急性肺损伤大鼠肺组织CC16的表达及其对肺局部炎症反应的调控

Expression of CC16 in the lung tissues of rats with acute lung injury and its modulation on the local inflammatory reactions in lungs

研究CC16在内毒素急性肺损伤(ALI)发病中的作用。取56只雄性SD大鼠随机分为对照组、内毒素致伤后0.5、1、2、4、6、24h组。用体视学方法测定大鼠肺泡隔面积密度(PASAD)和肺泡隔中性粒细胞(PMN)数。大鼠肺组织匀浆液中CC16蛋白含量测定用Westernblot法,促炎因子TNF和IL-6含量测定用放免分析法。肺组织CC16mRNA表达水平测定用半定量RT-PCR法。大鼠肺组织中核因子NF-κB的表达与活化用免疫荧光和Westernblot法。结果显示静脉注射内毒素后6h内,大鼠PASAD逐渐增大,于6h达最大,24h基本恢复至正常,肺泡隔PMN数进行性增多,于6h达峰值,24h恢复。伤后各时相点CC16蛋白水平分别为(0.740±0.212)、(0.630±0.148)、(0.603±0.111)、(0.570±0.125)、(0.529±0.124)、(0.614±0.185),均明显低于对照组(1.000),差异有高度统计学意义(P<0.01)。致伤后各时相点大鼠CC16mRNA表达水平均明显低于对照组水平,差异有统计学意义。致伤后各时相点肺组织中NF-κB阳性细胞数均明显多于对照组。致伤后各时相点大鼠肺匀浆液中IκB-α含量明显低于对照组。肺匀浆液中促炎因子TNF水平于致伤后0.5h开始迅速升高,于伤后1h达峰值,其后维持在一种高水平状态,于6h后开始恢复。肺匀浆液中IL-6水平于伤后1h开始明显升高,于伤后4h达峰值,6h后开始恢复。CC16与上述多种指标显著相关,提示CC16对肺局部炎症反应有调控作用。CC16表达变化在内毒素致急性肺损伤的发病中有重要作用。

To investigate the role of CC 16 on the pathogenesis of acute lung injury（ALI） induced by lipopolysaccharide （LPS）, 56 male Sprague-Dawley（SD） rats were randomly divided into control group and the 6 experimental groups, while in the experimental groups, ALI were induced by LPS at 0.5, 1, 2, 4, 6 and 24 hours respectively. The pulmonary alveolar septa area density （PASAD） and the numbers of polymorphonuclear leukocytes （PMN） and the contents of CC16 and pro-inflammatory cytokines TNF and IL-6 in rat lung tissues were analyzed morphometrically, and were assayed by Western blotting and immunoradioassay respectively. Semi-quantitative RT-PCR was used to detect the expression level of CC16 mRNA, and immunofluorescence and Western blotting were carried out to assess the expression and activation of NF-κB in rat lung tissues. The experimental results showed that the PASAD and numbers of PMN in the lung tissues of rats with ALI were significantly increased at 0.5 hours post-injury by LPS and both peaked after 6 hours of injury, but returned to values of baseline at 24 hours. The contents of CC16 in the lung tissues of rats with ALI at 0.5, 1, 2, 4, 6 and 24 hours were （0.74±0.212）, （0.630±0.148）, （0.603±0.111）, （0.570±0.125）, （0.529±0.124） and （0.614±0.185） respectively, that was significantly lower than that of the control group. Also, the levels of the CC16 mRNA in the lung tissues of ALI rats at different times were significantly lower than that the control group. Meanwhile, the numbers of the NF-κB- positive cells of ALI rats at different times were larger than that of the control group, but the contents of IκB-α at different times of the ALI rats were significantly lower than that of the control groups. In addition, the level of TNF in the lung homogenates showed an immediate elevation after LPS injection, attained to a higher level than that of the control group after 0.5 hours, reached to its peak value at one hour, maintained to high level until 6 hours and then gradually recovered. While, the level of IL-6 in the lung homogenates of ALI rats began to rise an hour postinjury, peaked at 4 hours and then gradually returned to normal level after 6 hours. It is evident that CC16 correlates significantly with all the indices mentioned above, suggesting that it could modulate the local inflammatory reactions in lungs and may play a critical role in the pathogenesis of acute lung injuries induced by LPS.