Effect of Fuzi Lizhong Pill on Intestinal Flora of Spleen Yang Deficiency IBS-D Rats Based on High-Throughput Sequencing Technique
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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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Key words:
- Fuzi Lizhong Pill /
- spleen Yang deficiency syndrome /
- diarrhea /
- IBS-D /
- intestinal flora
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图 1 稀释曲线图(a)和物种累积曲线图(b)
注：A1~A10.空白组10个样本；B1~B10.模型组10个样本；C1~C10. 附子理中丸低剂量组10个样本；D1~D10.附子理中丸高剂量组10个样本
图 2 距离矩阵热图和主坐标分析图
注：FLZP.附子理中丸; A1~A10.空白组10个样本；B1~B10.模型组10个样本；C1~C10.附子理中丸低剂量组10个样本；D1~D10.附子理中丸高剂量组10个样本
图 3 各组菌群相对丰度值柱形图
注：与模型组比较，*P < 0.05，**P < 0.01；与空白组比较，▲P < 0.05，▲▲P < 0.01；FLZP.附子理中丸。x±s, n=10。
表 1 造模后各组大鼠MDA含量和SOD活力比较(x±s，n=6)
组别 MDA/ (nmol·mL-1) SOD/ (U·mL-1) 空白大鼠(空白组) 7.000±4.274 137.436±19.006 造模大鼠(模型组) 13.833±4.065* 106.068±16.260* 造模大鼠(附子理中丸低剂量组) 17.333±9.400* 114.103±16.730* 造模大鼠(附子理中丸高剂量组) 17.000±9.612* 96.154±12.337** 注：与空白组比较，*P < 0.05，**P < 0.01。 
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表 2 α多样性指数统计分析结果表(x±s，n=10)
组别 Ace Chao1 Shannon Simpson 空白组 497.224±17.238 506.983±19.236 6.420±0.436 0.964±0.015 模型组 441.113±41.609** 450.960±41.695** 5.543±0.461** 0.933±0.030 附子理中丸低剂量组 469.790±34.140# 474.911±34.149* 5.825±0.839 0.923±0.086 附子理中丸高剂量组 457.677±27.031** 463.692±29.316** 5.136±0.929** 0.877±0.064**# 注：与空白组比较，*P < 0.05，**P < 0.01；与模型组比较，#P < 0.05。
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表 3 标识性菌群分析结果表(n=10)
菌群 LDA得分
(模型组vs空白组)变化趋势
(模型组vs空白组)变化趋势
(FLZP低剂量组vs模型组)变化趋势
(FLZP高剂量组vs模型组)拟杆菌门(Bacteroidetes) 3.850 ↓ ↑ ↑ 拟杆菌纲(Bacteroidia) 3.850 ↓ ↑ ↑ 梭菌_1科(Clostridiaceae_1) 3.403 ↑ ↓ ↓ Muribaculaceae 3.697 ↓ ↑ ↑ 狭义梭菌属(Clostridium_sensu_stricto_1) 3.406 ↑ ↓ ↓ 布劳特氏菌属(Blautia) 3.767 ↑ ↓ ↓ 苏黎世杆菌属(Turicibacter) 3.264 ↑ ↓ ↓ 乳杆菌属(Lactobacillus) 4.247 ↑ ↑ 注：FLZP.附子理中丸。
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