美洲大蠊促创面修复的药效物质解析及作用机制研究

Wound healing and tissue regeneration is implicated in a wide range of disorders. Although stem cell therapy and genetic engineering technology displays advantages and accounts for advanced therapy in this field, it is still a great challenge for chronic wound repair exemplified as diabetic foot and various chronic ulcers. Therefore, it is urgently necessary to make efforts on improving therapeutic approaches of chronic wound repair. In nature, some animals such as earthworms, geckoes, octopuses, and salamanders are unique in showing the high capacity for regeneration, including full limb and tail regeneration. Besides ancient maggot therapy receives great attentions nowadays due to its magic efficacy in wound repair. All these examples demonstrate that animals or animal-derived products might be promising for wound repair and tissue regeneration. Periplaneta americana, known as a kind of cockroach, and a patent drug (namely Kangfuxin Liquor) made of P. americana have been used for the treatment of various ulcers and wounds in China without scar, which is of great impression. However, what substances are responsible for its pronounced effects remains unknown, which prohibits further clinic applications of Kangfuxin Liquor. We have been interested in insect chemistry and made great achievements in this aspect in recent years. To secure the efficacy of P. americana, several extracts or fractions out of P. americana were prepared and biologically evaluated using several assays closely associated with wound repair. The results showed that some fractions are active and helpful for wound repair. With these in hand, our isolation of P. americana afforded more than 40 compounds with diverse or intriguing structures. Further bioassays revealed that some compounds are active against inflammation, collagen production, and Wnt/β-catenin expression, indicating that rational substances might be present in P. americana. HPLC profiling indicates that substances, in particular of polar substances, are rich in the extract of P. americana. It is therefore necessary to clarify their structures, their biological roles in wound repair focusing on inflammation, proliferation and granulation, and remodeling. It is also necessary to understand how these biologically active substances regulate wound healing and tissue regeneration. These will be the mission of this project. Our fruitful production on this insect will help to gain a deep insight into chemical profiling of P. americana and will probably provide a drug lead in wound healing field beneficial for future drug development.

针对创伤修复与组织再生，基因工程和干细胞技术虽然有效，但也仅能解决部分问题且价格高昂，糖尿病足、各种溃疡等慢性创伤修复仍面临挑战。著名的蚯蚓、壁虎、章鱼、蝾螈的断肢再生及近年兴起的蛆虫疗法均展现出动物对于创面修复的优势。由美洲大蠊（ML）制成的主要由小分子组成的康复新液临床上愈创效果突出，且不留疤痕，但药效物质基础长期不明。前期，我们发现ML的不同组分在不同细胞模型上确有愈创活性，继而从ML中鉴定40余个结构多样的化合物，并发现部分成分具有抗炎、促胶原分泌、调节Wnt/β-catenin等愈创活性，初步揭示了ML愈创有其物质基础。由于HPLC显示ML中成分尤其是高极性成分众多，但它们的结构特征、在炎症反应、肉芽形成及组织重建中的角色及作用特点等均有待阐明。因此本项目围绕上述科学问题深入挖掘，有望充分解析ML愈创物质基础，绘制成分/效应网络，支撑康复新液临床应用，并为开发愈创药物提供新视角。

美洲大蠊俗称蟑螂。美洲大蠊在云南大理白族地区用于烧烫伤效果突出且历史悠久，后来开发了康复新液新药（美洲大蠊为唯一药材）主要用于溃疡等创伤修复，民间也将其用作抗癌之用。但长期以来有关美洲大蠊和康复新液的药效物质却一直没有答案。鉴于此，本项目采用LC-UV/DAD-MS等分离策略手段共从美洲大蠊中获得了非肽类小分子化合物193个（新化合物55个），结构类型丰富多样，这应该是迄今为止对美洲大蠊药效物质研究最为彻底的一次。鉴于美洲大蠊主要适应症为创伤修复，基于创伤修复与炎症、肉芽生成及组织重建相关，我们采用与之相匹配的多维模型包括人皮肤成纤维细胞（HDFs）增殖、人脐静脉内皮细胞（HUVECs）血管生成、人脐静脉内皮细胞迁移、炎性物质NO生成、COX-2抑制等对大部分化合物进行了与创伤修复活性相关的研究，共发现活性化合物44个，其中创伤修复活性25个，抗炎活性17个。此外也是值得一提的是，发现了3个天然成分在纳摩尔级水平上可显著抑制COX-2活性，IC50值分别为768 nM, 617.7 nM和599.5 nM。同时对3个高活性化合物进行了体内抗口腔溃疡试验，验证其具有一定疗效。上述发现揭示美洲大蠊促创伤修复的成分结构具有多样性，愈创活性具有多途径性，也提示美洲大蠊较好的愈创疗效是多成分多途径协同起效的结果。该研究一定程度上解析了康复新液愈创的物质基础，将为助推其临床应用和实现国民对美洲大蠊（蟑螂）可治病的科普发挥积极作用。此外研究发现了ZL-49可以同时阻断三阴性乳腺癌细胞的 TGFβ/BMP 信号传导，有效抑制肿瘤生长、侵袭和转移。此外诞生了新的研究生长点：ZL-49及衍生物显示出很强的抗肾纤维化作用，目前正在进行其衍生物的抗肾纤维化深入研究；采用仿生合成方法，从苯乙醇合成了40多个结构新奇的聚合衍生物，给我们研究虫类小分子提供了新的思路。这些研究成果及继续的研究将对美洲大蠊的质量标准和临床应用及产品开发提供新的基础和依据。