激活Pink1/Parkin通路减轻劳力型热射病大鼠急性肺损伤

Activation of Pink1/Parkin pathway alleviates the acute lung injury in exertional heat stroke rats

目的探讨PTEN诱导酶1(PTEN-induced putative kinase 1,Pink1)/帕金蛋白(parkin)通路在劳力型热射病(exertional heat stroke,EHS)大鼠急性肺损伤中的作用。方法将60只SD大鼠随机分为4组,正常组(CON组)、正常Parkin过表达组(CON+Parkin组)、劳力型热射病组(EHS组)和劳力型热射病Parkin过表达组(EHS+Parkin组),每组大鼠15只。正常Parkin过表达组和EHS Parkin过表达组大鼠经尾静脉注射携带有Parkin基因的腺相关病毒0.15μL。20 d后,建立EHS大鼠模型,绘制生存曲线。检测肺系数和肺毛细血管通透性;HE染色观察肺组织病理变化;ELISA方法检测肺组织中白介素(interleukin,IL)-6、IL-1β、肿瘤坏死因子-α(tumour necrosis factor-α,TNF-α)和活性氧簇(reactive oxygen species,ROS)的含量;免疫组织化学观察肺组织凋亡;Western blot方法测定大鼠肺组织中Pink1、Parkin、泛素结合蛋白P62(Ubiquitin-binding protein p62)和微管相关蛋白1轻链3(microtubule-associated protein 1 light chain 3,LC3)的表达,并计算LC3-II/LC3-I比值。免疫荧光检测Pink1和Parkin共定位。单因素多水平组比较采用单因素方差分析,采用SNK-q法进一步进行组间两两比较。结果与正常组相比,EHS组大鼠生存率降低(P<0.001),肺系数和肺血管通透性均升高[(4.39±0.42)、(33.38±8.29)μg/g,P<0.05)],肺组织发生渗出和实变,炎症因子IL-6、IL-1β、TNF-α和ROS水平均显著升高[(34.31±5.34)pg/mL、(34.03±4.78)pg/mL、(91.64±8.16)pg/mL、(259.01±89.17)U/mg,P<0.05],细胞凋亡增加。Western及免疫荧光结果显示,劳力型热射病大鼠Pink1、Parkin表达减少,Pink1和Parkin结合减弱,LC3-II/LC3-I比值降低,P62表达增加。与EHS组相比,EHS Parkin过表达组的大鼠生存率明显升高(P<0.05),肺系数和肺血管通透性均降低[(3.83±0.62)、(22.49±7.90)μg/g,P<0.05],渗出和实变等病理改变明显减轻,上述炎症因子和ROS水平均显著降低[(14.09±3.24)pg/mL、(26.94±2.11)pg/mL、(63.35±11.62)pg/mL、(161.13±26.31)U/mg,P<0.05];肺组织凋亡减轻。肺组织中Parkin表达和LC3-II/LC3-I比值均升高(P<0.05),Pink1和Parkin的结合增强,P62表达减低(P<0.05),而Pink1表达差异无统计学意义(q=0.75)。正常组和正常Parkin过表达组之间差异无统计学意义(q=0.95)。结论激活Pink1/Parkin通路,增强线粒体自噬可减轻劳力型热射病大鼠急性肺损伤。

Objective To investigate the role of Pink1/Parkin-induced mitophagy in acute lung injury of exertional heat stroke rats.Methods Sixty SD rats were randomly divided into four groups,including normal group(CON group),normal Parkin overexpression group(CON+Parkin group),heat stroke group(EHS group)and heat stroke Parkin overexpression group(EHS+Parkin group),with fifteen rats in each group.The rat model of exertional heat stroke was established and the survival curve was drawn.Pulmonary coefficient and pulmonary capillary permeability were detected.HE staining was used to observe the pathological changes of lung tissue.ELISA was used to detect the contents of IL-6,IL-1β,TNF-αand ROS in lung tissue;immunohistochemistry was used to observe apoptosis in lung tissue;Western blot was used to determine the expression of Pink1,Parkin,P62 and LC3 in rat lung tissue,and the LC3II/LC3I ratio was calculated.Single factor multi-level group comparison was performed by single factor analysis of variance,SNK-q method was used to further pairwise comparison between groups.Results Compared with the normal group,the survival rate of EHS group was decreased(P<0.001),lung coefficient and pulmonary vascular permeability were increased[(4.39±0.42),(33.38±8.29)μg/g,P<0.05)],lung tissue was exudative and solid,the levels of inflammatory factors IL-6,IL-1β,TNF-αand ROS were significantly increased[(34.31±5.34)pg/mL,(34.03±4.78)pg/mL,(91.64±8.16)pg/mL,(259.01±89.17)U/mg,P<0.05)],and apoptosis was increased.Western and immunohistochemistry results showed that the expressions of Pink1 and Parkin were decreased,co-location of Pink1and Parkin was attenuated,LC3II/LC3I were decreased,and P62 expression was increased.Compared with the EHS group,the survival rate of EHS+Parkin group was significantly increased(P<0.05),lung coefficient and pulmonary vascular permeability were decreased[(3.83±0.62),(22.49±7.90)μg/g,P<0.05)],exudation and consolidation and other pathological changes were significantly reduced,and the levels of the above inflammatory factors and ROS were significantly decreased[(14.09±3.24)pg/mL,(26.94±2.11)pg/mL、(63.35±11.62)pg/mL,(161.13±26.31)U/mg,P<0.05].Lung tissue apoptosis was reduced.The co-location of Pink1and Parkin、Parkin expression and LC3II/LC3I ratio were increased(P<0.05),P62 expression was decreased(P<0.05),while Pink1 expression was not significant different(q=0.75).There was no difference between normal group and normal Parkin overexpression group(q=0.95).Conclusion Activation of Pink1/Parkin-induced mitophagy can alleviate the acute lung injury in exertional heat stroke rats.