潺槁木阿朴菲生物碱类PTP1B抑制剂的发现及其改善胰岛素抵抗作用机制研究

Natural inhibitors of Protein tyrosine phosphatase 1B (PTP1B) were important sources for developing agents in curing insulin resistance (IR). Litsea glutinosa, a natural medicinal plant with abundant resources in Hainan province, has been used in treating IR. Previously, our research group firstly found that the extract of Litsea glutinosa showed significant activities in ameliorating IR. Further pharmacological investigations have been successfully carried out by which the active fraction was determined. Tentatively chemical investigation displayed that the active fraction was rich in aporphine alkaloids (APA), and these chemical constituents exhibited significant activities in curing IR. Furthermore, the potential mechanisms were found to be closely related to inhibiting PTP1B. On the basis of those previous works, this project is aimed to systemically investigate the medicinal plant by integrating modern methods including natural product chemistry, and pharmacology, etc. By applying these approaches, the APA in the effective part will be fully isolated and structurally characterized. Next, the binding abilities between APA and PTP1B in silico, their activities in improving IR, and inhibiting the enzyme of PTP1B will be evaluated completely. By analyzing the results obtained from the above three aspects, the structure-activity relationship will be investigated. Compounds with interesting structures and significant biological activities will be selected and then prepared in large amount. Following, the target compounds will be orally taken by ob/ob mice. After checking the biochemical indexes and tissue pathology, their activities in improving IR will be deeply evaluated. Finally, by detecting protein and RNA expression levels of PTP1B and its down proteins and genes, applying antagonist and RNA interference, the potential mechanisms will be determined. In this way, our findings will establish solid scientific foundation, facilitating not only in finding new lead compounds, but also providing scientific data for investigating both Litsea glutinosa and new agents in curing IR.

蛋白酪氨酸磷酸酶1B（PTP1B）天然抑制剂是胰岛素抵抗（IR）治疗药物研究的重要源头。潺槁木为海南典型药用植物，资源丰富且被用于治疗IR。课题组首次发现其改善IR的有效部位富含阿朴菲生物碱类成分（APA），且APA发挥功效与抑制PTP1B密切相关。本项目拟在前期研究基础上，综合运用天然药物化学、药理学、分子生物学等方法，系统获得有效部位中的APA；体外评价APA改善IR作用、与PTP1B的结合能力和抑制PTP1B酶的活性，剖析构效关系；遴选结构新颖、活性显著的APA，足量制备；考察其对ob/ob小鼠生理病理指标的影响，评估其改善IR的功效；通过检测遴选APA对细胞和组织中PTP1B及其下游蛋白表达、磷酸化、基因表达水平的影响，结合选择性拮抗剂和RNA干扰验证其功效与抑制PTP1B的相关性，阐明其分子机制。最终，发现新型先导化合物，为潺槁木的研究提供科学依据，为IR治疗药物的研究奠定基础。

潺槁树是海南地区特色的热带药用植物，临床报道其具有抗糖尿病的功效。课题组通过调研及前期研究发现，潺槁树根皮中所含的阿朴菲生物碱类成分具有显著改善胰岛素抵抗的作用。本项目在前期工作的基础上，对潺槁树根皮中所含的阿朴菲生物碱类成分进行了系统的提取、分离和结构鉴定；并对所得阿朴菲生物碱类成分进行了葡萄糖消耗作用评价，剖析构效关系；对代表性活性成分新木姜子碱进行体内外活性评价，对其药代体内药代动力学进行了详细的研究，并对其改善胰岛素抵抗的潜在机制进行了研究。通过本项目的研究，从潺槁树中获得32个成分，其中生物碱类成分17个，阿朴菲生物碱类成分10个，新化合物7个。通过筛选发现5个促进葡萄糖消耗活性显著的阿朴菲生物碱类成分，确定氮原子上的取代基对活性影响显著。同时，课题组对潺槁树总生物碱改善胰岛素抵抗的作用进行了研究，发现灌胃给予潺槁树总生物碱50、100和200mg/kg，可显著降低血清胰岛素水平，其中200mg/kg 的效果甚至优于阳性药罗格列酮，使血清胰岛素水平恢复到接近正常动物水平。胰岛素敏感指数也显著降低，提示总生物碱具有优异的改善胰岛素抵抗作用。同时，作为表征胰岛素响应度的口服葡萄糖耐量试验结果亦显示，潺槁木总生物碱可以显著改善动物的葡萄糖耐量，增强动物的餐后胰岛素响应性。而且，课题组对代表性阿朴菲生物碱-新木姜子碱（50、100和200mg/kg）可显著降低空腹血糖、OGGT，改善胰岛素敏感指数，显著改善db/db小鼠胰岛素抵抗的情况。同时，其对db/db小鼠肝脏、胰腺、肾脏均有显著的改善。通过深入研究，明确新木姜子碱通过调控线粒体氧化磷酸化，激活AMPK通路，发挥功效的分子机制。通过上述研究，发现先导化合物1个，发表学术论文8篇，其中SCI收录的论文5篇，中文期刊3篇；授权国家发明专利1项。培养的硕士研究生4名，其中金燕同学荣获海南省2020年度优秀硕士论文。本项目的完成，基本阐明了潺槁树临床治疗糖尿病的有效成分，为潺槁树的深入研究提供基础。同时，从中发现了作用显著、安全的先导化合物新木姜子碱，并发现其通过调控线粒体氧化磷酸化，激活AMPK通路，发挥功效的分子机制，为新型改善胰岛素抵抗药物的研究提供科学依据。本项目还为基于调控线粒体生物能量改善胰岛素抵抗的机制研究提供工具化合物。