花姬蛙皮肤潜在药用多肽的结构与功能研究

Amphibian skin contains a large number of pharmacologically bioactive substances with diverse structure and function, which is a pending development giant resource treasure house and being studied widely at present. Although there are a variety of therapeutic effects of Microhyla pulchra as a traditional Chinese medicine for several hundred years, its drug components are not known to the people. Recently, we constructed and sequenced the cDNA library of Microhyla pulchra skin and predicted the functions of the products encoded by those genes from the library. As a result, there are lots of genes whose products are highly homolog to peptides or proteins with reported pharmacological functions. Among them, the predicted pharmacological activities of the peptides encoded by 28 genes are closely associated with the therapeutic effects of Microhyla pulchra as a traditional Chinese medicine. However, we are not sure what the pharmacological activities of 28 peptides are exactly, what the structural basis of their pharmacological activities are and whether these peptides constitute the basis for therapeutic effects of Microhyla pulchra as a traditional medicine. In this study, 28 peptides will be obtained by chemical synthesis or recombinant expression, their functions will be determined by extensive pharmacological activities screening, as well as their spatial structures will be identified by circular dichroism spectra, 3-D nuclear magnetic resonance, crystal diffraction, homology modeling and site mutation. This project aims to study the structure and function of 28 potential pharmaceutical peptides from Microhyla pulchra skin, explore the molecular basis of Microhyla pulchra as a taditional Chinese medicines, find some novel lead molecules with promising medicinal prospect and provide the ideas and basics for the protection and utilization of Microhyla pulchra.

两栖动物皮肤中含有大量结构新颖和功能复杂多样的药理活性物质，是有待开发利用的资源宝库，国际研究的热点。尽管花姬蛙被用作中药有几百年，但是其疗效分子的本质却不为人们所知。通过cDNA文库大规模测序和生物信息学分析，我们最近从花姬蛙的皮肤中发现许多与功能已知多肽/蛋白密切相关的基因，其中28个基因编码的多肽的生物功能可能与花姬蛙的疗效密切相关。但是我们不确定这28个多肽的药理活性、高级结构和它们与花姬蛙中药疗效间的具体关系。本研究拟通过化学合成或重组表达来获得这28个多肽，通过药理活性的广泛筛选来确定它们的生物功能，通过圆二色谱、3-D核磁、晶体衍射、同源模拟和定点突变来确定它们的高级结构。本课题旨在研究花姬蛙皮肤28个潜在药用多肽的结构与功能，探讨花姬蛙作为中药的物质基础，期望找到具有药用前景的生物活性先导分子，并为花姬蛙的利用和保护提供思路和基础。

两栖动物皮肤中含有大量结构新颖和功能复杂多样的药理活性物质，是有待开发利用的资源宝库，国际研究的热点。尽管花姬蛙被用作中药有几百年，但是其疗效分子的本质却不为人们所知。通过cDNA文库大规模测序和生物信息学分析，我们从花姬蛙的皮肤中发现许多与功能已知多肽/蛋白密切相关的基因；依据生物信息学预测，我们合成抗菌肽、铃蟾肽、神经介素样肽、缓激肽、速激肽等多肽并研究了这些多肽的药理学功能；通过深入研究Esc-1GN、Cath-MH、Cath-HG、Fejerlectin、Brevinin-2GHk、FM-CATH、Brevinin-2KP、Brevinin-1FL、Tachykinin-HR等肽的结构与功能。我们发现Esc-1GN、Cath-MH、Cath-HG等都是多功能抗菌肽，具有抗菌、抗炎和LPS/LTA中和等作用。此外，Esc-1GN还有抗氧化和抗流感病毒作用，而Cath-MH和Cath-HG还能调节蛋白酶活性，抑制血浆凝固。FM-CATH除有抗菌、抗炎功能外，也能调节蛋白酶活性，抑制血浆凝固；Fejerlectin是一个具有抗HIV和抗氧化作用的凝集素；皮肤修复肽Tachykinin-HR通过作用于NK1R受体调节多细胞功能而对伤口愈合有多方位的促进作用；Brevinin-2GHk在体外有抗菌和抗寨卡病毒活性；Brevinin-2MP拥有抗菌和抗炎作用；Brevinin-2KP拥有抗菌抗炎、促进肥大细胞脱颗粒和组胺释放作用；Brevinin-1FL作为抗氧化剂，在体内外均可以降低氧化损伤，保护细胞。尽管这些多肽序列各异，但是在SDS和膜模拟环境中的二级结构主要为α螺旋。抗菌机制都是与破坏细胞膜有关，而抗炎机制大都涉及抑制炎性细胞内的MAPK/NF-κB通路活化和炎性因子分泌。这些多肽的结构与功能鉴定使我们拥有大量治疗细菌感染性疾病、炎性疾病、神经退行性疾病和病毒性疾病的候选药物分子模板，尤其是在治疗脓毒血症、痤疮、创伤、流感、艾滋病和寨卡病毒病等方面有潜在价值。此外，研究结果也为理解花姬蛙作为中药的物质基础，蛙类生存的分子机制和多肽药物的设计改造提供帮助。