凉薯多糖的制备及其对羰基应激损伤的干预作用及机理

Oxidative stress and nonenzymatic glycosylation are two most important biological side reactions. Reactive carbonyl species (RCS) are the commen intermediate products, which will react with lipids, proteins and nucleic acid in cells, causing damages to the cell strucure and function. The term "carbonyl stress" describes the damaging processes and the elevated levels of modified lipids, proteins and nucleic acid induced by RCS. Many studies have provided convincing evidence that carbonyl stress is one of the mechanisams of aging and chronic diseases. Pachyrhizus erosus is an underutilized crop, with very high yield. The crude polysaccharides from the tubers of Pachyrhizus erosus showed significant inhibition effects on oxidative stress and nonenzymatic glycosylation in vitro. The present project will prepare the polysaccharide from the tubers of Pachyrhizus erosus, and investigate their inhibition effects on carbonyl stress induced by high glucose. The reseach focus on two objectices: 1) to extract the crude polysaccharides from the tubers of Pachyrhizus erosus, separate and purify the polysaccharides with the higher inhibition effects on oxidative stress and nonenzymatic glycosylation in vitro, and investigate the structure of the purified polysaccharides; 2) to determine the inhibition effects of the purified polysaccharides on carbonyl stress induced by high glucose in human mesanginal cells and diabetic rats, and investigate the action mechnisams from the points of their inhibition effects on oxidative stress and nonenzymatic glycosylation in cells and in renal and aorta tissues of diabetic rats. The present project will provide the scientific basis for the high-value development of Pachyrhizus erosus.

羰基应激是生命体内氧化应激和非酶糖基化互相促进的损伤过程，被认为是衰老及多种疾病的发生机制之一。凉薯是一种高产农作物，本课题组研究发现凉薯块茎粗多糖具有很强的体外非酶糖基化抑制作用和抗氧化活性。本项目以凉薯块茎为原料，采用定向制备技术，制备对高糖所致羰基应激损伤有干预作用的凉薯多糖。研究主要围绕两个方面进行：1）凉薯多糖的提取、定向制备和结构表征。包括从凉薯中提取粗多糖的最佳工艺条件优化；以体外非酶糖基化抑制活性和抗氧化活性为指标，定向分离、纯化目标多糖；对目标多糖进行结构表征。2）以人肾系膜细胞和糖尿病大鼠为实验模型，研究目标多糖对高糖环境所致羰基应激损伤的干预作用；从目标多糖对高糖环境所致人肾系膜细胞、糖尿病大鼠肾脏和主动脉组织中非酶糖基化和氧化应激抑制作用两个方面探讨目标多糖对高糖所致羰基应激损伤的干预作用机理。本课题为凉薯中活性多糖的制备和应用提供了理论基础和科学依据。

采用自然发酵技术发酵凉薯，制备凉薯多糖，并对多糖的提取过程进行优化，最高得率为4.73%；利用乙醇浓度梯度（95%、80%和60%）对凉薯多糖进行分级，得到三个凉薯多糖组分；利用光谱、色谱、质谱、核磁等分析技术初步解析了三个多糖组分的物理性质和基本结构；以清除自由基和抑制AGEs生成评价了三个凉薯多糖组分的体外抗氧化能力和抑制糖基化能力。利用DEAE和Sephedex G100对具有较高活性的95%组分进行了初步分离和纯化，得到目标多糖PT-2a；利用H2O2诱导的HepG2细胞氧化应激模型和D-gal诱导小鼠老化模型，评价了PT-2a在细胞水平上的抗氧化能力和抑制体内高糖导致的羰基化能力，初步分析了纯化后凉薯多糖干预羰基应激老化损伤的水平和机制。采用腹腔注射链脲佐菌素制备II型糖尿病模型小鼠，记录了三个凉薯多糖组分对小鼠饮食、体重、血糖、肝脏、胰脏和肾脏指数的影响，测定了对肝糖元、总蛋白、MDA及体内抗氧化酶的影响，分析了对肝脏、胰脏和肾脏的一般结构组织影响，并进一步分析肾脏系膜基质指数及TGF-β1表达，结果表明凉薯多糖具有抑制高糖氧化应激损伤的能力，组化分析表明凉薯多糖具有缓解糖尿病肾病病症的潜在应用价值。