大鼠间质性肺炎Ⅱ型肺泡上皮细胞自噬活性的变化及其机制研究

Changes of autophagy activity and its mechanism in type Ⅱ alveolar epithelial cells of rats with interstitial pneumonia

目的探讨大鼠间质性肺炎Ⅱ型肺泡上皮细胞自噬活性的变化及其机制。方法将16只Wistar雄性大鼠随机数字分为对照组(A组)和实验组(B组),每组8只。实验组雾化吸入博来霉素,对照组雾化吸入等量生理盐水。所有大鼠于28d处死,收集各组肺组织标本。苏木精-伊红染色(HE)观察肺组织病理改变;Masson三色染色法观察肺组织胶原纤维生成情况;采用免疫黏附法分离Ⅱ型肺泡上皮细胞后,采用蛋白质免疫印迹法(Western blot)检测-平滑肌肌动蛋白(α-SMA)、泛素结合蛋白p62、自噬微管相关蛋白轻链3(LC3Ⅰ/Ⅱ)、磷酸化的蛋白激酶B(p-Akt)、哺乳动物雷帕霉素靶蛋白(mTOR)及其磷酸化形式p-mTOR的表达变化。结果1)HE染色显示,A组大鼠肺组织呈正常表现,未见肺泡炎及纤维化改变;B组肺实质呈大片融合实变,其间有少量巨噬细胞、中性粒细胞等炎性细胞浸润,成纤维细胞增多,肺泡间隔明显增厚,可见胶原沉积。2)Masson染色显示,A和B组蓝色区域面积分别为(12.12±1.61)mm^2、(23.73±4.20)mm^2,B组蓝色面积大于A组,差异有统计学意义(P<0.05));A组蓝色区域主要集中在气管或血管周围,肺间质只有少量散在的点状着色,B组蓝色区域除气管和血管周围外,肺间质也可见大量蓝色着色区,呈片絮状。3)Western blot结果显示,B组α-SMA蛋白相对表达量高于A组;B组p62蛋白相对表达量高于A组;B组LC3Ⅱ/Ⅰ比值低于A组;B组p-Akt和p-mTOR相对表达量均高于A组,差异均有统计学意义(均P<0.05);A组和B组mTOR蛋白相对表达量差异无统计学意义(P>0.05)。结论大鼠间质性肺炎Ⅱ型肺泡上皮细胞出现细胞自噬活性抑制,Akt/mTOR信号通路激活是其可能的机制之一。

Objective To investigate the changes of autophagy activity and its mechanism in typeⅡ alveolar epithelial cells of rats with interstitial pneumonia.Methods Sixteen male rats were randomly and averagely assigned into control group(Group A)and experimental group(Group B).Experimental group received bleomycin inhalation,while the control group received saline inhalation.Rats were sacrificed on the 28 hdays and lung tissue were collected.Hematoxylin-eosin staining(HE)was used to observe the pathological changes of lung tissue.Collagen fibers were evaluated by Masson staining.TypeⅡalveolar epithelia were obtained by immune adhesion,and the expression ofα-SMA and autophagy markers(p62 and LC3Ⅰ/Ⅱ)as well as signaling pathway proteins(p-mTOR/mTOR and p-Akt)of typeⅡalveolar cells were assessed with Western blot.Results1)Lung tissue morphology of group A was normal.In group B,large consolidation of lung parenchyma with mild infiltration of inflammatory cells(macrophages and neutrophils)and fibroblasts were found,and the alveolar interval was thickened with collagen deposition.2)Masson staining showed that the blue area of Group B was significantly larger than Group A [(23.73±4.20)mm^2 vs(12.12±1.61)mm^2,P<0.05].The stained area of Group A was concentrated around trachea and vessels,while the stained area of Group B was further detected in the lung mesenchyme,presented as flake form.3)Western blot showed that the expression ofα-SMA of experimental group was higher than control group(P<0.05).The p62 levels of Group B were significantly higher than Group A(P<0.05).LC3Ⅱ/Ⅰratio of Group B was lower than Group A(P<0.05).The expression of pAkt and p-mTOR of experimental group was higher than control group(P<0.05).There was no significant difference in the expression of mTOR between the two groups(P>0.05).Conclusion Autophagy activity is inhibited in typeⅡ alveolar epithelial cells of rat with interstitial pneumonia,possibly due to the activation of Akt/mTOR signaling pathway.