目的:探讨Parkin过表达线粒体自噬在减轻劳力性热射病(EHS)大鼠急性肺损伤中的作用。
方法:80只SD大鼠随机分为4组:对照组(CON组),Parkin过表达对照组(CON+Parkin组),劳力性中暑组(EHS组),和劳力性中暑Parkin过表达组(EHS+Parkin组)。将携带Parkin基因的腺相关病毒静脉注射到大鼠体内,以在肺组织中过表达Parkin。建立劳力性中暑大鼠模型,并绘制了生存曲线。行肺部Micro-CT,测量肺系数和肺微血管通透性。酶联免疫吸附试验(ELISA)用于测定白细胞介素-6(IL-6)的水平,白细胞介素-1β(IL-1β),肿瘤坏死因子-α(TNF-α),和活性氧(ROS)。透射电镜观察肺组织Ⅱ型上皮细胞线粒体形态。肺组织的凋亡,线粒体自噬的水平,用免疫荧光法测定Pink1和Parkin的共定位。Pink1,Parkin,MFN2,PTEN-L,PTEN,用蛋白质印迹法测定大鼠肺组织p62和微管相关蛋白1轻链3(LC3)。
结果:与CON组相比,有更严重的肺损伤和更高水平的IL-6,IL-1β,TNF-α在EHS大鼠中的表达。LC3-II/LC3-I比值以及LC3和Tom20在EHS大鼠肺组织中的共定位均降低。与EHS组相比,EHS+Parkin过表达组大鼠的存活率显著提高,肺系数和肺微血管通透性降低,渗出和巩固等病理变化明显减轻。IL-6、IL-1β、TNF-α,和ROS显著降低;Ⅱ型肺泡上皮细胞线粒体肿胀程度降低,并且没有观察到空泡化。肺组织凋亡减少,以及Pink1和Parkin的共定位荧光,以及LC3和Tom20,都增加了。Parkin和LC3-II/LC3-I比值在肺组织中的表达均升高,而P62、Pink1、MFN2和PTEN-L的表达降低。
结论:Pink1/Parkin介导的线粒体自噬功能障碍是EHS大鼠急性肺损伤的机制之一,激活Parkin过表达诱导的线粒体自噬可以减轻EHS引起的急性肺损伤。
OBJECTIVE: To investigate the role of Parkin overexpression-induecd mitophagy in alleviating acute lung injury of exertional heat stroke(EHS) rats.
METHODS: Eighty SD rats were divided into four groups: Control group (CON group), Control Parkin overexpression group (CON + Parkin group), exertional heat stroke group (EHS group), and exertional heat stroke Parkin overexpression group (EHS + Parkin group). Adeno-associated virus carrying the Parkin gene was intravenously injected into the rats to overexpress Parkin in the lung tissue. An exertional heat stroke rat model was established, and survival curves were plotted. Lung Micro-CT was performed, and lung coefficient and pulmonary microvascular permeability were measured. Enzyme-linked immunosorbent assays(ELISA) were used to determine the levels of interleukin-6(IL-6), interleukin-1β(IL-1β), Tumor necrosis factor-α(TNF-α), and reactive oxygen species(ROS). The morphology of mitochondria in type II epithelial cells of lung tissue was observed using transmission electron microscopy. The apoptosis of lung tissue, the level of mitophagy, and the co-localization of Pink1 and Parkin were determined using immunofluorescence. The expression of Pink1, Parkin, MFN2, PTEN-L, PTEN, p62, and microtubule associated protein 1 light chain 3 (LC3) in rat lung tissue was measured by western blot.
RESULTS: Compared with the CON group, there were more severe lung injury and more higher levels of IL-6, IL-1β, TNF-α in EHS rats. Both of the LC3-II/LC3-I ratio and the co-localization of LC3 and Tom20 in the lung tissue of EHS rats decreased. Compared with the EHS group, the survival rate of rats in the EHS + Parkin overexpression group was significantly increased, lung coefficient and pulmonary microvascular permeability were reduced, and pathological changes such as exudation and consolidation were significantly alleviated. The levels of IL-6, IL-1β, TNF-α, and ROS were significantly decreased; the degree of mitochondrial swelling in type II alveolar epithelial cells was reduced, and no vacuolization was observed. Lung tissue apoptosis was reduced, and the colocalization fluorescence of Pink1 and Parkin, as well as LC3 and Tom20, were increased. The expression of Parkin and LC3-II/LC3-I ratio in lung tissue were both increased, while the expression of P62, Pink1, MFN2, and PTEN-L was decreased.
CONCLUSIONS: Pink1/Parkin-mediated mitophagy dysfunction is one of the mechanisms underlying acute lung injury in rats with EHS, and activation of Parkin overexpression induced-mitophagy can alleviate acute lung injury caused by EHS.