GSTpi调控血管内皮屏障功能抵御促炎刺激的分子机制研究

Endothelial cells control the passage of plasma constituents and circulating cells from blood to the underlying tissues. Inflammation-induced lost or impaired of endothelial barrier function is in several pathological conditions including atherosclerosis, ischemia and sepsis. However, The regulation of endothelial barrier function is unclear. The oxidative stress is the part of the inflammation, but the molecular mechanism of antioxidants surpress inflammation is unknown. GSTpi is a member of GSTs, which have properties of enzyme catalysis/detoxification, protein:protein interactions, and catalysis of the forward reaction of the S-glutathionylation cycle. GSTpi is an important molecule involved in resisting oxidative stress. We have reported the anti-inflammatory function of GSTpi. We also reported that GSTpi protects against angiotensin II-induced proliferation and migration of vascular smooth muscle cells by preventing signal transducer and activator of transcription 3 activation. Recently we found that GSTpi inhibited the increase of endothelial permeability induced by the pro-inflammatory factors. Further experiments showed that GSTpi inhibited the formation of actin stress fibres and destablization of membrane VE-cadherin induced by TNF suggested that GSTpi might regulate the endothelial barrier and stabilize endothelial junctions. We will investigate: (1) The protective effect of GSTpi on the endothelial barrier and the stabilization of endothelial junctions,(2) How GSTpi acts as a key factor involved in the quick auto-regulation of anti-inflammation through regulating the changes of endothelial permeability induced by pro-inflammatory mediators,(3) How GSTpi regulates endothelial barrier and stabilize endothelial junctions through affecting s-glutathionylation of VE-cadherin and actin. This project will help us to look insight in the understanding the regulation of endothelial barrier function and the relationship between antioxidant and antiinflammation .

血管内皮屏障功能的精确调节是维持循环稳态和器官生理的基础，炎症引起血管内皮通透性增高是动脉粥样硬化、败血症等疾病的病理基础。氧化应激与炎症关系密切，但机体抗氧化系统抵御促炎刺激的机制不明确。对内皮屏障功能的调控而维持其稳态是抗炎的重要方面。GSTpi是细胞内抵御氧化应激的重要蛋白，我们前期研究表明，GSTpi抑制促炎因子引起的血管内皮通透性的增加、减少应力纤维产生、抑制细胞膜VE-cadherin减少、促进蛋白谷胱甘肽化等。提示GSTpi是血管内皮屏障功能调控的重要参与者。本课题拟研究：GSTpi保护内皮细胞间连接、维持血管内皮正常屏障功能的作用；GSTpi对细胞连接蛋白和骨架蛋白的作用与调控血管内皮通透性的关系；GSTpi通过对激酶和骨架蛋白的谷胱甘肽化而分别影响其磷酸化和重构，实现对炎症反应控制的机制。本研究将抗氧化与抗炎相关联，使我们从全新角度认识血管稳态及相关疾病的病理生理。

血管内皮屏障功能的精准调节是维持循环稳态和器官生理的基础，炎症诱发的血管内皮屏障通透性增高会导致动脉粥样硬化、败血症等疾病的发生。研究显示GSTpi在炎症反应中扮演重要角色，GSTpi的酶催化/解毒、蛋白质结合作用、蛋白谷胱甘肽化的作用可能参与了炎症信号通路的级联调控，但其对血管内皮的调控作用有待进一步阐明。因此本课题研究GSTpi调控促炎因子诱导的血管内皮通透性异常的分子机制。结果表明，GSTpi抑制TNF-α诱导的血管内皮细胞通透性的增加，并且这种调控作用与其抑制 VE-cadherin的内化相关，提示GSTpi可调节内皮屏障，稳定内皮细胞间的连接。进一步的研究显示，GSTpi可抑制TNF-α诱导的原癌基因酪氨酸蛋白激酶（Src）/VE-cadherin通路的活化。GSTpi的抑制作用与选择性S-谷胱甘肽化Src激酶有关，而GSTpi的酶活突变体（Y7F）未能有效地逆转TNF-α诱导的血管内皮细胞通透性的增加，说明GSTpi抑制Src激酶的活性与GSTpi谷胱甘肽转移酶活性相关。利用计算机分子对接联合质谱的分析显示，Src 激酶可能在Cys185，Cys245，Cys400位点存在谷胱甘肽化修饰。点突变实验检测显示，GSTpi可能主要通过Src 激酶的Cys185位点的谷胱甘肽化调控其磷酸化。另外，GSTpi可通过p38/Hsp27调控actin的重构，以抑制促炎因子引起的应力纤维的形成，调控血管内皮的通透性。AAV-GSTpi在急性肺损伤动物模型中发挥了屏障保护作用。实现对炎症诱发的血管通透性增加的调控。本研究使我们从全新角度认识血管稳态及相关疾病的病理生理。.本研究的科学意义：揭示了 GSTpi 通过将炎症信号通路中重要激酶谷胱甘肽化影响其磷酸化而发挥抗炎的作用，通过研究 GSTpi 对血管内皮屏障功能的调控，阐明了GSTpi 促蛋白谷胱甘肽效应与抗炎作用的相互关系。研究表明，GSTpi 通过蛋白的谷胱甘肽化修饰可逆性地调控内皮细胞连接相关蛋白的磷酸化，从而对血管内皮屏障功能进行动态控制，以实现机体对抗促炎应激的快速反应。为研究血管内皮屏障功能开创新的道路，为相关疾病的防治提供新策略。

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