Polysulfides介导Caveolin-1硫巯基化修饰在LDL跨内皮细胞穿胞及早期AS中的作用

H2S has an anti-atherosclerotic effect, and H2S-induced S-sulfhydration of specific cysteine residues in target proteins has been implicated in cardiovascular diseases. However, a relatively small number of direct molecular targets of H2S have been identified. Recently, some study indicated that the oxidized products of H2S—Polysulfides are actually H2S-derived signaling molecules. The retention of apoB100-containing lipoproteins (e.g. Low density lipoprotein, LDL) in subendothelial space is the initiating process in AS, and is usually termed “response to retention” hypothesis. Our previous study found that caveolae mediated transcytosis is the major pathway for the LDL particles to traffic across endothelial cells (ECs). The preliminary experiments suggested that H2S and Polysulfides decreased the transcytosis of LDL across ECs, especially Polysulfides. Thus, we hypothesis that the protective role on AS is associated with H2S-Polysulfides-induced S-sulfhydration of caveolin-1(the marker of caveolae), which inhibits the transcytosis of LDL across ECs. This study aims to establish in-vitro transcytosis model to evaluate the effects of H2S-Polysulfides-induced S-sulfhydration on the transcytosis of LDL across ECs, and investigate the influence of S-sulfhydration on the subendothelial retention of LDL in human umbilical venous walls, and further analyze the effects of caveolin-1 S-sulfhydration on the formation of atherosclerotic lesions by establish AS animal model. Exploring the relation of H2S-Polysulfides-induced S-sulfhydration to the transcytosis of LDL, we will reveal a novel underlying mechanism of the protective role of H2S on cardiovascular diseases related to AS.

H2S具有抗AS作用，且其介导的硫巯基化修饰在心血管疾病中十分重要，但靶分子不明。近来报道，H2S通过其氧化产物Polysulfides发挥效应。“滞留-反应”学说认为，含ApoB脂蛋白(尤其LDL)在血管内皮下滞留是AS的始发环节。前期研究表明，LDL主要经小窝穿胞进入血管内皮下。预实验显示，H2S、Polysulfides可抑制LDL跨内皮细胞穿胞，且后者作用更明显。由此我们假设，H2S通过生成Polysulfides介导Caveolin-1(小窝标志蛋白)硫巯基化，抑制LDL穿胞进入血管内皮下，抑制AS发生。本项目拟以硫巯基化与穿胞的关系为出发点，通过建立LDL体外穿胞模型、培养人脐静脉、建立AS模型，在细胞、组织和动物水平探讨H2S-Polysulfides介导Caveolin-1硫巯基化在LDL跨内皮细胞穿胞及早期AS中的作用及机制，为H2S抗AS相关心血管疾病的研究提供新思路。

H2S介导的硫巯基化修饰在心血管疾病中发挥重要作用，但其抑制动脉粥样硬化（Atherosclerosis, AS）形成的相关机制尚需进一步研究。脂质“滞留-反应”学说认为，含ApoB脂蛋白（尤其是LDL）在血管内皮下沉积滞留是AS的始发环节。本项目旨在探讨H2S及其氧化产物Polysulfides介导的硫巯基化修饰在LDL跨内皮细胞穿胞及脂质沉积中的作用及可能机制。本项目还进一步探讨了NLRP3炎症小体是否参与H2S抑制LDL跨内皮细胞穿胞及C反应蛋白（C reactive protein, CRP）促进LDL跨内皮细胞穿胞过程。.通过建立Apoe-/-小鼠AS模型发现，H2S-Polysulfides可抑制主动脉根部脂质沉积，减小AS斑块面积；同时，H2S-Polysulfides对小鼠体重无明显影响，可轻度降低血TC、TG、LDL-c及H2S水平，轻度增加HDL-c水平。另外，H2S-Polysulfides可促进血清炎症介质TNF-α、IL-1β合成，可降低血清胰岛素水平，而对小鼠空腹血糖水平无明显影响。通过培养人脐静脉细胞株发现，H2S通过抑制内皮细胞内吞LDL，抑制穿胞关键蛋白Caveolin-1磷酸化修饰而促进其发生硫巯基化修饰，并抑制细胞中穿胞相关内吞分子（Cavin-1、Dynamin-2/DNM2）、胞吐分子（NSF、α-SNAP、VAMP3、Syntaxin4/STX4）表达，最终抑制LDL跨内皮细胞穿胞并在血管内皮下沉积滞留。Caveolin-1过表达以后，H2S通过促进Caveolin-1硫巯基化修饰，抑制Caveolin-1磷酸化修饰，抑制内皮细胞内吞LDL，抑制穿胞相关分子表达，进一步证实了H2S介导的硫巯基化修饰在LDL跨内皮细胞穿胞中具有重要作用。.另外，在细胞水平，H2S对NLRP3炎症小体相关分子（NLRP3、Caspase-1、ASC、IL-1β）表达具有明显抑制作用，且NLRP3炎症小体参与H2S抑制内皮细胞内吞LDL及LDL穿胞过程。同时，我们发现，CRP可以激活NLRP3炎症小体，NLRP3炎症小体也参与CRP促LDL跨内皮细胞穿胞过程。.H2S抗AS作用已十分明确，本项目发现，H2S介导的Caveolin-1硫巯基化修饰及NLRP3炎症小体在H2S抑制LDL跨内皮细胞穿胞、抑制脂质沉积及AS斑块形成中具有重要作用。