κB and TLR4 in ASM were increased and maintained at high level after LPS challenge throughout 24h observation period, both in vivo and in vitro. Sevoflurane reduced LPS-induced airway hyperresponsiveness, lung inflammatory cell infiltration and proinflammatory cytokines release in BALF as well as maximal isometric contractile force of ASM segments to acetylcholine, but it increased maximal relaxation response to isoproterenol. Treatment with specific NF-κB inhibitor produced similar protections as sevoflurane, including decreased expressions of TLR4 and NF-κB in cultured ASMCs and improved pharmacodynamic responsiveness of ASM to ACh and isoproterenol.
This study demonstrates the crucial role of TLR4 activation in ASMCs during ALI in response to LPS. Sevoflurane exerts direct relaxant and anti-inflammatory effects in vivo and in vitro via inhibition of TLR4/NF-κB pathway.
感染是急性肺损伤（ALI）的常见原因。本研究旨在探讨气道平滑肌细胞（ASMCs）的Toll样受体4（TLR4）是否在脂多糖（LPS）诱导的气道高反应性中发挥作用及其潜在机制。
体内实验：在对雌性小鼠腹腔注射3%七氟醚或腹腔注射苯巴比妥麻醉前，腹腔注射致敏剂量的LPS（50μg）。稳定后，用5μg气管内LPS对小鼠进行攻击以模拟炎症攻击。通过测量气道对乙酰甲胆碱的反应性、组织学检查以及支气管肺泡灌洗液（BALF）中IL-1、IL-6、TNF-α水平来评估七氟醚的作用。还评估了TLR4和NF-κB的蛋白质和基因表达。体外实验：在对ASMCs和ASM节段预致敏24小时后，在存在或不存在七氟醚的情况下，连续LPS暴露1、3、5、12和24小时后，测量培养的ASMCs中TLR4和NF-κB蛋白的水平。24小时后测量ASM的收缩和舒张反应性。
在整个24小时观察期内，体内和体外实验中，LPS攻击后ASM中NF-κB和TLR4的mRNA和蛋白水平均升高并维持在高水平。七氟醚降低了LPS诱导的气道高反应性、肺部炎性细胞浸润以及BALF中促炎细胞因子的释放，以及ASM节段对乙酰胆碱的最大等长收缩力，但增加了对异丙肾上腺素的最大舒张反应。用特异性NF-κB抑制剂治疗产生了与七氟醚类似的保护作用，包括培养的ASMCs中TLR4和NF-κB表达降低，以及ASM对乙酰胆碱和异丙肾上腺素的药效学反应性改善。
本研究证明了在ALI期间，LPS刺激下ASMCs中TLR4激活的关键作用。七氟醚通过抑制TLR4/NF-κB途径在体内和体外发挥直接舒张和抗炎作用。
