Calpain-Akt信号通路在哮喘气道平滑肌重建中的作用

发布时间：2018-09-05 20:13

【摘要】：目的：探讨钙蛋白酶（calpain）以及其下游的PI3K/Akt信号通路在气道炎症导致的哮喘气道平滑肌重建中的作用。方法：以卵清蛋白被动致敏的方法复制小鼠慢性支气管哮喘气道重构的模型，并腹腔注射calpain抑制剂calpeptin，检测肺组织calpain活性，并用Masson染色检测支气管胶原含量以及平滑肌厚度。用哮喘相关细胞因子IL-4，IL-5和TNF-α处理原代培养支气管平滑肌细胞，检测calpain活性， Western blot方法检测Ⅰ型胶原和磷酸化的Akt（p-Akt）的蛋白表达，以及BrdU-ELISA方法检测平滑肌细胞增殖。同时用calpain抑制剂MDL28170、PI3K抑制剂LY294002分别阻断calpain、Akt信号，进一步证实该通路在胶原生成和细胞增殖中的作用。结果：哮喘小鼠肺组织calpain活性明显高于对照组，Masson染色结果显示支气管有明显的胶原沉积以及平滑肌增厚，而使用calpain抑制剂的哮喘+calpeptin组小鼠肺组织calpain活性低于哮喘组，，而且胶原含量和平滑肌层厚度均低于哮喘组。哮喘细胞因子IL-4，IL-5及TNF-α处理，均可促使气道平滑肌细胞calpain活性增加、细胞增殖以及Ⅰ型胶原和p-Akt表达。 Calpain抑制剂MDL28170抑制Akt的磷酸化（p-Akt），而且，MDL28170及PI3K抑制剂LY294002均可减轻哮喘细胞因子引起的细胞增殖和Ⅰ型胶原表达。结论： Calpain在支气管哮喘气道平滑肌细胞重建中起重要作用，calpain活化PI3K/Akt信号通路是其机制之一。
[Abstract]:Aim: to investigate the role of calpain (calpain) and its downstream PI3K/Akt signaling pathway in airway smooth muscle remodeling induced by airway inflammation. Methods: the airway remodeling model of chronic bronchial asthma in mice was induced by passive sensitization of ovalbumin. The lung tissue calpain activity was detected by intraperitoneal injection of calpain inhibitor calpeptin, and the content of bronchial collagen and the thickness of smooth muscle were detected by Masson staining. Bronchial smooth muscle cells were treated with Asthma related cytokines (IL-4,IL-5 and TNF- 伪). The expression of type I collagen and phosphorylated Akt (p-Akt) protein was detected by calpain activity, Western blot method, and the proliferation of smooth muscle cells was detected by BrdU-ELISA method. At the same time, the calpain,Akt signal was blocked by LY294002, a calpain inhibitor, MDL28170,PI3K inhibitor, to further confirm the role of this pathway in collagen production and cell proliferation. Results: the calpain activity of lung tissue in asthmatic mice was significantly higher than that in control group. The results showed that there was obvious collagen deposition and smooth muscle thickening in the bronchus, while the calpain activity in lung tissue of asthmatic calpeptin group with calpain inhibitor was lower than that of asthmatic calpeptin group. And collagen content and smooth muscle layer thickness were lower than asthma group. The treatment of asthma cytokines IL-4,IL-5 and TNF- 伪 could increase the calpain activity, cell proliferation and the expression of type I collagen and p-Akt in airway smooth muscle cells. MDL28170, an inhibitor of Calpain, inhibited the phosphorylation of Akt (p-Akt), and both MDL28170 and LY294002, the inhibitor of PI3K, could attenuate the proliferation and type I collagen expression induced by cytokines of asthma. Conclusion: Calpain plays an important role in the remodeling of airway smooth muscle cells in bronchial asthma. Calpain activates PI3K/Akt signaling pathway is one of its mechanisms.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R562.25

【参考文献】