RKIP对脑卒中的保护作用及其影响机制

Stroke remains to be the primary cause of adult disability and ranks only behind cancer and cardiac disease as a cause of death worldwide. Interestingly, a similar variation in susceptibility to stroke occurs in spontaneously hypertensive rats (SHR), and thus a sub-strain of SHR was developed based on their susceptibility to stroke and named stroke-prone spontaneously hypertensive rats (SHR-SP),which develop a stroke at a mean age of 10 (males) to 14 months (females). We speculated that since both strains (SHR and SHR-SP) were hypertensive, a comparison of SHR-SP with SHR would provide new insights into the pathogenesis and development of stroke beyond hypertension. .Our results showed that the area of cerebral infarction after middle cerebral artery occlusion (MCAO) of SHR-SP was significantly larger than that of SHR, suggesting that SHR-SP is more susceptible to ischemic stroke than SHR. To further reveal the mechanisms of this difference, we examined the differentially expressed protein profiles in the brains of SHR-SP and SHR by using two dimensional fluorescent difference gel electrophoresis (2D-DIGE). We firstly found that Raf kinase inhibitor protein (RKIP) was significantly down-regulated in SHR-SP, and this result has been confirmed by Realtime-PCR. .Based on these findings; we speculated that the decreased RKIP in the brain of SHR-SP mabye plays very important roles in stroke. To verify this hypothesis, this project intends to study both in vitro and in vivo. The following experiments will be performed: 1. Confirm the exact relationship between the susceptibility to stroke of SHR-SP and the decrease of RKIP both in vitro and in vivo; 2. Explore the molecular mechanism of the effect of RKIP expression on stroke in SHR-SP. This project intends to provide an effective target for the prevention of stroke, and thus provide new ideas for the treatment of stroke.

脑卒中一旦发生后果严重，防止其发生非常重要。SHR-SP是从SHR定向培育的具自发性脑卒中倾向的亚种，到一定年龄就发生脑卒中并死亡。我们发现，大脑中动脉栓塞后SHR-SP脑梗死面积显著大于SHR，说明同样缺血状态下SHR-SP比SHR更敏感。为进一步探讨这种差异的机制，我们以蛋白质组学研究SHR-SP脑蛋白质表达变化，结果发现：与SHR相比，RKIP在SHR-SP脑中表达显著减少，进而我们以Realtime-PCR验证了该结果。据此，我们推测RKIP表达减少在SHR-SP脑卒中发生过程中很可能起重要作用。为验证该假设，本课题拟分别从离体细胞和整体动物水平进行以下研究：确定SHR-SP易发生脑卒中与RKIP表达减少有关；进一步探讨RKIP表达减少导致SHR-SP易发脑卒中的机制。本研究可为预防脑卒中发生提供有效作用靶点，为减少脑卒中发生探索一个行之有效的治疗方法，为脑卒中的治疗提供新的思路。

前期工作中，我们发现脑卒中发生后动物脑部RKIP蛋白和mRNA均比正常对照组表达显著减小。RKIP属于PEBP家族，广泛存在于各种生物中，参与了对细胞内MAPK信号转导通路、G蛋白偶联受体信号转导通路以及NF-κB等多种信号转导通路的调节作用，在膜的生物合成、细胞凋亡和细胞周期调控等生理过程中发挥重要作用，该结果提示我们：RKIP在脑卒中的发生过程中很可能起着重要作用。.目的：从动物水平和细胞水平验证RKIP的神经保护作用。.方法：a．体内实验.实验用SD大鼠被随机分入四个实验组：假手术组；MCAO组；RKIP干预组（实验动物给予皮层定点注射RKIP慢病毒载体，14天后行MCAO）。所有SD大鼠行MCAO或假手术24h后，收集血清进行代谢组学分析，并取全脑做TTC染色测定梗死面积和免疫组化。.b．体外实验.1.体外培养PC12细胞，分为五组：正常组、OGD模型组、空载病毒组、RKIP过表达病毒组和 SiRNA干扰组。模拟体内缺血缺氧状态（OGD模型），考察RKIP表达变化对PC12细胞功能的影响。.2.分离大鼠原代小胶质细胞做体外培养，分为五组：分为五组：正常组、OGD模型组、空载病毒组、RKIP过表达病毒组和 SiRNA干扰组。模拟体内缺血缺氧状态（OGD模型），考察RKIP表达变化对原代小胶质细胞功能的影响。 .结果：a.体外实验.1.RKIP过表达组梗死面积比MCAO组明显变小，免疫组化的结果发现RKIP过表达组小胶质细胞活化程度降低；2.由代谢组学轮廓分析，我们鉴定得出RKIP上调后对缺血性脑卒中的保护作用主要体现在能量代谢、氨基酸代谢、脂质代谢等通路上，其中棕榈酸和油酰胺的变化趋势提示神经保护作用可能是通过抑制炎症反应来实现的。.b．体外实验.1.RKIP上调后PC12细胞活力升高，凋亡率明显降低，细胞增殖能力升高，胞内活性氧降低，MAPK通路和NF-κB通路的活化被抑制。.2.RKIP上调后小胶质细胞的细胞活力变化不大，活化程度降低，transwell实验显示细胞的迁移能力降低，IL-1β，TNF-α，iNOS等炎性因子的mRNA水平降低。.结论：在发生脑卒中时，RKIP的上调降低了实验动物的脑梗面积，抑制小胶质细胞的活化；体外实验也表明RKIP的上调可以减轻缺糖缺氧条件下神经元细胞的损伤，同时抑制小胶质细胞的活化，进一步起到神经保护作用。