鱼皮源活性肽抑制皮肤光老化的结构表征及作用机制的研究

Many Studies have shown that the food-derived bioactive peptides have significant physiological activities. Previous studies showed bioactive peptides from fish skin protein had good free radical scavenging activities and high metal chelating capacities, and they also had significant effects on protecting the skin from photoaging. In order to carry out further researches on the activity peptides from fish skin on skin photoaging and their inhibition mechanism, this project will employ "biomimetic digestion system" for preparating the high digestion resistant hydrolyzates of fish skin protein and further isolate novel peptides, especially being rich in hydroxyproline. The information of novel and previous reported active peptides of fish skin will be collected. The structure characteristics of peptides will be analyzed, including the molecular weight, polarity, hydrophobicity, bond, binding of special amino acids and permeability of lipid membrane. Furthermore, the relationship between structure and free radical scavenging activity, speed and metal-chelating capacity will be investigated. Some peptides with high biological activities will be collected and analyzed by their relations between their structure and cell transmembrane transport mechanism. The project also study the interaction of collagen, hyaluronic acid and its related enzymes in the photoaging model. The inhibitive activities of the active peptides in the process of photoaging will be quantitatively and qualitatively analyzed by combining the structural characteristics and activity analysis of the active peptides, and exploring the corresponding mechanism of active peptides.

研究显示食品源活性肽对人体具有显著的生理活性。申请人前期研究从鱼皮蛋白中制备活性肽，活性肽具有较好的清除自由基和螯合金属能力，并在保护皮肤光老化中具有明显的效果。为进一步解析鱼皮源活性肽在抑制皮肤光老化中的作用及机制。本项目拟在前期的研究基础上，采用"高仿体内消化模型"制备鱼皮蛋白高抗消化性的水解产物，从中分离纯化新型的鱼皮活性肽，特别是富含羟脯氨酸的活性肽。收集新制备和以前报道的鱼皮活性肽信息，分析活性肽结构特点，包括分子量、极性、疏水性、成键性、固定氨基酸链接及与脂质膜的结合性和渗透性，探讨其结构与清除自由基的大小和快慢及螯合金属能力的关系。筛选高活性肽，进一步分析其结构性质与细胞跨膜转运机制的关系。形成光老化模型，讨论细胞和动物在紫外线照射下胶原蛋白、透明质酸及两者相关酶的相互影响，结合活性肽的结构和活性特点，定量或定位分析活性肽对光老化过程的抑制作用，并探讨其相应作用机制。

本项目建立了“高仿体内消化模型”，研究发现初始pH值具有关键性，而离子强度对胶原蛋白水解过程影响不明显。利用高分辨质谱结合Uniprot数据库和De-novo肽片段整合，分别从鳕鱼皮和罗非鱼皮中鉴定了7个关键活性肽，其中鳕鱼皮为YGCC、DSSCSG、NNAEYYK和PAGNVR；罗非鱼皮为GYTGL、LGATGL和 VLGL。关键活性肽分子量、极性、固定氨基酸链接和脂质透过性均符合抗氧化活性肽的特征。关键活性肽显示了较好的抑制光老化小鼠皮纤维细胞的氧化性和MMP酶的活性，分子对接显示关键活性肽能够与MMP-1和MMP-9相结合，形成氢键和结合Zn2+，从而抑制其酶活，关键活性肽的C端GL或GP在结合过程中有重要的作用。利用Caco-2单细胞层研究了胶原蛋白水解液的转运和吸收特性，从结果上看，水解液中大约有5%的活性肽能够透过细胞单层，一般是通过PepT1通路进入。利用小鼠肠膜和小鼠研究关键活性肽的体内吸收性，关键活性肽能够透过肠膜，通过率大约在2%左右。关键活性肽能够进入小鼠的血液循环系统，从血液、肝脏、肾脏和肺部均能检出完整的关键活性肽。利用无毛小鼠制备光老化模型，活性肽整体上显示了较好的抑制光老化作用，从金属基质蛋白酶、炎症因子、透明质酸及酶和氧化指标各方面显示出较好的保护作用。从组织切片上看，活性肽能够保护光老化小鼠皮肤的一般组织和胶原蛋白的结构，与活性肽抑制MMP和透明质酸酶活性有较好的一致性。