调控Toll样受体2/核转录因子-κB信号通路对呼吸机相关性肺损伤大鼠的影响

The impacts of regulating Toll-like receptor 2/nuclear factor-κB signal pathway on rats with ventilator-induced lung injury

目的 观察Toll样受体2/核转录因子-κB（TLR2/NF-κB）信号通路预处理在呼吸机相关性肺损伤（VILI）中的作用。方法 将30只雄性SD大鼠按随机数字表法分为3组，每组10只。A组：经气管导管缓慢滴入10μg/kg TLR2单克隆抗体（TLR2-mAb）200μL干预后，40 mL/kg潮气量（VT）行机械通气；B组：8 mL/kg VT行机械通气；C组：滴入10μg/kg无生物学活性的TLR2-mAb作为同型抗体干预后，40 mL/kg VT行机械通气。于通气4h计算肺湿/干质量（W/D）比值，镜下观察肺组织病理学及细胞超微结构改变；用酶联免疫吸附试验（ELISA）检测血清和支气管肺泡灌洗液（BALF）中白细胞介素（IL-1β、IL-6）、肿瘤坏死因子-α（TNF-α）水平；用实时荧光定量反转录-聚合酶链反应（RT-PCR）检测肺组织TLR2、NF-κB及髓样分化因子88（MyD88）的mRNA表达。结果 显微镜下观察显示：A、B组肺组织无明显病理学改变，肺泡巨噬细胞、Ⅰ型和Ⅱ型肺泡上皮细胞超微结构无明显损伤；C组可见明显肺泡腔融合，肺间隔增宽，大量炎性细胞聚集，肺泡巨噬细胞、Ⅰ型和Ⅱ型肺泡上皮细胞胞膜破坏，胞质大量空泡化，细胞器破坏严重，细胞核固缩，核周隙明显增宽。A、B组肺W/D比值以及血清和BALF中炎症细胞因子水平均较C组明显降低〔肺W/D比值：1.151±0.026、1.128±0.048比1.403±0.062；血清IL-1β（ng/L）：37.05±5.61、34.52±4.31比51.45±8.18，IL-6（ng/L）：53.65±5.16、55.77±5.62比89.96±7.08，TNF-α（ng/L）：71.93±13.29、67.36±11.42比96.20±11.60；BALF中 IL-1β（ng/L）：56.48±6.16、54.44±7.26比99.77±8.41，IL-6（ng/L）：172.44±21.26、163.47±18.70比216.22±23.90，TNF-α（ng/L）：235.81±42.75、231.72±40.38比374.85±69.61，均P＜0.01〕，而A组与B组上述指标差异均无统计学意义（均P＞0.05）。A、B组肺组织TLR2、MyD88和NF-κB的mRNA表达均明显低于C组〔TLR2 mRNA（2-ΔΔCt）：1.021±0.287、0.938±0.196比3.862±0.871，MyD88 mRNA（2-ΔΔCt）：1.235±0.277、1.300±0.306比3.618±1.107，NF-κB mRNA（2-ΔΔCt）：0.519±0.036、1.043±0.170比20.280±9.466，P＜0.05或P＜0.01〕，而A组与B组上述因子基因表达差异均无计学意义（均P＞0.05）。结论 TLR2-mAb预处理通过阻断TLR2/NF-κB信号通路可减轻VILI模型大鼠炎症细胞因子的释放，在一定程度上减轻VILI。

Objective To evaluate the role of Toll-like receptor 2/nuclear factor-κB（TLR2/NF-κB）signaling pathway pretreatment in ventilator-induced lung injury（VILI）. Methods Thirty male Sprague-Dawley（SD）rats were randomly divided into three groups by using random number scale,with 10 rats in each group. Group A：rats were given 200μL of TLR2 monoclonal antibodies（TLR2mAb,10μg/kg）by slow instillation through tracheal catheter, and then ventilated with a high tidal volume（VT）of 40 mL/kg. Group B：ventilated with a normal VT of 8 mL/kg. Group C：rats were tracheally instilled with 10 μg/kg of TLR2mAb devoid of biologic activity,and then ventilated with a high VT of 40 mL/kg. The rats were mechanically ventilated for 4 hours,the lung wet to dry weight ratio（W/D）was calculated. The changes in pathology and ultrastructure in lung tissue were observed with microscope. Enzyme linked immunosorbent assay（ELISA）was performed to determine the concentration of interleukins（IL-1β,IL-6）and tumor necrosis factor-α（TNF-α）in serum and brconchoalveolar lavage fluid（BALF）. Real-time fluorescent quantitation reverse transcription-polymerase chain reaction（RT-PCR）was used to assess the mRNA expressions of TLR2, NF-κB and myeloid differentiation factor 88（MyD88）in lung tissue. Results No obvious pathological changes in lungs were found in group A and group B,and no obvious damages to ultra-microstructure were found in lung macrophages, typeⅠepithelial cell and typeⅡepithelial cell. In group C,pathological changes were observed,including pulmonary alveoli fusion,alveoli septum thickening,inflammatory cells infiltration,and damages to ultrastructure of lung macrophage,damage to cell membrane of typeⅠepithelial cells and typeⅡepithelial cells,vacuoles in cytoplasm, damage to organelle,and even pyknosis and perinuclear cistern thickening. Compared with group C,W/D ratio and mean concentration of inflammatory cytokines in serum and BALF showed a significant decrease in group A and B〔W/D ratio：1.151±0.026,1.128±0.048 vs. 1.403±0.062;concentration of IL-1βin serum（ng/L）：37.05±5.61, 34.52±4.31 vs. 51.45±8.18;concentration of IL-6 in serum（ng/L）：53.65±5.16,55.77±5.62 vs. 89.96±7.08;concentration of TNF-αin serum（ng/L）：71.93±13.29,67.36±11.42 vs. 96.20±11.60;concentration of IL-1βin BALF（ng/L）：56.48±6.16,54.44±7.26 vs. 99.77±8.41;concentration of IL-6 in BALF（ng/L）：172.44±21.26, 163.47±18.70 vs. 216.22±23.90;concentration of TNF-α in BALF（ng/L）：235.81±42.75,231.72±40.38 vs. 374.85±69.61,all P〈0.01〕,but there were no significant differences between group A and group B（all P〉0.05）. The mRNA expressions of TLR2,MyD88,and NF-κB were significantly decreased in group A and group B compared with those in group C〔TLR2 mRNA（2-ΔΔCt）：1.021±0.287,0.938±0.196 vs. 3.862±0.871;MyD88 mRNA （2-ΔΔCt）：1.235±0.277,1.300±0.306 vs. 3.618±1.107;NF-κB mRNA（2-ΔΔCt）：0.519±0.036,1.043±0.170 vs. 20.280±9.466,P〈0.05 or P〈0.01〕,but there was no significant difference among the parameters mentioned above between group A and B（all P〉0.05）. Conclusion To some extent,pre-intervention with TLR2mAb to block the TLR2/NF-κB signal pathway can inhibit the release of pro-inflammatory factors,and regulate the VILI.