动式染尘构建矽肺大鼠模型及ACE2-Ang(1-7)-Mas轴动态变化规律

Establishment of a rat silicosis model by dynamic silica dust exposure and its dynamic variation rule of ACE2-Ang(1-7)-Mas axis

目的肾素-血管紧张素系统(renin-angiotesin system,RAS)与肺纤维化的发生发展密切相关,文中拟观察RAS系统重要靶向轴血管紧张素转换酶(angiotensin converting enzyme,ACE)2-血管紧张素(angiotensin,Ang)(1-7)-Mas轴在动式染尘法矽肺大鼠模型中动态变化规律。方法实验分为对照组以及染尘2、4、8、16、24周组,每组10只。实验组采用HOPE-MED8050动式染尘控制系统构建大鼠矽肺模型,分别染尘2、4、8、16、24周。对照组未行染尘处理。采用HE、Masson染色观察病理形态;免疫组化染色检测α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、波形蛋白(vimentin)、ACE2在肺组织中的定位和表达;Western blot法检测肺组织I型胶原、α-SMA、vimentin、ACE2和Mas的表达;ELISA检测肺组织中Ang(1-7)的含量。结果 HE、Masson染色结果显示染尘8周可出现细胞性矽结节,染尘16周可出现多个结节的融合,至染尘24周时可出现细胞纤维性结节。免疫组化染色结果显示,ACE2主要定位于支气管纤毛上皮细胞、肺泡上皮细胞,而在矽结节中ACE2表达明显减弱。Western blot结果显示,与对照组Ⅰ型胶原(0.05±0.01)、α-SMA(0.03±0.01)比较,染尘2、4、8、16、24周组Ⅰ型胶原(0.11±0.01,0.22±0.02,0.42±0.01,0.90±0.03,1.48±0.02)、α-SMA表达(0.08±0.01,0.17±0.02,0.28±0.01,0.43±0.01,0.42±0.01)均明显升高(P<0.05),染尘8、16、24周Vimentin表达明显升高(P<0.05)。与对照组比较,染尘4、8、16、24周组ACE2表达降低(P<0.05),染尘2、4、8、16、24周Mas表达降低(P<0.05)。ELISA法检测肺组织中Ang(1-7)含量结果显示,随着染尘时间的延长Ang(1-7)的表达逐渐降低。结论动式染尘能够构建矽肺大鼠模型,而ACE2-Ang(1-7)-Mas轴表达水平下调参与了矽肺大鼠纤维化的进展。

Objective It is reported that renin angiotensin system （ RAS） has an important role in the occurrence and development of pulmonary fibrosis. The study was to observe the dynamicvariation rule of ACE2-Ang（ 1-7） -Mas axis in the rat silicosis modelt ＇ t nestablished by dynamic silica dust exposure. Methods HOPE- -MED8050 dynamic silica dust exposure control system was applied inthe establishment of a rat silicosis model, in which rats were exposed in silica dust for 2w, 4w, 8w, 16w and 24w respectively. Routine HE and Masson staining were used to observe the pathomorphology. Im- munohistochemistry was used to examine the location and expression of a -SMA, vimentin and ACE2. Western blot was applied to analyzethe expression of collagen type I , a -SMA, vimentin, ACE2 and Mas in lung tissue and ELISA to detect the Ang（ 1-7） content in lung tissue. Results The results of HE and Masson staining showed that the appearance of cellular silicotic nodules at 8 w and confluent multinodular lesions at 16 w. Fibrous cellular nodules could be seen at 24 w. According to the results of immunohistochemistry, ACE2 were mainly located in the bronchial ciliated epithelium cells and alveo-lar epithelial cells, and significantly decreased in the silicon nodules. Moreover, the results of western blot and ELISA revealed that the levels of collagen type I, a-SMA protein in the lung tissue increased obviously with the prolonged exposure time which were respectively increased from 0.05±0.01、 0.03±0.01 in normal control group to 1.48±0.02、 0.42±0.01 in silicosis model rats at 24 weeks and that the levels of ACE2, Ang （1-7） and Mas reduced with the prolonged exposure time which were increased respectively from 0.33 ±0.01、 102.40±9.06、 0.19 ±0.02 in normal control group to 0.03 ±0.00、 45.22 ± 6 .66、 0.02 ±0.01 in silicosis model rats at 24 week. Conclusion The rat silicosis model can be established by the dynamic silica dust exposure and the down-regulation of ACE2-Ang（ 1- 7）-Mas axis expression is closely related to fibrosis in silicosis rats.