CHIP蛋白调控RIPK3介导的程序性坏死参与脑缺血再灌注损伤的分子机制及潜在治疗策略

Cerebral ischemia-reperfusion injury is one of the most prevalent causes for brain damage caused by ischemic stroke. Different from necrosis and apoptosis, necroptosis as a novel mode of cell death has been reported to be involved in the neuron damage induced by cerebral ischemia-reperfusion injury, and RIPK3 plays an important role in this molecular pathway. However, the related pathogenesis remains elusive. In our previous work, we identified the interactions between carboxyl terminus of Hsp70-interacting protein (CHIP) and receptor-interacting protein kinase 3 (RIPK3), and CHIP could regulate the ubiquitination of RIPK3 and mediate degradation of RIPK3 via the ubiquitin-proteasome pathway. Based on these findings, we put forward a hypothesis that CHIP may be involved in the RIPK3-mediated necroptosis via regulating the ubiquitination of RIPK3 in the cerebral ischemia-reperfusion injury models. Besides, upregulation of CHIP by small molecular drugs or gene modification may attenuate necroptosis in models of cerebral ischemia-reperfusion injury. In this study, we will investigate the pathogenesis of CHIP involved in the RIPK3-mediated necroptosis in the cerebral ischemia-reperfusion injury by the cell models and cerebral ischemia-reperfusion/CHIP transgenic mice models, and find the potential therapeutic strategy of cerebral ischemia-reperfusion injury. Based on the solid previous work in accordance with the scientific hypothesis and advanced experiment methods, this study is expected to provide novel strategies for the neuroprotective therapy in ischemic.

脑缺血再灌注损伤是引起缺血性卒中脑损伤的主要原因之一，近期研究显示：独立于坏死和凋亡之外的新型细胞死亡方式：“程序性坏死”参与了脑缺血再灌注损伤的发生，RIPK3蛋白则在程序性坏死启动过程中扮演关键作用，但相关分子通路尚不明了。前期工作显示：泛素E3连接酶CHIP与RIPK3存在相互作用，其能够泛素化RIPK3并介导其通过UPS通路降解，发挥类RIPK3抑制剂作用。基于以上发现，我们推测：在脑缺血再灌注损伤过程中，CHIP蛋白是否也可通过调控RIPK3泛素化过程参与调控程序性坏死？通过外源性小分子药物和基因修饰手段调控CHIP蛋白表达是否可能下调脑缺血再灌注损伤后程序性坏死水平从而起到脑保护作用？本实验拟从分子、细胞、动物多水平探讨CHIP蛋白调控RIPK3介导的程序性坏死参与脑缺血再灌注损伤的分子机制并探寻新的治疗策略，本研究的开展将为缺血性卒中脑保护研究提供新的思路，具有重要意义。

缺血性脑卒中是一类以高发病率、高致死率、高致残率为特点的疾病，脑卒中后血流再通可造成脑组织缺血再灌注损伤，是引起缺血性卒中脑损伤的主要原因之一。当前对缺血性脑卒中的治疗主要聚焦于尽早恢复缺血脑组织的血液供应以减少脑组织因缺乏氧气和能量供应所致的损害，对血液再灌注后引起的脑损伤的机制及治疗方法尚需进一步研究。导致脑组织缺血再灌注损伤的确切机制尚不清楚，既往研究表明，脑缺血再灌注损伤的发生与细胞兴奋性毒性作用、自由基的损伤作用、钙超载、一氧化氮毒性、细胞凋亡和炎症反应等密切相关。近年来研究表明，程序性坏死作为一种新的细胞死亡方式，也参与了脑缺血再灌注损伤的发生。程序性坏死主要受RIPK1、RIPK3和MLKL三种蛋白激酶介导且RIPK3在其中起着关键的作用。CHIP是一种兼具伴侣分子和E3泛素连接酶活性的蛋白，在细胞的蛋白质质量控制系统中起核心桥梁作用，主要在脑组织、心脏、肝脏等大量表达。前期工作显示泛素E3连接酶CHIP与RIPK3存在相互作用，其能够泛素化RIPK3并介导其通过UPS通路降解，发挥类RIPK3抑制剂作用，减轻程序性坏死水平。但在脑缺血再灌注损伤过程中，CHIP蛋白是否能够通过调控RIPK3介导的程序性坏死从而影响脑缺血再灌注损伤过程及CHIP蛋白在脑缺血再灌注损伤中的其它作用仍未得到全面的研究及阐述。本项目从分子、细胞、动物多水平全面深入探讨了CHIP蛋白调控RIPK3介导的程序性坏死参与脑缺血再灌注损伤的分子机制及CHIP蛋白对脑缺血再灌注损伤的神经保护作用。我们的研究结果表明，缺血再灌注后，细胞及动物模型程序性坏死水平升高，同时体外细胞模型进一步验证了CHIP蛋白与RIPK3存在相互作用。应用小分子激动剂茴香霉素及构建转基因小鼠模型诱导CHIP蛋白过表达可以减弱脑缺血再灌注损伤模型程序性坏死水平，减轻脑损伤。我们的研究结果充分揭示了CHIP蛋白对程序性坏死的调节方式及CHIP蛋白过表达对缺血再灌注脑损伤的保护作用。