黑灵芝多糖通过甘露糖受体（MR）调控单核巨噬细胞抗动脉粥样硬化的作用研究

It is common to know that atherosclerosis (AS) is an inflammatory disease, and monocyte/macrophage has been largely implicated in the pathogenesis of AS. Mannose receptor(MR) was showed to be mainly expressed on the surface of macrophage membrane and preliminary studies have demonstrated that Ganoderma atrum polysaccharide could inhibit macrophage-induced inflammation via the recognition of MR. Although Ganoderma atrum polysaccharide has been determined to possess the effect of ameliorating AS, it unknown whether MR-mediated anti-inflammatory effects could trigger the effect of Ganoderma atrum polysaccharide to alleviate AS. Recently, our data revealed that Ganoderma atrum polysaccharide could reduce the secretion of IL-1β via up-regulating MR expression in inflammatory macrophage. Strikingly, P62 protein is currently demonstrated as a “molecule brake” on the control of IL-1β-induced inflammation injury. Therefore, the aim of the present study is to investigate the effects of Ganoderma atrum polysaccharide on vascular endothelial function and monocyte/macrophages both in vivo and in vitro. Subsequently, MR inhibitor is employed to investigate the protective effect of Ganoderma atrum polysaccharide against AS and further examine the molecular mechanisms. Furthermore, si-p62 recombinant plasmids is constructed to inhibit p62 protein, and then explore the effect of p62 protein on AS-mediated macrophage inflammatory response. Moreover, further experiments on p62 intend to reveal the mechanisms of MR leading to the control of monocyte/macrophage caused by Ganoderma atrum polysaccharide, indicating the immunoregulatory mechanism of Ganoderma atrum polysaccharide involved in the improvement of AS. Taken together, this research will provide positive effects to illustrate the intrinsic mechanisms involved in the health benefits of Ganoderma atrum polysaccharide against AS, and thus offer a novel strategy for the pharmacological research on traditional Chinese medicine referring to the vascular protective effects to be against cardiovascular and cerebrovascular diseases.

动脉粥样硬化（AS）为炎症疾病, 单核巨噬细胞在动脉粥样硬化进程起关键作用。甘露糖受体（MR）主要存在巨噬细胞膜表面，前期已探寻出黑灵芝多糖可识别MR，抑制巨噬细胞炎症反应。尽管已证实黑灵芝多糖有抗AS作用，然而能否通过MR抑制血管炎症反应，继而改善AS仍需进一步研究。最近发现，黑灵芝多糖可通过上调MR，抑制致炎巨噬细胞IL-1β分泌。新近报道，p62蛋白在抑制IL-1β介导的炎症损伤扮演分子制动器角色。为此，本项目拟体内外检测黑灵芝多糖对血管内皮功能与单核巨噬细胞的作用；应用MR抑制剂研究黑灵芝多糖对AS影响及作用机制；构建si-p62重组质粒抑制p62蛋白，探究p62蛋白对AS介导巨噬细胞炎症反应的影响；最后基于p62蛋白揭示黑灵芝多糖通过MR调控单核巨噬细胞抗AS的免疫机理。本研究将有利于诠释黑灵芝多糖抗AS的内在分子机制，并为其他中药抗心脑血管疾病的血管保护作用的药理学研究拓展思路

动脉粥样硬化（AS）属于糖尿病患者比较常见的慢性的血管并发症，本项目构建大鼠2型糖尿病（T2DM）模型，收集血管，研究黑灵芝多糖（PSG）抗AS研究。结果总结如下：.1.探究PSG抗糖尿病作用：PSG能有效改善糖尿病引起的大鼠多食多饮多尿症状，抑制大鼠体重下降，并且在一定程度上恢复T2DM大鼠精神状态。PSG能显著降低T2DM大鼠血糖血脂水平，减轻由高糖高脂和STZ诱导的胰岛素抵抗。实验观察到PSG能改善胰腺组织受损和胰岛β细胞功能损伤情况，表明PSG具有良好的胰腺保护和抗糖尿病功能。 .2.探究PSG对大鼠AS的保护作用及可能机制：数据显示PSG能够改善动脉粥样硬化造成的血管组织损伤，减少LDH从心肌细胞中逃逸入血。PSG显著提高组织抗氧化酶活性，抑制T2DM大鼠血管氧化应激；并且通过抑制炎症信号通路NLRP3/Capase-1/IL-1β，从而降低血管炎症水平。表明PSG可能通过抑制T2DM大鼠血管氧化应激水平和炎症水平，发挥血管保护作用。.3.巨噬细胞可以分化为两个主要的亚群，包括经典活化型(M1)和选择活化型(M2)巨噬细胞。使用脂多糖(LPS) + IFN-γ和IL-4建立了两种极端状态的体外模型，即促炎M1和抗炎M2。结果显示，PSG对巨噬细胞极化的行为改变有影响，它减少了LPS＋IFN-γ诱导的M1巨噬细胞的CD80表达，并削弱了IL-4诱导的M2巨噬细胞的CD23表达。进一步的研究表明，PSG-1调节的M1和M2巨噬细胞极化与控制吞噬、活性氧的产生、NO和细胞因子(IL-1β、IL-6和IL-10)、MR和p62蛋白有关，最从炎症阐明黑灵芝多糖调控单核巨噬细胞在炎症反应中的作用，揭示MR和p62蛋白参与了黑灵芝多糖抗动脉粥样硬化的免疫机理。.4.探究PSG对T2DM大鼠肠道菌群紊乱的调节作用：应用高通量测序技术，分析PSG对T2DM大鼠肠道微生物的调节作用。研究发现，PSG可以通过减轻T2DM大鼠肠道病理损伤，降低肠道通透性，调节肠道菌群结构和肠道菌群代谢产物分泌，发挥肠道保护作用症。.总之，PSG可通过调控MR/p62介导信号通路介导的炎症水平发挥抗AS作用。此外，本文项目菌群代谢产物LPS、TMAO的分泌可能是PSG发挥抗AS血管保护作用的另一途径。