基于蛋白质组学技术探究无机氟致大脑损伤的机制  

Mechanisms of brain damage caused by inorganic fluoride using proteomics-based techniques

作　　者：周箫 万雯[1] 姜德文 艾福军 叶凌 刘明海 张艺 刘艳洁[1,3] ZHOU Xiao;WAN Wen;JIANG Dewen;AI Fujun;YE Ling;LIU Minghai;ZHANG Yi;LIU Yanjie(Department of Pathology,Guizhou Medical University,Guiyang,Guizhou 550004,China;Guiyang Huaxi District People's Hospital,Guiyang,Guizhou 550004,China;Department of Pathology,Affiliated Hospital of Guizhou Medical University,Guiyang,Guizhou 550004,China)

机构地区：[1]贵州医科大学病理学教研室,贵州贵阳550004 [2]贵阳市花溪区人民医院,贵州贵阳550004 [3]贵州医科大学附属医院病理科,贵州贵阳550004

出　　处：《环境与职业医学》2024年第1期34-40,共7页Journal of Environmental and Occupational Medicine

基　　金：国家自然科学基金项目(81960572);国家自然科学基金委员会-贵州省人民政府联合基金子项目(U1812403);贵州省卫健委科技基金项目(gzwkj2022-217);贵州省教育厅青年科技人才项目(黔教合KY字[2021]183)。

摘　　要：[背景]慢性过量氟暴露会导致中枢神经系统出现损伤,造成一定程度的学习记忆功能损害,然而其损伤机制尚不明确,仍需进一步探究。[目的]应用4D非标记定量蛋白质组学技术探究慢性过量氟暴露致大脑损伤的差异表达蛋白及其潜在的作用机制。[方法]选取SPF级成年SD大鼠24只,雌雄各半,采用随机数字表法分为对照组与染氟组,每组12只。其中,对照组饮用自来水(氟含量<1 mg·L^(-1)),染氟组饮用氟化钠溶液(氟含量10 mg·L^(-1)),两组均饲以普通鼠饲料(氟含量<0.6 mg·kg^(-1))。饲养180 d后称SD大鼠的体重,随后取部分脑组织分别行苏木素-伊红(HE)染色和尼氏体染色的病理学检查,其余脑组织速冻后于-80℃保存。每组脑组织样本随机挑选3份进行蛋白质组学检测,筛选出差异表达蛋白并进行亚细胞定位分析,随后进行基因本体(GO)功能分析、京都基因和基因百科全书(KEGG)通路分析、聚类分析及蛋白相互作用网络分析获得关键蛋白。最后选用脑组织样本提取蛋白行蛋白免疫印迹实验检测关键蛋白的表达水平。[结果]饲养180 d后,染氟组SPF级成年SD大鼠的体重较对照组明显降低(P<0.05)。HE染色结果显示染氟组大鼠脑皮质未见明显形态变化,海马区见神经元丢失、神经元排列层次不整齐、部分神经元嗜酸性变和胞体固缩。尼氏染色结果显示染氟组大鼠脑皮质及海马区神经元染色变浅(尼氏体减少)。蛋白质组学检测共鉴定出6927个蛋白,经过筛选在对照组与染氟组中获得差异表达蛋白206个,包括96个表达上调蛋白和110个表达下调蛋白,差异蛋白主要定位于细胞质(30.6%)、细胞核(27.2%)、线粒体(13.6%)、质膜(13.6%)和胞外(11.7%)。GO分析结果显示差异表达蛋白主要参与铁离子运输、多巴胺神经元分化的调控和炎症反应中呼吸爆发的负调控等生物过程,行使亚铁结合、铁氧化酶活性、细胞因子�[Background]Chronic excessive exposure to fluoride can cause damage to the central nervous system and a certain degree of learning and memory impairment.However,the associated mechanism is not yet clear and further exploration is needed.[Objective]Using 4D unlabelled quantitative proteomics techniques to explore differentially expressed proteins and their potential mechanisms of action in chronic excessive fluoride exposure induced brain injury.[Methods]Twenty-four SPF-grade adult SD rats,half male and half male,were selected and divided into a control group and a fluoride group by random number table method,with 12 rats in each group.Among them,the control group drank tap water(fluorine content<1 mg·L^(-1)),the fluoride group drank sodium fluoride solution(fluorine content 10 mg·L^(-1)),and both groups were fed with ordinary mouse feed(fluoride content<0.6 mg·kg^(-1)).After 180 d of feeding,the SD rats were weighed,and then part of the brain tissue was sampled for pathological examination by hematoxylin-eosin(HE)staining and Nissl staining.The rest of the brain tissue was frozen and stored at -80℃.Three brain tissue samples from each group were randomly selected for proteomics detection.Differentially expressed proteins were screened and subcellular localization analysis was performed,followed by Gene Ontology(GO)function analysis,Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis,cluster analysis,and protein-protein interaction analysis.Finally,Western blotting was used to detect the expression levels of key proteins extracted from the brain tissue samples.[Results]After 180 d of feeding,the average weight of the rats in the fluoride group was significantly lower than that in the control group(P<0.05).The brain tissue stained with HE showed no significant morphological changes in the cerebral cortex of the fluoride treated rats,and neuron loss,irregular arrangement of neurons,eosinophilic changes,and cell body pyknosis were observed in the hippocampus.The Nissl staining results showed that the s

关 键 词：慢性氟暴露 大脑损伤 蛋白质组学 差异表达蛋白 葡萄糖-6-磷酸异构酶 

分 类 号：R114[医药卫生—卫生毒理学]