SIRT1在MRTF-A促进PAEC损伤及功能紊乱中的作用及机制研究

Pulmonary vascular endothelial cell (PAEC) damage and dysfunction plays an important role in the initiation and development of hypoxic pulmonary hypertension (HPH). We found that hypoxia promoted MRTF-A expression and nuclear translocation in PAEC, MRTF-A can increase the expression of adhesion molecules and ET-1 in PAEC, it can decrease the expression of NO in PAEC, too. In hypoxic rats, MRTF-A promoted the PAEC damage and development of HPH, but the mechanism is not clear yet. Our preliminary experiment showed that MRTF-A could decrease SIRT1 promoter activity and PGC-1α mRNA expression in hypoxic PAEC, and it could increase the content of reactive oxygen species in PAEC. Combined with literature, we hypothesized that MRTF-A promoted PAEC damage and dysfunction by enhancing transcriptional activity of HIF-1 α and NF- κ B p65, while reducing activity of PGC-1 α and eNOS. The inhibition effect of MRTF-A on SIRT1 is the key to MRTF-A promoting PAEC damage and dysfunction. Adopting HPAEC and MRTF-A endothelial cell conditional knockout mice as the research objects, this project aims to study the effect of SIRT1in MRTF-A promoting hypoxia PAEC damage, dysfunction and the development of HPH, revealing the pathogenesis of HPH. This will shed new light on the pathogenesis of HPH.

肺动脉内皮细胞(PAEC)损伤及功能紊乱在缺氧肺动脉高压（HPH）发生发展中发挥重要作用。我们研究发现缺氧促进PAEC MRTF-A表达和核转位，MRTF-A上调PAEC ET-1和粘附分子表达、抑制NO表达，促进缺氧大鼠PAEC损伤及HPH发展，但机制均未阐明。我们预实验发现MRTF-A可抑制缺氧PAEC SIRT1启动子活性及PGC-1α mRNA表达、上调ROS生成。结合文献我们推测：MRTF-A通过抑制SIRT1表达，进而增强HIF-1α、NF-κB p65转录活性、降低PGC-1α、eNOS活性，促进PAEC损伤及功能紊乱。对SIRT1 的抑制作用是MRTF-A促进PAEC损伤及功能紊乱的关键。本项目拟采用HPAEC和MRTF-A内皮细胞条件基因敲除小鼠为研究对象，深入研究SIRT1在MRTF-A促PAEC损伤、功能紊乱及促进HPH发展中的作用，进一步揭示HPH的发病机制。

肺血管内皮细胞(PAEC)损伤及功能紊乱在缺氧肺动脉高压（HPH）发生发展中发挥重要作用。本项研究目前已经完成了低氧对HUVEC SIRT1表达及活性的影响及SIRT1对低氧 HUVEC细胞活力及功能的影响；并就SIRT1对低氧HUVEC细胞活力及功能影响的机制进行了深入探讨。进一步阐明了缺氧性肺动脉的发病机制，为缺氧性肺动脉高压的防治提供了新的思路和靶标。完成了以下研究：（1）低氧对HUVEC 细胞活力、SIRT1表达及活性的影响，发现低氧能抑制HUVECs 细胞活力、抑制SIRT1蛋白表达及活性。（2）Sirt1在低氧致血管内皮细胞损伤及功能紊乱中的作用。发现抑制Sirt1表达及活性可抑制低氧HUVECs活力、促进其功能紊乱，加剧氧化应激反应，上调炎症因子表达；上调SIRT1表达及活性可增强低氧HUVECs活力、抑制其功能紊乱，抑制氧化应激反应，下调炎症因子表达。运用过表达质粒上调SIRT1表达及SRT1720促进Sirt1表达及活性可升高HUVEC细胞活力，抑制IL-6、IL-1ɑ、MCP-1、ICAM-1，上调eNOS、NO表达。（3）Sirt1对低氧 HUVEC细胞活力及功能影响的机制研究。低氧培养HUVEC，运用siRNA干扰Sirt1表达及烟酰胺抑制Sirt1活性可降低HUVEC细胞PGC-1α、UCP-2表达，促进ROS生成、磷酸化NF-kappa B p65表达升高；运用过表达质粒上调SIRT1表达及SRT1720促进Sirt1活性可升高HUVEC PGC-1α、UCP-2表达表达，抑制ROS生成、磷酸化NF-kappa B p65表达降低。提示Sirt1增强低氧HUVEC 细胞活力，降低其黏附分子及炎症因子表达，逆转低氧所致的血管活性物质平衡“打破”的机制与其抑制低氧所致HUVEC 氧化应激反应有关。（4）白藜芦醇( RSV) 对低氧内皮细胞炎症反应的影响及机制，发现RSV能上调低氧 HUVECs 活力及SIRT1 表达； 抑制NF－κB p65激活，下调炎症因子、黏附分子表达；上调PGC-1α、UCP-2表达，抑制ROS 生成。表明RSV可促进低氧 HUVECs 细胞活力，其机制可能与RSV促进SIRT1 表达，抑制氧化应激，进而抑制炎症反应有关。

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