外源益生菌对肠道拟杆菌和乳杆菌调控及对肝细胞脂质氧化损伤拮抗机制研究

The function of improving intestinal flora and antagonism to oxidation damage of Lactobacillus fermentum grx08, Lactobacillus casei grx12 and Lactobacillus rhamnosus hsryfm 1301 and their group has been confirmed by our previous research, and the regulation of Bacteroides and Lactobacillus in intestinal and the effect mechanism of antagonism of lipid oxidation damage to hepatocyte of L. fermentum grx08, L. casei grx12 and L. rhamnosus hsryfm 1301 and their group will be studied by using models of hyperlipidemia rat. The antagonistic effect of lipid oxidation damage to the hepatocyte is to be clarified by the effect of probiotics on serum and the expressing levels of related proteins and genes of lipid oxidation to the hepatocyte of rat models based on genomics, proteomics and immunohistology methods, and then the antagonistic effect mechanism is to be explored by combining with the research of the main regulatory molecules and the induced bacteria of the antagonistic effect of lipid oxidation damage to hepatocyte in vitro by which external probiotics and the target bacteria of probiotics intervene. The aim of this project is to explore the mechanism of probiotics of antagonistic effect of lipid oxidation damage to hepatocyte at cellular, genetic and protein levels and the target bacteria, which will provide theoretical and technical support for fermented milk with probiotics and other functional food development.

本项目以筛选获得的具有改善肠道菌群结构和拮抗氧化损伤功能的益生菌发酵乳杆菌grx08、干酪乳杆菌grx12、鼠李糖乳杆菌hsryfm 1301及其组合为研究对象，利用高血脂大鼠动物模型，围绕外源益生菌对大鼠肠道拟杆菌和乳杆菌水平与构成的调控及肝细胞脂质氧化损伤拮抗效应相关机制问题，应用基因组学、蛋白质组学和免疫组学相结合的方法，研究益生菌对模型动物血清、肝细胞脂质氧化损伤相关蛋白、基因表达水平的影响，探明肝细胞脂质氧化损伤拮抗效应；结合外源益生菌及其作用的目标菌群对体外肝细胞脂质氧化损伤拮抗的主要调节分子及诱导菌群的研究，以期从肠道目标菌群和细胞、基因及蛋白水平上探索益生菌对肝细胞脂质氧化损伤拮抗效应的作用机制，为益生菌发酵乳等功能食品开发提供理论和技术支持。

本项目研究了外源益生菌（Lactobacillus fermentum grx08、L. casei grx12及L. rhamnosus LV108等）对高脂饮食模型大鼠肠道菌群、血液和肝脏脂肪水平、肝脏氧化指标、脂质代谢基因转录以及肝脏蛋白表达的影响。结果表明，外源益生菌及其发酵乳能明显缓解高脂饮食造成的不良影响，减轻大鼠肥胖，显著降低大鼠血清的TG、TC、LDL-C水平，并提高HDL-C水平。外源益生菌还能提高高脂饮食大鼠肠道菌群多样性，相关性分析表明大鼠肠道内梭菌属、大肠杆菌属的相对丰度与大鼠血清TC及LDL-C含量为显著正相关，乳酸杆菌属与大鼠血清TG和LDL-C显著负相关。RT-PCR结果表明，外源益生菌促进了胆固醇逆转运，抑制肝脏中糖类向脂肪的转化，降低脂质合成，减少了脂肪的堆积，减轻了肝脏细胞的脂肪变性，肝细胞脂肪空泡减少、变小；肝脏SOD、GSH、GSH-Px、CAT水平显著升高，抗氧阴离子自由基、羟自由基、NO自由基和MDA水平均显著低于模型组，表明外源益生菌发酵乳可有效降低大鼠肝脏的脂肪水平，提高大鼠肝脏部分抗氧化酶活力和非酶抗氧化物质的含量，清除自由基、脂质过氧化产物，缓解肝脏受损的程度。肝脏蛋白质组研究发现，外源乳酸菌发酵乳调控大鼠非酒精性脂肪肝病相关的26个蛋白，主要涉及多种氨基酸的合成与代谢信号通路、糖酵解/糖异生信号通路、过氧化物酶体信号通路及P53信号通路等37条信号通路，表明外源益生菌发酵乳还可以通过抑制细胞凋亡和平衡脂质代谢来拮抗肝脏脂质过氧化。本项目以高脂饮食大鼠为模型，证实了外源益生菌能够减少脂肪摄入、降低血脂和肝脏脂肪水平，同时调整肠道菌群、调控氧化酶活力和非酶抗氧化物质，抑制细胞凋亡和平衡脂质代谢，以多种机制拮抗肝细胞脂质氧化损伤。