TLR4/NF-κB在烟雾暴露大鼠肺血管重塑中的表达及意义∗

摘要

目的：：观察烟雾暴露环境下肺血管Toll样受体4(TLR4)、核因子-κB(NF-κB)亚基P65蛋白和增殖细胞核抗原( PCNA)的表达,探讨TLR4及NF-κB信号通路在大鼠肺血管重塑中的可能作用机制。方法： SPF级健康雄性SD大鼠48只,随机分为对照组( control组)、烟雾暴露4周组( S4组)、烟雾暴露8周组( S8组)和烟雾暴露12周组( S12组),每组各12只。制备烟雾暴露大鼠模型,HE染色法观察肺血管形态学变化,并测量各组大鼠肺血管管壁面积/血管总面积( WA%)和血管壁厚度/血管外径( WT%)。免疫组织化学染色法检测肺血管TLR4、P65和PCNA的表达,蛋白含量用平均积分吸光度来表示。 RT-qPCR检测肺血管TLR4的mRNA表达,并分析各组肺血管WA%、WT%、TLR4蛋白和TLR4 mRNA、P65蛋白、PCNA蛋白与肺血管重塑的相关性及TLR4蛋白与WA%、WT%、P65蛋白、PCNA蛋白之间的相关性。结果：与对照组比,烟雾暴露组大鼠肺血管WA%及WT%都明显增高,且WA%及WT%与烟雾暴露时间呈正相关关系( P<0．05)；烟雾暴露组肺血管TLR4蛋白、P65蛋白、PCNA蛋白和TLR4 mRNA的表达量较对照组比显著增加,且与烟雾暴露时间呈正相关关系(P<0．05)。结论：烟雾暴露上调大鼠肺血管TLR4蛋白的表达,TLR4和NF-κB P65蛋白的表达量与肺血管重塑程度呈正相关性。肺血管重塑可能与TLR4/NF-κB信号通路激活有关。%[ ABSTRACT] AIM: To observe the expression of Toll-like receptor 4 ( TLR4 ) , nuclear factor-κB subunit P65 protein (NF-κB P65) and proliferating cell nuclear antigen (PCNA) in the pulmonary vascular tissues of the rats exposed to smoke, and to explore the possible mechanism of TLR4/NF-κB signaling pathway in pulmonary vascular remodeling. METHODS:SPF male healthy rats (n=48) were randomly divided into control group, smoke exposure for 4 weeks group ( S4 group) , smoke exposure for 8 weeks group ( S8 group) and smoke exposure for 12 weeks group ( S12 group) , with 12 rats in each group. HE staining was used to observe the morphological changes of pulmonary vessels, and then the pulmo-nary vascular wall area/total vascular area ( WA%) and vascular wall thickness/vascular external diameter ( WT%) were measured by the medical image analysis system. The expression of TLR4, NF-κB P65 and PCNA in the pulmonary vascular tissues was detected by immunohistochemical staining. The protein content was expressed by the average integral absor-bance. The mRNA expression of TLR4 in the pulmonary vessels was detected by RT-qPCR. The relationships between WA%, WT%,TLR4 protein, TLR4 mRNA, P65 protein, PCNA protein and pulmonary vascular remodeling, and another relationships between WA%, WT%, P65 protein, PCNA protein and TLR4 protein were analyzed. RESULTS:The WA%and WT% in smoke exposure groups significantly increased compared with control group, and the ratio was proportional to the time of smoke exposure. The protein expression of TLR4, p65 and PCNA, and the mRNA expression of TLR4 in smoke exposure groups also increased significantly compared with control group. CONCLUSION:The extent of pulmonary vascu-lar remodeling in the rats increases when the protein expression of TLR4 is up-regulated. There is a positive correlation be-tween pulmonary vascular remodeling and the protein expression of TLR4 and NF-κB P65. Pulmonary vascular remodeling may be related to the activation of TLR4/NF-κB signaling pathway.