从PI3K/Akt/mTOR通路探讨田螺多糖干预动脉粥样硬化斑块内血管新生的作用机制

Angiogenesis is a key inducing factor in the formation and rupture of the atherosclerotic vulnerable plaques. PI3K/Akt/mTOR is an important pathway in intervening angiogenesis. The polysaccharide from Cipangopaludina chinensis (CP) can stabilize and decrease atherosclerotic plaques, but its mechanism is not recognized clearly at present. It has been reported that the polysaccharide, containing 1→3 or 1→6 link and sulfate radical, can inhibit angiogenesis. Our research team have confirmed that CP not only include (1→3)-glucose link and contains 7.42% of sulfate radical, but also can suppress the expression of key factor VEGF in pathways downstream. Therefore, we hypothesize that the mechanism of CP in atherosclerotic plaque stability may be inhibiting angiogenesis by adjusting PI3K/Akt/mTOR pathway. In order to verify this hypothesis, a combined method of in vivo and in vitro is used in the present research. Techniques of histopathology, microscopic imaging and immunohistochemistry are applied to observe the effect of CP on the atherosclerotic plaque area, internal composition and microvessel density. Methods of Matrigel, Transwell chamber and MTT are employed to investigate the effect of CP on transformation of endothelial cells to neovascularization. Techniques of Western Blot, qRT-PCR and ELISA are used to study the effect of CP on the expression of signal pathway and downstream key factor to promote the angiogenesis in the plaque and endothelial cells. The mechanism that CP inhibits angiogenesis in atherosclerotic plaque is discussed by regulating pathways from the whole, cell and molecular level. These studies will provide a new idea for preventing and treating atherosclerosis.

血管新生是动脉粥样硬化（AS）易损斑块形成及破裂的关键诱因。PI3K/Akt/mTOR是调控血管新生的重要通路。田螺多糖（CP）能稳定和消退斑块，但其机制不清楚。据报道，具1→3或1→6链接，且含硫酸基的多糖能抑制血管新生。课题组前期证实，CP具(1→3)-葡萄糖链接，含7.42%硫酸基，能抑制通路关键因子VEGF表达。故推测CP可能通过调控该通路抑制血管新生而稳定斑块。为验证该假说，本研究采用体内与体外实验相结合，用病理组织学、显微影像及免疫组化技术观察CP对斑块形态、组成、新生血管密度的影响；用Matrigel、Transwell小室及MTT法考察CP对内皮细胞新生血管转化的影响；用Western Blot、ELISA及qRT-PCR研究CP对斑块及内皮细胞通路信号和下游促血管新生关键因子表达的影响。从整体、细胞及分子水平探讨CP抑制斑块内血管新生的作用机制，为AS防治提供新的思路。

血管新生是动脉粥样硬化（AS）易损斑块形成及破裂的关键诱因。PI3K/Akt/mTOR是调控血管新生的重要通路。田螺多糖 (CP) 能稳定和消退斑块，但其机制不清楚。据报道，具1→3或1→6链接，且含硫酸基的多糖能抑制血管新生。课题组前期证实，CP具(1→3)-葡萄糖链接，含7.42%硫酸基，能抑制通路关键因子VEGF表达。故推测CP可能通过调控该通路抑制血管新生而稳定斑块。为验证该假说，课题组通过体内实验，应用病理组织学、显微影像技术观察田螺多糖对ApoE-/-小鼠AS模型的斑块面积、斑块内部组成和易损指数及其斑块内新生血管密度的影响，并利用免疫组织化学、qRT-PCR及Western Blot技术观察CP对斑块组织内PI3K/Akt/mTOR通路信号转导分子及促血管新生关键因子表达的影响，阐明了CP干预AS斑块内血管新生以增强斑块稳定性的作用及其基于PI3K/Akt/mTOR信号转导的相关调控机制。在此基础上，通过体外细胞模型，应用MTT法、Transwell小室法及Matrigel法，观察CP对内皮细胞新生血管转化的“增殖、迁移及管腔形成”三个主要步骤的影响，并用Western Blot、qRT-PCR及ELISA技术研究通路信号转导分子及下游促血管新生关键因子的蛋白及mRNA表达的影响，进一步明确了CP对内皮细胞新生血管转化过程的影响及其与PI3K/Akt/mTOR信号转导通路的关系。从整体、细胞及分子水平探讨了CP调节PI3K/Akt/mTOR通路抑制AS斑块内血管新生以稳定斑块的作用及分子机制，为AS相关疾病的防治提供新的思路和干预措施。此外，课题组基于该通路与巨噬细胞自噬的密切关系，采用免疫荧光法还测定了斑块内巨噬细胞自噬的特征蛋白原位表达情况。结果发现，CP对beclin1和p62等巨噬细胞自噬的特征蛋白表达具有明显促进作用，对LC3II/LC3I值也具有明显的升高作用，且各组各蛋白表达变化的总体趋势与斑块面积大小及易损指数的变化趋势具有相似性，据此，课题组推测CP稳定AS斑块的作用机制可能还与以斑块内巨噬细胞自噬事件为中心的其它相关调控机制有关，对此有待进一步研究。