内皮素-1诱导培养大鼠大脑皮质神经元凋亡的初步研究

目的观察内皮素(endothelin,ET)-1有无直接诱导原代培养神经元凋亡的作用,以及其作用通过的ET受体亚型。方法神经元培养取自新生SD大鼠大脑皮质。培养5 d后分别加入0.2 nmol/L,20 nmol/L ET-1处理24 h,用Annexin V、Hoechst 33258染色半定量测定细胞凋亡。再用流式细胞仪分别定量检测ET受体A拮抗剂(BQ123)或ET受体B拮抗剂(BQ788)对20 nmol/L ET-1诱导神经元凋亡的效果。结果0.2 nmol/L ET-1未显示诱导培养神经元凋亡的作用;20 nmol/L ET-1处理后24 h,培养神经元凋亡率明显高于对照组(P<0.01);BQ123和BQ788分别部分阻断了20 nmol/L ET-1诱导神经元凋亡的作用(P<0.01),但阻断效果不完全。结论20 nmol/L ET-1可直接诱导培养大鼠大脑皮质神经元凋亡,其作用可能是通过其A受体和B受体亚型共同实现的。

Endothelin-1 induces apoptosis of cultured rat brain cortical neurons via both endothelin A and B receptors

Objective To investigate whether endothelin (ET) -1 can directly induce apoptosis in primary cultured rat brain neurons, and which ET receptor subtype(s) is involved in this action. Methods Primary neuron cultures were obtained from cerebral cortex of neonatal rats, and treated with ET-1 (0.2 nmol/L or 20 nmol/L) for 24 h, 5 days after plantation. Apoptosis was semi-quantitatively evaluated with Annexin V and Hoechst33258 staining, respectively. Thereafter, ET-1 (20 nmol/L), with BQ123 or BQ788, respectively, was added into the culture medium simultaneously, and the neuron apoptosis was quantitatively measured with flow cytometer 24 h later. Results After been treated with 0.2 nmol/L ET-1, no obvious change was found in the rate of Annexin V or Hoechest 33258 positively stained cells. By contrast, after incubation with the higher dose of ET-1 (20 nmol/L), much higher rates of apoptosis were evidented when measured with Annexin V, Hoechest33258 staining and flow cytometer (P<0.01). BQ123 (selective antagonist for ET receptor A, 1 mmol/L) and BQ788 (selective antagonist for ET receptor B, 1mmol/L) partially blocked the effect of ET-1 inducing neuron apoptosis (P<0.01), respectively. Conclusion The higher dose of ET-1 (20 nmol/L) can directly induce apoptosis in primary cultured rat brain neurons, but the lower dose of ET-1. The effect of ET-1 inducing neuronal apoptosis may be mediated via both ET receptor A and B.