邻苯二甲酸二丁酯对原代培养海马神经元的毒性及机制  

Toxicity of dibutyl phthalate in primary cultured rat hippocampal neurons and the toxicological mechanism

作　　者：李洋[1] 李秀娟[1,2] 谢明丹[2] 程莉 陈恒胜 孙红 蒋莉[1] LI Yang;LI Xiujuan;XIE Mingdan;CHENG Li;CHEN Hengsheng;SUN Hong;JIANG Li(Department of Neurology,Children's Hospital of Chongqing Medical University;Ministry of Education Key Laboratory of Child Development and Disorders/National Clinical Research Center for Child Health and Disorders/China International Science and Technology Cooperation Base of Child Development and Critical Disorders/Chongqing Key Laboratory of Pediatrics,Chongqing 400014,China)

机构地区：[1]重庆医科大学附属儿童医院神经内科 [2]儿童发育疾病研究教育部重点实验室//国家儿童健康与疾病临床医学研究中心//儿童发育重大疾病国家国际科技合作基地//儿科学重庆市重点实验室,重庆400014

出　　处：《南方医科大学学报》2020年第2期225-232,共8页Journal of Southern Medical University

基　　金：国家自然科学基金(8140120259)~~

摘　　要：目的本研究探讨邻苯二甲酸二丁酯(DBP)暴露对原代培养海马神经元的神经毒性及可能机制。方法海马神经元原代培养4 d后将海马神经元暴露在含有终浓度为0.0(对照)、1 g/LDBP的培养基中,选取染毒24、48、96 h 3个时相点,免疫荧光及透射电子显微镜下观察DBP染毒后海马神经元轴突及超微结构的形态变化;膜片钳测定海马神经元的动作电位;cck-8检测DBP染毒后海马神经元活性。Western blot测定脑源性神经营养因子(BDNF)、神经肽Y(NPY)、雌激素受体β(ERβ)核酸及蛋白的表达情况。高效液相色谱串联质谱检测神经递质GABA的释放。结果DBP染毒96 h后神经元网络稀疏、轴突长度变短(P<0.01),电镜下细胞核呈圆形,染色质聚集,细胞质空泡化;膜片钳测定发现DBP染毒96 h后细胞出现去极化漂移、放电频率增加(P<0.01);cck-8检测发现DBP染毒24、48、96 h的细胞活性均低于同时间点正常对照组(P<0.01)。DBP染毒48、96 h的ERβ、BDNF、NPY蛋白表达显著低于对照组(P<0.05)。DBP染毒96 h后神经递质GABA的释放显著低于对照组(P<0.05)。结论DBP染毒后可导致海马神经细胞形态受损,并导致功能改变,这可能与神经递质GABA参与的ERβ-BDNF-NPY信号通道受阻相关。Objective To investigate the neurotoxicity and toxicological mechanism of dibutyl phthalate(DBP)in primary cultured rat hippocampal neurons.Methods Primary rat hippocampal neurons cultured for 4 days were exposed to 1 g/L DBP for 24,48,or 96 h.Immunofluorescence assay and transmission electron microscopy(TEM)were used to observe the morphological changes of the axons and the ultrastructure of DBP-treated neurons.The action potential(AP)of the hippocampal neurons was measured with patch-clamp electrophysiology.CCK-8 assay was used to detect the viability of the hippocampal neurons,and Western blotting was performed to determine the mRNA and protein expressions of brain-derived neurotrophic factor(BDNF),neuropeptide Y(NPY)and estrogen receptorβ(ERβ).High-performance liquid chromatography-tandem mass spectrometry(HPLC-MS)was employed to detect the release of the neurotransmitter GABA.Results After exposure to DBP for 96 h,the cellular network of the hippocampal neurons became sparse,and the neurons showed significantly decreased axonal length(P<0.01)and presented with round cell nuclei,chromatin aggregation and cytoplasmic vacuolization.Patch-clamp electrophysiology revealed depolarization drift and increased frequency of discharge in the exposed neurons(P<0.01).The neurons with DBP exposure for 24,48 and 96 h all showed significantly decreased cell viability(P<0.01).DBP exposure for 48 and 96 h significantly lowered the protein expressions of ERβ,BDNF and NPY,and a 96-h exposure significantly reduced the release of the neurotransmitter GABA in the neurons(P<0.05).Conclusion DBP exposure causes morphological and functional damages of primary cultured rat hippocampal neurons.DBP-induced neurotoxicity is probably associated with GABA-mediated blockage of the ERβ-BDNF-NPY signaling communication.

关 键 词：DBP 海马神经元 雌激素受体 脑源性神经营养因子 神经肽Y Γ-氨基丁酸 

分 类 号：R114[医药卫生—卫生毒理学]