冷休克通路在低温脑复苏中的作用及其线粒体机制

The cardiac arrest remains the leading cause of death in human. Therefore, how to effectively reduce the cerebral injury after return of spontaneous circulation and to improve the survival rate of patients is an area of tremendous importance. Therapeutic hypothermia is an effective intervention shown to improve outcome in patients following cardiac arrest, but its key mechanism still remains unclear. Cold inducible RNA binding protein (CIRP) is shown to be inducible sensitively by cold stress. The results in vitro indicated that CIRP could activate cold shock pathways and improve neuron survival by affecting apoptotic pathway. Our previous studies suggest that CIRP expression, which was up-regulated by therapeutic hypothermia, was closely related to mitochondrial apoptosis associated proteins in the cardiac arrest rat model. On the basis of these results, we speculate that cold-shock pathways enhanced by therapeutic hypothermia could resuscitating neurons by protecting mitochondria from ischemia reperfusion injury. To verify this assumption, we use well-established global ischemia reperfusion models (in vivo, cardiac arrest models in rats, and in vitro, model OGD-R models in neurons) and professional skills (adenovirus transfection, confocal laser, immunofluorescence double staining techniques and so on) to study from the whole to the cell: ① the role of CIRP induced by hypothermia in resuscitating cardiac arrest rats and reducing mitochondrial damage; ② the effect of exogenous administration of CIRP combined with therapeutic hypothermia on brain resuscitation in cardiac arrest rats. This results will provide a new target for brain resuscitation.

心跳骤停一直是人类死亡的首要原因。如何有效减轻自主循环恢复后脑损伤，提高成活率意义重大。治疗性低温是有效的脑复苏方法, 但关键机制尚未完全阐明。冷诱导RNA结合蛋白（CIRP）在冷应激时表达明显增加，且对低温高度敏感。离体研究发现，CIRP启动冷休克通路，通过抑制细胞凋亡，提高神经元存活率。我们的前期研究表明，低温上调全脑缺血再灌注后CIRP表达，并且低温诱导的CIRP与线粒体凋亡蛋白具有高度相关性。因此，我们推测，低温可能通过强化冷休克通路减轻线粒体损伤，进而复苏受累神经元。为了证明这一假说本实验构建在体（心跳停止）和离体（OGD-R）全脑缺血再灌注模型，采用腺病毒转染、激光共聚焦和免疫荧光双染等先进技术，从整体到细胞研究：①CIRP在低温复苏神经元中的作用和所涉及的线粒体机制；②外源性给予CIRP制剂联合低温治疗，对心跳骤停大鼠的复苏效果。本结果将为脑复苏提供新靶点。

背景：心搏骤停一直是人类死亡的首要原因。虽然近年来自主循环恢复率有所提高，但是出院存活率依然很低。因此，如何提高出院存活率是研究的重点。浅低温是临床上公认的有效治疗方法，但其作用机制尚不十分明确。浅低温可诱导机体产生冷休克蛋白，而高等生物体中最常见的一种冷休克蛋白是冷诱导RNA结合蛋白（CIRP）。CIRP能够通过多种途径抑制细胞凋亡，包括活化 ERK1/2 和 PI3K/Akt途径，抑制DNA损伤上调的p53等，活化的这些蛋白又可通过调控 Bcl-2 超家族成员作用于线粒体。研究内容：本课题拟建立大鼠海马神经元氧糖剥夺/复氧模型和经食道诱颤致心搏骤停模型，运用基因沉默技术，分别从离体、在体两个方面探讨CIRP在低温复苏自主循环恢复大鼠中的作用和线粒体机制；并通过外源性注入CIRP制剂，探讨CIRP联合低温对线粒体的复苏效果、对心搏骤停大鼠神经功能预后和生存率的影响。结果：建立了接近临床现实、稳定可靠、可重复性高的全脑缺血/再灌注(ischemia／reperfusion，I／R)模型。我们证实32℃-34℃治疗性浅低温能够明显改善心搏骤停大鼠的神经功能和提高生存率。浅低温能够上调海马CIRP的表达，抑制线粒体途径的细胞凋亡；而下调海马CIRP的表达，将削弱浅低温对心搏骤停大鼠神经元的保护作用，其机制可能与线粒体分裂有关。除此外，浅低温还可通过上调CIRP表达来抑制硫氧还蛋白1通路的相关蛋白表达，从而减轻心肺复苏后大鼠海马神经元的氧化应激损伤。我们还证实硫化氢和富氢液也能够改善心搏骤停大鼠的神经功能和提高生存率，且其与浅低温联合治疗效果更优。富氢液的主要作用位点在抑制氧化应激后进而减轻内质网应激；硫化氢的主要作用位点可能是激活核因子E2相关因子（Nrf2/血氧素氧合酶（HO-1）通路，抑制MMP-9蛋白表达、减少Occludin蛋白丢失。科学意义 建立了模拟临床实际情况的、稳定的心搏骤停心肺复苏模型，为后续治疗方法的研究打下基础。而本课题中关于CIRP及其线粒体机制的研究可为脑复苏提供新靶点。富氢液和硫化氢可有望作为浅低温治疗的辅助治疗，减轻心肺复苏患者脑损伤，进而提高出院存活率。