破壁预处理方式影响黑木耳多糖结构与抗氧化应激损伤能力的分子机制

Apoptosis of islet β-cells induced by oxidative stress is an important mechanism of T2DM. In our previous study, we found that the polysaccharide of Auricularia auricula could effectively protect the oxidative stress injury of rat islet β-cells. On the basis of the research, first of all, the influence of wall-breaking methods (mechanical, physical, chemical, biological enzyme method) on the structural characteristics of Auricularia auricula polysaccharides will be analyzed aiming at the tough cell wall of Auricularia auricula, the difficult separation of the intramural polysaccharides and the complex structure of the polysaccharides. Then, combined with the antioxidant stress model of Caenorhabditis elegans and the controllable degradation of polysaccharides, the “active fragment” of AAPF will be identified and its molecular structure information will be analyzed. And then, combined with the whole animal and rat insulinoma cell strain INS-1 model, the effect of AAPF on insulin secretion dysfunction and the key molecules links of its downstream signal transduction will be studied. That is, the effects of AAPF on the insulin gene transcription key factor PDX-1/MafA, insulin signal transduction key protein IRS-1/IRS-2 and PI3K/Akt/FoxO1 signaling pathway, etc. Based on the above results, the molecular mechanism of antioxidant stress injury in AAPF will be explored, and these might provide a new molecular target for the nutritional intervention of oxidative stress injury, meanwhile, might have an important scientific significance on further development of Auricularia auricularia polysaccharide.

氧化应激所致胰岛β细胞凋亡是T2DM发生的重要机制。本课题组在前期研究发现黑木耳多糖可有效保护大鼠胰岛β细胞氧化损伤的基础上，针对黑木耳细胞壁质地坚韧、壁内多糖难以分离、结构复杂等特点，首先探析破壁方式（机械、物理、化学、生物酶法破壁）对黑木耳多糖结构特征的影响规律；然后结合秀丽隐杆线虫的抗氧化应激模型与多糖的可控降解探明其“活性片段”（AAPF），解读其分子结构信息；进而结合整体动物与大鼠胰岛素瘤细胞株INS-1细胞模型，研究AAPF对氧化应激所致胰岛素分泌功能障碍及其下游信号转导关键分子环节的影响，即对胰岛素基因转录关键因子PDX-1/MafA、胰岛素转导关键信号蛋白IRS-1/IRS-2、调控PI3K/Akt/FoxO1信号通路等的影响，试图阐明AAPF抗氧化应激损伤的分子机制，为寻找氧化应激损伤的营养干预提供新的分子靶点，亦为黑木耳多糖的深入开发提供支撑。

在“氧化应激-胰岛素抵抗-糖代谢失控”致病链中，氧化应激是致因，而抗氧化能有效阻断这个致病链。多糖是黑木耳子实体中重要的营养物质，其在降低体机氧化应激损伤、改善糖脂代谢紊乱等方面发挥重要作用，但关于其深层次作用机制的研究数据有限。此外，担子菌亚门的黑木耳细胞壁异常坚韧，常规手段很难达到有效破壁效果，多糖得率低、损失大。.我们的研究内容及结果如下：① 采用化学法（碱处理），机械法（4种）以及生物酶法（5种酶）等方式破壁，发现酶法（尤其纤维素酶）在多糖溶出率与清除自由基能力上具有理想效果。② 该法制备的多糖由甘露糖、葡萄糖、半乳糖、葡萄糖醛酸和木糖组成，分子量1148.15 kDa，具有三股螺旋结构。该多糖能显著延长氧化应激状态下线虫的寿命，减轻细胞凋亡，上调daf-16、sod-3、sir-2.1和skn-1等基因的表达量。③ 多糖可控酸水解物（AAPs-H）由甘露糖、葡萄糖和半乳糖组成，以β构型为主，存在C-3的β-Glc(1→3)-糖苷键、C-6的β-Glc(1→4)-和α-Glc-糖苷键。AAPs-H能使应激状态线虫体内SOD、CAT、GSH分别增加78.67%、98.52%与28.12%（p<0.05）。进而，AAPs-H能显著提高急性糖尿病小鼠的血清GLP-1水平，增强肝脏CAT与SOD活力与GSH水平，降低血清TC和TG含量，调节糖代谢关键酶（PEPCK、PK）活力，改善糖代谢紊乱。④ 透射电镜表明，AAPs-H能减缓应激损伤的大鼠INS-1空泡现象及细胞核弥散程度。qPCR表明，AAPs-H能显著上调INS-1细胞中Akt/FOXO1信号通路相关基因表达，但对转录关键因子PDX-1/MafA及信号蛋白IRS-1/IRS-2的mRNA表达无影响。线虫的转录组学表明，细胞内snRNA转录后RNA剪接体spliceosome被显著富集，表明可通过小核仁RNA的调节发挥抗氧化应激作用。糖尿病小鼠肝脏转录组学表明，TRP通道炎症介质调节、花生四烯酸代谢、亚油酸代谢、谷胱甘肽代谢、PPAR信号途径等显著富集，其中前三者均与PLA2G6基因（敲除后葡萄糖耐量及胰岛素分泌能力受损）相关。上述研究为解决黑木耳多糖在制备应用中面临的问题，以及在预防并逆转糖脂代谢紊乱的营养干预提供了新的依据。