大蒜素减缓膳食丙烯酰胺及其代谢产物环氧丙酰胺基因损伤机制研究

Acrylamide (AA) is an important endogenous contaminant in food, which has neurotoxic to human body, and has reproductive toxicity, genotoxicity and carcinogenicity to experimental animals. The in vivo metabolite glycidamide (GA) is more carcinogenicity, mutagenicity to experimentals animals. In our previous study, we found that allicin could significant reduce the damage induced by AA and GA, especially in oxidative stress, immune systems and DNA damage. But the mechanisms are not clear. Based on the results of genome microarray expression technology, we found that AA and GA had significant influence on the gene regarding to the cell death, oxidative damage, immune system process and cancers. Is it possible for allicin to relieve the gene damages induced by AA and GA through the expression of the corresponding genes? On the basis of the preliminary studies, the project aims to screen and verify the target genes of AA and GA induced genotoxicity based on the whole genome microarray expression technology, Real-time PCR and Western-blot technology, to analyze the change of adducts of hemoglobin and DNA by HPLC-MS/MS method, to explore the intevention mechanisms of allicin against AA and GA induced gene damage from gene and protein aspects based on gene silence technology, Western-blot and molecular dynamics simulations technology. These results can reveal the intevention mechanisms of allicin against AA and GA induced gene damages and lay the foundation to further develop and utilize allicin as a natural AA antidote, and meanwhile to provide the theory and new strategies to study the protective mechanisms of the other natural AA antidote.

丙烯酰胺(AA)是一种重要的食品内源污染物，与其体内环氧化代谢产物环氧丙酰胺(GA)均对人体健康具有潜在影响。项目组前期发现，大蒜素对AA及GA引起的毒性损伤具有明显的抑制效果，特别是在氧化应激、免疫、DNA损伤等方面作用尤为突出，但其作用机制尚不明确。通过基因芯片扫描显示，AA及GA对细胞凋亡、氧化损伤、癌症、免疫等相关基因产生明显的上调或下调作用，大蒜素是否通过影响这些基因的表达，进而缓解AA/GA引起的基因损伤呢？因此，本项目在此基础上，借助全基因组芯片表达谱、Real-time荧光定量PCR、Western-blot等技术对AA及GA致基因毒性靶基因进行筛选及验证；利用HPLC-MS/MS从加合物角度解释大蒜素作用机制；利用基因敲除、Western-blot、分子动力学模拟等方法从基因和蛋白水平共同验证大蒜素作用的分子机制，全面揭示大蒜素对AA及GA致基因损伤的干预机制及调控机理。

天然植物源成分对食品中潜在致癌物——丙烯酰胺（Acrylamide，AA）的毒性保护是现今食品安全研究的热点之一。本项目按照计划书内容，研究发现AA及其代谢产物环氧丙酰胺（glycidamide，GA）对小鼠机体具有明显的氧化损伤作用，并介导了炎症的发生，AA对机体产生的氧化损伤和致癌性与GA生成过程及强氧化性都有着密不可分的关系。GA致氧化损伤、致癌作用可能主要通过抑制抗氧化物酶活性、直接攻击DNA、通过基因调控降低DNA修复能力和修复效率以及降低基因稳定性等几个方面实现。通过大鼠BRL-3A肝细胞实验发现，AA可通过降低大鼠肝细胞BRL-3A中SOD、GST及GSH-Px的活力使其产生明显的氧化损伤；通过显著升高BRL-3A细胞中ROS、8-OHdG含量而对细胞造成DNA氧化损伤；同时，显著促进大鼠肝细胞BRL-3A促炎因子TNF-α和IL-6的分泌并抑制抗炎因子IL-10的分泌，致使肝细胞发生炎症反应。在AA和GA调控的差异基因中，我们发现细胞色素P450的外源化合物代谢、MAPK信号通路、NF-κB信号通路等与氧化应激、免疫应答等相关的重点通路显著受到了AA和GA的调控。进一步研究Ras/MAPK信号转导通路发现AA对肝细胞的损伤机制可能通过调控其生长增殖，引发炎症反应并导致DNA损伤断裂和过度应激反应，从而导致肝细胞损伤。大蒜素在发挥对AA所致大鼠肝细胞BRL-3A毒性保护作用时，大蒜素明显调控了细胞色素P450的外源化合物代谢及MAPK信号通路，大蒜素的保护机制可能通过调控Ras/MAPK信号转导通路，进而调控细胞生长增殖，降低DNA和氧化损伤，减轻应激反应，从而发挥对肝细胞损伤的保护，最终减缓肝细胞损伤的发生和发展。该研究是国内外率先从基因损伤角度开展对大蒜素抑制AA毒性作用的研究，是今后深入挖掘大蒜素的潜在作用机制的基础。项目共发表论文9篇，其中SCI论文6篇，EI收录论文1篇，中文核心论文2篇；获学术奖励1项；培养研究生6人。