基于生物信息学手段桑椹中PPARs激动剂类抗糖尿病活性成分的发现与作用机制研究

The incidence of diabetes mellitus (DM) increased year by year and 90% of people with DM have type 2 diabetes mellitus (T2DM). DM has become an important disease threat to human health. Since insulin resistance (IR) is the main pathogenesis of T2DM, targeted to active peroxisome proliferater-activated receptor γ (PPARγ) and reverse IR is an important strategy for the treatment of T2DM. TCM holds that DM belongs to the category of "Xiaoke", Mori Fructus are commonly used in clinical treatment of “Xiaoke”, but its chemical base is not clear. In our provious study, chemoinformatics approach and molecular docking technology were combined to screen anti-diabetic compounds from Mori Fructus, and benzofurans were found as potential PPARα/γ agonists. The present research is conducted to enrich, isolate and identify benzofurans from Mori Fructus with the guidance of bioassay and LC-MS. The potential targets and signal pathway of the obtained compounds will be predicted using bioinformatics approach, then their anti-diabetic activities and structure-activity relationship (SAR) will be studied to obtain the effective and low toxicity leading compounds. Antidiabetic mechanism of the leading compounds will be further investigated and validated with molecular biology methods. The study of this project is of great important significance to clarify the chemical base of Mori fructus as a TCM for the treatment of DM and it will also provide the leading compounds for developing safety and effectively IR-reversal in the treatment of DM.

糖尿病的发病率逐年上升，其中90%是2型糖尿病（T2DM），已成为严重威胁人类健康的重大疾病。胰岛素抵抗是T2DM的主要发病机制，靶向激活过氧化物酶体增殖物激活受体γ（PPARγ）、逆转胰岛素抵抗是治疗T2DM的重要策略。中医认为糖尿病属“消渴”范畴，桑椹临床常用于治疗消渴症，但其药效物质尚不明确。在前期对桑椹化学成分进行系统研究中，利用化学信息学和分子对接技术为导向结合活性实验验证，发现其苯并呋喃类化合物为潜在的PPARα/γ激动剂，本项目拟进一步采用活性跟踪和液质联用指导富集并追踪分离该类化合物，利用生物信息学手段预测其潜在的作用靶点和信号通路，研究其降糖活性和构效关系，采用分子生物学方法验证其作用靶点和通路，阐明其作用机制，优选出高效低毒的抗糖尿病先导化合物，以期阐明中药桑椹抗糖尿病药效物质，并为高效低毒的胰岛素增敏剂类降糖药物的开发提供先导化合物。

中医认为糖尿病属“消渴”范畴，桑椹临床常用于治疗消渴症，但其药效物质尚不明确。本研究综合运用各种色谱学手段对桑椹种子的70%乙醇提取物进行了系统分离，通过化学和波谱学方法鉴定了各单体化合物的结构。本项目共分离鉴定化合物86个，其中苯并呋喃类化合物11个，黄酮类化合物12个（含新化合物1个），苯丙素类化合物23个（含新化合物1个），生物碱类化合物9个（含新化合物1个），其他类化合物31个。利用体外酶和细胞模型系统研究了单体化合物抑制α-葡萄糖苷酶活性，激动PPARγ表达活性和抗神经炎症活性，还评价了一些化合物的抗氧化活性。结果显示苯并呋喃类化合物BF2化合物BF4和化合物BF5具有显著的激动PPARγ活性，同时显著增加FASN的表达，化合物8-11具有显著的抑制α-葡萄糖苷酶活性，化合物1，5，7，10，14具有显著的抗神经炎活性且没有细胞毒性，化合物1，5，7和14能显著下调iNOS, COX-2和IL-1β mRNA的表达，同时下调iNOS和COX-2蛋白的表达，并能有效抑制LPS诱导的NF-κB向细胞核转移。. 项目研究结果提示桑椹可能从抑制碳水化合物的消化和吸收，促进糖脂代谢、抑制炎症和氧化应激等多种途径来实现降糖和减少糖尿病并发症的作用。本项目研究成果已发表研究论文3篇，申请国家发明专利1项，还有1篇文章已投稿。通过本项研究，进一步阐明了桑椹降糖的药效物质和作用机制，为其进一步开发奠定了基础。