文章摘要
赵跃恒,常姗,史瑞玲,等.麦门冬汤对间质性肺炎小鼠Ⅱ型肺泡上皮细胞自噬及肺水清除的作用机制研究[J].浙江中医药大学学报,2021,(2):116-123.
麦门冬汤对间质性肺炎小鼠Ⅱ型肺泡上皮细胞自噬及肺水清除的作用机制研究
Study on the Mechanism of Maimendong Decoction on Autophagy of Type Ⅱ Alveolar Epithelial Cells and Lung Fluid Clearance in Mice with Interstitial Pneumonia
投稿时间:2020-07-20  
DOI:10.16466/j.issn1005-5509.2021.02.003
中文关键词: 间质性肺炎  麦门冬汤  强的松  Ⅱ型肺泡上皮细胞  自噬  肺水清除  肺纤维化  PI3K/AKT/mTOR信号通路
英文关键词: interstitial pneumonia  Maimendong decoction  prednisone  type Ⅱ alveolar epithelial cells  autophagy  lung fluid clearance  pulmonary fibrosis  PI3K/AKT/mTOR signaling pathway
基金项目:2018年度河南省高等学校重点科研项目(18A320036)
作者单位
赵跃恒 河南省荣军医院 河南新乡 453002 
常姗 河南省荣军医院 河南新乡 453002 
史瑞玲 河南省荣军医院 河南新乡 453002 
张华 郑州大学附属郑州中心医院 
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中文摘要:
      [目的]观察麦门冬汤对间质性肺炎小鼠Ⅱ型肺泡上皮细胞自噬及对肺水清除的影响,并探究其作用机制。[方法]将120只无特定病原体(specific pathogen free,SPF)级C57BL/6小鼠随机分为6组:正常组、模型组、强的松组及麦门冬汤高、中、低剂量组,每组20只。除正常组小鼠经鼻腔滴注0.9%氯化钠溶液之外,其余各组小鼠均通过鼻腔滴注博来霉素(bleomycin,BLM)建立间质性肺炎模型。造模后,麦门冬汤高、中、低剂量组小鼠分别灌胃给予17.16g·kg-1、8.58g·kg-1、4.29g·kg-1麦门冬汤,强的松组小鼠灌胃给予0.46g·kg-1强的松,正常组和模型组小鼠灌胃给予等量蒸馏水,均为1次/d,连续28d。实验结束分离小鼠肺组织,计算肺组织湿干重比(wet/dry weight ratio,W/D);以Evans Blue法计算肺泡液体清除率(alveolar fluid clearance,AFC);苏木精-伊红(hematoxylin-eosin,HE)染色观察小鼠肺组织病理变化;Masson染色观察肺组织纤维化情况,羟脯氨酸测定胶原表达水平;免疫黏附法分离Ⅱ型肺泡上皮细胞,Western blot法检测α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、p62、自噬微管相关蛋白轻链3-Ⅱ(microtubule-associated protein light chain 3-Ⅱ,LC3-Ⅱ)、磷酸化磷酯酰肌醇-3-激酶(phosphorylated-phosphatidylinositol-3-kinase,p-PI3K)、磷酸化蛋白激酶B(phosphophorylated-protein kinase B,p-AKT)和磷酸化哺乳动物雷帕霉素靶蛋白(phosphorylated-mammalian target of rapamycin,p-mTOR)表达水平。[结果]与正常组比较,模型组小鼠肺组织W/D值显著升高,AFC降低(P<0.05)。HE染色和Masson染色显示模型组小鼠肺组织正常结构基本消失,纤维化显著,肺组织羟脯氨酸含量显著升高,胶原蛋白显著增加,肺泡Ⅱ型肺泡上皮细胞α-SMA、p62、p-PI3K、p-AKT和p-mTOR的表达均显著增高,LC3-Ⅱ表达降低(P<0.05)。与模型组比较,麦门冬汤各剂量组和强的松组小鼠肺组织W/D值均降低,AFC均升高,其中麦门冬汤高、中剂量组W/D值低于强的松组,AFC高于强的松组(P<0.05)。麦门冬汤高、中剂量组小鼠肺组织肺泡结构相对清晰,肺纤维化得到改善,胶原蛋白减少,但麦门冬汤低剂量组和强的松组小鼠肺组织仍存在明显纤维化情况,胶原蛋白沉积明显。与模型组比较,麦门冬汤各剂量组和强的松组小鼠Ⅱ型肺泡上皮细胞α-SMA、p62、p-PI3K、p-AKT和p-mTOR蛋白表达均降低,LC3-Ⅱ蛋白表达均增加(P<0.05),其中麦门冬汤高、中剂量组小鼠Ⅱ型肺泡上皮细胞α-SMA、p62、p-PI3K、p-AKT和p-mTOR蛋白表达低于强的松组,LC3-Ⅱ蛋白表达高于强的松组(P<0.05),而麦门冬汤低剂量组上述蛋白表达与强的松组比较,差异无统计学意义(P>0.05)。[结论]麦门冬汤对小鼠间质性肺炎和肺水清除有改善作用,其作用机制可能与抑制PI3K/AKT/mTOR信号通路,增强肺组织细胞自噬活性有关。
英文摘要:
      [Objective]To observe the effect of Maimendong decoction on autophagy of type Ⅱ alveolar epithelial cells and lung fluid clearance in mice with interstitial pneumonia and explore its mechanism of action. [Methods]One hundred and twenty specific pathogen free(SPF) C57BL/6 mice were randomly divided into 6 groups: Normal group, model group, prednisone group, Maimendong decoction high-dose group, medium-dose group and low-dose group, with 20 animals per group. Except for normal group of mice instilled with 0.9% sodium chloride solution, the other groups of mice were instilled with bleomycin(BLM) to establish interstitial pneumonia model. After the model was built, the mice in Maimendong decoction high-dose group, medium-dose group and low-dose group were given 17.16g·kg-1, 8.58g·kg-1, 4.29g·kg-1 Maimendong decoction by gavage respectively, the mice in prednisone group were given 0.46g·kg-1 prednisone by gavage, and mice in normal and model groups were