体外培育牛黄配伍冰片激活PI3K/Akt通路抑制脑缺血再灌注模型大鼠神经元凋亡

In Vitro Cultured Calculus Bovis Combined with Borneol Activates PI3K/Akt Pathway and Inhibit Neuronal Apoptosis in Cerebral Ischemia-Reperfusion Model Rats

目的从抗凋亡角度探讨体外培育牛黄(ICCB)、冰片及其配伍对脑缺血再灌注模型大鼠脑保护作用的分子机制。方法采用改良线栓法制备短暂大脑中动脉阻塞(MCAO)模型,缺血90 min后再灌注,苏木精-伊红(HE)染色观察缺血侧脑组织病理损伤情况;Tunel染色观察缺血侧大脑皮层神经元凋亡;RT-PCR检测相关蛋白mRNA的表达;Western blot及免疫组织化学方法检测PI3K/Akt通路相关蛋白的表达。结果与溶剂模型组比较,体外培育牛黄、冰片及两药配伍可不同程度地降低MCAO模型大鼠缺血侧脑组织病理损伤,抑制缺血侧皮层神经元凋亡,升高缺血侧脑组织中p-PI3K、p-Akt及Bcl-2蛋白表达,降低Caspase-3、Bax蛋白表达以及Bax/Bcl-2比值。结论体外培育牛黄、冰片及二者配伍的脑神经保护作用与激活PI3K/Akt通路,抑制脑缺血再灌注后神经元凋亡有关,进而发挥“开窍醒神”功效。

Objective Based on PI3K/Akt signaling pathway,the molecular mechanism of brain protective effect of in vitro cultured Calculus Bovis(ICCB),borneol and their combination on cerebral ischemia-reperfusion model rats was discussed from the perspective of anti-apoptosis.Methods The model of transient middle cerebral artery occlusion(MCAO)was established by modified suture and reperfusion 90 minutes after ischemia.HE staining and Tunel staining were used to observe the pathological injury of brain tissue and the apoptosis of cerebral cortical neurons.The expression levels of PI3K/Akt pathway related proteins were detected by Western blotting and immunohistochemistry.Expression of mRNA was detected by RT-PCR.Results Compared with the solvent model group,the pathological injury of brain tissue was reduced and apoptosis of cortical neurons on the ischemic side was inhibited;the expressions of p-PI3K,p-Akt and Bcl-2 protein in the ischemic brain tissue were increased;the expression of Caspase-3,Bax protein and Bax/Bcl-2 were decreased in ICCB group,borneol group and combination group.Conclusion The brain protective effect of ICCB,borneol and their combination is related to activation of PI3K/Akt pathway and inhibition of neuronal apoptosis after cerebral ischemia-reperfusion,so as to play the role of"opening the orifices and waking the mind".