苹果多酚基于GLP-1受体途径调节胰岛β细胞功能的物质基础及作用机制研究

The increasing population of diabetes, dietary adjustment recognized by the medical profession is one of the most cost-effective means. Edible apple is restricted for the population of diabetes because of a lot of sugar. Polyphenols are the main functional components of apple and have become the research focus of the health food ingredients at home and abroad with a variety of biological effects such as anti-oxidation. Apple polyphenols were refined by chromatography and purified from sugar, so it can be edible for diabetics. In our preliminary work, it was found for the first time that apple polyphenols can protect pancreatic β cells, promote the high expression of the GFP gene in NIT-1 cells with RIP1-CRE transiently transfected, which showed that apple polyphenols play the role as a GLP-1 receptor agonists. In order to clarify the material foundation and receptor mechanism of apple polyphenols to regulate the pancreatic β cell based on GLP-1 pathway, we hope to research this project. In this project, using the high-throughput of GLP-1 target screening as a guide, the functional components to protect the pancreatic βcells were tracked and isolated by a variety of chromatographic separation methods. At the same time, the mechanisms of anti-apoptosis, proliferation and promoting the secretion of functional components in apple polyphenols were studied by using the DNA-ladder and western-blot in the NIT-1 pancreatic β cells. In addition, the protected pancreatic function of components of apple polyphenols based on GLP -1 receptor way were studied using immunohistochemical methods in high glucose and fat rat model. The results of this project will provide theoretical and experimental basis for the functional foods (apple polyphenols) to protection of pancreas for the diabetic population.

糖尿病人群与日俱增，医学界公认饮食调节是对该人群最经济有效的手段之一。苹果由于含有大量糖分而使其食用受限，多酚是其主要功能成分，具有抗氧化等多种功效，经过层析脱糖精制，已成为国内外健康食品原料的研究热点。前期工作首次发现苹果多酚能够保护胰岛β细胞，调节RIP1-CRE瞬时转染的NIT-1细胞中GFP报告基因高表达，发挥GLP-1受体激动剂作用。为阐明苹果多酚基于GLP-1受体途径调节胰岛β细胞功能的物质基础和作用机理，本课题以GLP-1靶点高通量筛选为指导，运用多种色谱方法追踪分离苹果多酚保护胰岛β细胞的功能成分；利用DNA-ladder、western-blot等方法研究苹果多酚功能成分对NIT-1胰岛β细胞抗凋亡、促增殖和促分泌作用机制，利用免疫组化等方法研究其基于GLP-1受体途径对高糖高脂大鼠模型胰腺的保护调节功能。研究结果将为糖尿病人群获得辅助保护胰腺的功能食品提供理论和试验依据

苹果多酚具有抗氧化等多种功效，经过层析脱糖精制，已成为国内外健康食品原料的研究热点。本课题以GLP-1靶点进行筛选验证了苹果多酚能调节RIP1-CRE瞬时转染的胰岛β细胞中报告基因GFP的高表达，发挥GLP-1受体激动剂作用，同时预防性给药可以改善多次小剂量注射STZ诱导的糖尿病ICR小鼠多饮、多食、多尿的“三多一少”症状，改善糖耐量，促进糖尿病ICR小鼠胰岛素的分泌，增强对胰岛素的敏感性，保护胰岛β细胞，减少胰腺组织的损伤，使胰腺组织中PDX-1蛋白表达上调，从而明确了苹果多酚基于GLP-1受体途径对胰岛β具有保护作用。离体胰岛细胞实验也确认苹果多酚有促胰岛细胞增殖、通过发挥抗氧化作用而对H2O2诱导的胰岛细胞损伤具有显著的保护作用。通过体内外活性筛选确认苹果多酚50%乙醇提取物为其保护胰岛β细胞的有效部位，其通过上调PDX-1、Akt的表达，抑制caspase-3的表达来改善胰岛功能，保护胰岛细胞，发挥降血糖作用。运用多种色谱方法从中共追踪分离鉴定了34个化合物，其中有15个奎宁酸、香豆酸类成分；10个黄酮类成分，9个儿茶素类成分。发现了一个新化合物，11个化合物首次从苹果多酚中发现。对所有的化合物首先利用体外胰岛细胞进行了促增殖实验测试，有12个化合物具有促增殖作用，5个化合物作用显著；特别发现了一类聚合儿茶素类成分具有很好地促胰岛细胞增殖的作用。体外细胞实验显示该类化合物能够保护胰岛细胞免受氧化应激损伤，体内实验显示该化合物显著增加正常小鼠的糖耐量，利用DNA-ladder、western-blot等方法确定34显著促进胰腺分泌胰岛素和增加血清胰岛素含量，且能促进GLP-1受体下游重要分子PDX-1的高表达，从而发挥调节胰岛组织细胞功能的作用。建立了有效部位指纹图谱，指认14个色谱峰，占总面积的82.1%，并测定主要成分含量。研究结果为阐明苹果多酚保护胰岛细胞，促进胰岛素分泌，降低血糖的物质基础和作用机制提供有利支撑。