基于转录组联合代谢组的抗氧化剂原花青素调节钙代谢异常机理的研究

Aging-induced abnormal calcium metabolism can cause osteoporosis, which is one of important health problems in ageing society. Abnormal calcium metabolism is closely related with oxidative stress. Antioxidant proanthocyanidins in which fruits and vegetables are rich could prevent low bone mass. However, its preventive mechanism is still not clear, especially molecular mechanism, and the explanation for its mechanism based on antioxidant effect has rarely been addressed. Calcium metabolism is intricate and can be affected by many factors. Unfortunately, it is difficult to understand fully the mechanism for the regulation of proanthocyanidin on abnormal calcium metabolism m. Therefore, this study will explore the regulation of the short- and long-term prevention of proanthocyanidin B2 on abnormal calcium metabolism as a whole and its molecular mechanism in aging mice induced by D-galactose, based on free radical biology theoretics. This regulation will mainly involve in the intestinal calcium absorption and the bone calcium metabolism. One important aim is to analyze the relationship between antioxidant of proanthocyanidin and calcium metabolism regulation and to clarify target tissue regulated by proanthocyanidin. The quantitative reverse transcription PCR and western blotting will be used to explore its elementary molecular mechanism. Furthermore, the transcriptomics technology (gene expression chips) will be used to screen the key modified genes and the related single transduction pathways, and metabolomics approach will also be used to search for potential metabolite biomarkers. It is important to take confluence analysis of the data from transcriptomics and metabolomics approach in order to try to elucidate synthetically its complicated molecular mechanism at multilevel. Subsequently, some key results from transcriptomics and metabolomics approach will be validated. The results form this project will provide a certain theoretical direction for nutritional prevention of low bone mass in the old people as well as for developing the related functional foods.

衰老性钙代谢异常引发骨质疏松是老龄化社会面临的健康问题。钙代谢异常与氧化应激密切相关，果蔬中强抗氧化剂原花青素可干预骨量降低，但机理尚不明确，特别是分子机制，且鲜有研究从抗氧化角度阐释其机理。钙代谢复杂且影响因素多，单凭某个研究技术很难较全面地认识其调节机理。本项目通过小鼠衰老模型从抗氧化角度整体性（主要关注肠钙吸收与骨钙代谢等相关的多个环节）地探索原花青素二聚体B2短、长期干预钙代谢异常的调控作用，解析原花青素抗氧化作用与钙代谢调控之间关系，明确其作用的重要调节环节。通过定量RT-PCR与蛋白印迹技术先初探其分子机制，再重点通过转录组技术（基因表达芯片）与代谢组技术（LC-MS）分别进行基因功能富集分析与代谢标志物筛选，并进行多组学的整合解析与验证，从而多层次较全面综合地阐释相关调节作用的可能分子机理。本研究为老年人骨量减少的营养干预及有关保健食品的开发提供理论依据。

抗氧化剂原花青素可干预骨量降低，但其机理尚不完全明确。本项目以D-半乳糖诱导衰老小鼠为模型，通过原花青素B2干预发现其具有提高D-半乳糖小鼠的抗氧化能力抵御氧化损伤，降低炎症因子水平，且可增加股骨钙含量与股骨机械强度，改善D-半乳糖诱导的钙代谢异常。钙代谢复杂且影响因素多，单凭某个研究技术很难较全面地认识其调节机理。为了进一步探究其作用机理，本项目从代谢产物鉴别、差异代谢通路、差异表达基因、肠道菌群结构变化等方面进行了研究。采用IT-TOF-MSn多级质谱技术分析了短期补充原花青素B2在小鼠体内的代谢产物及其组织中的分布，发现了45 种原花青素B2代谢产物（其中16种为新产物）。B2在小鼠体内代谢形式复杂多样，且组织分布广，在肠、肝等组织中发现代谢产物种类最多，原花青素B2 一部分直接被吸收进入肝脏代谢，另一部分亦可通过肠道微生物分解为小分子后被吸收利用，且主要通过尿液途径排泄。采用代谢组学技术对小鼠血清进行数据采集并结合多元统计分析结果显示泛酸、丙酮酸、亚油酸、α-酮戊二酸、胆酸等标志性代谢产物发生变化，其抗衰老机制涉及糖代谢、脂类代谢、胆酸代谢等多条代谢通路。采用Illumina Hiseq测序技术采集了小鼠股骨头RNA转录组学数据，发现原花青素干预组、衰老模型组与对照组之间的差异表达的基因主要涉及成骨细胞功能、细胞基质与MMP、IGF、RANK/RANKL/OPG等信号通路。采用16S高通量测序技术发现，原花青素干预组可显著降低衰老模型小鼠肠道菌群的厚壁菌门/拟杆菌门比值，显著增加Roseburia、 Lachnospiraceae与Bacteroides的相对丰度，减少Ruminiclostridium、 Bifidobacterium与 Blautia的相对丰度。通过对代谢组、转录组、肠道菌群结构等综合解析发现，原花青素B2具有显著干预衰老性钙代谢异常的作用，这种作用可能与调节肠道菌群抑制炎症反应，调控能量代谢，干预骨形成作用相关基因表达有关。本项目研究发现加深对原花青素抗衰老与调控钙代谢机理的认识，为老年人钙代谢异常的营养干预及有关保健食品的开发提供理论依据。