Li Yang, Li Xiujuan, Xie Mingdan, Cheng Li, Chen Hengsheng, Sun Hong, Jiang Li
Department of Neurology, Children's Hospital of Chongqing Medical University, Chongqing 400014, China.
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.
Nan Fang Yi Ke Da Xue Xue Bao. 2020 Feb 29;40(2):225-232. doi: 10.12122/j.issn.1673-4254.2020.02.14.
To investigate the neurotoxicity and toxicological mechanism of dibutyl phthalate (DBP) in primary cultured rat hippocampal neurons.
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.
After exposure to DBP for 96 h, the cellular network of the hippocampal neurons became sparse, and the neurons showed significantly decreased axonal length ( < 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 ( < 0.01). The neurons with DBP exposure for 24, 48 and 96 h all showed significantly decreased cell viability ( < 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 ( < 0.05).
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.
探讨邻苯二甲酸二丁酯（DBP）对原代培养大鼠海马神经元的神经毒性及毒理学机制。
将培养4天的原代大鼠海马神经元暴露于1 g/L DBP中24、48或96小时。采用免疫荧光法和透射电子显微镜（TEM）观察轴突的形态变化及DBP处理神经元的超微结构。用膜片钳电生理学方法测量海马神经元的动作电位（AP）。采用CCK-8法检测海马神经元的活力，并用蛋白质免疫印迹法检测脑源性神经营养因子（BDNF）、神经肽Y（NPY）和雌激素受体β（ERβ）的mRNA和蛋白表达。采用高效液相色谱-串联质谱法（HPLC-MS）检测神经递质γ-氨基丁酸（GABA）的释放。
暴露于DBP 96小时后，海马神经元的细胞网络变得稀疏，神经元轴突长度显著缩短（<0.01），细胞核呈圆形，染色质聚集，细胞质空泡化。膜片钳电生理学显示，暴露神经元出现去极化漂移，放电频率增加（<0.01）。暴露于DBP 24、48和96小时的神经元细胞活力均显著降低（<0.01）。暴露于DBP 48和96小时显著降低了ERβ、BDNF和NPY的蛋白表达，暴露96小时显著降低了神经元中神经递质GABA的释放（<0.05）。
DBP暴露可导致原代培养大鼠海马神经元的形态和功能损伤。DBP诱导的神经毒性可能与GABA介导的ERβ-BDNF-NPY信号通讯阻断有关。
