高通量测序技术研究附子理中丸对脾阳虚IBS-D大鼠肠道菌群的影响

Effect of Fuzi Lizhong Pill on Intestinal Flora of Spleen Yang Deficiency IBS-D Rats Based on High-Throughput Sequencing Technique

    摘要
  • 摘要: 目的   研究附子理中丸对脾阳虚腹泻型肠易激综合征(IBS-D)大鼠肠道菌群的影响和作用机制，同时探讨脾阳虚证与肠道菌群的关系。方法   选取60只雄性SD大鼠随机平均分为空白组、模型组、附子理中丸低剂量组、附子理中丸高剂量组。空白组正常饲喂，模型组和附子理中丸低、高剂量组采用“饮食失节+劳倦过度+番泻叶灌胃”复合因素法造模21 d，建立大鼠脾阳虚IBS-D模型。造模成功后，附子理中丸低、高剂量组给予附子理中丸21 d。给药结束后采集粪便样本，提取粪便样本总DNA，根据细菌16S rRNA V3~V4区设计引物进行扩增，利用Illumina Miseq平台进行高通量测序。结果   与空白组相比，模型组拟杆菌门(Bacteroidetes)、拟杆菌纲(Bacteroidia)、Muribaculaceae的相对丰度显著下降(P < 0.05)，梭菌科(Clostridiaceae)、狭义梭菌属(Clostridium_sensu_stricto_1)、苏黎世杆菌属(Turicibacter)、布劳特氏菌属(Blautia)、乳杆菌属(Lactobacillus)的相对丰度显著升高(P < 0.01)。与模型组相比，附子理中丸给药组的拟杆菌门(Bacteroidetes)、拟杆菌纲(Bacteroidia)、Muribaculaceae的相对丰度显著升高(P < 0.05)，梭菌科(Clostridiaceae)、狭义梭菌属(Clostridium_sensu_stricto_1)、苏黎世杆菌属(Turicibacter)、布劳特氏菌属(Blautia)的相对丰度显著下降(P < 0.01)。附子理中丸给药组乳杆菌属(Lactobacillus)相对丰度升高，附子理中丸高剂量组具有显著性差异(P < 0.05)。结论  附子理中丸可以促进益生菌乳杆菌的生长和拟杆菌、苏黎世杆菌、布劳特氏菌向正常组回调。对肠道菌群特别是与炎症/免疫反应密切相关菌群的调节，是附子理中丸治疗脾阳虚IBS-D腹泻的重要作用机制。

     

    Abstract: OBJECTIVE  To research the effect and mechanism of Fuzi Lizhong Pill (FLZP) on intestinal flora of spleen Yang deficiency IBS-D rats, and to explore the relationship between intestinal flora and spleen Yang deficiency in rats. METHODS  60 male SD rats were selected and randomly divided into normal group, model group, FLZP low dose group and FLZP high dose group. Rats in normal group were fed normally. The spleen Yang deficiency IBS-D model were established by using the combined factor modeling method of "irregular diet + tired excessive + senna lavage" for 21 days. After successful modeling, FLZP was administrated in rats of FLZP low and high dose groups for 21 days. Then feces samples were collected from rats. Total DNA was extracted from these feces samples. Primers were designed according to the V3-V4 region of bacterial 16S rRNA for amplification. Finally, Illumina Miseq platform was used for high-throughput sequencing. RESULTS  Compared with normal group, the relative abundance of Bacteroidetes, Bacteroidia and Muribaculaceae in model group significantly reduced (P < 0.05) and the relative abundance of Clostridiaceae, Clostridium_sensu_stricto_1, Turicibacter, Blautia and Lactobacillus in model group significantly increased (P < 0.01). Compared with model group, the relative abundance of Bacteroidetes, Bacteroidia and Muribaculaceae in FLZP low and high dose groups significantly increased (P < 0.05) and the relative abundance of Clostridiaceae, Clostridium_sensu_stricto_1, Turicibacter, Blautia in FLZP low and high dose groups significantly reduced (P < 0.01). The relative abundance of Lactobacillus increased in FLZP low and high dose groups, and significantly in FLZP high dose group (P < 0.05). CONCLUSION   FLZP can increase the relative abundance of probiotics Lactobacillus and recover the relative abundance of Bacteroidetes, Turicibacter and Blautia. The regulation of intestinal flora that is closely related to inflammation and immune system, is the important mechanism of FLZP in treating spleen Yang deficiency IBS-D diarrhea.

     

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