自由基ONOO介导的蛋白质酪氨酸硝化参与脑缺血损伤，但尚不清楚哪些蛋白质被硝化及对功能的影响。课题组近年来主要从事脑缺血中核因子PPARg的作用及调节机制研究，已发表的成果及前期研究表明：1. PPARg激活保护脑缺血损伤（抗炎作用等）；2. 缺血脑组织中分子量约55kDa的蛋白质硝化最显著，而PPARg分子量与55kDa接近，且含有多个酪氨酸残基，是ONOO的潜在硝化靶点；3. ONOO可使培养的皮层神经元PPARg硝化。据此，本项目将首先在原代神经元或小胶质细胞中，直接或间接诱导ONOO生成，观察PPARg硝化，分析对PPARg活性的影响（如配体依赖的核移位、PPRE结合活性、靶基因表达等）；之后利用体内外脑缺血模型验证缺血诱导ONOO-PPARg硝化，并明确该硝化将抑制PPARg的脑保护活性，从而揭示缺血损伤对PPARg的调节修饰作用、为建立基于PPARg的脑保护策略提供实验依据。

Nitration of tyrosine residues in proteins，a kind of protein post-translation modification, has been proven to be implicated in cerebral ischemia-reperfusion injury. Peroxynitrite (ONOO-)，the potent oxidant which is formed by the rapid reaction of nitric oxide(NO) with superoxide, is thought to be predominantly responsible for tyrosine nitration of protein in the brain. The formation of nitrotyrosine in protein can result in the alteration of protein activity and function. Although several studies have been carried out, it is still unclear what type of protein is nitrated and whether the nitrated protein interferes with protein function in the setting of cerebral ischemia/reperfusion. Our team has been focusing on the study of the role of peroxisome proliferator activated receptor-gamma (PPARg) and related regulatory mechanisms in cerebral ischemia/reperfusion. Our published data showed that the activation of the nuclear transcriptional factor PPARg is neuroprotective against the ischemic injury of the brain by its anti-inflammatory properties. We also demonstrated in our published data that in ischemic brain the molecular weight of the most strongly nitrated protein was 55kDa protein, which is close to that of PPARg. In addition, PPARg possesses several tyrosine residues, which might be potential targets for nitration by peroxynitrite during cerebral ischemia/reperfusion. Additionally, in our preliminary study, we observed that PPARg was nitrated by authentic ONOO- in primary cultured cortical neurons . In this proposal, we will continue to investigate whether nitration of PPARg interferes with its function in terms of ligand-dependent translocation from cytosol into nucleus, PPAR-DNA (PPRE) binding activity, and its target genes expression in cultured primary cortical neurons or microglia. Then, the above results will be further evaluated in cerebral ischemia model in vitro and in vivo to confirm that ONOO-mediated PPARg nitration is involved in ischemic injury, and the nitrated-PPARg loses its neuroprotective effects especially the anti-inflammatory actions in ischemic brain. The demonstration that PPARg is one of the targets of ONOO- and ONOO-induced PPARg nitration is involved in the pathogenesis of brain ischemia is believed to provide insight into the understanding of PPARg post-translational modification and the therapeutic implications for stroke treatment.