RIP3巯基亚硝基化在缺血性脑中风中的作用及机制研究

Neuronal NO synthase (nNOS) is activated by Ca2+ influx via NMDA receptors which increases the release of endogenous NO in the stroke induced by cerebral ischemia. The accumulated NO may S-nitrosylate proteins in cells by adding NO to the thiol moiety of reactive cysteine residues, which finally exacerbates ischemic brain injury. The receptor-interacting protein 3 (RIP3) has been demonstrated the prominence in regulating apoptotic and necrotic cell death as a stress signaling molecular. Whether RIP3 participate the cerebral ischemia and its mechanisms, however, remain unclear. Our preliminary experiments indicate that RIP3 is S-nitrosylated and activated after 3 hours of rat brain ischemia/reperfusion. So we hypothesize that the S-Nitrosylation of RIP3 contributes to its dimerization and phosphorylation (activation), which in turn facilitates the activation of the downstream signaling pathway and finally exacerbates ischemic neuron death. The models and technique we will use in the project are as follows: Sprague-Dawley rat four-vessel occluded transient whole cerebral ischemia model, Oxygen and Glucose Deprivation (OGD) of cultural hippocampal neurons and SH-SY5Y cell, technique of molecular biology and biochenmistry, immunohistochemistry and histological analysis. Our objects are to study the effects of S-Nitrosylation of RIP3 on regulating neuronal cell death in the ischemia stroke and reveal a potential new therapeutic approach to the future treatment of transient cerebral ischemia injury.

在缺血性脑中风中，NMDA受体介导钙离子内流，快速作用于nNOS，产生自由基NO，使胞内靶蛋白巯基亚硝基化，加重脑损伤。RIP3作为细胞应激传感分子，在调控细胞凋亡、细胞坏死通路中发挥重要作用。但是RIP3在脑缺血中是否引起细胞死亡及其机制尚不清楚。我们的预实验结果显示，大鼠脑缺血再灌注3小时，RIP3巯基亚硝基化明显升高，并导致了其酶活化。因此，我们提出在脑缺血中, RIP3的活化可能受蛋白质巯基亚硝基化调控的设想：RIP3巯基亚硝基化可促进其二聚化引起自身磷酸化而活化，活化后进一步激活其下游信号分子，加重缺血性脑损伤。本项目拟以SD大鼠四动脉结扎全脑缺血模型和培养的海马神经元、SH-SY5Y细胞缺氧缺糖模型，采用分子生物学、生物化学以及免疫组化和组织学等方法，进一步探索RIP3巯基亚硝基化在缺血性脑中风的作用，以期为治疗缺血性脑损伤提供新的对策和药物作用的靶点。

在缺血性脑中风中，NMDA受体介导钙离子内流，快速作用于神经性一氧化氮合酶（nNOS，Neuronal NO synthase），产生自由基NO，使胞内靶蛋白受体相互作用蛋白3（RIP3，Receptor-Interacting Protein 3）巯基亚硝基化，可促进其磷酸化而活化，活化后进一步激活其下游信号分子RIP1等，加重缺血性脑损伤；在培养的HEK293细胞中，富含脯氨酸的蛋白激酶2（PYK2，Proline-rich tyrosine kinase 2）能被NO供体GSNO亚硝基化，PYK2的534位半胱氨酸（Cys534）为其承担亚硝基化的位点，OGD后内源性的NO介导PYK2的亚硝基化，亚硝基化的PYK2参与了其活化，导致神经细胞损伤；缺血/复灌或OGD/reoxygenation可诱导神经元中nNOS发生去亚硝基化而使酶激活，nNOS去亚硝基化过程主要由Trx/TrxR系统介导；脑缺血再灌注中胱天蛋白酶原9（procaspase-9）与XIAP（X-linked inhibitor of apoptosis）发生了转亚硝基化，Trx系统介导了二者的转亚硝基化，procaspase-9因去亚硝基化而水解活化进而促进神经元凋亡，小肽TAT-AVPY通过阻止二者的结合，能够抑制缺血再灌注诱导的XIAP的亚硝基化及procaspase-9的去亚硝基化和水解活化，从而在脑缺血时起保护作用。SD大鼠肾缺血/复灌后，活化的去乙酰化酶2（SIRT2，sirtuin 2）丝氨酸发生磷酸化，SIRT2去乙酰化激活FOXO3a，FasL表达上调，caspase8 and caspase3激活，增加肾小管上皮细胞的凋亡，最终加剧缺血性肾损伤。以上研究结果为治疗缺血性脑损伤和肾损伤提供了新的对策。