氟中毒对神经细胞损伤及自噬的影响  

Effects of fluorosis on thedamages and autopany of cerebral cortical neurons of mice

作　　者：王正薇 高霄 蔡娜 何雯雯 唐智 齐晓岚[1] 官志忠[1] 肖雁[1] WANG Zhengwei;GAO Xiao;CAI Na;HE Wenwen;TANG Zhi;QI Xiaolan;GUAN Zhizhong;XIAO Yan(Key Laboratory of Endemic and Minority Disease of Minority of Education,Guizhou Medical University,Guiyang 550004,Guizhou,China,Key Laboratory of Medical Biology,Guizhou Medical University,Guiyang 550004,Guizhou,China)

机构地区：[1]贵州医科大学地方病与少数民族性疾病教育部重点实验室,贵州医科大学分子生物学重点实验室,贵州贵阳550004

出　　处：《贵州医科大学学报》2024年第5期625-635,共11页Journal of Guizhou Medical University

基　　金：国家自然科学基金项目(82260245);国家自然科学基金委,贵州喀斯特中心项目子课题(U1812403-6);贵州医科大学国基培育项目(20NSP069)。

摘　　要：目的探讨氟中毒对小鼠小胶质细胞株BV2和小鼠来源神经母细胞瘤细胞株N2a及小鼠大脑皮质神经细胞损伤及自噬的影响。方法8周龄清洁级雄性C57BL/6J野生型小鼠,随机分为对照组(饮水含氟量<0.5 mg/L)、低氟组(饮水含氟量10.0 mg/L)和高氟组(饮水含氟量50.0 mg/L),每组10只喂养12周;BV2及N2a正常培养后分对照组、低氟组(培养基含氟量1.0 mmol/L)和高氟组(培养基含氟量1.5 mmol/L);采用CCK8检测不同浓度氟处理下BV2和N2a细胞增殖活力变化,氟离子选择电极法检测造模小鼠尿、血、骨氟含量变化;蛋白免疫印迹法(Western blot)检测BV2、小鼠大脑皮质中小胶质细胞活化标志物离子钙结合衔接分子1(Iba-1)的表达变化及N2a细胞、BV2细胞和小鼠大脑皮质中自噬相关微管相关蛋白1轻链3(LC3)及自噬受体蛋白P62蛋白(SQSTM1/P62)的表达水平;线粒体动力学及自噬融合分裂相关蛋白线粒体融合蛋白1(Mfn1)、线粒体融合蛋白2(Mfn2)、动力相关蛋白1(Drp1)、张力蛋白同源物诱导的假定激酶1(Pink1)及E3泛素连接酶(Parkin)的表达水平。结果CCK8结果显示,1.5 mmol/LNaF处理的BV2、N2a细胞增殖活力明显下降(P<0.001,);低氟组小鼠氟斑牙发生率为60%,高氟组小鼠氟斑牙发生率为80%;与对照组比较,染氟组小鼠尿、血、骨氟含量明显上升(P<0.001);蛋白免疫印迹结果显示,与对照组比较,高氟组的小鼠大脑皮质和BV2的小胶质细胞活化标志物Iba-1表达水平显著升高(P<0.001,P<0.01),高氟组的小鼠大脑皮质和BV2及N2a细胞自噬相关蛋白LC3、Parkin、Pink1表达量显著增加(P<0.05)、P62水平显著下降(P<0.01);线粒体融合蛋白Mfn1、Mfn2水平显著增加(P<0.001),分裂蛋白Drp1无明显变化。结论氟中毒可导致线粒体损伤、破坏线粒体融合分裂平衡并引起细胞自噬,为阐明氟中毒神经系统损伤与细胞自噬之间相互关系提供新思路。Objective To investigate the effect of fluorosis on microglia BV2 and N2a cells,and on the damages and autopany of cerebral cortical neurons of mice.Methods Eight-week-old clean-grademale mice of C57BL/6J wild-type were randomly divided intothe control group(fluoridecontent in drinking water was<0.5 mg/L),the low-fluoride group(fluoride in drinking water was 10.0 mg/L)andthehigh-fluoride group(fluoride in drinking water was 50.0 mg/L),All the mice(10 cases in each group)were fed for 12 weeks.BV2 and N2a cells were divided into the control group,thelow-fluoride group(medium fluoride 1.0 mmol/L)and the high-fluoride group(medium fluoride 1.5 mmol/L)after normal culture.CCK8 was used to detect thechanges in proliferative viability of BV2 and N2a cells under different concentrations of fluorine treatment.Fluoride ion-selective electrode method was used to detect the changes offluoride contentsin urine,blood and bone of the modeled mice.Protein immunoblotting was used to detect the changes of the expression of ion-calcium-binding bridging molecule-1(Iba-1),which served as a marker of microglia activation,in the cortex of the mouse.Western blot test(WB)was used to detect the expression levels of autophagy-related microtubule-associated protein 1 light chain 3(LC3),and autophagy receptor protein P62(SQSTM1/P62)in N2a cells,BV2 cells and mouse cerebral cortex,and further to detectmitochondrial dynamics and autophagy fusion and division-associated proteins Mitochondrial Fusion Protein 1(Mfn1),Mitochondrial Fusion Protein 2(Mfn2),and Dynamic related protein 1(Drp1),tension protein homologue-induced putative kinase 1(Pink1),and E3 ubiquitin ligase(E3 ubiquitin-protein ligase,Parkin)expression levels.Results CCK8 showed that the value-added viability of BV2 and N2a cells treated with 1.0 mmol/L and 1.5 mmol/L NaF decreasedsignificantly(P<0.01,P<0.0001);the incidence of dental fluorosis in mice in the low-fluoride group was 60%(6/10),and that in the high-fluoride group was 80%(8/10),whilethe fluoride contentinurine,blood,and

关 键 词：氟中毒 细胞自噬 线粒体分裂融合 线粒体自噬 小胶质细胞 小鼠 

分 类 号：R338.62[医药卫生—人体生理学]