甘丙肽2型受体对氧化应激海马神经元自噬的调控作用及机制研究

The Regulatory Effect and Mechanism of Galanin Receptor on Autophagy in Hippocampal Neurons Under Oxidative Stress

本研究旨在解析仔猪脑海马甘丙肽2型受体(galanin receptors type 2,GALR2)参与氧化应激调节的分子机制.本研究基于成功构建的仔猪活体和大鼠海马神经元氧化应激模型,采用实时PCR技术考察仔猪脑海马和大鼠海马神经元GALR2的表达变化.并采用实时PCR、蛋白质印迹法及透射电镜技术进一步探索GALR2介导的信号途径和自噬之间的关系.结果发现,与对照组相比,氧化应激仔猪脑海马与大鼠海马神经元GALR2的转录水平上调(P<0.01;P<0.05).同时,氧化应激神经元自噬相关基因LC3、ATG5与Beclin-1的转录水平均上调(P<0.05;P<0.05;P<0.01).相关性分析结果显示,GALR2与LC3、ATG5及Beclin-1的转录水平正相关(P<0.05;P<0.05;P<0.01).GALR2特异性抑制剂M871的处理降低氧化应激海马神经元的活力(P<0.01)、抑制氧化应激引起的自噬小体数量增多(P<0.01)、自噬相关基因LC3、Beclin-1及ATG5转录水平的上调(均P<0.01)以及LC3-Ⅱ/β-actin比值与P62蛋白质水平的升高(均P<0.05),表明伴随GALR2表达水平的抑制,被氧化应激上调的海马神经元自噬效应被抑制,从而削弱对氧化损伤的抵抗,降低了神经元的活力.与此同时,M871的处理也降低了被氧化应激上调的JNK蛋白表达量(P<0.01)及磷酸化水平(P<0.05),表明JNK是GALR2调控海马神经元氧化应激的下游靶酶.而JNK特异性抑制剂SP600125的处理则下调了被氧化应激上调的自噬标记蛋白LC3-Ⅱ/β-actin比值(P<0.01),表明氧化应激状态下,抑制的JNK阻碍了神经元中上调的GALR2对自噬信号通路的激活.综上可见:氧化应激状态下,海马神经元中上调的GALR2可通过调节JNK信号途径激活细胞自噬途径,从而降低神经元的氧化应激损伤,保护神经元.

The aim of this study is to elucidate the molecular mechanism of Galanin receptors type 2(GALR2)which involved in regulation of oxidative stress in the hippocampus. We used real-time PCR technique to investigate the change of GALR2 expression in hippocampus of piglets and hippocampal neurons of rats, based on the successfully constructed oxidative stress model. Real-time PCR, Western blotting and transmission electron microscopy were used to further explore the relationship between the signal pathway mediated by GALR2 and autophagy. The results showed that the transcription levels of GALR2 were up-regulated in the hippocampus of oxidative stressed piglets and rat hippocampal neurons compared with the control group(P<0.01;P<0.05). At the same time, the transcription levels of LC3, ATG5 and Beclin-1 in oxidative stressed neurons were up-regulated(P<0.05;P<0.05;P<0.01). Correlation analysis showed that GALR2 was positively correlated with LC3, ATG5 and Beclin-1(P<0.05;P<0.05;P<0.01). The treatment with M871, a specific inhibitor of GALR2, decreased the activity of hippocampal neurons under oxidative stress(P<0.01), increased the number of autophagosomes(P<0.01) and transcription levels of LC3, Beclin-1 and ATG5(P<0.01), and increased the ratio of LC3-Ⅱ/actin and P62 protein level(P<0.05), showing that the autophagy of hippocampal neurons, which was up-regulated by oxidative stress, was inhibited with the inhibition of GALR2 expression, thus weakening the resistance to oxidative damage and decreasing the viability of neurons. Simultaneously, M871 treatment also decreased the upregulated protein level(P<0.01) and the phosphorylation level of JNK(P<0.05) in oxidative stressed neurons,indicating that JNK is the downstream target enzyme of GALR2 in hippocampal neurons under oxidative stress.However, treatment with JNK specific inhibitor SP600125 lowered the ratio of LC3-Ⅱ/actin, which was upregulated by oxidative stress(P<0.01), showing the inhibition of JNK blocks the activation of the autophagic pathway by an up-regulated GALR2 in neurons. To sum up: under oxidative stress, the up-regulated GALR2 in hippocampal neurons can activate the autophagy pathway by up-regulating JNK signal pathway, thus attenuate oxidative stress injury and protect neurons.