小胶质细胞NLRP3炎症体激活在锰诱导神经功能损伤中的作用  

Role of microglia NLRP3 inflammasome activation in manganese-induced neurological impairment

作　　者：姜雨昕 冯鉴 宋腾 陈语轩 王迪雅 陈景元 JIANG Yuxin;FENG Jian;SONG Teng;CHEN Yuxuan;WANG Diya;CHEN Jingyuan(Department of Military Occupational and Environmental Health,Ministry of Education Key Laboratory of Hazard Assessment and Control in Special Operational Environment,School of Military Preventive Medicine,Xi an 710032,China;Shaanxi University of Chinese Medicine,Xianyang 712046,China;No.4 Cadet Regiment,School of Basic Medical Sciences,Air Force Medical University,Xi an 710032,China;No.3 Cadet Regiment,School of Basic Medical Sciences,Air Force Medical University,Xi an 710032,China)

机构地区：[1]空军军医大学军事预防医学系军队劳动与环境卫生学教研室,特殊作业环境危害评估与防治教育部重点实验室,陕西西安710032 [2]陕西中医药大学,陕西咸阳712046 [3]空军军医大学基础医学院学员四大队,陕西西安710032 [4]空军军医大学基础医学院学员三大队,陕西西安710032

出　　处：《空军军医大学学报》2024年第5期491-497,共7页Journal of Air Force Medical University

基　　金：国家自然科学基金国际合作项目(82020108026)。

摘　　要：目的揭示亚急性锰染毒诱导的小胶质细胞对小鼠中枢神经的损伤效应,阐明核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)炎性小体在其中的关键作用及机制。方法将C57BL/6小鼠和NLRP3^(-/-)小鼠各随机分为2组,每组12只,共4组,即对照组、锰组(Mn组)、NLRP3^(-/-)组和NLRP3^(-/-)+Mn组。Mn组和NLRP3^(-/-)+Mn组在第1、4、7、10、13日时,采用皮下注射(100 mg/kg)的方式进行锰暴露,对照组和NLRP3^(-/-)组给予相同体积生理盐水。运用行为学检测法观察小鼠运动能力改变情况;运用免疫组织化学TH染色法观察小鼠脑组织神经元形态学的改变;运用原子吸收法对各组小鼠血锰含量进行检测;通过qRT-PCR法检测小胶质细胞活化相关标志物IL-18、IL-1β的mRNA表达水平;通过免疫荧光染色实验检测纹状体区内小胶质细胞的激活情况。使用Western blotting检测NLRP3相关蛋白NLRP3、Caspase-1、IL-18、IL-1β的表达情况。结果与对照组相比,Mn组小鼠运动协调能力明显损伤(P<0.05),黑质区神经元细胞数量明显降低(P<0.05),小胶质细胞明显发生活化(P<0.05),NLRP3及其相关分子NLRP3、Caspase-1、IL-18、IL-1β蛋白表达水平增高(P<0.05)。NLRP3敲除后,锰暴露所诱导小鼠运动协调能力下降显著回升(P<0.05),且可以逆转锰暴露所诱导的神经元损伤、小胶质细胞活化和NLRP3相关分子蛋白水平表达增加(P<0.05)。结论锰暴露可能通过诱导小鼠纹状体区小胶质细胞活化,促神经功能损伤;特异性敲除NLRP3基因后,可以抑制锰暴露引起的小胶质细胞活化和神经功能的损伤,起到显著的神经功能保护作用。Objective To reveal the damaging effects of subacute manganese exposure-induced microglia on the central nervous system of mice,and to elucidate the key role and mechanism of nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing protein 3(NLRP3)inflammasome.Methods C57BL/6 mice and NLRP3^(-/-)mice were randomly divided into 2 groups,respectively,with a total of 4 groups(12 mice in each group):control group,manganese group(Mn group),NLRP3^(-/-)group,and NLRP3^(-/-)+Mn group.Mn group and NLRP3^(-/-)+Mn group were exposed to manganese using subcutaneous injection(100 mg/kg)on day 1,4,7,10,and 13.Control group and NLRP3^(-/-)group were given the same volume of normal saline.Behavioral tests were used to observe the changes in motor ability of mice;immunohistochemical TH staining was used to observe the morphological changes of neurons in the brain tissue of mice;blood manganese levels of mice in each group were detected by atomic absorption spectrophotometry;mRNA expression levels of microglia activation-related markers IL-18 and IL-1βwere detected by qRT-PCR;and the activation of microglia in the striatum was detected by immunofluorescence staining.Western blotting was used to detect the expression of NLRP3-related proteins NLRP3,Caspase-1,IL-18,and IL-1β.Results Compared with the control group,mice in the Mn group showed significantly lower motor coordination ability(P<0.05),significantly lower number of neuronal cells in the substantia nigra(P<0.05),significant activation of microglia(P<0.05),and increased protein expression levels of NLRP3 and its related molecules NLRP3,Caspase-1,IL-18,and IL-1β(P<0.05).After NLRP3 knockout,the decrease in motor coordination ability induced by manganese exposure in mice was significantly rebounded(P<0.05),and the neuronal damage,microglia activation,and increased expression of NLRP3-related proteins induced by manganese exposure could be reversed(P<0.05).Conclusion Manganese exposure may induce microglia activation in the striatal region of mi

关 键 词：锰暴露 小胶质细胞 神经功能损伤 NLRP3炎症体 

分 类 号：R114[医药卫生—卫生毒理学]