中药更年春方通过ERβ及PI3K/Akt信号通路对Aβ25-35致大鼠胎鼠海马神经元损伤的保护作用

 目的 观察中药更年春方（Gengnianchun formula，GNC）通过雌激素受体β（estrogen receptor β，ERβ）及PI3K/Akt信号通路对β淀粉样蛋白25-35片段（Aβ_25-35）致大鼠胎鼠海马神经元细胞损伤的保护作用。方法 采用Aβ_25-35作用人神经母细胞瘤SH-SY5Y细胞系，通过CCK-8法检测细胞活力摸索Aβ_25-35及GNC含药血清（GNC serum，GS）处理神经细胞的合适浓度及时间。采用Aβ_25-35作用大鼠胎鼠海马神经元构建阿尔茨海默病（Alzheimer’s disease，AD）的体外模型，分为无血清对照组、模型组、GS组、空白血清对照（normal saline serum，NS）组和PHTPP（选择性ERβ拮抗剂）组。利用正置显微镜观察细胞形态、CCK-8法检测细胞活力及LDH释放法检测细胞损伤。利用Hoest33258/PI双重荧光染色实验、qRT-PCR及Western blot实验检测细胞凋亡相关指标。结果 与无血清对照组相比，模型组大鼠胎鼠海马神经元胞体及突触出现明显的收缩崩解和细胞碎片、细胞活力下降、LDH释放升高。与模型组和NS组相比，GS组大鼠胎鼠海马神经元胞体及突触的收缩崩解和细胞碎片减少、细胞活力上升、LDH释放减少、PI阳性神经元细胞数目下降、神经元bax/bcl-2 mRNA、cyt-c mRNA水平及bax/bcl-2、cleaved-caspase 3、cyt-c蛋白表达水平下降、p-Akt蛋白表达水平上升。与GS组相比，PHTPP组大鼠胎鼠海马神经元bax/bcl-2 mRNA、cyt-c mRNA水平及bax/bcl-2、cleaved-caspase 3、cyt-c蛋白表达水平上升、p-Akt蛋白表达水平下降。结论 GNC对Aβ_25-35致大鼠胎鼠海马神经元的损伤具有保护作用，此作用是通过ERβ及PI3K/Akt信号通路介导的。

Neuroprotective effect of herbal medicine Gengnianchun formula on primary cultured rat fetal hippocampal neurons injury induced by Aβ25-35 through ERβ and PI3K/Akt signaling pathway

Objective To observe the neuroprotective effect of herbal medicine Gengnianchun formula (GNC) on primary cultured rat fetal hippocampal neurons injury induced by amyloid beta 25-35 fragment (Aβ_25-35)through ERβ and PI3K/Akt signaling pathway. Methods Human neuroblastoma cell line (SH-SY5Y) were treated with Aβ_25-35 to establish an in vitro cell model of Alzheimer's disease (AD) and were cultured with different concentration of GNC serum (GS) and Aβ_25-35 to determine the appropriate treatment concentration and duration of these drugs by cell viability test (CCK-8) method. Primary cultured rat fetal hippocampal neurons were treated with Aβ_25-35 to establish an in vitro cell model of AD and were divided into 5 groups,including control group,model group,GS group,normal saline serum(NS) group and PHTPP(a selective ERβ antagonist) group. Morphological observation,CCK-8 test and LDH release assay were used to evaluate the cell injury of primary cultured rat fetal hippocampal neurons. Hoest33258/PI double fluorescence staining,qRT-PCT and Western blot experiments were used to evaluate the cell apoptosis of primary cultured rat fetal hippocampal neurons. Results Compared with the control group,synaptic shrink,collapse and cell debris of primary cultured rat fetal hippocampal neurons were more obvious,cell viability decreased and LDH release increased in model group. Compared with the model group and NS group,synaptic shrink,collapse and cell debris of primary cultured rat fetal hippocampal neurons reduced,cell viability increased,LDH release decreased,number of PI positive primary cultured rat fetal hippocampal neurons were decreased,levels of bax/bcl-2 and cyt-c mRNA and the protein levels of bax/bcl-2,cleaved-caspase-3 and cyt-c were down-regulated and level of p-Akt was up-regulated in GS group. Compared with GS group,levels of bax/bcl-2 and cyt-c mRNA and the protein levels of bax/bcl-2,cleaved-caspase-3 and cyt-c were up-regulated and level of p-Akt was down-regulated in PHTPP group.Conclusions Neuroprotective effect of GNC on primary cultured rat fetal hippocampal neurons injury was mediated by or at least partly by ERβ and PI3K/Akt signaling pathway.