钙稳态在DBP诱导胚胎大鼠皮层神经元凋亡中的作用

王文娟; 杨光红; 曾奇兵; 喻馨兰; 汪文云

doi:10.13213/j.cnki.jeom.2016.15442

钙稳态在DBP诱导胚胎大鼠皮层神经元凋亡中的作用

Role of Calcium Homeostasis in Apoptosis of Embryonic Rat Cortical Neurons Induced by Dibutylphthalate

摘要

摘要: 目的
研究钙稳态在邻苯二甲酸二丁酯（DBP）诱导大鼠皮层神经元凋亡中的作用。
方法
取胚胎大鼠皮层神经元，用25、50、100 mg/L的DBP进行细胞染毒，同时用加入10 μmol/L钙络合剂（BAPTA-AM）和上述各浓度的DBP进行细胞培养，检测各组细胞的凋亡率、细胞内钙浓度（Fluo-3/AM荧光强度）、活性氧浓度以及线粒体膜电位。
结果
25、50、100 mg/L DBP染毒组的神经元凋亡率（12.27±0.93）%、（18.53±0.87）%、（27.63±1.06）%，荧光强度（10 623±826）%、（14 815±732）%、（18 328±708）%，细胞内活性氧的发生率（10.21±1.05）%、（29.23±1.35）%、（34.68±1.03）%及神经元线粒体膜电位下降率（30.71±1.81）%、（45.30±3.25）%、（53.00±4.29）%均高于对照组，差异均有统计学意义（P < 0.05）。随着DBP染毒剂量的增加，上述各项指标均呈剂量-效应关系（r=0.977，r=0.970，r=0.962，r=0.914，均P < 0.05）；BAPTA-AM干预后，与DBP同剂量组比较皮层神经元凋亡率有所降低，荧光强度明显下降，线粒体膜电位下降率降低，细胞内活性氧的发生率降低（均P < 0.05）。
结论
DBP可诱导大鼠皮层神经元凋亡，钙络合剂通过降低钙离子浓度，缓解DBP诱导的皮神经元的凋亡，表明钙稳态失衡促进DBP诱导的大鼠皮层神经元凋亡。

Abstract: Objective
To study the role of calcium homeostasis in the apoptosis of rat cortical neurons induced by dibutylphthalate (DBP).
Methods
Neurons were sampled from the cortex of pregnant rats. The cortical neurons were treated with 25, 50, and 100 mg/L DBP; meanwhile, 10 μmol/L calcium chelator (BAPTA-AM) was added into the above combinations. The apoptosis rates, intracellular free calcium concentrations Ca²⁺_i, reactive oxygen species (ROS), and mitochondrial membrane potential of the cortical neurons were detected by flow cytometry.
Results
After treated with 25, 50, and 100 mg/L DBP, the apoptosis rate (12.27±0.93)%, (18.53±0.87)%, and (27.63±1.06)%, respectively, Fluo-3/AM fluorescence intensity (10623±826)%, (14 815±732)%, and (18 328±708)%, level of ROS (10.21±1.05)%, (29.23±1.35)%, and (34.68±1.03)%, and decrease rate of mitochondrial membrane potential (30.71±1.81)%, (45.30±3.25)%, and (53.00±4.29)% were all higher than those of the control group (all Ps < 0.05) in a dose-response manner (r=0.977, r=0.970, r=0.962, r=0.914, all Ps < 0.05). After treated with BAPTAAM, the apoptosis rates, Fluo-3/AM fluorescence intensity, level of ROS, and decrease rate of mitochondrial membrane potential of the cortical neurons were decreased compared with corresponding DBP only groups (all Ps < 0.05).
Conclusion
DBP could in duce rat cortical neuron apoptosis, and BAPTA-AM could blunt the apoptosis through decreasing Ca²⁺_i, suggesting that the disequilibrium of calcium homeostasis might promote the apoptosis of rat cortical neurons induced by BBP.

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