Lai Jie, Tang Zhanhong, Hu Juntao, Zhou Wei, Zhang Chi, Chen Xianfeng
Department of Critical Care Medicine, the First Affiliated Hospital of Guangxi Medical University, Nanning 530021, Guangxi, China, Corresponding author: Tang Zhanhong, Email:
Zhonghua Wei Zhong Bing Ji Jiu Yi Xue. 2014 Nov;26(11):815-20. doi: 10.3760/cma.j.issn.2095-4352.2014.11.010.
OBJECTIVE: To investigate the effect of hypothermia on the expression Toll-like receptor 2 (TLR2), myeloid differentiation factor 88 (MyD88), nuclear factor-ΚBp65 (NF-ΚBp65), plasminogen activator inhibitor-1 (PAI-1) in the TLR2/MyD88 pathway in rats with acute lung injury (ALI) induced by lipopolysaccharide (LPS) inhalation. METHODS: Ninety male Sprague-Dawley (SD) rats were randomly divided into control group (n=18), hypothermia group (n=24), temperature controlled group (n=24), and temperature-uncontrolled group (n=24). The ALI model was reproduced by 0.5 mL/kg LPS intratracheal instillation, while only normal saline was instilled intratracheally for control group. Arterial blood was collected and physical cooling was started 1 hour after instillation. The body temperature was lowered to 32-34 centigradein hypothermia group and 36-37 centigradein temperature controlled group, and no intervention was used for temperature-uncontrolled group and control group. The arterial blood gas was determined in all the groups before and 1 hour after instillation of saline or LPS and 1, 6, 12 hours after intervention. Rats were sacrificed respectively at 1, 6 and 12 hours after temperature control therapy, the morphological changes in lung tissue were observed under light microscope. The protein expression of PAI-1 in bronchoalveolar lavage fluid (BALF) was determined by enzyme linked immunosorbent assay (ELISA). TLR2 mRNA and MyD88 mRNA transcriptional level were determined by reverse transcription-polymeras chain reaction (RT-PCR). NF-ΚBp65 protein level was determined by Western Blot. RESULTS: After instillation of LPS, the oxygenation index (PaO₂/FiO₂) of each group was decreased obviously, the damage of lung tissues was aggravating, the lung injury score was increased significantly, PAI-1 protein in BALF and the expressions of TLR2 mRNA, MyD88 mRNA, NF-ΚBp65 protein in lung tissues were increased obviously. Each index was improved by therapeutic Hypothermia, the effect of which was best in using a cooling period in the 1-6 hours, while might be benefit at 6-12 hours. Compared with temperature controlled group, PaO2/FiO2 (mmHg, 1 mmHg=0.133 kPa) at 1 hour and 6 hours of hypothermia group was improved(1 hour: 402.49 ± 38.61 vs. 324.36 ± 28.93, 6 hours: 349.72 ± 98.20 vs. 284.35 ± 13.68, both P<0.01), the lung injury score at 1, 6 and 12 hours were significantly decreased (1 hour: 6.04 ± 0.74 vs. 7.96 ± 0.65, 6 hours: 9.09 ± 0.80 vs. 13.13 ± 1.02, 12 hours: 10.79 ± 1.42 vs. 13.42 ± 0.68, all P<0.01), the PAI-1 protein (ng/L) in BALF at 1, 6 and 12 hours were significantly decreased(1 hour: 121.36 ± 4.62 vs. 197.74 ± 9.42, 6 hours: 230.53 ± 10.76 vs. 294.06 ± 16.60, 12 hours: 270.48 ± 13.20 vs. 319.40 ± 10.24, all P<0.01), TLR2 mRNA and MyD88 mRNA expressions(2 (-Δ ΔCt)) in the lung tissues at 1, 6 and 12 hours were significantly decreased(TLR2 mRNA 1 hour: 2.18 ± 0.26 vs. 3.04 ± 0.39, 6 hours: 4.09 ± 0.29 vs. 4.90 ± 0.35, 12 hours: 6.02 ± 0.43 vs. 7.10 ± 0.54; MyD88 mRNA 1 hour: 2.25 ± 0.41 vs. 3.04 ± 0.30, 6 hours: 5.67 ± 0.55 vs. 7.01 ± 0.76, 12 hours: 7.14 ± 0.60 vs. 8.87 ± 0.54, all P<0.01), NF-ΚBp65 protein expression (A value) at 6 hours and 12 hours was significantly decreased(6 hours: 0.31 ± 0.08 vs. 0.53 ± 0.12, 12 hours: 1.05 ± 0.17 vs. 1.76 ± 0.35, both P<0.01). There was no difference in each index between temperature controlled group and temperature-uncontrolled group. CONCLUSIONS: Hypothermia can down-regulate the expression of TLR2 mRNA, MyD88 mRNA, NF-ΚBp65 protein and PAI-1 in the TLR2/MyD88 pathway to protect lung tissue of rats with ALI induced by LPS inhalation from injury.
