MMP-2/9活性与TIMP-1/2蛋白表达在大鼠肺纤维化形成中的变化特点

Feature changes of MMP-2/9 activities and TIMP-1/2 protein expressions during the progression of pulmonary fibrosis in rats

目的：观察大鼠肺纤维化形成过程中基质金属蛋白酶（matrix metalloproteinase，MMP）-2／9的活性及其活性抑制物——基质金属蛋白酶组织抑制因子（tissue inhibitor of matrix metalloproteinase，TIMP）-1／2蛋白表达的动态变化，探讨肺纤维化形成过程中细胞外基质（extracellular matrix，ECM）降解的调控规律。方法：SD大鼠48只，随机分为模型组（30只）与正常对照组（18只）。模型组大鼠予以1次性气管内注射博来霉素诱导肺纤维化模型，正常对照组大鼠于气管内注入等量生理盐水。每组再各设1d、3d和1、2、3、4周共6个时间观察点，动态观察大鼠肺系数的变化。HE染色观察大鼠肺组织病理学的改变；Masson染色观察大鼠肺组织胶原沉积情况；Jamall氏法测定肺组织羟脯氨酸（hydroxyproline，Hyp）的含量；Western印迹法分析肺组织MMP-2、MMP-9、TIMP-1和TIMP-2蛋白的表达变化；明胶酶图法检测肺组织MMP-2与MMP-9的活性。结果：与正常对照组大鼠比较，模型组大鼠染毒后2周其肺组织发生局灶性病变，肺泡壁与间隔增厚，肺泡腔与间质内有大量炎细胞浸润，伴有少量成纤维细胞；肺系数明显增高；肺组织内Hyp含量稍升高；MMP-2／9蛋白的表达与活性增加，以活化型MMP-2增加更为明显；TIMP-1／2蛋白的表达则逐渐增加。模型组大鼠染毒后3～4周时，肺泡结构遭到破坏，部分肺泡腔塌陷消失，由胶原纤维和成纤维细胞占据，肺泡壁与间隔进一步增厚，形成严重的肺纤维化；肺系数较2周时有所下降，肺组织Hyp含量和TIMP-1／2蛋白的表达明显增加，至染毒后4周时最为明显；MMP-2／9活性较2周时有所下降，尤以活化型MMP-2的活性下降较为明显。结论：博来霉素诱导肺纤维化形成过程中，早期肺组织的病理学变化以肺泡炎症为主，肺组织内MMP-2／9活性增高；后期以纤维化为主，肺组织TIMP-1／2表达明显增加，MMP-2／9活性则有所下降。

Objective ： To investigate the dynamic trends of activities of matrix metalloproteinase （MMP）-2/9 and protein expressions of their inhibitors-tissue inhibitor of matrix metalloproteinase （TIMP）-1/ 2 during the progression of pulmonary fibrosis in rats so as to get insight of the roles played by MMP-2 /9 in lung injury and fibrogenesis. Methods. Forty-eight SD rats were randomly divided into normal control group （n = 18） and bleomycin （BLM）-treated group （n =30）. The pulmonary fibrosis was induced by intratracheal injection of BLM once. At the consecutive time of 1 day, 3 days, 1 week, 2, 3, and 4 weeks after intoxica- tion, the lung-to-body weight ratio was calculated and the inflammation and collagen deposition in lung tissue were checked by HE and Masson stainings respectively. Meanwhile, the content of hypdroxyproline （Hyp） in lung tissue was assayed with Jamall＇s method, the protein expressions of MMP-2/9, TIMP-1/2 were examined by Western blotting, and the activities of MMP-2/9 were detected by gelatin zymography. Results： The histopathological changes in lung tissue in the BLM-treated group from 1 day to 2 weeks after intoxication presented local lesions, broadened alveolar wall and septum, infiltration with lots of inflammatory cells and few of fibroblasts inside alveolar space and septum. At this early stage in the BLM-treated group, the lung-tobody weight ratio was increased significantly, the protein expressions and activities of MMP-2/9 were obviously increased especially for activity of active MMP-2, and the protein expressions of TIMP-1/2 were also increased gradually, as compared with those in the normal control group. From 3 to 4 weeks after intoxication in the BLM-treated group, the alveolar structure was damaged, parts of the alveolar space collapsed and replaced by collagens and fibroblasts, and the alveolar wall and septum obviously widened with remarkable fibrotic characteristics, as compared with those in the normal control group. Meanwhile, the lung-to-body weight ratio and the activities of MMP-2/9 were decreased in the BLM-treated group as compared with those in the same group at 2 weeks after intoxication, but the content of Hyp and the protein expressions of TIMP-1/ 2 were both increased dramatically, especially at 4 weeks after intoxication. Conclusions： During the lung fibrogenesis induced by BLM in rats, the alveolar inflammation is the most important alteration with enhanced MMP-2/9 activities in the early stage. While in the late stage, the main change is displayed as pulmonary fibrosis, characterized by increased TIMP-1/2 and declined MMP-2/9 activities.