钙结合蛋白S100A8/A9在慢性阻塞性肺疾病大鼠中的作用及机制研究

The role and mechanism of S100A8/A9 in rat model of chronic obstructive pulmonary disease

目的探讨钙结合蛋白S100A8/A9在慢性阻塞性肺疾病(简称慢阻肺)大鼠中的作用及机制。方法将12只Wistar大鼠随机分为慢阻肺组和正常对照组。烟熏联合内毒素处理1个月建立慢阻肺大鼠模型。在光镜下观察大鼠的肺组织病理形态,采用图像分析系统检测肺气肿指标平均肺泡数(MAN)、肺平均内衬间隔(MLI)和肺泡腔面积占总面积的比值(PAA),酶联免疫吸附法测量大鼠支气管肺泡灌洗液(BALF)和血浆中S100A8/A9蛋白的浓度,荧光定量聚合酶链反应检测大鼠肺组织中S100A8、S100A9、Toll样受体4(TLR4)和髓样分化因子88(MyD88)mRNA的表达情况,免疫组织化学方法检测大鼠肺组织中S100A8/A9、TLR4和MyD88蛋白的表达情况。结果烟熏联合内毒素处理1个月后,大鼠肺组织出现了典型的慢阻肺病理改变,反映肺气肿的指标MAN、MLI和PAA较正常对照组有显著性差异(均P<0.05)。与正常对照组比较,慢阻肺组BALF及血浆中S100A8/A9蛋白的浓度均显著升高(均P<0.05),慢阻肺组大鼠肺组织中S100A8、S100A9、TLR4和MyD88 mRNA的表达均显著增加(均P<0.05)。S100A8、S100A9 mRNA表达量分别与TLR4、MyD88 mRNA的表达量呈正相关(均P<0.05),S100A8/A9蛋白主要在细胞膜和细胞浆中表达,而MyD88主要表达于细胞浆,TLR4则主要表达于细胞膜。慢阻肺组大鼠肺组织中S100A8/A9、MyD88和TLR4蛋白表达较正常对照组显著增加(均P<0.05),S100A8/A9蛋白表达量分别与MyD88、TLR4蛋白表达量呈正相关(均P<0.05)。结论 S100A8/A9作为新的炎症介质可能参与了慢阻肺的发生发展。其作用机制为通过上调TLR4和MyD88的表达,从而启动经典TLR4-MyD88炎症通路,导致并促进了慢阻肺的发生发展。

Objective To investigate the role and mechanism of S100A8/A9 in rat model of chronic obstructive pulmonary disease(COPD). Methods Twelve Wistar rats were randomly divided into a normal control group and a COPD group. The COPD model was established by exposing the rats to cigarette smoke and injected lipopolysaccharide(LPS) in bronchus for 1 month. The pathological changes of the lung tissue were observed under light microscope, and the emphysema indexes of pulmonary mean linear intercept(MLI), mean alveolar numbers(MAN) and pulmonary alveolar area(PAA) were analyzed by image analysis system. The concentrations of S100A8/A9 in serum and bronchoalveolar lavage fluid(BALF) were measured by enzyme linked immunosorbent assay. The mRNA expression levels of S100A8, S100A9, Toll-like receptor-4(TLR4) and myeloid differentiation factor 88(MyD88) of lung tissues were measured by real time polymerase chain reaction. The protein expressions of S100A8/A9, TLR4 and MyD88 of lung tissues were detected by immunohistochemistry. Results After cigarette smoking and LPS injection for 1 month, the rat lung tissue appeared in accordance with the typical pathological changes of COPD. The MLI, MAN and PAA had obvious difference compared with the normal control group(P<0.05). The concentrations of S100A8/A9 protein in BALF and serum of the COPD group were obviously higher than those of the normal control group(P<0.05). The levels of S100A8,S100A9, TLR4 and MyD88 mRNA of lung tissues in the COPD group were obviously higher than those in the normal control group(P<0.05), and the expression levels of S100A8 and S100A9 mRNA were positively correlated with the expression levels of TLR4 and MyD88 mRNA respectively(P<0.05). The levels of S100A8/A9, TLR4 and MyD88 protein of lung tissues in COPD group were obviously higher than those in normal control group(P<0.05), and the levels of S100A8/A9 protein were positively correlated with the levels of MyD88 and TLR4 protein(P<0.05). Conclusions As a new inflammatory mediator, S100A8/A9 may be involved in the occurrence and development of COPD. By up-regulating the expression of TLR4 and MyD88, the classical TLR4-MyD88 inflammatory pathway is activated, thus promotes the occurrence and development of COPD.