低切应力通过β1AR-mTOR信号转导通路影响内皮细胞eNOS功能的分子机制研究

Reduction of engothelial shear stress is associated with endothelial cell injury, leading to the formation and development of atheroscleosis. Unfortunately, there is a lack of data regarding the underlying mechanism on how endothelial cell sensing the low shear stress and resulting in cellular dysfunction. β1sympathetical adrenergetic receptor (β1AR), one of the G protein coupled receptors, is extensively distributed in endothelial cells and smooth muscle cells. But the interplay between low shear stress and endothelial cell injury via mudulation of β1AR is still to be unknown.Our previous work demonstrated that low shear stress upregulated the activity of mTOR and downregulated the eNOS mRNA level through β1AR activation, and the further activation of the mTOR pathway was associated with the increment of reactive oxygen substance( ROS ) and the devrement of the eNOS activity. Accordingly, the hypothesis is: low shear stress activates the β1AR by coupling with its G?i or G?s subunit, enhances the mTOR activity,leading to the eNOS mRNA downregulation and dysfunction, with resultant endothelial cell dysfunction. The present study aimes to investigate the mechanisms and signal transduction pathway through which low shear stress regulates the eNOS activity via activating β1AR, by using modern techniques including protein overexpression , receptor function inhibiton, activation and inhibition of the targets in signal transduction pathway. The results from the current study would be helpful to clarify the mechanisms contributed to the endothelial cell injury by low shear stress, and would provide more detalied information with clinical interventions.

低切应力导致内皮细胞损伤，进而促进动脉粥样硬化形成和发展。但是对于内皮细胞如何感知低切应力继而出现功能障碍的机制目前仍不清楚。交感肾上腺素能β1受体（β1AR）是一种G蛋白偶联受体，广泛分布于血管内皮细胞和平滑肌细胞表面，但低切应力是否及如何通过β1AR导致内皮细胞损伤的机制尚未见报道。我们的前期工作表明低切应力通过β1AR上调mTOR的活性，降低eNOS的mRNA水平，且mTOR通路的激活导致细胞内活性氧（ROS）水平升高及eNOS活性降低。由此提出的科学假说为：低切应力激活β1AR，通过相应的G?亚基上调mTOR活性，进而导致eNOS mRNA水平降低及功能障碍，进而造成内皮细胞功能受损。本研究拟通过蛋白过表达、受体功能抑制、信号通路靶点激动与抑制等技术，研究低切应力通过β1AR调节eNOS活性的机制和信号转导通路。本研究有助于阐明低切应力损伤内皮细胞的机制，为防病治病提供理论及实验依

低剪切力在动脉粥样硬化的发生发展中扮演着重要的角色，它直接作用于内皮细胞导致细胞损伤，另外还能够诱导炎症因子趋化聚集到损伤内皮表面加速氧化应激反应，从而导致内皮进一步损伤并促进了粥样硬化的发展，但是低剪切力是如何将机械作用力转化为化学信号传入细胞并最终影响内皮功能尚没有明确的研究阐明。本课题应用平板流动腔系统给予内皮细胞梯度时间(0, 5, 15, 30, 60, 120分钟)的低剪切力作用，检测炎症因子、一氧化氮以及mTOR、eNOS、β1肾上腺素能受体（β1AR）等信号通路蛋白的表达情况，并使用以上蛋白的抑制剂验证其在低剪切力诱导的内皮损伤中的作用，结果发现：（1）低剪切力导致内皮细胞发生皱缩、脱落并诱导内皮细胞炎症因子VCAM-1和IL-6表达增加，而一氧化氮生成减少；（2）低剪切力诱导mTORC2表达减少以及eNOS-Ser1177和eNOS-Thr495表达增加；（3）低剪切力通过激活β1AR的Gαi亚基导致mTOR及其下游信号的改变。本项课题的研究结果揭示了低剪切力导致内皮功能损伤的分子生物学机制，为动脉粥样硬化的治疗提供了新的思路，即激活内皮细胞中mTORC2信号通路或者抑制β1AR的Gαi亚基的激活有助于延缓动脉粥样硬化的发展。