基于“肝－胆汁酸－肠道菌群”轴研究白头翁汤治疗湿热泄泻的作用机制

Diarrhea is an important subject to be solved in the breeding. Baitouweng Tang is famous prescription for veterinary clinical treatment diarrhea, widely used, but its mechanism for the treatment of dampness-heat diarrhea syndrome is not clear. According to Traditional Chinese veterinary Medicine theory and clinical practice and the previous study which discover Baitouweng Tang treat the Diarrhea by adjusting the bile acid，the Baitouweng Tang as object, the intervention on dampness-heat diarrhea syndrome animal model, we analyze bile acid pathway related gene expression in liver tissue, bile acid quantitative monitoring and intestinal flora changes by using the use of Gene expression spectrum analysis, target metabolomics and macro genomics. After analyzing several of the integration of the above data study bile acid metabolism related gene expression in liver tissue, bile acid in the serum and liver tissue and intestinal flora between inner rule changes, we except to reveal the gene expression patterns changes, bile acid metabolism pathway, the relationship between intestinal flora changes and diarrhea, to clarify Baitouweng Tang in the treatment of diarrhea liver-bile acid-intestinal axis mechanism, focus on the future basic experiments and clinical experiments on the Baitouweng Tang treatment of intestinal flora as target accurately and single optimization, making it more effective pulsatilla decoction, giving full play to the advantages of Traditional Chinese Veterinary Medicine.

腹泻是目前养殖业中亟待解决的一个重要课题，白头翁汤是临床治疗湿热型腹泻的名方，但作用机制不明确。前期研究发现白头翁汤可以通过调节胆汁酸的代谢治疗湿热型腹泻，但具体胆汁酸如何调节机体以及与肠道菌群的相互作用不清楚。因此，本项目以白头翁汤为研究对象，建立湿热泄泻动物模型，以“胆汁酸靶标代谢监测”为切入点，拟采用基因表达谱分析、靶标代谢组学、宏基因组学等方法分析白头翁干预湿热泄泻大鼠肝组织中胆汁酸通路相关基因的表达，血清、肝及肠组织中胆汁酸组分的含量，肠道菌群的变化。通过对以上数据的相关及多重比较揭示胆汁酸通路相关基因表达、胆汁酸组分的含量及肠道菌群变化之间内在的规律及与湿热泄泻的关系，以阐明白头翁汤治疗湿热泄泻的 "肝－胆汁酸－肠道菌群"轴生物效应机制，为系统、全面阐释白头翁汤的作用机制、临床应用和湿热型腹泻治疗等方面提供依据。

本项目建立腹泻模型，通过疾病活动指数、炎性细胞因子、总胆汁酸、总胆固醇、组织病理学观察等指标评价白头翁汤对其治疗作用，白头翁汤组对细胞因子的回调效果接近空白对照组。腹泻模型组小鼠结肠部杯状细胞脱落坏死丢失，白头翁汤治疗能明显改善修复杯状细胞的坏死脱落，促进结肠部黏膜屏障的修复，可恢复结肠超微结构上的病理变化，与结肠组织病理切片的结果相符。运用UHPLC-MS/MS分析空白对照组、模型组、阳性对照组和白头翁汤组的33种胆汁酸水平，血清中，与空白组相比，模型组小鼠UDCA、HDCA、ωMCA、αMCA、βMCA、CA、GLCA、TβMCA水平显著升高，而DCA、TUDCA、THDCA、TCDCA、TDCA含量显著降低。白头翁汤治疗后，UDCA、HDCA、αMCA、βMCA、CA、GLCA浓度下降，部分胆汁酸特别是CA、αMCA 、βMCA恢复正常。模型组血清、胆汁、肝脏、粪便中的TCDCA、TUDCA、TωMCA、 TαMCA、TβMCA均升高，白头翁汤可降低TCDCA、TUDCA、TωMCA、TαMCA、TβMCA水平。白头翁汤能显著降低肝脏中CA:CDCA、CA:βMCA、CA:αMCA和CA:DCA比值，恢复肠道菌群中主要的优势菌群厚壁菌门（Firmicutes）、拟杆菌门（Bacteroidetes）、变形菌门（Proteobacteria）三个主要的门在门水平上的占比。对胆汁酸含量较高的16种胆汁酸和7个门水平的肠道菌进行相关性分析。厚壁菌门与拟杆菌门的负相关最为显著，同时厚壁菌门、软壁菌门、放线菌门及TM7门还与αMCA、βMCA、UDCA及CA等表现出不同程度的负相关，拟杆菌和脱铁杆菌门则相反。白头翁汤治疗可上调紧密连接蛋白，恢复遭破坏的结肠上皮细胞间的紧密连接，同时上调与短链脂肪酸代谢相关的G偶联蛋白受体蛋白，促进短链脂肪酸代谢。白头翁汤治疗后，相比模型组显著恢复了胆汁酸通路中25个基因，上调9个基因，显著恢复了FXR、TGR5、CYP7A1、FGF15各蛋白的表达量，ABCB11和RXRα表达量有升高趋势。本项目通过上述结果表明白头翁汤可以通过激活胆汁酸受体FXR及其通路基因，影响肠道细菌丰度、活性从而调节胆汁酸代谢以治疗湿热泄泻。