脂多糖诱导下慢性阻塞性肺疾病大鼠模型远端肺动脉平滑肌细胞中Toll样受体4表达情况研究

Expression of the Toll-like Receptor-4 in Distal Pulmonary Artery Smooth Muscle Cells of Lipopolysaccharide-induced Chronic Obstructive Pulmonary Disease Model Rats

背景慢性阻塞性肺疾病(COPD)是以不完全可逆的气流受限为特征的慢性支气管炎和肺气肿,也是导致肺动脉高压的主要原因之一,但其发生发展机制尚未完全清楚。目的探讨脂多糖(LPS)诱导下COPD大鼠模型远端肺动脉平滑肌细胞(PASMCs)中Toll样受体(TLR)4表达情况,以期为探讨TLR4信号通路在COPD炎性反应及免疫应答中的作用提供理论基础。方法 2015年12月—2016年3月,选取SPF级Wistar大鼠24只,雌雄对半,6~8周龄。适应性饲养大鼠1周后,将其随机分为模型组和正常组,各12只。模型组大鼠于实验第1、14天经气道注入1μg/ml的LPS 200μl,置于自制有机玻璃舱,烟熏1 h/d,共8周;正常组大鼠于实验第1、14天经气道注入等量0.9%氯化钠溶液,与模型组大鼠在同等条件下饲养8周。8周后,两组分别随机选取2只大鼠,开胸取肺组织,分别进行HE染色观察肺组织病理学改变和免疫组织化学染色观察肺动脉平滑肌层TLR4表达情况。从模型组剩余大鼠中随机选取6只,分离、培养远端PASMCs,选用第3~6代(对数生长期)细胞,光镜下(×100)观察PASMCs形态,采用免疫荧光法(荧光显微镜下,×200)观察其α-肌动蛋白表达情况,并随机分为对照组(不进行干预)、LPS 12 h组(加入1μg/ml的LPS 10μl作用12 h)、LPS 24 h组(加入1μg/ml的LPS 10μl作用24 h)、LPS 48 h组(加入1μg/ml的LPS 10μl作用48 h)、LPS 72 h组(加入1μg/ml的LPS 10μl作用72 h),采用Western blotting法检测各组PASMCs中TLR4表达水平。结果肺组织病理学改变:模型组肺泡壁断裂,肺泡融合,肺大泡形成,肺动脉平滑肌层明显增厚,大量炎性细胞浸润,病理表现符合典型的COPD病理改变。肺动脉平滑肌层TLR4表达情况:模型组倒置相差显微镜下可见TLR4表达阳性,且肺动脉平滑肌层染成黄色较正常组颜色明显加深。PASMCs形态及其α-肌动蛋白表达情况:光镜下PASMCs呈梭形、不规则生长,随着培养时间的延长,层数增多,堆积形成"峰-谷"状;荧光显微镜下可见PASMCs胞质α-肌动蛋白表达阳性,胞质中有向细胞两极呈放射状分布的条丝状物,与细胞长轴平行,形态清晰。LPS 12 h组、LPS 24 h组、LPS 48 h组、LPS 72 h组PASMCs中TLR4表达水平均高于对照组(P<0.05);LPS 24 h组、LPS 48 h组、LPS 72 h组PASMCs中TLR4表达水平均高于LPS 12 h组(P<0.05);LPS 48 h组、LPS 72 h组PASMCs中TLR4表达水平均高于LPS 24 h组(P<0.05)。结论 LPS诱导下COPD大鼠模型远端PASMCs中TLR4表达水平升高,猜测LPS可能通过TLR4信号通路诱导PASMCs的合成分泌功能,从而加重炎性反应及肺血管重塑。

Background Chronic obstructive pulmonary disease（ COPD） is a lung disease that includes two main types,chronic bronchitis and emphysema,characterized by chronic incompletely reversible airflow. It is one of the leading causes of pulmonary hypertension. However,its occurrence and development mechanism is not yet fully clear. Objective To explore the TLR-4 expression in distal pulmonary artery smooth muscle cells（ PASMCs） of COPD model rats induced by lipopolysaccharide（ LPS）,so as to provide a theoretical basis for studying the role of TLR-4 signaling pathway in inflammation and immune response to COPD. Methods This study was conducted from December 2015 to March 2016. Twenty-four SPF grade Wistar rats（ half males and half females） aging between 6 and 8 weeks were randomly divided into normal group and model group,12 rats in each after feeding adaptively for 1 week. Rats in model group and normal group were respectively administered with 200 μl LPS（ 1μg/ml）,and 200 μl sodium chloride（ 0. 9%） solutions,via the trachea on the 1st and the 14 th day after intervention,and treated by passive inhaling of cigarette smoke in plexiglass cabin for 1 h once daily for 8 weeks. After the intervention,2 rats respectively randomly selected from both groups were sacrificed and the lung tissues were took out for observing the pathological changes via HE staining,and TLR-4 expression in PASMCs determined with immunohistochemical（ IHC） staining. Six model rats ranomly selected from remaining rats were sacrificed and the distal PASMCs were isolated and cultured. Cultured cells at passages 3-6（ logarithmic phase） were used for experiments. The morphology of PASMCs was observed under the optical microscope（ the images were magnified 100 times） and α-SM-actin expression in PASMCs stained by immunohistochemistry was observed under the immunofluorescence microscope（ the images were magnified 200 times）,then they were randomly divided into control group（ no intervention）,LPS 12 h group（ treatment with 10 μl LPS of 1 μg/ml for 12 h）,LPS 24 h group（ treatment with 10 μl LPS of 1 μg/ml for 24 h）,LPS 48 h group（ treatment with 10 μl LPS of 1 μg/ml for 48 h） and LPS 72 h group（ treatment with 10 μl LPS of 1 μg/ml for 72 h）. The TLR-4 protein expression in PASMCs in these 5 groups were measured with Western blotting. Results The pathological changes in lung tissues found in the model group were rupture of alveolar walls, alveolar fusion,formation of bullae,dramatically thickened PASMCs layer in artery,a large number of inflammatory cell infiltration,all these were consistent with COPD typical features. TLR-4 expression in pulmonary artery smooth muscle layer： under the inverted phase contrast microscope,in model group,the TLR-4 expression in pulmonary artery smooth muscle layer was seen to be positive,the pulmonary artery smooth muscle layer was yellow,and it was much deeper than that in the normal group. PASMCs morphology and their α-SM-actin expression： under optical microscope,PASMCs were spindle shaped and presented irregular growth. With the extension of culture time,the number of layers were increased,leading to the formation of ＂ peak valley＂;under immunofluorescence microscopy,it could be seen that α-SM-actin expression in PASMCs was positive,the cytoplasm had a radial distribution of filaments to the cell poles,parallel to the long axis of the cell,the morphology was clear. TLR-4 expression in PASMCs of LPS 12 h group,LPS 24 h group,LPS 48 h group and LPS 72 h group was higher than that of control group（ P〈0. 05）; TLR4 expression in PASMCs of LPS 24 h group,LPS 48 h group and LPS 72 h group was higher than that of LPS 12 h group（ P〈0. 05）; TLR4 expression in PASMCs of LPS 48 h group and LPS 72 h group was higher than that of LPS24 h group（ P〈0. 05）. Conclusion TLR-4 expression level increases in distal PASMCs of COPD model rats induced by LPS; it is speculated that LPS could induce the synthesis and secretion of PASMCs through the TLR4 signaling pathway,which aggravated the inflammatory response and pulmonary vascular remodeling.