氟暴露对NG108-15细胞自噬及AMPK/mTOR/ULK1通路的影响  

Effects of fluoride exposure on autophagy and AMPK/mTOR/ULK1 pathway in NG108-15 cells

作　　者：张露文 张露 石小雪 谢春[1] Zhang Luwen;Zhang Lu;Shi Xiaoxue;Xie Chun(School of Public Health,Key Laboratory for Environmental Pollution Monitoring and Disease Control,Ministry of Education,Guizhou Medical University,Guiyang 550025,China)

机构地区：[1]贵州医科大学公共卫生与健康学院、环境污染与疾病监控教育部重点实验室,贵阳550025

出　　处：《中华地方病学杂志》2023年第2期99-105,共7页Chinese Journal of Endemiology

基　　金：国家自然科学基金(81860563)。

摘　　要：目的探讨氟暴露对小鼠神经母细胞瘤与大鼠神经胶质瘤融合细胞(NG108-15细胞)自噬及腺苷酸活化蛋白激酶(AMPK)、哺乳动物雷帕霉素靶蛋白(mTOR)、Unc-51样激酶1(ULK1)表达的影响。方法体外培养NG108-15细胞,按照氟化钠终浓度分为对照组(0 mg/L)、低氟组(20 mg/L)、中氟组(40 mg/L)、高氟组(80 mg/L),处理24 h后收集细胞备用。采用免疫荧光(IF)/免疫细胞化学(ICC)法检测细胞自噬情况(以磷酸氯喹处理为自噬阳性对照);实时荧光定量PCR(qRT-PCR)检测各组细胞AMPK、mTOR、ULK1 mRNA表达水平;蛋白质印迹法(Western blot)检测各组细胞自噬相关蛋白微管相关蛋白1轻链3B(LC3B)、AMPK、mTOR、ULK1、磷酸化(p)-AMPK、p-mTOR、p-ULK1表达水平。结果对照组未检出自噬体,各染氟组均检出自噬体;且低、中、高氟组LC3B蛋白表达水平(1.80±0.59、2.16±0.60、2.30±0.57)均明显高于对照组(1.00±0.29,均P<0.05)。qRT-PCR检测结果显示,与对照组比较,中、高氟组AMPK mRNA表达水平均较高(2.30±0.57、4.41±1.05比1.00±0.01,均P<0.05);低、中、高氟组mTOR mRNA表达水平均较低(0.79±0.04、0.76±0.09、0.64±0.10比1.00±0.01,均P<0.05),ULK1 mRNA表达水平均较高(1.81±0.39、1.96±0.35、4.22±1.03比1.00±0.01,均P<0.05)。Western blot检测结果显示,与对照组比较,低、中、高氟组AMPK(1.21±0.05、1.20±0.04、1.30±0.07比1.00±0.03),p-AMPK(1.12±0.05、1.20±0.06、1.49±0.07比1.00±0.02),ULK1(1.16±0.05、1.26±0.05、1.15±0.05比1.00±0.04)及p-ULK1蛋白表达水平均较高(1.19±0.04、1.17±0.02,1.24±0.05比1.00±0.05,均P<0.05),mTOR蛋白表达水平均较低(0.77±0.03、0.60±0.03、0.55±0.04比1.00±0.04,均P<0.05);中、高氟组p-mTOR蛋白表达水平均较低(0.93±0.05、0.48±0.02比1.00±0.02,均P<0.05)。结论氟暴露可致NG108-15细胞发生自噬,AMPK、ULK1表达上调,而mTOR表达下调。Objective To investigate the effects of fluoride exposure on autophagy and the expression levels of adenosine monophosphate activated protein kinase(AMPK),mammalian target of rapamycin(mTOR)and Unc-51-like kinase 1(ULK1)in mouse neuroblastoma and rat glioma fusion cells(NG108-15 cells).Methods NG108-15 cells were cultured in vitro and divided into control group(0 mg/L),low fluoride group(20 mg/L),medium fluoride group(40 mg/L)and high fluoride group(80 mg/L)according to the final concentration of sodium fluoride,and the cells were collected after 24 h of treatment for standby.NG108-15 cells autophagy was detected by immunofluorescence/immunocytochemistry(IF/ICC method,the autophagy positive control group was treated with chloroquine phosphate);the mRNA expression levels of AMPK,mTOR and ULK1 in each group were detected by real-time fluorescent quantitative polymerase chain reaction(qRT-PCR);the protein expression levels of autophagy related protein microtubule-associated protein 1 light chain 3B(LC3B),AMPK,mTOR,ULK1,phosphorylation(p)-AMPK,p-mTOR,p-ULK1 in each group were detected by Western blotting.Results No autophagosome was detected in the control group,and autophagosomes were detected in all the fluoride groups.The protein expression level of LC3B in the low,medium and high fluoride groups(1.80±0.59,2.16±0.60,2.30±0.57)was significantly higher than that in the control group(1.00±0.29,P<0.05).The results of qRT-PCR showed that compared with the control group,the mRNA expression levels of AMPK in medium and high fluoride groups were higher(2.30±0.57,4.41±1.05 vs 1.00±0.01,P<0.05);the mRNA expression levels of mTOR in the low,medium and high fluoride groups were lower(0.79±0.04,0.76±0.09,0.64±0.10 vs 1.00±0.01,P<0.05),and the mRNA expression levels of ULK1 were higher(1.81±0.39,1.96±0.35,4.22±1.03 vs 1.00±0.01,P<0.05).The results of Western blotting showed that compared with the control group,the protein expression levels of AMPK(1.21±0.05,1.20±0.04,1.30±0.07 vs 1.00±0.03),p-AMPK(1.12±0.05,

关 键 词：氟 NG108-15细胞 自噬 腺苷酸活化蛋白激酶 哺乳动物雷帕霉素靶蛋白 Unc-51样激酶1 

分 类 号：R114[医药卫生—卫生毒理学]