鱼皮抗氧化肽对PM2.5中铅锰联合致神经元氧化应激的保护作用机制

Owing to PM 2.5 contains the heavy metals such as Lead and Manganese which cause the production of neurons oxidative stresses, air pollution has arisen increasing attentions of people on PM 2.5. It is press to find a solution of decreasing oxidative damages of organisms arising from the stresses. The existing research found that antioxidant peptide from fish skin plays a protective role in oxidative stress of neurons leading by joint Lead and Manganese. However, the protective mechanism has been rarely reported. This work prepared antioxidant peptide from fish skin with grass carp skin as experimental material, screened active components using evaluations systems of in vitro antioxidant activity and neuroprotective activity. Furthermore, the authors constructed a Lead and Manganese joint exposure based SH-SY5Y cell oxidative stress model. By using this model, the capabilities that antioxidant peptide from fish skin resists oxidative stresses of neurons inducing by joint Lead and Manganese were investigated. In addition, The differences of oxidative stresses of neurons induced by Lead , Manganese, and joint Lead and Manganese were compared respectively by using techniques including transmission electron microscope(TEM), flow cytometry, confocal immunofluorescence and reverse transcription-polymerase chain reaction(RT-PCR) based on removal of free radicals, activation of Nrf2/ARE pathways, protection of mitochondria structures and functions , and restriction of apoptosis. Besides, this study revealed the protective mechanism of antioxidant peptide on oxidative stresses of neurons induced by joint Lead and Manganese containing in PM 2.5 in terms of cell, protein and gene. This research provides a scientific basis for realizing high waste utilization in aquatic processing industry and application of antioxidant peptide in the harms control of PM 2.5.

近年来PM2.5污染肆虐，据研究其上所附重金属如铅锰可诱导神经元产生氧化应激，如何降低应激对机体的氧化损伤是亟需解决的问题。前期研究发现鱼皮抗氧化肽对铅锰联合诱导的神经元氧化应激有保护作用，但其作用机制尚未明晰。本项目以草鱼鱼皮为实验材料制备鱼皮抗氧化肽，利用体外抗氧化活性和神经保护活性评价体系筛选活性组分；进一步构建以铅锰联合染毒的SH-SY5Y细胞氧化应激模型，研究鱼皮抗氧化肽抗铅锰联合诱导神经元氧化应激的能力；应用透射电镜、流式细胞术、免疫荧光共聚焦、RT-PCR等技术，从清除自由基、激活Nrf2/ARE通路、保护线粒体结构与功能、抑制细胞凋亡四个方面，研究比较铅锰联合和单独诱导神经元氧化应激的差异，在细胞-蛋白-基因三个层次上阐明抗氧化肽对PM2.5中铅锰联合致神经细胞氧化应激的保护机制。研究可为抗氧化肽在PM2.5危害控制中的应用提供科学依据，并有利水产加工废弃物的高值化利用。

针对PM2.5中重金属危害所导致的氧化应激，应采取何种措施进行干预，从而减少环境污染对人体的损害，是一个迫切需要解决的问题。本项目以草鱼皮为实验材料，通过碱性蛋白酶酶解及多种分离纯化技术获得三种抗氧化肽，鉴定GPH-E21、GPH-E4和GPH-E5的一级序列结构和相对分子量分别为：Pro-Tyr-Ser-Phe-Lys (640.54 u)、Gly-Phe-Gly-Pro-Glu-Leu (618.50 u) 和Val-Gly-Gly-Arg-Pro (484.11 u)。定期收集测试大气PM2.5中重金属铅、锰浓度，在此基础构建以铅锰联合染毒的SH-SY5Y细胞氧化应激模型，选取草鱼皮中体外抗氧化较强的抗氧化肽GPH-E21，作为拮抗铅锰联合诱导神经元氧化应激的保护剂。通过检测细胞凋亡率和ROS水平确定铅锰单独染毒和联合染毒的浓度，设计具体实验处理组，结合细胞存活率、ROS水平、SOD活力和GSH-Px活力指标比较分析铅锰联合染毒和单独染毒以及抗氧化肽GPH-E21保护处理在清除自由基方面的差异，说明中高浓度的GPH-E21抗氧化肽能够有效清除自由基，保护细胞免受氧化损伤，减少对细胞增殖的抑制。结果表明GPH-E21抗氧化肽对细胞的保护可能是上调了体系中HO-1、NQO1和Nrf2的基因，促进其表达，同时能够激活Nrf2核转位；结合线粒体内MDA、NO、iNOS、Ca2+、细胞色素C水平和线粒体膜电位指标发现铅锰染毒对线粒体的功能造成严重损伤，影响细胞的正常活动，经GPH-E21抗氧化肽预处理后实现线粒体功能的保护，减少了对线粒体的损伤；通过研究凋亡相关蛋白的表达发现铅锰染毒使胞浆中Bax和Caspase-3的表达升高，Bcl-2的表达降低，GPH-E21抗氧化肽保护组胞浆中Bax和Caspase-3的表达低于铅锰染毒组。结合以上三个方面，在细胞-蛋白-基因三个层次上说明铅锰联合染毒相比单独染毒对神经元造成损伤显著，并阐明抗氧化肽GPH-E21对PM2.5中铅锰联合染毒致神经氧化应激的保护机制，抗氧化肽可抑制细胞内ROS的产生和脂质过氧化，提高细胞内SOD和GSH-Px活力，降低细胞色素C表达水平，改善线粒体活动，促进Nrf2核转位以及NQO1和HO-1蛋白的表达，从而抑制细胞凋亡，减少细胞损伤。本研究可为食源性抗氧化剂干预环境带来的健康影响提供科学依据。