蛹虫草高分子多糖通过调控肠道脱硫弧菌减轻高脂饮食作用的机制研究

The change of gut condition could regulate the cell motility and the structure formation of lipopolysaccharide (LPS) of opportunistic pathogen The change of gut condition could regulate the cell motility and the structure formation of lipopolysaccharide (LPS) of opportunistic pathogenic Desulfovibrio, resulting in a change of the inflammatory immune response in the circulation system due to the trans-location of Desulfovibrio. Our previous study showed that addition of a high molecular weight Cordyceps militaris polysaccharide CMP-Ⅲ to the high-fat diet (HFD) mice could reduce the concentration of pro-inflammatory cytokines in blood plasma. At the same time, fermentation of the CMP-Ⅲ could significantly change the gut bio-chemical condition in HFD mice and inhibit the growth of Desulfovibrio spp. Based on these facts and results, here we want to further explore the effect of CMP-Ⅲ intestinal fermentation condition on the regulation of cell motility and the structure formation of LPS in Desulfovibrio, as well the subsequently immune response in the circulation system due to the trans-location of Desulfovibrio. In this study, we will use microscopic tracking technique coupled with the expression of genes that regulate cell motility, to analyze the effect of CMP-Ⅲ colon fermentation on motility of Desulfovibrio desulfuricans 003, which has been isolated from obese mice. We will then use mass spectra, NMR, as well as transcriptional analysis of genes that regulate the bio-synthesis and structural modification of lipid A, a toxic center of LPS, to analyze the effect of CMP-Ⅲ colon fermentation on the structure formation of lipid A in Desulfovibrio desulfuricans 003. After that, macrophage will be used to detect the inflammatory immune response via the lipid A/TLR4 recognition and the MyD88 dependent immune response. The completion of this project could help us to get a deeper understanding of health supporting mechanism of CMP-Ⅲ on HFD individual through regulation of opportunistic pathogen Desulfovibrio desulfuricans 003. And this project will also provide a theoretical basis on using CMP to balance HFD through the regulation of gut micro-organisms.

肠道环境可通过调控脱硫弧菌的运动及内毒素LPS结构来影响其在高脂饮食机体中移位肠外诱发的炎性免疫反应。前期研究显示，蛹虫草新型高分子多糖CMP-Ⅲ能降低高脂饮食小鼠促炎因子浓度，改变其肠道环境，并抑制脱硫弧菌生长。本项目拟进一步探讨该环境变化对脱硫弧菌运动和LPS结构生成的影响，以及由此诱导肠外免疫反应的改变。本项目通过显微追踪结合运动相关基因转录表达分析，研究CMP-Ⅲ体外肠酵解环境对脱硫弧菌运动性的影响；通过质谱、核磁多层级表征技术结合类脂A合成及结构修饰相关基因转录表达分析，探讨CMP-Ⅲ肠酵解环境下脱硫弧菌LPS毒性中心类脂A结构的改变；进一步分析该环境下脱硫弧菌对肠外单核巨噬细胞TLR4受体识别及其下游信号通路免疫应答的影响，从而揭示CMP-Ⅲ通过调控肠道脱硫弧菌影响高脂饮食机体炎性免疫应答发生的机制，为利用蛹虫草多糖调控肠道细菌进行高脂饮食调节奠定科学理论基础。

随着生活节奏的加快，高脂饮食在国人的膳食比例中日益加重，这导致了热量摄入与消耗长期不平衡，多余的热量转变为脂肪，进一步为肥胖症的泛滥提供了可乘之机。肥胖是一种代谢性综合征，与血脂异常、2型糖尿病、动脉粥样硬化、心脑血管疾病密切相关，还通常伴随有胰岛素抵抗、炎症生物标志物增加和肠道菌群紊乱等症状。蛹虫草是一种著名的食药两用真菌，前期研究提示其水提物具有一定的降低体内脂肪水平的作用，但尚无深入的蛹虫草多糖（CMP）对高脂饮食（HFD）机体的作用研究。为了进一步验证和探究蛹虫草多糖改善肠道菌群紊乱，预防肥胖的机理，本研究先在前人研究的基础上，筛选蛹虫草中最具有功能活性的组分蛹虫草高分子多糖（CMP40），对高脂饮食小鼠进行同步干预，探究其健康调节作用；继而在体外水平上，模拟肠道环境，探索CMP40肠酵解对肠道生化环境的影响，以及该环境的变化对常见的肠道致病菌或LPS产生菌的运动性和LPS毒性中心类脂A结构的影响，从而揭示CMP40通过调控肠道微生物影响机体健康的机制。.本文的主要研究结果如下：.（1）CMP40可以显著减少肥胖小鼠的脂肪形成，减轻体质量，改善肝脏脂肪变性，抑制血糖、血脂的升高，改善胰岛素抵抗等；.（2）CMP40可以显著改善代谢性内毒素血症，缓解肥胖小鼠的炎症状态改善肝脏和结肠组织的淋巴细胞浸润、结构异常和坏死；.（3）CMP40能够增加肠道菌群物种丰度及改变其含量，可增加肠道SCFAs的含量，促进ZO-1和Occludin的mRNA和蛋白表达，增强肠道屏障功能，有效阻止LPS进入外周循环系统，抵抗高脂饮食小鼠的肥胖发展及其引起的代谢紊乱综合征；.（4）CMP40肠道酵解可使得乙酸、丙酸、丁酸、异戊酸、戊酸和总的短链脂肪酸的浓度显著高于对照组；.（5）CMP40能够降低肠道致病菌和条件致病菌的运动能力和易位能力；.（6）CMP40肠道酵解引起的环境改变能够使LPS结构发生修饰，从而引起炎性免疫应答的改变，进而影响机体健康。