健脾补土组方对体外神经元低氧/复氧后PI3K、Akt、Caspase-3表达的影响

摘要

目的 观察健脾补土组方对新生SD鼠神经元体外培养低氧/复氧后的凋亡情况以及PI3K、Akt、Caspase-3表达的影响,探讨其减少神经元凋亡的作用机制.方法 原代分离培养新生SD鼠神经元,随机分为正常血清对照组、低氧模型组、神经生长因子组、健脾补土组.将标本低氧诱导24 h,再复氧培养4 h进行造模.用流式细胞术检测神经元凋亡情况,RT-qPCR法检测PI3K、Akt、caspase-3 mRNA的表达,免疫细胞化学法和Western blot法检测PI3K、Akt、caspase-3蛋白的表达.结果 与正常对照组相比,低氧模型组神经元凋亡率显著增加,PI3K、Akt mRNA与蛋白表达显著减少,Caspase-3 mRNA与蛋白表达显著增加;与低氧模型组比较,神经生长因子组、健脾补土组神经元凋亡率均显著减少,PI3K、Akt mRNA及蛋白表达显著增强,Caspase-3 mRNA及蛋白表达显著较少(P<0.01);与神经生长因子组比较,健脾补土组神经元凋亡率显著减少,PI3K、Akt mRNA及蛋白表达显著增加,Caspase-3 mRNA及蛋白表达显著降低.结论 健脾补土组方能减少脑缺血后神经元的凋亡,其作用机制可能与其通过上调PI3K和Akt的表达水平,最终抑制在细胞凋亡中起关键作用的因子Caspase-3的表达有关.%Objective To observe the effect of Jianpi Butu (tonifying spleen) formula on apoptosis and expression of PI3K, Akt and Caspase3 in neonatal SD neurons cultured in vitro induced by hypoxia/reoxygenation, to explore the mechanism of reducing neuronal apoptosis. Methods Primary cultured neurons of newborn SD rats were randomly divided into normal serum control group, hypoxia model group, NGF group, Jianpi Butu formula group. Specimens to modeling were induced by hypoxia for 24 h, reoxygenation for 4 h. The neuronal apoptosis was induced by flow cytometry. The expression of PI3K,Akt and Caspase3 mRNA was determined by qPCR. The expression of PI3K, Akt and Caspase3 protein was detected by im-munocytochemistry and Western blot. Results Compared with normal control group, the rate of neuronal apoptosis in hypoxia model group increased significantly, the expression of Akt mRNA and protein reduced obviously, and the expres-sion of Caspase3 protein and mRNA increased significantly. Compared with the hypoxia model group, the rate of neuronal apoptosis in NGF group, Jianpi Butu formula group decreased significantly, and the expression of Akt mRNA and protein in-creased obviously, and the expression of Caspase3 mRNA and protein decreased significantly (P<0.01). Compared with the nerve growth factor group, the neuronal apoptosis rate in Jianpi Butu group decreased significantly, the expression of Akt mRNA and protein increased obviously, and the expression of Caspase3 mRNA and protein decreased significantly. Con-clusion Jianpi Butu formula could reduce the apoptosis of neuron after cerebral ischemia. Its mechanism may be related to the up-regulated expression of PI3K and Akt, the ultimate expression of inhibitory factor Caspase3 plays a key role in cell apoptosis.