基于肠道微生物群研究水飞蓟宾治疗非酒精性脂肪肝的作用机制

Being related to the pathogenesis of non-alcoholic fatty liver disease (NAFLD), gut microbiota modulation may act as a possible new therapeutic strategy for NAFLD. As a flavonolignan, silybin(Sil) is therapeutically used for treating NAFLD in china, due to its anti-oxidant and hepatocyte protection activity. Previous studies have described its potent antibacterial activity, whether Sil has any effects to gut microbial is still unknown. Our previous study demonstrated that, supplementation of FOS could significantly improve pharmacological activity of Sil in prohibiting the progression of NAFLD; Sil also exhibited inhibitory effect to Bacteroides, Bifidobacterium in vitro. To study effects of Sil on intestinal microbial ecology in mice, we would perform a variety of techniques (PCR-DGGE, real-time quantitative PCR, etc). The new knowledge associated with regulation of Sil in gut microbiota, we would be in a better position to improve therapies for NAFLD, with combined prebiotics, probiotics. To elucidate molecular mechanism of Sil in mediating endotoxin-induced inflammation, we hope to further study effects of Sil on the protective LPS tolerance, LPS- TLR4 and CpG-DNA-TLR9 signaling pathway. These findings would provide new theoretical basis for development of Sil in NAFLD treatment.

通过调控肠道微生态平衡治疗非酒精性脂肪肝（NAFLD）是具有可行性的新途径。一种黄酮类化合物-水飞蓟宾（silybin, Sil），具有抗氧化、保护肝细胞膜作用而广泛用于NAFLD治疗，有研究报道Sil对某些细菌具有抑制作用，但是其对肠道菌群的影响尚未报道。前期工作我们发现微生物调节剂果寡糖（FOS）能明显提高Sil对NAFLD的疗效，体外试验表明Sil能够影响双歧杆菌、拟杆菌等细菌的生长。本项目拟针对肠道微生物这一新的靶点，通过PCR/变性梯度凝胶电泳(DGGE)、实时荧光定量PCR等技术探讨Sil治疗NAFLD新的作用机制。指导Sil临床应用中，选择适宜的联用药物。并进一步研究Sil对内毒素耐受、LPS-TLR4和CpG-DNA-TLR9下游信号通路的调节作用，以阐明Sil干扰肠源性内毒素信号通路抑制炎性病变的分子机制，最终为Sil临床应用和结构优化提供实验和理论依据。

肠道菌群紊乱，特别是拟杆菌门与厚壁菌门比例失调，与非酒精性脂肪肝(Nonalcoholic fatty liver disease, NAFLD)的发生密切相关，另外，该类疾病个体也表现出的肠道微生物代谢功能的变化，和内毒素水平增加。一种黄酮类化合物-水飞蓟宾（Silybin, Sil），广泛用于NAFLD治疗，其在治疗NAFLD过程中是否能影响肠道微生态平衡，尚未报道。因此，本项目研究了Sil对疾病治疗个体肠道菌群的影响，以及其对肠道细菌代谢物和内毒素激活的信号通路的影响，揭示了新的作用机制。.取药物干预的小鼠盲肠内容物，采用 illumina miseq平台进行测序后，运用 QIIME、R、RDPTools 等程序完成数据分析。发现低剂量(30mg/kg)给药干预时，小鼠肠道微生物群多样性与丰富度均显著增高；中高剂量Sil（100 mg/kg和300mg/kg）干预后，高脂饮食小鼠肠道菌群多样性与丰富度均变化不显著。Sil对NAFLD小鼠肠道菌群组成发挥显著调节作用。例如，Sil给药处理均显著降低了高脂饮食小鼠体内厚壁菌门丰富度，而显著增加了拟杆菌门比例。属水平上，相对丰度排在前10的属中，Sil显著降低了高脂饮食小鼠肠道内的优势菌Firmcutes spp (Lachnoclostridium、Lactobacillus、Lachnospiraceae)丰富度，且与正常组差异不显著；剂量依赖的增加了高脂饮食小鼠体内益生菌Bacteroidales_ S24-7_group、Bacteroidetes丰富度。.Sil可能通过调节初级胆酸转变为去氧胆酸、以及长链脂肪酸的代谢，使肠道微生态趋向平衡；促进肠内菌群代谢产生短链脂肪酸，抑制血脂的合成，改善胰岛素的敏感等，从而有利于疾病恢复。Sil不能通过调节TLR4、MyD88和TRIF蛋白表达而抑制LPS激活引起的NF-κB活性。.调节肠道微生态平衡是治疗NAFLD有效可行的新途径，本项目通过高脂饮食诱导小鼠肠道微生物群紊乱，选择益生元果寡糖(Fructooligosaccharide, FOS)优化NAFLD小鼠肠道微生物群，研究FOS对Sil治疗高脂诱导的NAFLD的增效作用。发现FOS（2000 mg/kg)对Sil增效作用最具有经济性、有效性和可行性。为临床有效提高Sil疗效而选择合适的FOS剂量提供理论依据.