雷帕霉素上调NIX表达保护劳力性热射病大鼠肺功能

Rapamycin up-regulates NIX for protecting exertional heat stroke rats against acute lung injury

目的探讨雷帕霉素(Rapa)对劳力性热射病(EHS)大鼠肺功能的保护作用及其机制。方法将60只健康雄性SD大鼠随机分为对照组(Ctrl)、雷帕霉素组(Rapa)、EHS组和EHS+雷帕霉素干预组(EHS+Rapa),每组15只。建立EHS模型后,测量大鼠直肠温度变化。取动脉血进行血气分析,伊文氏蓝(EB)染色测定肺血管通透性,HE染色观察大鼠肺组织病理学改变,原位末端转移酶标记法(Tunel)观察大鼠肺组织凋亡,透射电镜下观察大鼠肺Ⅱ型上皮细胞中线粒体的形态,Western blot方法测定大鼠肺组织中NIX和微管相关蛋白1轻链3(LC3)的表达,并计算LC3-Ⅱ/LC3-Ⅰ比值,免疫荧光双标观察肺组织中LC3与Tom20的共定位情况。结果EHS和EHS+RAPA组大鼠核心温度均显著升高至42℃以上,雷帕霉素改善EHS大鼠肺血管通透性和氧合,减轻肺组织病理损伤和细胞凋亡。透射电镜下,雷帕霉素保护了EHS大鼠肺Ⅱ型上皮细胞中的线粒体形态。Western blot和免疫荧光结果显示,雷帕霉素干预上调EHS大鼠肺组织中NIX水平,增加LC3Ⅱ/LC3I比值,增强LC3和Tom20在肺组织中的相互结合。结论雷帕霉素通过上调NIX激活线粒体自噬,从而减轻EHS导致的肺损伤,保护肺功能。

Objective To investigate the mechanism of rapamycin(Rapa)on alleviating acute lung injury of rats with exertional heat stroke(EHS).Methods Sixty heathy male SD rates were randomly divided into four groups:control group(Ctrl group),Rapa group,EHS group,and EHS+Rapa group,with 15 rats in each group.After establishing the EHS models,the core temperature and the arterial blood gas analysis were measured.The pulmonary vascular permeability was measured by Evans blue(EB)staining.The pulmonary histopathological changes were analyzed by hematoxylin eosin(HE)staining.The apoptosis in rat lung tissue was observed by terminal-deoxynucleoitidyl transferase mediated nick end labeling(TUNEL).Mitochondrial morphology in typeⅡepithelial cells of rat lung was observed by transmission electron microscopy.Western blot was used to detect the expressions of NIX and LC3,and the ratio of LC3-Ⅱto LC3-Ⅰwas calculated.Immunofluorescence was used to detect the co-localization of LC3 and Tom20 in lung tissue.Results The core temperature of EHS group and EHS+Rapa group increased sharply to 42℃.Rapamycin alleviated the pathological injury,decreased the apoptosis of lung tissues,reduced pulmonary vascular permeability,and improved the oxygenation of arterial blood.Under transmission electron microscopy,the mitochondrial morphology in typeⅡepithelial cells of rat lung was maintained in EHS+Rapa group.Western blot and immunofluorescence results showed that rapamycin up-regulated the expressions of NIX proteins,increased the ratio of LC3-Ⅱ/LC3-Ⅰin lung tissues of EHS rats,and enhanced the co-localization of LC3 and Tom20 in EHS rat′s lung tissue.Conclusions Rapamycin up-regulates NIX expression and activates mitochondrial autophagy,thereby alleviating EHS-induced lung injury and protecting lung function.