早期应用泛素-蛋白酶体抑制剂对脑缺血再灌注中线粒体损伤保护机制的研究

Mitochondrial dysfunction is an important cause of neurological deficit in cerebral ischemia-reperfusion injury. Protection of the structure and function of mitochondria is of great significance against cerebral ischemia-reperfusion injury. Mitochondrial dynamic-related proteins play a key role in maintaining mitochondrial function. Our preliminary data showed that ubiquitin-proteasome activity was increased and mitochondrial dynamic-related proteins were rapidly degraded, which was the early events of ischemia-reperfusion injury. Overexpression mitochondrial dynamic-related protein MFN2 ameliorated mitochondrial dysfunction induced by oxygen-glucose deprivation/reperfusion. Therefore, based on preliminary findings, in this proposal, we will real-time investigate activation of ubiquitin-proteasome system, ubiquitylation and degradation of mitochondrial dynamic-related proteins at the early stage of ischemia-reperfusion injury both in vitro and in vivo experiment. We will also determine the neuroprotective time window of ubiquitin-proteasome inhibitor at the early stage of cerebral ischemia-reperfusion. Finally, we will study whether ubiquitin-proteasome inhibitors exert neuroprotective effects by regulating the expression level of mitochondrial dynamic-related proteins. The successful implementation of this project will contribute to expand understanding of the pathogenesis of cerebral ischemia reperfusion injury, while providing potential targets for intervention and treatment.

脑缺血再灌注中线粒体功能损伤是造成神经功能缺损的重要原因。保护线粒体的结构及功能，在脑缺血再灌注损伤后的神经保护治疗中具有重要意义。线粒体动力相关蛋白对维持线粒体功能起着关键作用。我们前期研究发现，在缺血再灌注早期，泛素-蛋白酶体活性增加，快速降解线粒体动力相关蛋白，可能是缺血再灌注损伤的早期发生事件。而过表达线粒体动力相关蛋白MFN2，能减轻氧糖剥夺再灌注对线粒体的损伤。因此，本研究中，我们在前期研究的基础上，通过体内、外实验，实时检测缺血再灌注早期泛素-蛋白酶体通路激活、线粒体相关蛋白泛素化及降解情况；确定泛素-蛋白酶体抑制剂在脑缺血再灌注早期神经保护作用的时间窗；并进一步证实泛素-蛋白酶体抑制剂通过调节线粒体动力相关蛋白的水平而发挥神经保护作用。本项目的顺利实施将有助于扩展对脑缺血再灌注损伤发生机制的认识，同时为干预和治疗提供潜在的靶点和手段。

脑缺血再灌注中线粒体功能损伤是造成神经功能缺损的重要原因。保护线粒体的结构及功能，在脑缺血再灌注损伤后的神经保护治疗中具有重要意义。线粒体动力相关蛋白对维持线粒体功能起着关键作用。本项目的主要研究内容是通过体内、外实验，实时检测缺血再灌注早期泛素-蛋白酶体通路激活、线粒体相关蛋白泛素化及降解情况；确定泛素-蛋白酶体抑制剂在脑缺血再灌注早期神经保护作用的时间窗；并进一步证实泛素-蛋白酶体抑制剂通过调节线粒体动力相关蛋白的水平而发挥神经保护作用。通过实施本项目，我们发现：1.脑缺血再灌注早期泛素-蛋白酶体活性先高后低，线粒体蛋白被快速泛素化，并降解。2.确定了脑缺血再灌注早期泛素-蛋白酶体活性在缺血再灌注0-1小时升高，1小时后蛋白酶体活性降低。3.确定了泛素-蛋白酶体抑制剂（去泛素化蛋白USP30）对缺血再灌注损伤具有神经保护作用。4.分离了缺血再灌注组织的线粒体，定量质谱确定了蛋白量改变的线粒体相关蛋白，证实了线粒体蛋白MFN2和线粒体动力相关蛋白Drp1对缺血再灌注损伤具有神经保护作用。我们的研究扩展了对脑缺血再灌注损伤发生机制的认识，研究结果提示靶向抑制泛素蛋白酶体，或增加去泛素化酶USP30活性可能为脑缺血再灌注损伤相关疾病的治疗提供一种新的策略。