CagA阳性幽门螺杆菌通过外泌体/SHP2调控血管内皮细胞ZO-1和VCAM-1导致动脉粥样硬化斑块不稳定

It is reported that CagA-positive Helicobacter pylori (HP) infection is associated with atherosclerotic stroke. However, the underline mechanisms remain unclear. Our previous results showed that CagA-positive Helicobacter pylori infection induced human gastric epithelial cells to release the exosomes containing CagA protein into the blood, and the exosomes could regulate the vascular endothelial cells permeability and adhesion. In the present project, we will use gene-knockout mice, transmission electron microscopy, transwell, adhesion assay, co-immunoprecipitation and immunofluorescence staining to verify our working hypothesis. Firstly, we will verify that CagA-positive HP infection releases the CagA-containing exosomes into the blood, and promotes atherosclerotic plaque instability of ApoE knockout mice. Secondly, we will verify that CagA disturbs the location of endothelial cell tight junction protein ZO-1 by SHP-2/PAR3/aPKC signaling, which results in damaged endothelial cell barrier function. Thirdly, we will verify that CagA regulates the expression of VCAM-1 in endothelial cells by activating the SHP-2/Akt/P65 pathway, which increased the aggregation of mononuclear cells and involved in the instability of atherosclerotic plaques. This project will elucidate the molecular mechanisms of CagA-positive exosomes on plaque instability and provide a new target for the treatment of atherosclerotic plaque instability.

研究表明CagA阳性幽门螺杆菌（HP）感染与动脉粥样硬化性脑梗死有关，但其机制不清。我们发现：CagA阳性幽门螺杆菌感染人胃上皮细胞后，可释放含有CagA蛋白的外泌体入血，作用于血管内皮细胞，调控内皮细胞通透性和粘附能力。本项目拟采用基因敲除小鼠、透射电子显微镜、Transwell实验、单核细胞粘附实验、免疫共沉淀、免疫荧光等技术，阐明CagA阳性外泌体对斑块不稳定性的影响及分子机制，验证工作假说：①CagA阳性 HP感染释释放CagA外泌体入血，促进ApoE敲除小鼠动脉粥样硬化斑块不稳定；②CagA通过SHP-2/PAR3 /aPKC通路调控内皮细胞紧密连接蛋白ZO-1的胞内定位，导致内皮细胞屏障功能破坏；③ CagA通过SHP-2/Akt/P65通路，调控内皮细胞粘附分子VCAM-1表达，参与动脉粥样硬化斑块的不稳定。通过本项目明确CagA为抑制动脉粥样硬化斑块不稳定性的新治疗靶点。

动脉粥样硬化是一种慢性炎症性疾病。慢性感染和动脉粥样硬化之间的病理生理学相似性引发了人们对这些疾病的兴趣。在本研究中，通过酶联免疫吸附试验（ELISA）评估的幽门螺杆菌感染和表达细胞毒素相关基因A（CagA）菌株感染的血清流行率表明，患者中CagA菌株而非幽门螺杆菌的流行与动脉粥样硬化的发生呈正相关。相应地，我们发现CagA增加了ApoE−/− 小鼠动脉粥样硬化早期斑块的生长，促进小鼠主动脉内皮细胞（MAECs）粘附分子和炎性细胞因子的表达。在机制上，si-NLRP3和si-IL-1β都减轻了CagA对血管内皮细胞炎症激活的促进作用。在体内，MCC950对NLRP3的抑制显著减弱了CagA对ApoE−/− 老鼠斑块生长的促进作用。我们进一步为探讨幽门螺杆菌感染的胃上皮细胞源性外泌体(Hp-GES-EVs)和外泌体 CagA 在动脉粥样硬化中的作用，给予 ApoE-/-小鼠静脉或腹腔注射生理盐水、 GES-EVs、 Hp-GES-EVs 和重组 CagA 蛋白(rCagA)。两种动物模型中，CagA阳性的幽门螺杆菌PMSS1感染不诱导而是促进巨噬细胞源性泡沫细胞的形成，并增加动脉粥样硬化斑块的生长和不稳定性。同时，CagA分泌在Hp-GES-EVs中被释放入血，可被摄取至主动脉斑块中。此外，含CagA的 EVs 和 rCagA 在体内外都能加速巨噬细胞源性泡沫细胞的形成和损伤的发展，证明了CagA阳性的幽门螺杆菌促动脉粥样硬化的作用。CagA 通过下调转录因子 PPARγ 和 LXRα 的表达抑制胆固醇外排转运蛋白的转录，从而促进泡沫细胞的形成。以上结果表明，可以通过抑制CagA的作用从而早期和晚期逆转斑块进展。

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