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目的观察促肾上腺皮质激素释放激素(CRH)受体1拮抗剂CP154526对海马神经元凋亡的影响。方法原代培养大鼠海马神经元,噻唑蓝(MTT)法测定细胞存活率,然后分为4组:正常对照组(Con)、CRH刺激组(CRH)、CRH和CP154526共同刺激组(CRH+CP)、CP154526刺激组(CP)。TUNEL、流式细胞术AnnexinⅤ-PI法检测神经元凋亡率;Western blot检测凋亡蛋白Bax、Bcl-2、caspase-3表达水平。结果 10-8mol·L-1的CRH作用于海马神经元后,细胞存活率下降(P<0.05);50 mmol·L-1的CP154526可明显提升神经元存活率(P<0.05)。与正常对照组相比,CRH刺激后神经元凋亡率增加,Bax/Bcl-2比值增高,caspase-3表达增加;加用CP154526可明显降低神经元凋亡率、Bax/Bcl-2比值及caspase-3表达水平;而单独应用CP154526对凋亡没有明显影响。结论一定浓度的CRH可诱导海马神经元细胞凋亡,其受体1拮抗剂CP154526可有效减轻细胞凋亡,发挥神经保护作用。
Objective To observe the effects of corticotropin-releasing hormone (CRH) receptor 1 antagonist CP154526 on the apoptosis of hippocampal neurons. Methods Primary cultured rat hippocampal neurons were cultured with MTT method and then divided into 4 groups: control group, CRH stimulation group, CRH + CP154526 co-stimulation group, CRH + CP), CP154526 stimulation group (CP). TUNEL and flow cytometry Annexin Ⅴ-PI were used to detect the apoptosis rate of neurons. Western blot was used to detect the expression of Bax, Bcl-2 and caspase-3. Results After treated with 10-8mol·L-1 of CRH, the cell viability decreased (P <0.05). CP154526 (50 mmol·L-1) significantly increased the survival rate of neurons (P <0.05). Compared with the normal control group, the apoptosis rate of neurons increased, the ratio of Bax / Bcl-2 increased and the expression of caspase-3 increased after stimulation with CRH; CP154526 could obviously decrease the ratio of neuronal apoptosis, Bax / Bcl-2 caspase-3 expression level; CP154526 alone had no significant effect on apoptosis. Conclusion CRH can induce apoptosis in hippocampal neurons. Its receptor 1 antagonist CP154526 can effectively reduce apoptosis and exert neuroprotection.