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目的观察表没食子儿茶素没食子酸酯(EGCG)对体外应激海马神经元的保护作用,并探讨其相关作用机制。方法体外培养新生大鼠海马神经元,于原代培养D13时加入不同浓度的皮质酮,采用CCK8(cell counting kit-8)法检测对神经元存活率的影响;加入EGCG(0、0.1、1、5、10μmol/L)预处理24h后,加入皮质酮(CORT,1×10-5mol/L)建立应激损伤模型,采用CCK8法检测神经元的存活率;然后加入LY294002(PI3K/AKT特异性阻断剂)和U012(6ERK1/2特异性阻断剂),检测细胞存活率的改变,并以Hoechst33342核染色法检测细胞凋亡情况。结果皮质酮在10-6～10-4mol/L范围内对海马神经元的神经毒性作用呈现浓度-时间依赖性;0.1μmol/L EGCG处理对皮质酮造成的海马神经元损伤具有保护作用,能够增加细胞存活率,降低细胞凋亡的发生;而加入信号通路阻断剂LY294002(10 mol/L)和U0126(10 mol/L)后,此保护作用受到抑制,其能抑制EGCG对抗CORT引起的神经元细胞存活率下降,细胞凋亡增多。结论 EGCG处理对皮质酮造成的大鼠海马神经元损伤具有保护作用,其作用机制可能与EGCG调控PI3K/AKT、ERK1/2信号转导通路相关。
Objective To observe the protective effect of epigallocatechin-3-gallate (EGCG) on hippocampal neurons in vitro and its related mechanisms. Methods The hippocampal neurons of neonatal rats were cultured in vitro. Different concentrations of corticosterone were added to primary cultured D13 cells. The viability of neurons was detected by CCK8 (cell counting kit-8) , 5,10μmol / L) for 24 hours, and then cortisone (CORT, 1 × 10-5mol / L) was used to establish the model of stress injury. The survival rate of neurons was detected by CCK8 assay. Then, LY294002 (PI3K / AKT specific (UER) and U012 (6ERK1 / 2 specific inhibitor) were used to detect cell viability. Cell apoptosis was detected by Hoechst33342 nuclear staining. Results The neurotoxic effects of corticosterone on hippocampal neurons were concentration-time dependent in the range of 10-6 ~ 10-4mol / L. 0.1μmol / L EGCG could protect the hippocampal neurons induced by corticosterone, And increased the cell survival rate and decreased the apoptosis rate. However, the protective effects were inhibited by adding LY294002 (10 mol / L) and U0126 (10 mol / L), which inhibited the inhibitory effect of EGCG against CORT Neuronal cell survival rate decreased, apoptosis increased. Conclusions EGCG treatment can protect corticosterone-induced hippocampal neuron injury in rats, and its mechanism may be related to the regulation of PI3K / AKT and ERK1 / 2 signal transduction pathway by EGCG.