藏药绿萝花中作用于2型糖尿病PTP-1B，PPARs多靶标的活性成分及作用机制研究

Type 2 diabetes is a chronic disease characterized by hyperglycemia which is resulted by the lack of insulin or insulin resistance, its harm is huge and difficult to cure, so we should look for drugs with efficiency and low toxicity against type 2 diabetes.In our previous research,Edgeworthia gardneri Meissn showed significant activity against type 2 diabetes,we use type 2 diabetes model cells and PPAR-γ activating factor experiments to determine the effective part of the PE and green dill flower EA, and in EA part 12 compounds were isolated . The project intends to adopt a variety of chromatographic , spectroscopic techniques and in vitro activation of PPAR-γ factor experimental activity tracking method for the effective parts of green dill flower composition isolated and identified ; With HepG2, 3T3-L1 cell model of insulin resistance , the use of Western blot study chemical composition / group against the impact of type 2 diabetes target gene PTP-1B, PPAR-γ / α expression ; through Caco-2 cell monolayers in vitro model to predict the absorption of each active ingredient ; using gene technology research active ingredient and type 2 diabetes multiple complications affect gene expression, and ultimately reveal its mechanism of action, clear material basis , provide laboratory evidence for the discovery of new drug resistant type 2 diabetes and in-depth research and development of Edgeworthia gardneri Meissn.

2型糖尿病是一种胰岛素相对缺乏或胰岛素抵抗导致的以血糖增高为特征的慢性疾病，危害巨大，治愈难，寻找高效低毒的抗2型糖尿病药物刻不容缓。藏药绿萝花为瑞香科结香属植物滇结香的干燥花蕾。本课题组前期利用2型糖尿病模型细胞和PPAR-γ因子激活实验确定了绿萝花的有效部位PE及EA，并于EA部位中分离得到12个单体化合物。本项目拟采用多种色谱、光谱学技术及体外PPAR-γ因子激活实验活性跟踪法对绿萝花有效部位进行成分分离鉴定；借助HepG2,3T3-L1胰岛素抵抗细胞模型，利用Western blot研究化学成分/群对抗2型糖尿病靶点基因PTP-1B，PPAR-γ/α表达的影响；并通过Caco-2单层细胞模型预测各体外活性成分的吸收情况；利用基因技术研究活性成分对2型糖尿病及其并发症多重基因表达谱的影响，最终揭示其作用机制，明确物质基础，为新的抗2型糖尿病药物的发现及绿萝花的深入研究提供实验室依据。

基于PTP-1B,PPARs研究藏药绿萝花抗2型糖尿病的物质基础及作用机制具有重要科学意义。本项目采用多种色谱方法对绿萝花化学成分进行分离纯化，得松脂素、罗汉松脂素、(S)-(-)-N-苯甲酰苯丙氨醇、腺苷、2,3,4,9-四氢-1H-吡啶骈-[3,4-b]吲哚-3-羧酸、银锻苷、山奈酚、紫云英苷、gardenrd A、gardnerol B 、西瑞香素、西瑞香素-5-O-β-D-吡喃葡萄糖基-(1→4)-α-L-鼠李糖糖苷、西瑞香素-5-O-β-D-吡喃葡萄糖基-(1→2)-β-D-吡喃葡萄糖苷、结香苷 C 、结香酸、去甲基丁香色原酮、伞形花内酯、十五烷酸、邻羟基苯甲酸等19个化合物。 应用标准酶测活体系研究了绿萝花单体化合物对PTP-1B的体外抑制活性，各给药组中PTP-1B的增值率均大于96%，说明绿萝花降糖有效部位中单体化合物对PTP-1B均无抑制作用，PTP-1B并非绿萝花降糖的作用靶点。建立HepG2和3T3-L1胰岛素抵抗细胞模型，WB评价绿萝花中单体化合物对PPAR-γ（α）蛋白表达量的影响，结果显示：西瑞香素、银锻苷在80μmol/L剂量浓度下可以显著提高PPAR-γ/α蛋白表达，是潜在的PPARs双重激动剂。利用Caco-2单层细胞预测西瑞香素、银锻苷的体内吸收、转运情况；结果表明西瑞香度吸收难度中等，在体内转运方式以被动转运为主，可能存在肠道转运蛋白的外排机制；银锻苷Papp<10-7cm/s，PDR>10，为难吸收药物，生物利用度低。基因芯片技术考察西瑞香素发挥降糖作用的作用机制，结果显示西瑞香素可能通过影响PPAR、胰岛素分泌、脂肪酸代谢等多个通路发挥降糖作用。