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 receptor β (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).
Conclusions: 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.
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。
Keywords: brain-derived neurotrophic factor; dibutyl phthalate; estrogen receptor; hippocampal neurons; neuropeptide Y; γ-amino butyric acid.