基于蛋白质组学研究黄连效应物质群治疗2型糖尿病的多靶点作用机制

Coptis chinensis has a long history to treat diabetes for 2000 years. As the main components of Coptis chinensis, berberine, coptisine and ferulic acid have been proved to possess anti-hyperglycemia and anti-dyslipidemia properties. The mechanisms underlying this efficacy are related to different action targets and signaling pathways. We previously found that Coptis powder was superior to the berberine and Coptis decoction with the corresponding dose respectively in the treatment of type 2 diabetes (T2DM). However, the mechanism remains unclear. Since multiple pathophysiological dysfunctions are involved in the development of T2DM,we try to test our hypothesis that the "special" materials consisted of effective substances in Coptis chinensis act synergistically via multiple targets in the treatment of T2DM. Experiments have been designed to extract the effective materials by the way of supercritical fluid extraction(SFE) as well as to select and identify the technical conditions on SFE by comparing the effects on diabetic mice/rats model. Meanwhile,the fingerprint of the effective materials will be established and the composition and structural characteristics of effective materials will be tested by HPLC/LC-MS and NMR assays. To explore the mechanisms underlying the anti-diabetic efficacy of these effective materials on multiple targets, we will apply proteomics and bioinformatics techniques. The successful completion of the proposed research will illustrate the anti-diabetic mechanisms of the effective materials contained in Coptis chinensis on multiple targets, based on which we will try to develop a 1.2 class new drug of traditional Chinese medicine.

黄连用于治疗消渴症已有2000余年历史。现代研究表明，其主要成分小檗碱、黄连碱、阿魏酸等均具有一定的降糖调脂作用，且其作用靶点、环节各异，与 DeFronzo教授提出的联合使用多种药物以纠正多重病理生理缺陷的T2DM用药原则相吻合。基于课题组前期研究成果：黄连粉末给药治疗T2DM效果佳、起效快、作用稳定，优于等同剂量的小檗碱和黄连煎剂，提出"构建黄连'特定'效应物质群以治疗多重病理生理缺陷之T2DM"的设想，本课题采用超临界萃取方法、科学运用超临界介质提取黄连效应物质群；比较提取物对糖尿病小鼠/大鼠模型的疗效以筛选确定超临界萃取条件；借助HPLC/LC-MS、NMR等方法建立效应成分群指纹图谱、测试其组成比例、研究其结构特征；运用蛋白质组学和生物信息学技术，从终端研究该效应物质群治疗T2DM的多靶点作用机制，探讨'特定'效应物质群降糖调脂的作用特点，为将其开发成中药1.2类新药奠定基础。

黄连用于治疗消渴症已有2000余年历史。现代研究表明，其主要成分小檗碱、黄连碱、阿魏酸等均具有一定的降糖调脂作用，且其作用靶点、环节各异，与DeFronzo教授提出的联合使用多种药物以纠正多重病理生理缺陷的T2DM用药原则相吻合。基于课题组前期研究成果：黄连粉末给药治疗T2DM效果佳、起效快、作用稳定，优于等同剂量的小檗碱和黄连煎剂，提出"构建黄连'特定'效应物质群以治疗多重病理生理缺陷之T2DM"的设想。. 本研究采用L9(3^4)正交试验对黄连进行超临界萃取，利用1型糖尿病小鼠初步筛选出具有良好的降糖作用黄连的超临界提取物，继而采用2型糖尿病大鼠模型验证2萃取物的降糖调脂作用，同时进行血浆蛋白质组学分析，初步筛选出黄连超临界萃取物、模拟胃液萃取物治疗2型糖尿病的14种差异蛋白,即Pla2g7、G6PD、PSMA6、C4、C2、APOC3、APOC1、PRDX2、PKM2、APOE、GPX3、IGF-1、IGFBP4、IGFBP6，即其治疗2型糖尿病的血浆作用蛋白，提示其作用机制可能是通过直接或者间接促进抗氧化酶的活性，使胰岛β细胞受氧化应激损伤减轻，从而改善胰岛素抵抗；借此捕捉到的14种作用蛋白，结合建立的黄连超临界萃取物指纹图谱及其主要成分及其含量分析，运用计算机反向分子对接技术将差异蛋白与黄连萃取物中主要的单体成分进行一一对接计算，根据结合能力运算，进一步推断这14种差异蛋白可能是黄连治疗2型糖尿病的作用靶点，同时也推断黄连的5种主要成分（黄连碱、药根碱、小檗碱、表小檗碱及巴马汀）可能是该有效靶点的有效成分，而且黄连碱、药根碱及小檗碱是主要的有效成分；采用HepG2细胞高糖高脂模型及RT-PCR及Western blot手段验证了前述的推断结论，且体内、外研究表明：黄连碱、小檗碱及药根碱单体具有恢复内源性胰高血糖素样肽（GLP-1)分泌作用，且其混合物作用效果优于其单体。. 该研究从终端研究该效应物质群治疗T2DM的多靶点作用机制，探讨'特定'效应物质群降糖调脂的作用特点，为将其开发成中药1.2类新药奠定基础。