细胞色素p450 EET/sEH通路参与Nox4调控的血管平滑肌细胞炎症反应的机制研究

Oxidative stress initiates arteriogenesis and exaggerates the progress of atherosclerosis. It is of great importance to investigate the role of reactive oxidative species(ROS), oxidative stress and atherosclerosis and the corresponding mechanisms. Among many reactive oxidative species generating enzymes, Nox family plays a pivotal role in the modulation of ROS production. Nox4, one member of Nox family, is consecutively active and account for the production of mainly H2O2.However, it remains putative as to how Nox4 acts in the process of vascular diseases. According to reports and our previous research, we observed that in primary Nox4 dominant negative VSMCs, the expression of VCAM1 were significantly decreased compared with wide type group, which the underlying mechanisms are still unknown. Cytochrome p450 epoxyeicosatrienoic acids(EET)/ Soluble epoxide hydrolase(sEH) has been proved to be involved in many vascular diseases and VCAM1 were regulated by sEH. And also, ROS including H2O2 were also a stimulant to induce sEH expression. Meanwhile, Nox4 and sEH were both involved and coordinated to exert effects pathological process such as homocysteine induced aneurysms. Based on the mentioned-above, we hypothesized that Nox4 H2O2 has a cross talk with p450 EETs/sEH pathway and Nox4 might regulate VCAM1 and other inflammatory response through p450 EETs/sEH pathway. We planned to determine the expression of Nox4, sEH and VCAM1 utilizing real time PCR, western blot, chromatin immunoprecipitation. Hopefully, we could illustrate the involvement of sEH during the regulation of Nox4 on inflammation response and it might be helpful to explain the role of Nox4 in vascular diseases.

氧化应激启动和加剧动脉粥样硬化，探讨氧化应激和活性氧参与血管病变的相关机制意义重大。NADPH氧化酶Nox4是氧化应激生成H2O2的关键酶，其参与血管病变的具体过程尚未完全阐明。基于文献和前期实验，我们发现在血管平滑肌显性负突变Nox4可减弱炎症因子VCAM1的水平，但其机制不清。而已知细胞色素p450通路EETs代谢关键酶sEH可调控VCAM1的表达；活性氧ROS可调控sEH的表达；且Nox4和sEH在多种血管病变中协同作用；提示Nox4及其产生的H2O2可能是调控sEH的上游信号，我们提出Nox4通过sEH调控VCAM1等炎症反应的假说。拟通过qPCR、蛋白印记、co-Chip 等方法检测Nox4-sEH-VCAM1轴表达调控关系，以及Nox4可否转录调控sEH。研究结果有望揭示氧化应激Nox4、细胞色素p450通路和炎症反应的交互对话关系，进一步阐明Nox4参与血管疾病的相关机制。

氧化应激是动脉粥样硬化及内膜增殖的普遍机制，探讨氧化应激分子机制对于揭示上述疾病的病理生理机制及药物治疗靶点具有重要意义。Nox4是血管平滑肌（VSMCs）产生H202的关键酶。我们在本课题资助下，探讨了Nox4表达变化的外在条件；明确了细胞色素p450通路EETs的代谢酶sEH与Nox4介导的氧化应激之间的调控关系；进一步将Nox4、sEH与血管炎症分子VCAM1的表达调控轴建立。此外，还对Nox4是否转录调控sEH进行了研究。. 在本研究中，我们初步发现在ApoE-/-小鼠血管部分结扎模型中，Nox4在增殖的内膜中表达上调；而高脂饮食饲养的普通小鼠主动脉中Nox4未见明显增高；Nox4显性负突变的转基因小鼠在血管内皮剥脱后，内膜增殖受到明显抑制。Nox4显性负突变可降低血管sEH、VCAM1的蛋白质表达；同样地在离体培养的原代VSMCs中，Nox4抑制后sEH 和VCAM1的表达下调；过表达Nox4则上调sEH 和VCAM1。基因敲减sEH可抑制炎症因子VCAM1的表达水平；敲减sEH或化学抑制剂均可抑制VSMCs的增殖、迁移和粘附。同样地，基因敲减Nox4可抑制VSMCs的增殖和迁移，减少内源性H2O2水平；过表达Nox4则促进H2O2的生成。进一步采用荧光素酶报告基因系统证实，敲减Nox4可抑制NFκB和sEH的基因转录活性；提示Nox4可参与sEH的转录激表达；但Nox4调控sEH的具体转录机制尚需进一步研究。综合上述研究结果，我们初步揭示了Nox4通过sEH调控VCAM1的机制，为通过干预或联合干预Nox4、she进行抑制血管病变提供了实验基础；揭示了上述靶点作为治疗血管内膜增殖和动脉粥样硬化的潜在价值。. 此外，在本课题资助下，我们对调控Nox4的潜在表观遗传学机制进行了初步的研究，初步揭示了表观遗传分子Brd4可参与Nox4的表达，相关结果发表在Redox Biology上， 为后续课题申报和深入研究提供了基础。. 在上述课题的资助下，已培养在职博士一名（在读），相关结果还在整理投稿中。基于研究结果，项目负责人以第一作者或者通讯作者到目前已经发表SCI论文5篇，1篇Redox Biology上，3篇发表在Journal of Molecular and Cellular Cardiology；1篇Atherosclerosis。