| 郭靖宁,朱 静,张金虎,史艳平,吉 慧.布地奈德通过干预线粒体钙单转运蛋白影响哮喘大鼠气道上皮细胞自噬和屏障功能的机制[J].现代生物医学进展英文版,2021,(24):4641-4645. |
| 布地奈德通过干预线粒体钙单转运蛋白影响哮喘大鼠气道上皮细胞自噬和屏障功能的机制 |
| Budesonide Interferes with the Mechanism of Mitochondrial Calcium Single Transporter in Affecting Autophagy and Barrier Function of Airway Epithelial Cells in Asthmatic Rats |
| Received:April 06, 2021 Revised:April 28, 2021 |
| DOI：10.13241/j.cnki.pmb.2021.24.008 |
| 中文关键词: 布地奈德 MCU 哮喘大鼠 细胞屏障 自噬 |
| 英文关键词: Budesonide MCU Asthmatic rats Cell barrier Autophagy |
| 基金项目:陕西省自然科学基础研究计划项目（2018JQ-487） |
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| 中文摘要: |
| 摘要 目的：探究布地奈德通过干预线粒体钙单转运蛋白影响哮喘大鼠气道上皮细胞自噬和屏障功能的机制。方法：30只雄性SD大鼠作为研究对象，并根据实验目的分为3组：对照组（正常大鼠，生理盐水干预，n=10），哮喘组（通过OVA诱导大鼠哮喘模型，n=10），布地奈德组（气雾剂布地奈德用于治疗过敏性哮喘的大鼠，n=10）。通过钙测定试剂盒和蛋白印迹分析大鼠气道上皮细胞中Ca2+的吸收和MCU蛋白表达；TEER和TRITC 荧光分析检测大鼠气道上皮中的屏障功能；免疫组化分析分气道上皮细胞屏障功能相关因子ZO-1、E-cadherin的蛋白表达；ELISAF分析BALF上清液中炎性因子IL-4、IL-5和IL-13的水平；二氢乙锭衍生物和蛋白印迹分析BALF中ROS含量和caspase-3活性。结果：哮喘组较对照组Ca2+浓度降低，MCU蛋白表达升高（P<0.05），布地奈德组较哮喘组Ca2+浓度升高，MCU蛋白表达降低（P<0.05）。哮喘组较对照组TEER降低，TRITC升高（P<0.05），布地奈德组较哮喘组TEER升高，TRITC降低（P<0.05）。哮喘组较对照组ZO-1、E-cadherin的蛋白表达降低（P<0.05），布地奈德组较哮喘组ZO-1、E-cadherin的蛋白表达升高（P<0.05）。哮喘组较对照组IL-4、IL-5和IL-13的水平升高（P<0.05），布地奈德组较哮喘组IL-4、IL-5和IL-13的水平降低（P<0.05）。对照组组支气管和肺泡结构未见异常，与对照组相比哮喘组大鼠表现出肺泡间隔增厚，可见的肺毛细血管水肿，以及肺毛细血管和肺泡间隙中的大量炎性细胞浸润（P<0.05），与哮喘组相比，布地奈德组显著减轻肺部病变的严重程度（P<0.05）。哮喘组较对照组LC3B II/I、ATG5、Beclin-1 和LC3II 的表达升高（P<0.05），布地奈德组较哮喘组LC3B II/I、ATG5、Beclin-1 和LC3II 的表达降低（P<0.05）。哮喘组较对照组ROS含量和caspase-3活性升高（P<0.05），布地奈德组较哮喘组ROS含量和caspase-3活性降低（P<0.05）。结论：布地奈德通过调节MCU表达介导气道上皮细胞的屏障完整性和自噬水平，缓解哮喘气道炎症，改善哮喘症状。 |
| 英文摘要: |
| ABSTRACT Objective: To explore the mechanism of budesonide's influence on autophagy and barrier function of airway epithelial cells in asthmatic rats by interfering with mitochondrial calcium single transport protein. Methods: Thirty male SD rats were used as the research objects and were divided into 3 groups according to the purpose of the experiment: control group (healthy reared rats as a control, normal saline as a vehicle for control treatment, n=10), asthma group (a rat asthma model induced by OVA, n=10), budesine German group (aerosol budesonide used to treat rats with allergic asthma, n=10). Calcium assay kit and Western blot were used to analyze Ca<2+/sup> uptake and MCU protein expression in rat airway epithelial cells. TEER and TRITC fluorescence analysis were used to detect the barrier function in rat airway epithelium. The protein expressions of ZO-1 and E-cadherin, which are related to the barrier function of airway epithelial cells, were analyzed by immunohistochemistry. The levels of inflammatory factors IL-4, IL-5 and IL-13 in the BALF supernatant were analyzed by ELISAF. ROS content and caspase-3 activity in BALF were analyzed by dihydroethidium derivatives and Western blotting. Results: Compared with the control group, the Ca<2+/sup> concentration of the asthma group was lower, and the expression of MCU protein was increased(P<0.05). Compared with the asthma group, the Ca<2+/sup> concentration of the budesonide group was higher, and the expression of MCU protein was lower(P<0.05). Compared with the control group, the asthma group had lower TEER and higher TRITC (P<0. 05), and the budesonide group had higher TEER and lower TRITC than the asthma group(P<0.05). The protein expression of ZO-1 and E-cadherin in the asthma group was lower than that in the control group(P<0.05), and the protein expression of ZO-1 and E-cadherin in the budesonide group was higher than that in the asthma group(P<0.05). The levels of IL-4, IL-5 and IL-13 in the asthma group were higher than those in the control group(P<0.05), and the levels of IL-4, IL-5 and IL-13 in the budesonide group were lower than those in the asthma group (P<0.05). The bronchi and alveolar structure of the control group were not abnormal. Compared with the control group, rats in the asthma group showed thickened alveolar septum, visible pulmonary capillary edema, and a large number of inflammatory cell infiltrations in pulmonary capillaries and alveolar spaces(P<0.05). Compared with the asthma group, the budesonide group significantly reduced the severity of lung lesions(P<0.05). The expression of LC3B II/I, ATG5, Beclin-1 and LC3II in the asthma group was higher than that in the control group(P<0.05), and the expression of LC3B II/I, ATG5, Beclin-1 and LC3II in the budesonide group was lower than that in the asthma group(P<0.05). Compared with the control group, the ROS content and caspase-3 activity were higher in the asthma group(P<0.05), and the budesonide group was lower than the asthma group ROS content and caspase-3 activity (P<0.05). Conclusion: Budesonide mediates the barrier integrity and autophagy level of airway epithelial cells by regulating the expression of MCU, so as to alleviate airway inflammation and improve asthma symptoms. |
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