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

[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、细胞因子活性等分子功能,位于滑面内质网膜、膜的固定成分、氯化物通道复合体等细胞组分。KEGG显著富集通路为次级代谢产物的生物合成、碳代谢和多样环境中的微生物代谢等。差异蛋白相互作用网络分析结果显示葡萄糖-6-磷酸异构酶(Gpi)连接度最高。经蛋白免疫印迹实验检测,Gpi在染氟组成年SD大鼠脑组织中表达水平较对照组降低(P<0.05)。[结论]慢性氟中毒大鼠脑组织中存在多种差异表达蛋白,其功能与次级代谢产物的生物合成、碳代谢和多样环境中的微生物代谢有关;Gpi可能参与慢性过量氟暴露致脑神经损伤。

[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 staining of neurons in the cerebral cortex and hippocampus of rats exposed to fluoride decreased(Nissl bodies decreased).The proteomics results showed that a total of 6927 proteins were identified.After screening,206 differentially expressed proteins were obtained between the control group and the fluoride group,including 96 up-regulated proteins and 110 downregulated proteins.The differential proteins were mainly located in cytoplasm(30.6%),nucleus(27.2%),mitochondria(13.6%),plasma membrane(13.6%),and extracellular domain(11.7%).The GO analysis results showed that differentially expressed proteins mainly participated in biological processes such as iron ion transport,regulation of dopamine neuron differentiation,and negative regulation of respiratory burst in inflammatory response,exercised molecular functions such as ferrous binding,iron oxidase activity,and cytokine activity,and were located in the smooth endoplasmic reticulum membrane,fixed components of the membrane,chloride channel complexes,and other cellular components.The KEGG significantly enriched pathways included biosynthesis of secondary metabolites,carbon metabolism,and microbial metabolism in diverse environments.The results of differential protein-protein interaction analysis showed that the highest connectivity was found in glucose-6-phosphate isomerase(Gpi).The expression level of Gpi in the brain tissue of the rats in the fluoride group was lower than that in the control group by Western blotting(P<0.05).[Conclusion]Multiple differentially expressed proteins are present in the brain tissue of rats with chronic fluorosis,and their functions are related to biosynthesis of secondary metabolites,carbon metabolism,and microbial metabolism in diverse environments;Gpi may be involved in cerebral neurological damage caused by chronic overdose fluoride exposure.