海参多肽与锌螯合的分子机制及其促锌吸收机理研究

Zinc plays a significant role in maintaining nutrition and health of humans as a human essential trace element. Studies reveal that peptides derived from sea cucumber are structurally stable, easily abortable and capable of chelating Zinc, the peptides could promote intestinal zinc absorption after chelation with zinc. However, which mechanism is involved in chelation of sea cucumber peptides and zinc? What cellular pathway is associated with intestinal zinc absorption promoted by sea cucumber peptides? It still remains unknown. For the questions above, the structure of sea cucumber peptide will be identified and filtrated through UPLC-Q-TOF-MS/MS. The mechanism of chelation will be illustrated by utilization of UV-Vis, FTIR and SEC-HPLC; Moreover, the digestively stable sea cucumber peptide-zinc complex (PZC) will be filtrated by In vitro simulated digestive system mode. The intestinal absorption of PZCs will be investigated through UPLC and AAS by Mice everted gut sac with use of absorption inhibitors. The technology of Western blot and Inflorescences mark will help the analysis of cellular signaling pathway mechanism to understand the connection between endocytosis and transcytosis involved in peptide-promoted zinc absorption. Our project will understand the mechanism of zinc-chelating action and promotive zinc absorption of peptide derived from sea cucumber and provide theoretical ideas in improving intestinal zinc absorption.

锌作为人体必要微量元素，对人体的营养和健康发挥重要作用。研究发现，海参多肽结构稳定，具有较好的吸收性和锌螯合能力，与锌螯合后能够促进锌在肠道的吸收作用。然而，海参多肽与锌的螯合涉及哪些机制？海参多肽促锌吸收与哪些细胞通路相关？目前仍未确定。针对上述问题，本项目通过UPLC-Q-TOF-MS/MS鉴定和筛选海参锌螯合多肽结构，并利用UV-Vis，FTIR和SEC-HPLC明确螯合机制；通过体外模拟消化模型筛选具有消化稳定性的海参多肽-锌螯合物；采用大鼠翻转肠囊法，利用UPLC和AAS，结合吸收抑制剂，分析海参多肽-锌螯合物的吸收作用；结合Western blot和荧光标记等技术，分析肠道细胞通路中的生物信号传导机制，明确海参多肽促锌吸收与胞吞作用和转胞吞作用的相关性。通过本研究，明确海参多肽与锌螯合的分子机制，揭示其促锌吸收机理，为改善肠道锌的吸收奠定理论基础。

锌作为人体必要微量元素，对人体的营养和健康发挥重要作用。海参多肽结构稳定，具有较好的吸收性和锌螯合能力。然而，海参多肽与锌的螯合涉及哪些机制？海参多肽-锌复配物在人体胃肠道的消化吸收作用如何？本项目以此为研究背景，以海参多肽为研究对象，将海参体壁蛋白进行酶解，制备海参多肽酶解物；通过离子交换层析，凝胶色谱分子筛层析及高效液相色谱等手段，结合间苯二酚锌法对螯合能力进行测定，分离纯化了锌螯合肽组分，并探究了肽组分与锌螯合的机制；通过UPLC-Q-TOF-MS/MS，鉴定出了三条锌螯合多肽序列；将三条海参锌螯合多肽序列进行人工合成，通过间苯二酚法、傅里叶红外光谱法、原子火焰吸收法、圆二色谱法、ITC法、拉曼光谱法、核磁共振法等，综合分析探究了海参锌螯合多肽与锌螯合的分子机制；最后，将此三条海参锌螯合与锌进行最优配比螯合，制备了海参多肽-锌复配物，建立了体外人体模拟胃肠道消化模型、Caco-2细胞及大鼠翻转肠囊法吸收模型，通过高效液相色谱法，原子火焰吸收、激光共聚焦显微镜、荧光倒置显微镜、扫描电镜、组织原味染色、TSQ锌离子荧光探针等方法，研究了海参锌螯合多肽-锌复配物在人体胃肠道消化吸收作用以及初步探究了其在肠道细胞吸收作用通路。截止到目前，项目负责人已发表含有项目标注的SCI论文6篇（4篇见刊，2篇接收），申请专利1项；此外，以锌螯合肽为研究根基，与大连非得生物产业有限公司共同申请了省级项目1项，市级项目1项。在本项目的研究中，首次确定了海参锌螯合多肽与锌螯合的螯合位点（分子基团）以及与锌螯合的分子键；通过人胃肠道消化吸收实验，验证了海参锌螯合多肽的胃肠道稳定性及肠道跨膜协同吸收作用。本项目旨在揭示海参多肽的促锌吸收作用，为锌元素营养食品产品开发奠定一定的理论基础和指导意义。