高通量测序技术研究蚕沙对湿阻中焦证大鼠肠道菌群的影响

Investigating effect of Faeces Bombycis on intestinal microflora in rats with syndrome of damp retention in middle-jiao by high-throughput sequencing

研究蚕沙对湿阻中焦证大鼠肠道菌群的影响,并从蚕沙含有的微生物角度探讨其调节肠道菌群的作用机制。采用酶联免疫吸附试验(ELISA)法测定血清中ADH,CRP及空肠中AQP3的含量;利用Illumina Miseq平台对大鼠粪便及蚕沙药材进行高通量测序。结果显示,与正常组比较,模型组大鼠ADH,CRP含量明显升高(P<0.05),AQP3含量明显降低(P<0.05),给予蚕沙后ADH,CRP,AQP3均回调。大鼠粪便测序结果显示,模型组大鼠肠道菌群ACE,Chao1,Shannon指数最低;同时,与正常组比较,模型组从门到属水平均发生显著变化,且模型组变形菌门、不动杆菌属、厌氧杆菌属、绿脓杆菌属、副杆状菌属明显增加(P<0.05),而Marvinbryantia菌属明显减少(P<0.05);与模型组比较,蚕沙低、高剂量组变形菌门明显降低(P<0.05),蚕沙低、中、高剂量组不动杆菌属、厌氧杆菌属、绿脓杆菌属、副杆状菌属明显降低(P<0.05),且蚕沙高剂量组毛绒厌氧杆菌属、Intestinimonas、Marvinbryantia明显增加(P<0.05);蚕沙药材测序显示,勒克菌属、泛菌属、短状杆菌属、西姆惠菌属、哈特曼杆属、克雷伯菌属、沙雷菌属、橙单胞菌属、类芽孢杆菌属、芽孢杆菌属在蚕沙药材中相对丰度高,而在大鼠粪便中基本不存在或存在较少。因此,蚕沙可能通过平衡肠道菌群发挥治疗湿阻中焦的作用,而这种作用可能与蚕沙药材中微生物的代谢产物有关。

The aim of this paper was to investigate the effect of Faeces Bombycis(FB)on the intestinal microflora in rats with syndrome of damp retention in middle-jiao,and to explore its mechanism in regulating intestinal microflora from the perspective of microorganisms contained in FB.The contents of antidiuretic hormone(ADH)and C-reactive protein(CRP)in serum and aquaporin 3(AQP3)in jejunum were determined by enzyme-linked immunosorbent assay(ELISA).Illumina Miseq platform was used for high-throughput sequencing of the rat feces and FB.The ELISA results showed that as compared with the normal control group,the contents of ADH and CRP in the model group were significantly increased(P<0.05),and the content of AQP3 was significantly decreased(P<0.05).After drug administration,the ADH,CRP and AQP3 contents were recovered.Sequencing of rat feces showed that the ACE,Chao1 and Shannon indexes of the intestinal microflora were the lowest in the model group.As compared with the normal control group,the levels from phylum to genus were all significantly changed in model group,and Proteobacteria,Acinetobacter,Anaerobacter,Pseudomonas,and Parabacteroides levels were significantly increased(P<0.05),while Marvinbryantia level was significantly decreased(P<0.05).As compared with the model group,Proteobacteria was significantly decreased in the FB low and high dose groups(P<0.05),and Acinetobacter,Anaerobacter,Pseudomonas,Parabacteroides levels were significantly decreased in the low,medium and high dose groups(P<0.05),while Lachnoanaerobaculum,Intestinimonas and Marvinbryantia were increased significantly in the high dose group(P<0.05).Sequencing analysis of FB showed that the relative abundance of Leclercia,Pantoea,Brachybacterium,Shimwellia,Hartmannibacter,Klebsiella,Serratia,Aurantimonas,Paenibacillus and Bacillus was high in the FB,but they were basically not present or little in the rat feces.In conclusion,FB may play a role in the treatment of"syndrome of damp retention in middle-jiao"by balancing the intestinal microflora,and this effect may be related to the metabolites of microorganisms in the FB.