目的：探讨低温对脂多糖（LPS）吸入诱导的急性肺损伤（ALI）大鼠TLR2/MyD88通路中Toll样受体2（TLR2）、髓样分化因子88（MyD88）、核因子-κBp65（NF-κBp65）、纤溶酶原激活物抑制剂-1（PAI-1）表达的影响。 方法：将90只雄性Sprague-Dawley（SD）大鼠随机分为对照组（n=18）、低温组（n=24）、温度控制组（n=24）和温度非控制组（n=24）。通过气管内滴注0.5 mL/kg LPS建立ALI模型，对照组仅气管内滴注生理盐水。滴注后1小时采集动脉血并开始物理降温。低温组体温降至32-34℃，温度控制组体温降至36-37℃，温度非控制组和对照组不进行干预。在滴注生理盐水或LPS前、滴注后1小时以及干预后1、6、12小时测定所有组的动脉血气。在温度控制治疗后1、6和12小时分别处死大鼠，光镜下观察肺组织形态学变化。采用酶联免疫吸附测定（ELISA）法测定支气管肺泡灌洗液（BALF）中PAI-1的蛋白表达。采用逆转录-聚合酶链反应（RT-PCR）法测定TLR2 mRNA和MyD88 mRNA转录水平。采用蛋白质免疫印迹法测定NF-κBp65蛋白水平。 结果：滴注LPS后，各组氧合指数（PaO₂/FiO₂）明显降低，肺组织损伤加重，肺损伤评分显著升高，BALF中PAI-1蛋白及肺组织中TLR2 mRNA、MyD88 mRNA、NF-κBp65蛋白表达明显增加。低温治疗可改善各项指标，在1-6小时降温效果最佳，6-12小时也可能有益。与温度控制组相比，低温组1小时和6小时的PaO2/FiO2（mmHg，1 mmHg=0.133 kPa）有所改善（1小时：402.49±38.61 vs. 324.36±28.93，6小时：349.72±98.20 vs. 284.35±13.68，均P<0.01），1、6和12小时的肺损伤评分显著降低（1小时：6.04±0.74 vs. 7.96±0.65，6小时：9.09±0.80 vs. 13.13±1.02，12小时：10.79±1.42 vs. 13.42±0.68，均P<0.01），1、6和12小时BALF中PAI-1蛋白（ng/L）显著降低（1小时：121.36±4.62 vs. 197.74±9.42，6小时：230.53±10.76 vs. 294.06±16.60，12小时：270.48±13.20 vs. 319.40±10.24，均P<0.01），1、6和12小时肺组织中TLR2 mRNA和MyD88 mRNA表达（2（-ΔΔCt））显著降低（TLR2 mRNA 1小时：2.18±0.26 vs. 3.04±0.39，6小时：4.09±0.29 vs. 4.90±0.35，12小时：6.02±0.43 vs. 7.10±0.54；MyD88 mRNA 1小时：2.25±0.41 vs. 3.04±0.30，6小时：5.67±0.55 vs. 7.01±0.76，12小时：7.14±0.60 vs. 8.87±0.54，均P<0.01），6小时和12小时NF-κBp65蛋白表达（A值）显著降低（6小时：0.31±0.08 vs. 0.53±0.12，12小时：1.05±0.17 vs. 1.76±0.35，均P<0.01）。温度控制组和温度非控制组各项指标无差异。 结论：低温可下调TLR2/MyD88通路中TLR2 mRNA、MyD88 mRNA、NF-κBp65蛋白和PAI-1的表达，保护LPS吸入诱导的ALI大鼠肺组织免受损伤。
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