百草枯促进小胶质细胞分泌IL-1β对神经干细胞增殖和神经元生成的影响  

Effects of paraquat on proliferation and neurogenesis of neural stem cells by promoting secretion of IL-1βby microglia

作　　者：李倩 肖洪喜 李睿[3] 唐黎明[3] 常秀丽[2] 周志俊[2] LI Qian;XIAO Hongxi;LI Rui;TANG Liming;CHANG Xiuli;ZHOU Zhijun(Department of Preventive Medicine,School of Public Health,Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China;School of Public Health/Key Laboratory of Public Health Safety of Ministry of Education,Fudan University,Shanghai 200032,China;Pharmacology and Toxicology Department,Shanghai Institute for Drug and Food Control,Shanghai 201203,China)

机构地区：[1]上海中医药大学公共健康学院预防医学教研室,上海201203 [2]复旦大学,公共卫生学院/公共卫生安全教育部重点实验室,上海200032 [3]上海市食品药品检验所药理毒理室,上海201203

出　　处：《环境与职业医学》2022年第9期1045-1050,共6页Journal of Environmental and Occupational Medicine

基　　金：国家自然科学基金项目(81673202,81773472)

摘　　要：[背景]百草枯(PQ)是广泛使用的除草剂,具有神经毒性。PQ介导的神经炎症对神经干细胞的影响研究仍然有限。[目的]探讨PQ通过小胶质细胞介导的神经炎症对神经干细胞增殖和神经元生成的影响。[方法]选用小胶质细胞系(BV2细胞)和原代神经干细胞作为研究对象。将BV2细胞置于PQ终浓度分别为0、1、3.3、10、33、100μmol·L^(−1)的完全培养基中培养6 h,检测其细胞存活率,选择对细胞活力没有影响的最高PQ浓度作为最终染毒浓度(33μmol·L^(−1))。为了排除PQ对神经干细胞的直接影响,将BV2细胞置于PQ终浓度为33μmol·L^(−1)的完全培养基中培养6 h,再将培养基换成不含PQ的神经干细胞完全培养基继续培养24 h,用酶联免疫吸附法检测上清液中白细胞介素-1β(IL^(-1)β)的浓度。此外,为了检测IL^(-1)β对神经干细胞增殖和神经元生成的影响,从成年小鼠海马区分离提取的神经干细胞用上述BV2细胞上清液培养,分为4组:对照组上清+对照抗体组,对照组上清+IL^(-1)β中和抗体组(10 ng·mL^(−1)),PQ组上清+对照抗体组,PQ组上清+IL^(-1)β中和抗体组(10 ng·mL^(−1)),培养24 h后用流式细胞和免疫荧光法检测Ki67阳性的神经干细胞的比例,培养3~7 d后用流式细胞和免疫荧光法检测新生神经元的比例。[结果]与对照组相比,BV2细胞经过33μmol·L^(−1) PQ处理后上清液中IL^(-1)β含量上升(t=3.020,P<0.05)。用PQ处理过的BV2细胞上清液培养小鼠神经干细胞,与对照组相比,Ki67阳性的神经干细胞的比例下降(t=9.129,P<0.01),新生神经元的比例与对照组相比下降(t=4.638,P<0.01)。中和IL^(-1)β后,共培养的神经干细胞中Ki67阳性的神经干细胞百分比高于未中和组(t=22.05,P<0.01),新生神经元的比例也高于未中和组(t=11.09,P<0.01)。免疫荧光技术检测同样显示,中和33μmol·L^(−1) PQ处理的BV2细胞上清液中的IL^(-1)β后,Ki67阳性的神经干细胞数量�[Background]Paraquat(PQ)is a widely used herbicide that exerts neurotoxicity.The effects of PQ on neural stem cells(NSCs)through microglia mediated neuroinflammation remain limitedly studied.[Objective]To investigate the effects of PQ on the proliferation and neurogenesis of NSCs through neuroinflammation mediated by microglia.[Methods]Microglial cell lines(BV2 cells)and primary NSCs were used.BV2 cells were exposed to 0,1,3.3,10,33,and 100μmol·L^(−1) of PQ for 6 h followed by viability assessment.The highest PQ concentration that had no effect on cell viability was selected as the final exposure concentration(33μmol·L^(−1)).To exclude the direct effect of PQ on NSCs,after the BV2 cells were cultured in complete medium containing 33μmol·L^(−1) PQ for 6 h,the BV2 culture medium was replaced by NSCs complete medium without PQ for 24 h.The concentration of interleukin-1β(IL^(-1)β)in supernatant was detected by enzyme-linked immune sorbent assay.Besides,in order to detect the effects of IL^(-1)βon NSCs proliferation and neurogenesis,NSCs isolated from hippocampus of adult mice were cultured in the supernatant obtained above and divided into four groups:control supernatant+control antibody,control supernatant+IL^(-1)βneutralizing antibody(10 ng·mL^(−1)),PQ supernatant+control antibody,PQ supernatant+IL^(-1)βneutralizing antibody(10 ng·mL^(−1)).Proportion of Ki67-positive NSCs was detected by flow cytometry(FCS)and immunofluorescence after 24 h culture,and neurogenesis was detected by FCS and immunofluorescence after 3-7 d of culture.[Results]The IL^(-1)βconcentration in the supernatant of BV2 cells was significantly increased after the 33μmol·L^(−1) PQ exposure compared with the control group(t=3.020,P<0.05).After the NSCs were cultured with the supernatant of PQ-treated BV2 cells,the proportion of Ki67-positive NSCs(t=9.129,P<0.01)and the proportion of newborn neurons(t=4.638,P<0.01)were significantly decreased compared to the control group.After neutralizing IL^(-1)β,the proportion of Ki

关 键 词：百草枯 小胶质细胞 白介素-1Β 神经干细胞 神经元生成 

分 类 号：R114[医药卫生—卫生毒理学]