源自美洲大蠊多肽类物质对溃疡性结肠炎免疫功能调节的作用机制及物质基础研究

The pilot study of the proposed project on the basis of a series studies on the pharmaceutical development of medicinal insect Periplaneta americana, provided a novel bioactive fraction from the crude extract of title insect for the treatment of ulcerative colitis. Preliminary experiments showed that: the active fraction possessed satisfactory therapeutic effects towards acute-, chronic-, and immuno-ulcerative colitis rats, in addition, the main components of the active fraction were polypeptides. It is proposed to further study the active fraction and purified polypeptides and UC rats are induced by 2,4,6-trinitrobenzene sulfonic acid, with bioactivity evaluation and mechanism study on ulcer wound healing effect, the distribution pattern of inflammatory cell subsets, and the influence on the secretion and expression of inflammatory cytokines and the expression levels of ulcer wound repair related signal factors, and the activation of nuclear factor kappa B predominate on ulcerative colitis（NF-κB） signal pathways regulating role. Moreover, separation, analysis, and elucidation of the structures of the active components existed in active fraction of title insect will be also thoroughly performed, by means of cluster of modern proteomic analytic techniques such as ultrafiltration, electrophoresis, and mass spectrometry. In terms of mechanism study, it aims to explore the ways peptides from title insect intervening local inflammation of colon, regulating inflammatory cytokines regulate the local community and wound repair mechanism. It is expected that the results of the proposed research will evaluate the multi-components, multi-targets and combinative network effects of bioactive polypeptides from the active fraction of Periplaneta americana against ulcerative colitis. This research will be able to build up a firm basis for further development of the active fraction into a novel colon-targeted formulations of Chinese medicine and a theoretical basuis for its clinical application. It is likely to help on providing a scientific basis for better developing insect resources of Yunnan specialty.

本项目在药用昆虫美洲大蠊系列药物研发多年的基础上，从全虫总提物中进一步精制了对溃疡性结肠炎有治疗作用的活性部位，前期研究显示：该活性部位对大鼠急、慢性及免疫性溃疡性结肠炎有较好的治疗作用，该活性部位以多肽类为主要组分。现拟进一步研究该活性部位对2,4,6-三硝基苯磺酸诱导的UC大鼠结肠溃疡创面修复的效果，对其炎性细胞亚群分布格局、炎性因子分泌和机体免疫的影响，以及对溃疡性结肠炎激活核转录因子κB（NF-κB）信号传导途径的调节作用。本研究还将结合超滤、电泳和质谱等现代分离分析技术对多肽类物质基础进行纯化与结构解析，阐述美洲大蠊多肽类物质干预结肠局部炎症，调节炎症因子群落及局部创面修复的作用机制。本研究结果预期可为制备该活性部位的结肠靶向制剂临床应用提供理论基础，为更好的开发云南特色昆虫药物提供科学依据。

溃疡性结肠炎 (Ulcerative Colitis，UC)是一种复杂、慢性、免疫介导的炎症性疾病，发病率逐年上升。防治UC的药物研发仍是一项重大的挑战。前期研究显示美洲大蠊（Periplaneta americana L.）提取物可应用于防治UC。本项目从美洲大蠊全虫总提物中进一步精制了对溃疡性结肠炎有较好治疗效果的多肽活性组分为主的活性部位，以该活性部位作为研究对象，对其多肽类物质基础进行了初步的纯化分离与结构解析。通过建立2,4,6-三硝基苯磺酸（TNBS）诱导UC大鼠模型，评价大鼠UC模型的病理变化并确定药效研究实验周期。同时以灌肠和灌胃的2种不同的给药途径探索其对TNBS诱导的溃疡性结肠炎大鼠的药效作用，进一步研究该活性部位对TNBS诱导的UC大鼠结肠溃疡创面修复的效果，通过对各实验组大鼠DAI评分、结肠指数、CMDI评分、结肠HS评分等指标进行评价，通过炎性因子分泌、炎性细胞亚群分布格局和对溃疡性结肠炎激活核转录因子κB（NF-κB）信号传导途径中相关因子进行检测探究美洲大蠊多肽类的活性部位应用于大鼠溃疡性结肠炎的作用效果及作用机制。.从美洲大蠊多肽类活性部位中分离到12个多肽单体并解析其中5个小肽；凝胶分离并鉴定5个大分子蛋白质，N末端氨基酸序列检索表明均为未报道的新蛋白。美洲大蠊多肽类活性部位不同给药方式的结果表明其均对UC大鼠均具有较好的治疗效果，其作用方式可能与上调血清中的炎症因子IL-10水平，下调TNF-α、IFN-γ水平和上调结肠黏膜的EGF、TGF-β1水平，下调IL-17水平和COX-2表达水平，恢复CD3+、CD4+和CD8+的表达量，调节T淋巴细胞亚群的趋向平衡，能调节活动期UC大鼠TLR4/MyD88/NF-κB信号通路中的NF-κB p65等蛋白的表达水平，并调节 UC大鼠的肠道菌群有恢复为正常菌群的趋势。.推测其作用机制可能是通过多组分多靶点的网络调控效应减轻炎症和促进细胞生长因子促创面修复，调节机体免疫反应同时恢复肠道菌群有关。本项目的研究结果为进一步将美洲大蠊多肽类活性部位应用于溃疡性结肠制剂制剂的应用提供理论基础。