核桃源神经保护肽调控TRPV1缓解线粒体氧化应激机制研究

Transient receptor potential channel vanilloid 1 (TRPV1) is an important regulatory pathway for mitochondrial oxidative stress, but the regulation mechanism of exogenous peptide has not been fully elucidated. The walnut-derived neuroprotective peptide EVSGPGLSPN has been screened. Using quantitative proteomics, it was found that the intervention of peptide significantly down-regulated the expression of four proteins in PC12 cells, which were enriched in TRPV1 pathway by KEGG analysis, suggesting that peptide may mediate the regulation of TRPV1 to relieve mitochondrial oxidative stress. In this research, immunofluorescence staining and western blotting are used to verify the localization of TRPV1 and inhibition of differential proteins by peptides. Furthermore, based on specific antagonism/shRNA knockdown to block the expression of differential proteins respectively, specific activation or overexpression will be used to regulate the activity of the rest of proteins, combining intracellular and mitochondrial oxidative stress sensitivity, calcium homeostasis, the expression of inflammatory factors, mitochondrial membrane potential and respiratory function to explore a key signal pathway of oxidative stress regulated by TRPV1. The functions of proteins in the pathway will be analyzed to reveal the interaction of proteins and mechanisms of exogenous peptide regulating TRPV1 to relieve mitochondrial oxidative stress, which is of great importance to the development of neuroprotective peptide and delaying cognitive decline in the elderly.

瞬时受体电位通道香草酸亚型1（TRPV1）是线粒体氧化应激的重要调节通道，但外源活性肽对其调控机制尚未完全阐明。申请人前期筛选得到核桃源神经保护肽EVSGPGLSPN，利用定量蛋白质组学研究发现其干预PC12细胞使4种重要蛋白表达显著下调，KEGG分析富集到TRPV1通路，提示其可能介导TRPV1的调节缓解线粒体氧化应激。本研究拟利用免疫荧光染色和免疫蛋白印迹验证活性肽对差异蛋白的表达抑制及TRPV1的定位；通过特异性拮抗/shRNA敲减使差异蛋白分别阻断的基础上，利用特异性激活或过表达调控其余蛋白活性，结合胞内及线粒体氧化应激敏感性，钙稳态，炎症因子，线粒体膜电位及呼吸功能的变化，建立TRPV1调控的氧化应激关键通路，分析通路相关蛋白功能，明确蛋白的互作关系，挖掘外源活性肽介导TRPV1缓解线粒体氧化应激的作用机制。研究结果为开发肽类神经保护因子，延缓老年人认知功能下降具有重要意义。

瞬时受体电位通道香草酸亚型1（TRPV1）是线粒体氧化应激的重要调节通道，但外源活性肽对其调控机制尚未完全阐明。本研究构建过氧化氢诱导的氧化应激模型及TRPV1敲除细胞，通过外源核桃肽EVSGPGLSPN（WNP-10）的干预调节，结合胞内及线粒体氧化应激敏感性、钙稳态、炎症因子、线粒体膜电位及呼吸功能的变化，探索外源核桃肽介导TRPV1缓解线粒体氧化应激的模式及动态平衡的维持机制，同时验证核桃肽是否能够改善东莨菪碱诱导的小鼠记忆损伤。本研究证明核桃源活性肽WNP-10能够显著提高PC12细胞中IPR3、Gnas、CaMKⅡδ、TRPV1的蛋白表达（P<0.05）；TRPV1敲除细胞证明WNP-10可能介导TRPV1调节胞内钙离子含量变化，结合TRPV1激活剂（辣椒素CAP）和抑制剂（辣椒平CPZ）的处理同样能够证明活性肽WNP-10能够促进胞内钙离子含量上升，但是WNP-10与激活剂CAP无协同效果。基于对TRPV1的调节，活性肽WNP-10能够缓解PC12细胞内氧化应激损伤，显著提高线粒体ATP水平，减少线粒体膜电位的降低，提高线粒体融合蛋白（MFN2和OPA1）的表达，降低线粒体分裂蛋白（MFF和DRP1）的表达（P<0.05）；同时，WNP-10对于过氧化氢引起的线粒体呼吸链损伤有良好的恢复作用。进一步地，WNP-10能够显著降低促炎因子（IL-8、TNF-α、IFN-γ、IL-18）的产生，提高抑炎因子（IL-10、TGF-β）含量，但结合CAP，CPZ及敲除TRPV1细胞的炎症因子变化可知，WNP-10对炎症因子的调节可能涉及到其他途径。小鼠体内试验证明核桃肽WNP-10能够改善东莨菪碱诱导的小鼠记忆损伤。研究结果对开发肽类神经保护因子具有重要科学意义。