槲皮黄酮对原代皮层神经元细胞氧糖剥夺损伤的保护作用

Protective effects of quercetin on the oxygen and glucose deprivation injury in primary cortical neuronal cells

目的探讨槲皮黄酮对原代皮层神经元细胞氧糖剥夺损伤的保护作用及其作用机制。方法体外原代培养小鼠皮层神经元细胞,氧糖剥夺方法构建原代皮层神经元细胞的局部缺血/再灌注损伤,低(10μg/mL)、中(20μg/mL)、高(40μg/mL)剂量槲皮黄酮治疗24 h后,采用CCK-8试剂盒检测神经元细胞的相对存活率;免疫印迹技术检测凋亡相关蛋白及Traf6/TAK1信号通路蛋白表达水平;Traf6质粒及空白质粒瞬时转染神经元细胞,使Traf6蛋白在细胞中发生过表达,然后利用槲皮黄酮处理细胞,观察凋亡蛋白及Traf6/TAK1信号通路相关蛋白的表达情况。结果低、中、高剂量槲皮黄酮处理后细胞相对存活率显著增加(P<0.05),表现出浓度依赖性;促凋亡蛋白Bax、Traf6及p-TAK1表达显著降低,而抗凋亡蛋白Bcl-2表达水平显著升高(P<0.05);瞬时转染了Traf6重组质粒的细胞中,Traf6蛋白发生过表达,经槲皮黄酮处理后,Bax、Traf6、p-TAK1蛋白水平显著降低,而Bcl-2表达水平显著升高(P<0.05)。结论槲皮黄酮对原代皮层神经元细胞氧糖剥夺损伤具有较好的保护作用,其作用机制可能与抑制Traf6/TAK1信号通路活化有关。

Objective To investigate the protective effects of quercetin on the oxygen and glucose deprivation （OGD） injuries in primary cortical neuronal cells and its functional mechanism. Methods Primary cortical neuronal cells were cultured in vitro. The ischemia/reperfusion （I/R） injury model in neuron was established via oxygen and glucose deprivation. Then the low-, medium-, and high-dose quercetin were used to intervene primary cortical neuron for 24 h. CCK-8 kit was used to detect the relative survival rate of neuron and Western blot was used to determine the expression levels of apoptosis proteins and other proteins related to Traf6/TAK1 signaling pathway. The Traf6 plasmid and control plasmid were transiently transfected into neuronal ceUs to stimulate the over-expression of Traf6 protein. Moreover, the transfected cells were intervened by quercetin and the expression levels of related proteins were investiga- ted. Results The neuronal relative survival rates after treatment with quercetin were significantly increased compared to model group （ P 〈 0.05 ）, and showed to be concentration-dependent. The expression levels of Bax, Traf6 and p- TAK1 were significantly decreased while the level of Bcl-2 was significantly increased in drug groups compared with model group（ P 〈 0. 05 ）. The expression levels of Bax, Traf6 and p-TAK1 in transfected cells were significantly de- creased after intervention by quercetin compared with before drug intervention, while the expression level of Bcl-2 was significantly increased（ P 〈 0. 05 ）. Conclusion Quercetin reduces the neuronal apoptotic injury in primary cortical neuronal ceUs induced by the oxygen and glucose deprivation through inhibiting the activation of Traf6/TAK1 signaling pathway, which is the possible molecular mechanism.