目的:建立百草枯(paraquat,PQ)中毒大鼠模型,观察不同阶段肺线粒体膜电位(JC-1染色)、调控线粒体自噬关键性蛋白PINK1、Parkin及活性氧(reactive oxygen species,ROS)的变化,探讨线粒体自噬是否参与PQ中毒肺纤维化的发生.方法:成年雄性SD大鼠42只,随机分为正常对照组(n=6)、模型组(n=36),模型组下设2 h、12 h、1 d、3 d、7 d和14 d共6个时间点,每个时间点各6只大鼠.使用20%PQ溶液50 mg/kg一次性灌胃大鼠建立PQ中毒模型.通过流式细胞仪检测血红细胞ROS浓度;HE染色和Masson染色观察PQ中毒后肺组织病理损害;JC-1染色检测肺组织线粒体膜电位变化;Western blot检测PINK1、Parkin蛋白变化.结果:与对照组相比,随着PQ中毒时间的延长,肺纤维化病理评分较对照组升高,差异有统计学意义(P<0.05);PQ组ROS荧光阳性强度率比值在中毒后2 h显著升高,中毒后12 h达高峰后逐渐下降(P<0.05),中毒后14 d与对照组相比差异无统计学意义(P>0.05);PQ组肺组织JC-1红绿荧光比均较对照组降低,Western blot提示随中毒时间延长,PINK1及Parkin蛋白表达均升高,差异有统计学意义(P<0.01).PQ组肺组织JC-1红绿荧光比与PINK1及Parkin、肺纤维化病理评分均呈负相关(r=-0.890,P<0.01;r=-0.845, P<0.01;r=-0.794,P<0.01).在PQ中毒12 h内,血红细胞ROS荧光强度阳性率与JC-1红绿荧光比呈负相关(r=-0.712,P<0.01),与PINK1及Parkin、肺纤维化病理评分呈正相关(r=0.571,P<0.01;r=0.484,P<0.01;r=0.602,P<0.05).结论:PQ中毒导致大鼠产生了明显氧化应激,诱发了线粒体自噬.ROS的产生与线粒体自噬的发生密切相关,共同参与了PQ大鼠肺纤维化的发生发展.%Objective:To investigate the effect of mitophagy on the occurrence of pulmonary fibrosis in paraquat poisoning by observe the changes of JC-1, PINK1,Parkin and the reactive oxygen species(ROS)in different stages of paraquat-induced rat pulmonary fibrosis model.Methods:42 adult male sprague-dawley (SD) rats were randomly divided into normal group(n=6) and PQ group(n=36).PQ group rats were divided into six time points:2 h,12 h,24 h,72 h,7 d,14 d,each time point includes 6 rats.20% PQ solution with adose of 50 mg/kg were selected for once administration gavage to establish PQ poisoning model,through the detection of red blood cell ROS concentration by flow cytometry,HE staining and Masson staining of lung tissue damage was observed after PQ poisoning,JC-1 staining was used to detect the changes of mitochondrial membrane potential changes in lung tissue,PINK1,Parkin protein was detected by Western blot.Results:Compared with normal group,the degrees of pulmonary fibrosis was gradually increased with the increase of PQ poisoning time,the difference was significant (P<0.05).In the PQ groups, the positive rate of ROS fluorescence intensity increased significantly at 2h after poisoning,and peaked at 12 h after poisoning, and then decrease gradually(P<0.05),the difference between normal group and 14 d after poisoning has no statistical significance (P>0.05).The ratio of JC-1,red green fluorescence in lung tissue of PQ group was lower than that of control group,and Western and blot indicated that the expression of PINK1 and Parkin protein increased with the duration of poisoning, and the difference was statistically significant (P<0.01).Pearson test shows there is a negative correlation between the red green fluorescence ratio and PINK1,Parkin and the pathology score of pulmonary fibrosis.(r=-0.890,P<0.01;r=-0.845,P<0.01;r=-0.794,P<0.01).In 12 h after PQ poisoning,there is a negative correlation between the ratio of the positive rate of ROS fluorescence intensity in erythrocyte and the red green fluorescence (r=-0.712,P<0.01),and a positive correlation between the positive rate of ROS fluorescence intensity and PINK1,Parkin and the pathology score of pulmonary fibrosis(r=0.571,P<0.01;r=0.484,P<0.01;r=0.602,P<0.05).Conclusion:The mitophagy and oxidative stress were induced by PQ poisoning.The generation of ROS closely related to the development of autophagy in mitochondria and is involved in the development of pulmonary fibrosis in PQ rats.
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