鳕鱼皮肽对光老化皮肤基质胶原降解的拮抗机制及活性中心结构解析

Photoaging induced by ultraviolet threatens the health of humans seriously, and the degradation of skin matrix collagen is the main cause of photoaging. Although some marine bioactive peptides show the advantages of anti-photoaging, the mechanism and the active center of preventing skin matrix collagen degradation are unclear, which become an urgent problem to be solved. Pollock skin bioactive peptides will be prepared by the technology of biosensor and artificial neural network model in this study. The key enzymes of degradation of photoaging skin matrix collagen——matrix metalloproteinases (MMPs) are used as the crux of anti-photoaging. The antagonistic mechanism is explored through MAPK/AP-1 singnal pathway and related factors of the direct and indirect regulation of matrix metalloproteinase, which is clarified on the basis of the molecular level and metabolic pathway. The fragments of the active center of pollock skin bioactive peptides are tracked by MMPs inhibitory activity in fibroblasts induced by ultraviolet, and they are isolated and obtained using the chromatography technology. Then the target peptide fractions are identified by Nano-ESI-Ms/Ms and the structure- activity relationship is further clarified. It will provide scientific evidence for the novel application of pollock skin peptides and the development of anti-photoaging product.

紫外线诱导的光老化严重威胁着人们健康，皮肤基质胶原降解是导致光老化发生的重要原因。一些海洋活性肽显示了抗光老化优势，然而其如何阻断基质胶原降解尚不清楚，活性中心位点尚待进一步探讨，成为目前亟待解决的热点课题。本研究采用生物传感器与人工神经网络模型靶向制备鳕鱼皮活性肽，以基质胶原降解的关键酶——基质金属蛋白酶（MMPs）为突破点，从直接与间接调控MMPs的MAPK/AP-1通路及相关因子的角度，在分子水平与代谢途径上阐释其对光老化皮肤MMPs的拮抗机制。在此基础上，利用UV诱导成纤维细胞中MMPs的抑制活性示踪析因，通过色谱技术分离纯化，寻找并确定抑制MMPs的鳕鱼皮肽的活性中心片段，并利用纳升电喷雾串联质谱技术（Nano- ESI-MS/MS）对其结构解析，进一步阐明其化学结构与作用机制的相互关系，为鳕鱼皮肽的新应用提供科学依据，为新型抗光老化产品的开发奠定基础。

UV诱导的皮肤光老化的发病率逐年增加，严重威胁着人们健康，皮肤基质胶原降解是导致光老化发生的重要原因。尽管鳕鱼皮肽显示了抗光老化优势，然而其如何阻断基质胶原降解尚不清楚，活性中心位点尚待进一步探讨。本研究主要集中在：鳕鱼皮BIO-ANNs靶向活性肽（CH）对光老化小鼠皮肤基质胶原特征的影响；CH对光老化小鼠皮肤基质金属蛋白酶MMPs与基质金属蛋白酶抑制剂TIMPs的影响；基于MAPK/AP-1通路的CH对光老化小鼠皮肤MMPs的拮抗途径与作用机理；拮抗MMPs的鳕鱼皮肽活性中心片段的纯化、结构解析及机制研究。所取得重要结果：以GLU-LYS-ANN等模型在线监控制备CH；建立了UV诱导的光老化模型，系统评价CH干预基质胶原降解机制。CH可以减缓UV诱导的皮肤光损伤，抑制皮肤中III/I胶原比例的增加，提高皮肤中抗氧化酶的活力；CH可以显著的降低MMP-1、MMP-3含量和活力，提高MMPs 内源性的抑制剂TIMP-1含量来抑制MMPs活性。CH通过介导MAPK/AP-1通路中ERK、c-Jun和c-Fos，下调关键细胞因子IL-1α和TNF-α，拮抗光老化小鼠皮肤中的MMPs，减缓光老化皮肤中基质胶原的降解。利用UV诱导成纤维细胞中MMPs的抑制活性示踪析因，通过色谱分离纯化鳕鱼皮目标肽活性中心片段M1与M2，并利用串联质谱进行鉴定，M1与M2的序列分别为GEIGPSGGRGKPGKDGDA GPK、GFSGLDGAKGD，并在细胞水平上系统研究M1与M2的作用机制。研究表明M1与M2可以减缓UV诱导的体外成纤维细胞的光损伤，显著地干预UV诱导的体外成纤维细胞中MMP含量的增加与酶活力的提高，其作用机制为：鳕鱼皮活性中心肽段M1与M2能够通过提高TIMP-1与TIMP-2的水平，抑制MMP含量的增加；M1能够通过干预MAPK通路中JNK、p38、ERK mRNA及其磷酸化蛋白表达，下调AP-1通路中AP-1、c-Fos水平，阻断关键细胞因子IL-6的升高，进而拮抗MMP；M2能够通过干预MAPK通路中p38、ERK mRNA及其磷酸化蛋白表达，下调AP-1通路中AP-1、c-Fos水平，抑制关键细胞因子IL-1α的升高，拮抗MMP。本研究揭示了鳕鱼皮肽如何阻断光老化皮肤基质胶原降解，解析了鳕鱼皮活性中心片段，明确了其对MMP的拮抗途径与机制，这为鳕鱼皮活性肽的深入开