摘要
目的观察慢性氟中毒大鼠脑组织胶质纤维酸性蛋白(GFAP)、β-微管蛋白Ⅲ(β-tubulinⅢ)和突触素(synaptophsin)表达水平,探索三复合体突触在慢性氟中毒大鼠中枢神经系统(CNS)损伤机制中的作用,以及硫酸软骨素(CS)的神经保护作用。方法选择1月龄清洁级SD大鼠雌雄各48只,按体重(90~120 g)采用随机数字表法分为8组,每组12只,雌雄各半。采用含有不同浓度氟化钠(NaF)的自来水[<0.5 mg/L(对照,CN),10.0 mg/L(低剂量染氟,LF),50.0 mg/L(高剂量染氟,HF)]喂养,其中部分大鼠直接喂养185 d(CN、LF、HF组),其余大鼠于喂养180 d后连续5 d腹腔注射生理盐水(NS)或染氟大鼠腹腔注射0.66 mg/kg CS,分别为CN+NS、LF+NS、HF+NS组和LF+CS、HF+CS组。实验结束后采用苏木素-伊红染色于光镜下观察各组大鼠脑组织海马CA4区病理改变,免疫组织化学法检测海马CA4区GFAP、β-tubulinⅢ和synaptophsin的表达及分布,蛋白免疫印迹法检测海马GFAP、β-tubulinⅢ和synaptophsin蛋白表达水平。结果光镜下LF、HF、LF+NS、HF+NS组雌、雄性大鼠海马CA4区均可见神经元嗜酸性变、丢失和排列层次不整齐,LF+CS、HF+CS组较CN组形态学未见明显改变,而较LF、HF、LF+NS、HF+NS组有明显改善。免疫组织化学法检测结果显示,LF、HF组雌、雄性大鼠GFAP、β-tubulinⅢ、synaptophsin阳性面积率均低于CN组(P均<0.05);而LF+CS、HF+CS组分别高于LF、HF组(P均<0.05)。蛋白免疫印迹法检测结果显示,LF、HF组雌、雄性大鼠GFAP、β-tubulinⅢ、synaptophsin蛋白表达水平(LF组:0.90±0.09、0.82±0.08,1.43±0.14、0.92±0.02,1.21±0.15、0.87±0.02,HF组:0.58±0.14、0.73±0.03,0.63±0.06、0.67±0.03,0.87±0.04、0.70±0.05)均低于CN组(1.24±0.08、1.09±0.10,2.64±0.30、1.54±0.09,1.72±0.10、1.13±0.06,P均<0.05)。结论慢性氟中毒引起的CNS损伤可能与三复合体突触以及细胞外基质有关,CS可能在一定程度上降低损伤。
Objective To observe the expression levels of glial fibrillary acidic protein(GFAP),β-tubulin Ⅲ and synaptophsin,and explore the role of tripartite synapse in the mechanism of central nervous system(CNS)injury and the neuroprotective effect of chondroitin sulfate(CS).Methods One month old clean grade,48 female Sparague-Dawley rats and 48 male Sparague-Dawley rats,were randomly divided into 8 groups according to body weight(90-120 g)by random number table method,with 12 rats in each group,half male and half female.These rats were fed with water containing different concentrations of sodium fluoride(NaF)[<0.5 mg/L(control,CN),10.0 mg/L(low dose fluoride,LF)and 50.0 mg/L(high dose fluoride,HF)].Some rats were fed directly for 185 days(CN,LF and HF groups).In addition,rats of CN+normal saline(NS),LF+NS,HF+NS groups and LF+CS,HF+CS groups,were intraperitoneally injected with NS or 0.66 mg/kg CS for 5 consecutive days after 180 days of feeding.After the experiment,the pathological changes of hippocampal CA4 of brain tissue in each group were observed by hematoxylin eosin staining under light microscope,and the expression and distribution of GFAP,β-tubulin Ⅲ and synaptophsin in hippocampal CA4 of rats were detected by immunohistochemistry,the expression of GFAP,β-tubulin Ⅲ and synaptophsin at protein level in hippocampus of rats were detected by Western blotting.Results Under light microscope,eosinophilic change,loss and irregular arrangement of neuron in the hippocampal CA4 were observed in LF,HF,LF+NS and HF+NS groups.The morphology of LF+CS and HF+CS groups was not significantly changed compared with CN group,but was significant changed compared with LF,HF,LF+NS and HF+NS groups.Immunohistochemical results showed that the rates of positive area of GFAP,β-tubulin Ⅲ and synaptophsin in female and male rats in LF and HF groups were significantly decreased than those in CN group(P<0.05);the positive area rates of female and male rats in LF+CS and HF+CS groups were higher than those in LF and HF groups,respectively(P<0.05).Western blotting results showed that the proten expression levels of GFAP,β-tubulin Ⅲ and synaptophsin of female and male rats in LF and HF groups(LF group:0.90±0.09,0.82±0.08,1.43±0.14,0.92±0.02,1.21±0.15,0.87±0.02,HF group:0.58±0.14,0.73±0.03,0.63±0.06,0.67±0.03,0.87±0.04,0.70±0.05)were lower than those in CN group(1.24±0.08,1.09±0.10,2.64±0.30,1.54±0.09,1.72±0.10,1.13±0.06,P<0.05).Conclusions The tripartite synapse and extracellular matrix may take part in pathogenesis of the damages of CNS results from chronic fluorosis;CS may reduce the injury to a certain extent.
作者
王纯
梁璐璐
叶凌
何琪
钟毅
王胜远
何冬玲
官志忠
刘艳洁
Wang Chun;Liang Lulu;Ye Ling;He Qi;Zhong Yi;Wang Shengyuan;He Dongling;Guan Zhizhong;Liu Yanjie(Zigong Gongjing Hospital of Traditional Chinese Medicine,Zigong 643020,China;Department of Pathology,Affiliated Hospital of Guizhou Medical University,Guiyang 550004,China;Department of Neurology,Guiyang Huaxi District People's Hospital,Guiyang 550004,China;Key Laboratory of Endemic and Ethnic Diseases,Ministry of Enducation,Guiyang 550004,China)
出处
《中华地方病学杂志》
CAS
北大核心
2021年第10期781-789,共9页
Chinese Journal of Endemiology
基金
国家自然科学基金(81960572、U1812403)
地方病与少数民族性疾病教育部重点实验室(贵州医科大学)开放课题基金(黔教合KY字[2018]480)
贵州省科技厅贵阳医学院联合基金(黔科合LG[2012]006号)。
关键词
氟化物中毒
三复合体突触
细胞外基质
硫酸软骨素
性别差异
Fluoride poisoning
Tripartite synapse
Extracellular matrix
Chondroitin sulfate
Gender difference
