基于CREB/Bcl-2信号通路探究当归芍药散改善Aβ诱导的胆碱能神经元凋亡的作用机制

Mechanism of Danggui Shaoyao San on ameliorating Aβ-induced apoptosis of cholinergic neurons based on CREB/Bcl-2 signaling pathway

目的探究当归芍药散对β淀粉样蛋白(amyloidβ-protein,Aβ)诱导的胆碱能神经元损伤的保护作用及相关机制。方法以30μmol/L Aβ;诱导基底前脑胆碱能神经元损伤模型,给予2.5%、5.0%和10.0%当归芍药散含药血清进行干预,采用MTT法检测细胞存活率;TUNEL法和碘化丙啶(PI)/Hoechst荧光双染检测神经元凋亡情况;采用胆碱乙酰转移酶(choline acetyltransferase,ChAT)/磷酸化环磷腺苷效应元件结合蛋白(phosphorylated cAMP-response element binding protein,p-CREB)和ChAT/剪切型半胱氨酸天冬氨酸蛋白酶-3(cleaved cystein-asparate protease-3,cleaved Caspase-3)荧光双染检测当归芍药散对胆碱能神经元的抗凋亡情况;采用Western blotting法检测CREB/B淋巴细胞瘤2(B-cell lymphoma 2,Bcl-2)信号通路相关蛋白的表达情况。结果5.0%和10.0%当归芍药散含药血清可显著增加Aβ诱导的胆碱能神经元存活率(P<0.01),改善Aβ诱导的胆碱能神经元凋亡(P<0.01),降低Caspase-3活化水平(P<0.01);10%当归芍药散含药血清能逆转Aβ诱导p-CREB水平的降低(P<0.01),增加抗凋亡蛋白Bcl-2的表达(P<0.05)。结论当归芍药散可能通过CREB/Bcl-2信号通路改善Aβ诱导的胆碱能神经元凋亡。

Objective To study the protective effect and mechanism of Danggui Shaoyao San(当归芍药散,DSS)on amyloidβ-protein(Aβ)-induced cholinergic neuron injury.Methods Basal forebrain neuron injury model was induced by 30μmol/L Aβ25-35,2.5%,5.0%and 10.0%DSS containing serum were given for intervention,MTT assay was used to detect cell survival rate;TUNEL and PI/Hoechst fluorescence staining were used to detect neuronal apoptosis;Choline acetyltransferase(ChAT)/phosphorylated cAMPresponse element binding protein(p-CREB)and ChAT/cleaved cystein-asparate protease-3(Caspase-3)fluorescence double staining were used to detect the anti-apoptotic effect of DSS on cholinergic neurons;Western blotting was used to detect the expressions of CREB/B-cell lymphoma 2(Bcl-2)signaling pathway related proteins.Results DSS serum(5.0%and 10.0%)significantly increased the cell survival rate of Aβ-injured cholinergic neurons(P<0.01),improved Aβ-induced apoptosis(P<0.01)and decreased activation level of Caspase-3(P<0.01).DSS serum(10%)reversed Aβ-induced decrease in p-CREB level(P<0.01)and increased expression of anti-apoptotic protein Bcl-2(P<0.05).Conclusion DSS may ameliorate Aβ-induced apoptosis of cholinergic neurons through CREB/Bcl-2 signaling pathway.