given the same amount of distilled water. The mice were administered once a day for 28 consecutive days. At the end of the experiment, the lung tissues of mice were separated and the wet/dry weight ratio(W/D) of lung tissues was calculated. The alveolar fluid clearance(AFC) was calculated by Evans Blue method. The pathological changes of lung tissues of mice were observed by hematoxylin-eosin(HE) staining. Masson staining was used to observe the fibrosis of lung tissues. Hydroxyproline was used to determine the expression levels of collagen. Immune adhesion method was used to isolate type Ⅱ alveolar epithelial cells, and Western blot was used to detect α-smooth muscle actin(α-SMA), p62, microtubule-associated protein light chain 3-Ⅱ(LC3-Ⅱ), phosphorylated-phosphatidylinositol-3-kinase(p-PI3K), phosphorylated-protein kinase B(p-AKT) and phosphorylated-mammalian target of rapamycin(p-mTOR) expression level. [Results]Compared with normal group, the W/D value of the lung tissues of model group were significantly increased, and the AFC decreased(P<0.05). HE staining and Masson staining showed that the normal structure of the lung tissues basically disappeared in model group, the fibrosis of the lung tissues was significant, the content of hydroxyproline in the lung tissues were significantly increased, the collagen protein was significantly increased. The expression levels of α-SMA, p62, p-PI3K, p-AKT and p-mTOR in type Ⅱ alveolar epithelial cells were significantly up-regulated, and the expression of LC3-Ⅱ was significantly down-regulated(P<0.05). Compared with model group, the W/D values of lung tissues of mice in Maimendong decoction each dose group and prednisone group decreased, and the AFC increased. The W/D values of Maimendong decoction high-dose and medium-dose groups were lower than that in prednisone group, and the AFC was higher than that in prednisone group(P<0.05). The alveolar structure of the lung tissues of the mice in Maimendong decoction high-dose and medium-dose groups were relatively clear, and fibrosis was improved, while there were still obvious fibrosis and collagen deposition in the lung tissue in Maimendong decoction low-dose group and prednisone group. Western blot results showed that compared with model group, the expression levels of α-SMA, p62, p-PI3K, p-AKT and p-mTOR protein in type Ⅱ alveolar epithelial cells of Maimendong decoction each dose group and prednisone group were reduced, and the expression of LC3-Ⅱ protein was increased(P<0.05). The expression levels of α-SMA, p62, p-PI3K, p-AKT and p-mTOR protein in Maimendong decoction high-dose and medium-dose groups were lower than that of prednisone group, and the expression of LC3-Ⅱ protein was higher than that of prednisone group(P<0.05), while the Maimendong decoction low-dose group showed no significant difference in protein expression levels compared with prednisone group(P>0.05). [Conclusion]Maimendong decoction can improve interstitial pneumonia and lung fluid clearance in mice. The mechanism may be related to the inhibition of PI3K/AKT/mTOR signaling pathway and the enhancement of autophagy activity of lung tissue cells.
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