自噬与凋亡的转换与调控在脑卒中后神经保护中的作用及机制研究

It is vital to rescue autophagic neurons in treatment for cerebral stroke, but appropriate activated autophagy is neuroprotective. Our previous study illustrated that autophagy was markedly promoted but apoptosis were significantly decreased in neurons in the ischemic penumbra at 4 days after cerebral stroke, we theorized that there might be a transition between autophagy and apoptosis, and the transition and its modulation between them determined the fate of injured neurons, however, this mechanism is currently not understood. Bcl2-Beclin1 is an important compound involved in autophagy and apoptosis, promoting their dissociation can improve autophagy but decrease apoptosis. JNK is a main phosphorylating kinase for Bcl2, we predict phosphorylated Bcl2 leads to dissociation of Bcl2-Beclin1, sequentially, autophagy is promoted. In order to test this hypothesis, first, gene expression profiles and celluar localization of autophagy and apoptosis are performed; Second, autophagy or apoptosis is inhibited at characteristic time points, to understand the optimal timewindow for intervention. Meanwhile, the model of neuron ischemia is prepared in vitro, the transition and its regulatory mechanisms between autophagy and apoptosis are investigated after adding activator and inhibitor for JNK. Finally, using animal experiments to investigate molecule mechanisms of neuron survival determined by the stabilization of Bcl2-Beclin1, which modulated by JNK. The efficacy of the transition between autophagy and apoptosis in neuroprotection and its mechanisms following cerebral stroke are to be understood by this study.

挽救自噬神经元对脑卒中治疗至关重要，而适度激活的自噬具有神经保护作用。我们前期研究结果显示在脑卒中后4天时，缺血半影区神经元自噬显著升高而凋亡明显降低，提示二者间可能存在相互转换，其转换及调控决定损伤神经元的命运，目前该转换机制并不清楚。Bcl2-Beclin1是参与自噬与凋亡的重要复合物，促进其解离可增加自噬而减少凋亡。JNK是Bcl2的主要磷酸化激酶，我们推测JNK磷酸化Bcl2导致Bcl2-Beclin1解离，进而促进自噬。为验证此假说，首先检测脑卒中后凋亡与自噬表达时间谱及细胞定位；其次在特征时间点抑制凋亡或自噬，阐明脑卒中后最佳干预时间窗；同时建立体外神经元缺血模型，通过抑制或激活JNK活性，探明其对自噬与凋亡的转换与调控机制；最后通过动物实验验证JNK调控Bcl2-Beclin1稳定性决定损伤神经元命运的分子机制。通过本研究阐明自噬与凋亡的转换在脑卒中后神经保护中的作用及机制。

Bcl-2-Beclin1是介导自噬与凋亡间转换的复合物，而JNK是Bcl-2的主要磷酸化激酶。我们推测JNK磷酸化Bcl-2导致Bcl-2-Beclin1解离，进而诱导缺血半影区神经元凋亡向自噬转换，从而发挥脑卒中后的神经保护作用。为验证此假说，本项目的主要研究内容与结果如下：.（1）分别建立永久性和短暂性脑缺血大鼠模型，在脑卒中后自噬的特征变化时间点干预自噬活性，结果表明无论增强还是抑制自噬活性均不能诱导永久性脑缺血后的神经保护，而增强自噬可显著减轻脑缺血/再灌注后的神经损伤，因而本项目采用短暂性脑缺血模型进行后续研究；.（2）建立大鼠短暂性脑缺血模型，检测脑卒中后缺血半影区自噬与凋亡表达时间谱及细胞定位，结果表明脑卒中后12小时及2天为二者的特征变化时间点；.（3）在上述自噬与凋亡的特征变化时间点增强JNK活性，结果表明增强JNK活性可通过提高BCL-2磷酸化水平促进Bcl-2-Beclin1复合物解离，从而诱导缺血半影区神经元凋亡向自噬转换，且该转换机制可提高脑卒中后12小时的神经保护，但不能减轻脑卒中后2天时的神经损伤；.（4）为探究增强JNK活性在脑卒中不同阶段产生不同效应的原因，我们进一步检测自噬流通路中关键蛋白的表达，结果显示在脑卒中后2天时出现明显的自噬流障碍，该结果提示增强JNK活性可诱导神经元凋亡向自噬转换，进而通过加强自噬流提高脑卒中后12小时的神经保护，而该保护效应在脑卒中后2天时被受损的自噬流抵消；.（5）同时，利用体外培养神经元氧糖剥夺（OGD）细胞模型进一步验证上述结论。结果发现神经元OGD后5小时及10小时为凋亡与自噬的特征变化时间点。增强JNK活性通过提高OGD神经元的BCL-2磷酸化水平促进Bcl-2-Beclin1复合物解离，从而诱导OGD神经元凋亡向自噬转换，该转换机制可提高神经元OGD后5小时的细胞活性和存活，而该保护机制在OGD后10小时被受损的自噬流消减。