线粒体活性氧在氟化钠诱导PC12细胞NF-κB/NLRP3信号通路活化中的作用  

Role of mtROS on activation of NF-κB/NLRP3 signaling pathway induced by NaF in PC12 cells

作　　者：陈德良 张磊[2] 付长其 舒适 王国文 彭巍[3] CHEN Deliang;ZHANG Lei;FU Changqi;SHU Shi;WANG Guowen;PENG Wei(Institute of Animal Quarantine Supervision,Guangshan Agricultural and Rural Bureau,Xinyang,Henan 465450,China;Zhengzhou Key Laboratory of Animal Nutrition Metabolic and Poisoning Diseases,College of Veterinary Medicine,Henan University of Animal Husbandry and Economy,Zhengzhou 450000,China;Qinghai Academy of Animal Science and Veterinary Science,Xining 810000,China)

机构地区：[1]光山县农业农村局动物检疫监督所,河南信阳465450 [2]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 [3]青海省畜牧兽医科学院,青海西宁810000

出　　处：《中国兽医学报》2023年第10期2116-2122,共7页Chinese Journal of Veterinary Science

基　　金：青海省科技计划资助项目(2021-ZJ-736);河南牧业经济学院博士启动基金资助项目(M4030067)。

摘　　要：旨在探究氟化钠(NaF)对PC12细胞NOD样受体热蛋白结构域相关蛋白3(NLRP3)炎性小体的影响以及线粒体活性氧(mtROS)/核因子κB(NF-κB)信号在其中的调节作用。以PC12细胞为研究对象,分别转染NLRP3 siRNA、孵育NF-κB通路抑制剂PDTC(50μmol/L)或mtROS清除剂Mito-TEMPO(500μmol/L),并给予质量浓度为80 mg/L的NaF刺激24 h。通过CCK-8法检测细胞存活率,mitoSOX荧光探针检测mtROS水平,比色法测定乳酸脱氢酶(LDH)活性,ELISA法检测白细胞介素(IL)-18和IL-1β分泌,Western blot检测磷酸化NF-κB p65、磷酸化I-κB激酶β(iKKβ)、NOD样受体热蛋白结构域相关蛋白3(NLRP3)、胱天蛋白酶1(Caspase-1)p20蛋白表达,免疫荧光测定NF-κB p65的入核。与NaF组比较,NLRP3 siRNA干预显著降低了NaF暴露引起的NLRP3和Caspase-1 p20蛋白表达水平升高(P<0.01),缓解了NaF诱导的细胞存活率降低(P<0.01)、减弱了培养液上清中LDH活性以及IL-1β和IL-18的分泌水平(P<0.01)。与NaF组比较,PDTC+NaF组细胞中p-NF-κB p65和p-iKKβ水平降低(P<0.01),NF-κB p65的入核被阻断。此外,Mito-TEMPO干预后,NaF处理细胞中的mtROS、p-NF-κB p65、p-iKKβ、NLRP3、IL-1β和IL-18水平均降低(P<0.01)。NaF引起的PC12细胞NF-κB/NLRP3信号通路活化与mtROS水平升高有关。The aim of this study was to investigate the effect of sodium fluoride(NaF)on the Nodlike receptor heat protein domain associated protein 3(NLRP3)inflammasome and the regulatory role of mitochondrial reactive oxygen species(mtROS)/nuclear factorκB(NF-κB)signaling pathway in PC12 cells.PC12 cells were transfected with NLRP3 siRNA,incubated with NF-κB inhibitor(PDTC,50μmol/L)or mtROS scavenger(mito-TEMPO,500μmol/L),and then stimulated with80 mg/L NaF for 24 h.The cell viability,mtROS level,LDH activity,as well as the IL-1βand IL-18secretion were measured by CCK-8 assay,colorimetric method,and ELISA respectively.The protein expressions of p-NF-κB p65,p-iKKβ,NLRP3,Caspase-1 p20 and the nuclear translocation of NF-κB p65 were determined by Western blot and immunofluorescence.Compared with the NaF group,NLRP3 siRNA transfection significantly reduced the expressions levels of NLRP3 and Caspase-1 p20 protein(P<0.01),alleviated the NaF induced low cell survival rate(P<0.01),decreased the activity of LDH in culture medium supernatant,and decreased the secretion levels of IL-1βand IL-18(P<0.01).Compared with NaF group,the phosphorylation levels of NF-κB p65and iKKβwere decreased(P<0.01),and the nuclear translocation of NF-κB p65 was blocked in PDTC+NaF group(P<0.01).Additionally,Mito-TEMPO intervention decreased the levels of mtROS,p-NF-κB p65,p-IKKβ,NLRP3,IL-1β and IL-18 cells(P<0.01).As a result,the NaF-induced activation of NF-κB/NLRP3 signaling pathway in PC12 cells was associated with the increased mtROS levels.

关 键 词：氟化钠 线粒体活性氧 核因子-ΚB NOD样受体热蛋白结构域相关蛋白3 

分 类 号：S856.9[农业科学—临床兽医学]