蓝靛果花色苷C-3-G抑制辐射诱导氧化损伤作用机制研究

Ionizing radiation can cause a variety of damages on the body, even induce chromosomal aberration and cancer. Hence it is urgent need to strengthen the protection against radiation induced damage and relieve radiation sickness, screening and evaluation efficient and harmfulless radiation protective factors from natural product resources have become a hot research area. Our previous studies indicated that anthocyanins of Lonicera caerulea var. edulis (ALC) have obvious protective effect against oxidative damage induced by ionizing radiation, and cyanidin-3-glucoside (C-3-G) was the best functional component of ALC. Based on the results of our previous studies, the objective of this project is to reveal the molecular mechanism of C-3-G’s radiation protective activity. Both in vivo model and in vitro model will be created, and studies focus on the regulation effect of C-3-G on important way of radiation induced oxidant damage, especially cell apoptosis and survival related signaling pathway. This project will determine the key signaling pathway which regulated by C-3-G to protect cells, the regulation pattern, the key signaling proteins and their position in the signaling pathway, through using of techniques of RNAi, Western blot and Real-time PCR. All the studies will completely reveal the protection mechanism of C-3-G on radiation induced oxidant damage. The results of this project will provide the theoretical foundation for the application research of anthocyanins as radiation protection agents, and promote the research on the effect of phytochemicals on health promotion and development of functional foods.

电离辐射可对机体造成多种损伤，严重时甚至引发染色体畸变，导致癌症等恶性疾病的发生。加强辐射防护，减轻放射病是当今亟待解决的重要课题。从天然产物活性成分中开发高效、低毒的新型防护因子已成为研究的热点。申请人前期研究发现蓝靛果花色苷对辐射诱导氧化损伤具有良好的防护作用，并经分离鉴定发现其最佳活性成分为矢车菊素-3-葡萄糖苷（C-3-G）。本项目以C-3-G为研究对象，重点围绕辐射损伤中“氧化应激诱导细胞凋亡”这一重要损伤防护途径，通过体内和体外模型，结合RNAi、WB和qPCR技术，明确C-3-G辐射防护的关键信号传导通路、调控模式、作用靶蛋白在信号传导通路中所处位置，从整体、组织、细胞和分子水平揭示C-3-G防护辐射诱导氧化损伤作用的机制。该研究将为花色苷类植物化学物在天然产物辐射防护剂的应用研究提供理论依据，对推动植物化学物在促进健康和功能性食品的开发研究起到积极的作用。

随着核工业和放射技术的发展，电离辐射与人们的关系越来越密切。电离辐射可对机体的造血系统、免疫系统、神经系统造成多种损伤。天然产物作为无毒无害的辐射防护剂，前景广阔。本研究以矢车菊素-3-O-葡萄糖苷为研究对象，对蓝靛果花色苷复合组分进行分离纯化得到C-3-G单一花色苷组分，对其进行体外清除自由基的测定，并对C-3-G的辐射防护功能及机制进行研究。（1）C-3-G的分离纯化：采用聚酰胺树脂层析和高效液相制备色谱技术分离纯化蓝靛果花色苷复合组分，得到C-3-G单一组分。通过高效液相分析测得制备样品纯度为95.74%。（2）C-3-G体外清除自由基活性研究。C-3-G对ABTS自由基、DPPH自由基、羟基自由基和超氧阴离子自由基清除活性均高于蓝靛果花色苷复合组分。其中，C-3-G对ABTS自由基和羟自由基的清除能力高于抗坏血酸。研究表明，C-3-G是一种良好的天然抗氧化剂和自由基清除剂。（3）C-3-G对辐射诱导小鼠氧化应激损伤的防护作用研究。通过研究小鼠体重、脏器指数、外周血白细胞数、骨髓细胞微核数和肝脏组织形态学变化分析以及血清中T-SOD活力、CAT活力、GSH水平和MDA含量的测定，研究C-3-G对辐射诱导小鼠氧化损伤的防护作用。结果表明，C-3-G可有效地提高辐射小鼠的免疫器官指数，改善小鼠肝脏形态学变化，增加小鼠外周血象白细胞数降低骨髓细胞微核率。C-3-G显著提高小鼠血清中抗氧化酶T-SOD、CAT活性，增加内源抗氧化物GSH水平，降低脂质过氧化物MDA的含量，从而抑制辐射诱导的氧化应激对细胞及组织造成的损伤。（4）C-3-G对小鼠辐射防护用机制研究。研究发现，辐射导致小鼠肝细胞核内Nrf2基因和蛋白及其下游的HO-1、GST、NQO1的基因和蛋白表达降低。与辐射组相比，C-3-G干预组小鼠总Nrf2及核内Nrf2基因和蛋白表达均显著增加，同时，HO-1、GST、NQO1的基因和蛋白表达也显著增加。结果表明，C-3-G上调Nrf2基因和蛋白表达，促进Nrf2的入核转运，激活下游抗氧化蛋白及二相解毒酶HO-1、GST及NQO1的基因和蛋白表达，激活Nrf2通路，从而抑制辐射对小鼠造成的氧化应激损伤。