Huang Cuiyuan, Pan Linghui, Lin Fei, Qian Wei, Li Wei
Department of Anesthesiology, Affiliated Tumor Hospital, Guangxi Medical University, Nanning 530021, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2015 Feb;31(2):182-5, 189.
To investigate the role of alveolar macrophages Toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88) signaling in ventilator-induced lung injury (VILI) in rat model.
Thirty adult male Sprague-Dawley rats were ventilated with high tidal volume (HTV) (40 ml/kg) for 240 minutes to establish VILI model after oral intubation. Then 4DegreesCelsius PBS was infused through the endotracheal tube and alveolar macrophages (AMs) were purified from the bronchoalveolar lavage fluid (BALF). The AMs were randomly divided into 3 groups (n=8 each): PBS stimulating group (group CON), TNF-α stimulating combined with PBS blocking group (STI group), and TNF-α stimulating combined with TLR4 monoclonal antibodies (TLR4 mAb) blocking group (ANT group). CON group was cultured for 16 hours after blocked by PBS for 2 hours. STI group was cultured with TNF-α (20 ng/mL) for 16 hours after blocked by PBS for 2 hours. ANT group was cultured with TNF-α (20 ng/mL) for 16 hours after blocked by PBS and TLR4 mAb for 2 hours. The cell culture supernatants were collected for determination of the expressions of TNF-α, IL-1β and IL-6 with ELISA. The expressions of TLR4, TLR9, MyD88 and nuclear factor κB (NF-κB) at both mRNA and protein levels were detected by reverse transcription PCR and Western blotting, respectively.
Compared with CON group, concentrations of TNF-α, IL-1β and IL-6 in cell culture supernatants increased significantly in STI group and ANT; the mRNA and protein levels of TLR4, MyD88 and NF-κB in AMs rose significantly in STI group, but there was no significant difference in the mRNA and protein levels of TLR4, MyD88 and NF-κB in ANT group. Compared with STI group, concentrations of TNF-α, IL-1β and IL-6 in cell culture supernatants, the mRNA and protein levels of TLR4, MyD88 and NF-κB in AMs decreased significantly in ANT group. There was no significant difference in TLR9 mRNA and protein levels among the three groups.
The stimulation of inflammatory cytokines can up-regulate the secretion of TLR4, MyD88 and NF-κB in AMs. TLR4-MyD88 signaling plays an important role in the development of ventilator-induced lung injury in rats.
探讨肺泡巨噬细胞Toll样受体4（TLR4）/髓样分化因子88（MyD88）信号通路在大鼠呼吸机诱导性肺损伤（VILI）中的作用。
30只成年雄性Sprague-Dawley大鼠经口插管后用高潮气量（HTV，40 ml/kg）通气240分钟以建立VILI模型。然后经气管内导管注入4℃ PBS，并从支气管肺泡灌洗液（BALF）中纯化肺泡巨噬细胞（AMs）。将AMs随机分为3组（每组n = 8）：PBS刺激组（CON组）、TNF-α刺激联合PBS阻断组（STI组）和TNF-α刺激联合TLR4单克隆抗体（TLR4 mAb）阻断组（ANT组）。CON组在经PBS阻断2小时后培养16小时。STI组在经PBS阻断2小时后用TNF-α（20 ng/mL）培养16小时。ANT组在经PBS和TLR4 mAb阻断2小时后用TNF-α（20 ng/mL）培养16小时。收集细胞培养上清液，用酶联免疫吸附测定法（ELISA）检测TNF-α、IL-1β和IL-6的表达。分别用逆转录PCR和蛋白质印迹法检测TLR4、TLR9、MyD88和核因子κB（NF-κB）在mRNA和蛋白质水平的表达。
与CON组相比，STI组和ANT组细胞培养上清液中TNF-α、IL-1β和IL-6的浓度显著升高；STI组AMs中TLR4、MyD88和NF-κB的mRNA和蛋白质水平显著升高，但ANT组中TLR4、MyD88和NF-κB的mRNA和蛋白质水平无显著差异。与STI组相比，ANT组细胞培养上清液中TNF-α、IL-1β和IL-6的浓度以及AMs中TLR4、MyD88和NF-κB的mRNA和蛋白质水平显著降低。三组之间TLR9 mRNA和蛋白质水平无显著差异。
炎性细胞因子刺激可上调AMs中TLR4、MyD88和NF-κB的分泌。TLR4-MyD88信号通路在大鼠呼吸机诱导性肺损伤的发生发展中起重要作用。
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