PINK1敲除对氟化钠处理小鼠肺脏线粒体损伤、氧化应激和炎症反应的影响

Effects of PINK1knockout on sodium fluoride(NaF)-induced mitochondrial injury,oxidative stress,and inflammation in mice lungs

以6周龄雄性野生型(WT)C57BL/6J小鼠和磷酸酶与张力蛋白同源物诱导激酶1(PINK1)基因敲除(PINK1^(-/-))小鼠为对象,将小鼠随机分为WT对照组,WT模型组和PINK1^(-/-)模型组。WT模型组和PINK1^(-/-)模型组小鼠饮用含100mg/L氟化钠(NaF)的蒸馏水,WT对照组小鼠饮用蒸馏水,处理时间为12周。通过免疫印迹(Western blot)研究线粒体自噬相关蛋白表达水平;用透射电镜观察肺脏线粒体超微结构损伤;ATP通过检测试剂盒和RT-qPCR分别测定ATP水平和mtDNA拷贝数;DHE染色检测肺脏活性氧(ROS)水平;比色法测定超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活力以及丙二醛(MDA)的含量;TUNEL染色检测肺脏细胞凋亡;酶联免疫吸附法(ELISA)测定肺泡灌洗液中肿瘤坏死因子(TNF-α)和白介素6(IL-6)的水平。结果显示,与WT模型组比较,PINK1^(-/-)模型组小鼠肺脏中帕金森病蛋白2(Parkin)和微管相关蛋白1轻链3Ⅱ(LC3-Ⅱ)的蛋白表达水平显著降低(P<0.05),p62/SQSTM1和线粒体外膜转运孔蛋白20(TOM20)蛋白表达水平显著增加(P<0.05);与WT模型组比较,PINK1^(-/-)模型组小鼠肺脏受损线粒体百分比增加(P<0.05),ATP水平和mtDNA拷贝数减少(P<0.05)。PINK1^(-/-)模型组小鼠肺脏ROS水平、MDA含量和TUNEL阳性细胞数升高,SOD和CAT活力降低,较WT模型组有显著性差异(P<0.05)。此外,与WT模型组比较,PINK1^(-/-)模型组小鼠肺泡灌洗液中TNF-α和IL-6的水平显著增加(P<0.05)。结果表明,PINK1介导的线粒体自噬可减弱NaF引起的小鼠肺脏损伤。

Six-week-old male wild-type(WT)and PTEN-induced kinase 1(PINK1)knockout mice at C57BL/6Jbackground were adopted and were randomly divided into the WT control group,WT model group,and PINK1^(-/-)model group.Mice in the WT model group and PINK1^(-/-)model group were given distilled water containing 100 mg/L sodium fluoride(NaF),mice in the WT control group were given distilled water for 12weeks.The expressions of mitophagy-related proteins were investigated by Western blot.Ultrastructural damages of lung mitochondria were observed by transmission electron microscopy.ATP levels and mtDNA copy numbers were determined by ATP detection kit and RT-qPCR,respectively.Reactive oxygen species(ROS)were detected by DHE staining.The activities of superoxide dismutase(SOD),catalase(CAT),and malondialdehyde(MDA)were determined by colorimetric method.The apoptosis of lung cells was detected by TUNEL staining.The levels of tumor necrosis factor(TNF-α)and interleukin-6(IL-6)in alveolar lavage fluid were determined by enzyme linked immunosorbent assay(ELISA).The results showed that compared with the WT model group,the protein changes of Parkinson disease protein 2(Parkin)and microtubule-associated protein 1light chain 3Ⅱ(LC3-Ⅱ)were markedly decreased(P<0.05),while the protein expressions of p62/SQSTM1and mitochondrial 20kDa outer membrane protein(TOM20)were significantly decreased(P<0.05).The percentages of damaged mitochondria in lung cells were increased,while the ATP levels and mtDNA copy number were decreased in the PINK1^(-/-)model group when compared with those in the WT model group(P<0.05).The ROS level,MDA content,and TUNEL-positive cell numbers were significantly increased,while the activities of SOD and CAT were decreased in the lungs of PINK1^(-/-)model group,which were significantly different from those in WT model group(P<0.05).Additionally,compared with the WT model group,the levels of TNF-αand IL-6in alveolar lavage fluid of PINK1^(-/-)model group were significantly increased by PINK1knockout(P<0.05).The results indicated that PINK1-mediated mitophagy could attenuate NaF-induced lung injury in mice.