线粒体/ox-CaMKII通路在活性氮介导血管内皮细胞损伤中的作用及药物调控

Endothelial dysfunction and resulting vascular pathology have been identified as an early hallmark of multiple diseases. The initiating loop of cardiovascular disease can be eliminated by endothelial function rescue but no effective approaches are available now. Oxidative stress has been considered one of the major contributors of endothelial cell injury while the protective effects of broad spectrum of non-specific antioxidants have been disappointing, so the further mechanisms need to be elucidated. Previously,we reported that RNS donor S-Nitrosoglutathione (GSNO)-mediated nitration stress can directly induce mitochondrial-dependent apoptosis in endothelial cells, but its upstream activator and intervention mechanisms remains to be explored. Our latest research indicates that GNSO-induced endothelial cells insult associated with increased ox-CaMKII expression which inhibited can lead to significant reduction of eNOS phosphorylation and nitrotyrosine generation.Therfore, we speculate that mitochondrial/ox-CaMKII pathway was a critical upstream signal associated with the pathological nitric oxide(NO) activation in endothelial cells and is likely to be a pivotol pathway leading to endothelial cell damage under nitrosative stress. However, little is known about the molecular and cellular mechanisms that cause suggesting that ox-CaMKII-triggered endothelial cell death contributed to endothelial dysfunction. To verify this hypothesis, GSNO and oxygen-glucose deprivation induced endothelial dysfunction model will be used to evaluate the regulational effects of mitochondrial/ox-CaMKII pathway in RNS-mediated endothelial damage simultaneously. The purpose of this research attempts to explore the relationship between the involved signaling pathways associated with endothelial cell injury and identify what we believe to be a novel approach to reduce endothelial cells dysfunction by mitochondria-targeted ROS scavengers and ox-CaMKII inhibition. Hopefully, it will be helpful to provide further insights into endothelial cells dysfunction and validate novel intervenable targets and drug candidates.

血管内皮细胞（ECs）损伤导致的病理性改变是许多疾病的早期标志，但至今尚无有效保护手段。ECs损伤多认为与氧化应激有关，但广谱抗氧化剂的保护作用非常有限，亟需探究其深层次损伤机制。前期我们曾报道活性氮（RNS）供体GSNO介导的硝化应激可直接诱导ECs线粒体依赖性凋亡，但其上游激活和干预机制仍有待进一步阐明。课题组最新研究发现，GNSO诱导的内皮细胞损伤早期伴随ox-CaMKII增加，抑制其表达可显著减轻eNOS磷酸化和硝基酪氨酸生成，因此推测线粒体/ox-CaMKII激活很可能是导致ECs硝化应激损伤的关键信号通路。为验证这一假说，我们拟采用GSNO和缺氧缺糖诱导的ECs损伤模型系统考察线粒体/ox-CaMKII通路在RNS介导ECs损伤中的作用，同时评价特异性线粒体活性氧清除剂及ox-CaMKII抑制剂的调控作用。本课题将有助于进一步阐明ECs的损伤机制，验证新的干预靶点和候选药物。

血管内皮细胞（ECs）损伤导致的病理性改变是许多心脑血管疾病的早期标志，保护ECs功能有望消除病变的始动环节，但至今尚无有效手段。ECs损伤多认为与氧化应激有关，但广谱抗氧化剂的保护作用非常有限，亟需探究其深层次损伤机制。近年来，硝化应激在内皮细胞损伤中的作用备受关注，但其如何引起凋亡损伤，有哪些重要的通路或信号蛋白参与，其调控机制如何仍不明确。前期假说认为，线粒体/ox-CaMKII激活与下游活性氮介导的病理性信号通路存在着较大关联，很可能是ECs硝化应激损伤的关键通路。因此在本课题的开展过程中，我们以血管内皮为切入点，试图重点阐明血管内皮损伤相关信号途径介导的分子事件在心脑血管疾病发生、发展和转归中的关键意义重点，围绕活性氮所致的内皮细胞的损伤机制及药物干预作用开展了探索性研究工作。采用了包括活性氮供体GSNO、ONOO-致ECs损伤模型、缺氧缺糖（OGD）损伤模型、大鼠肠系膜血管缺血再灌注模型、大鼠MCAO模型及小鼠AS模型，进行了线粒体/ox-CaMKII通路与eNOS及ONOO-的关联性研究，同时考察了候选药物芒果苷、葫芦茶苷、Mito TEMPO、KN93在内皮细胞损伤中的干预作用。重要结果包括：（1）ox-CaMKII参与了活性氮（硝化应激）介导的内皮细胞损伤，芒果苷可通过抑制eNOS的脱偶联和异常活性，进而抑制ONOO-的产生和减少ox-CaMKII的表达，起到内皮细胞保护作用（2）CaMKII是一种节点信号，其中多个上游输入（例如ROS，高血糖，脱偶联的一氧化氮合酶）可以将CaMKII转化为参与心脑血管疾病的ox-CaMKII，有望作为潜在药物调控靶点。（3）G蛋白偶联受体124过表达、肽基-脯氨酰顺/反异构酶FKBP25的核移位及受体酪氨酸激酶ErbB4缺血均与皮细胞硝化应激损伤关系密切。本课题为研发血管内皮细胞/微血管硝化损伤机制及保护药物提供了实验理论依据，将有助于进一步阐明ECs的深层次损伤机制，同时验证了新的干预靶点和候选药物。