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

作　　者：宋超 张爱国 王坤丽 宋予震[1] 董青[1] 靳双星[1] 米俊宪[1] 王宏魁[1] 石冬梅[1] 王俊东 彭巍[3] 罗琴 SONG Chao;ZHANG Aiguo;WANG Kunli;SONG Yuzhen;DONG Qing;JIN Shuangxing;MI Junxian;WANG Hongkui;SHI Dongmei;WANG Jundong;PENG Wei;LUO Qin(Zhengzhou City Key Laboratory of Animal Nutrition Metabolic and Poisoning Diseases,College of Veterinary Medicine,Henan University of Animal Husbandry and Economy,Zhengzhou 450000,China;Huanong(Zhaoqing)Institute of Biotechnology Co.,Ltd.,Zhaoqing,Guangdong 526000,China;Qinghai Academy of Animal Science and Veterinary Science,Xining 810000,China)

机构地区：[1]河南牧业经济学院动物医药学院,郑州市动物营养代谢病与中毒病重点实验室,河南郑州450000 [2]华农(肇庆)生物产业技术研究院有限公司,广东肇庆526000 [3]青海省畜牧兽医科学院,青海西宁810000

出　　处：《中国兽医学报》2024年第4期712-718,共7页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(31902285,32102631);青海省科技计划资助项目(2021-ZJ-736);河南省自然科学基金青年科学基金资助项目(232300420218);河南省高等学校重点科研资助项目(21A230005);河南省科技攻关资助项目(222102110155)。

摘　　要：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

关 键 词：氟化钠 肺脏 磷酸酶与张力蛋白同源物诱导激酶1 线粒体自噬 氧化应激 炎症 

分 类 号：S852.3[农业科学—基础兽医学]