吉马酮通过调控miR-297表达对H2O2诱导的小鼠海马神经元细胞凋亡和氧化应激的影响

Effects of Germacrone on the Apoptosis and Oxidative Stress of Mouse Hippocampal Neurons Induced by H_(2)O_(2) by the Expression Regulation of miR-297

目的:探讨吉马酮对H_(2)O_(2)诱导的小鼠海马神经元细胞凋亡和氧化应激的影响及分子机制。方法:分离培养小鼠海马神经元,将其分为对照组、H_(2)O_(2)组、H_(2)O_(2)+吉马酮低、中、高(50,100,200μmol·L^(-1))浓度组、H_(2)O_(2)+miR-NC组、H_(2)O_(2)+miR-297组、H_(2)O_(2)+吉马酮(200μmol·L^(-1))+anti-miR-NC组、H_(2)O_(2)+吉马酮(200μmol·L^(-1))+anti-miR-297组。流式细胞术检测细胞凋亡;蛋白质印迹(Western blot)法检测B细胞淋巴瘤/白血病-2(Bcl-2)、Bcl-2相关X(Bax)蛋白表达;丙二醛(MDA)和超氧化物歧化酶(SOD)试剂盒分别检测MDA含量和SOD活性;实时荧光定量PCR(RT-qPCR)检测miR-297表达水平。结果:吉马酮低、中、高浓度处理后,H_(2)O_(2)诱导的小鼠海马神经元中细胞凋亡率、Bax表达水平和MDA含量降低,Bcl-2表达水平、SOD活性和miR-297表达水平升高,且呈浓度依赖性(P<0.05)。miR-297过表达可降低H_(2)O_(2)诱导的海马神经元细胞凋亡率和MDA含量,提高SOD活性(P<0.05)。抑制miR-297表达逆转了吉马酮对H_(2)O_(2)诱导的海马神经元细胞凋亡和氧化应激的作用。结论:吉马酮可能通过上调miR-297表达抑制H_(2)O_(2)诱导的小鼠海马神经元细胞凋亡和氧化应激。

Objective:To investigate the effect and molecular mechanism of germacrone on the apoptosis and oxidative stress in mouse hippocampal neurons induced by H_(2)O_(2).Methods:Mouse hippocampal neurons were isolated and cultured,and then divided into control group,H_(2)O_(2) group,H_(2)O_(2)+germacrone low,medium and high(50,100,200μmol·L^(-1))concentration groups,H_(2)O_(2)+miRNC group,H_(2)O_(2)+miR-297 group,H_(2)O_(2)+germacrone(200μmol·L^(-1))+anti-miR-NC group and H_(2)O_(2)+germacrone(200μmol·L^(-1))+anti-miR-297 group.Flow cytometry was used to detect the apoptosis;Western blot was used to detect B cell lymphoma/leukemia-2(Bcl-2)and Bcl-2 related X(Bax)protein expressions;malondialdehyde(MDA)and superoxide dismutase(SOD)kits were used to detect MDA content and SOD activity;real-time fluorescence quantitative PCR(RT-qPCR)was used to detect miR-297 expression.Results:After treatment with low,medium and high concentrations of germacrone,the apoptosis of H_(2)O_(2)-induced hippocampal neurons,Bax expression and MDA content were decreased,Bcl-2 expression,SOD activity and expression of miR-297 were increased,and all were in a concentration-dependent manner(P<0.05).Overexpression of miR-297 could reduce the apoptosis rate of H_(2)O_(2)-induced hippocampal neurons and MDA content,and increase SOD activity(P<0.05).Inhibiting the expression of miR-297 reversed the effects of germacrone on the apoptosis and oxidative stress of hippocampal neuronal induced by H_(2)O_(2).Conclusion:Germacrone may inhibit the apoptosis and oxidative stress of mouse hippocampal neurons induced by H_(2)O_(2) through up-regulating miR-297 expression.