寒凝血瘀证大鼠的肠道菌群变化与粪便代谢特征分析

目的:探讨寒凝血瘀状态下大鼠肠道菌群多样性及粪便中内源性代谢物的变化,并考察与寒凝血瘀证密切相关的肠道菌群、代谢物、代谢通路。方法:采用冰水浴诱导的大鼠寒凝血瘀证模型,利用16S rRNA基因测序技术联合UPLC-TOFMS的粪便代谢组学技术,将扰动的肠道菌群与内源性生物标记物进行关联分析。结果:基于Illumina Mi Seq平台,发现与空白组大鼠相比,寒凝血瘀证模型组大鼠中Firmicutes显著上调(P0.05),Bacteroidetes显著下调(P0.01),显著差异的属有23个。确定了7个与寒凝血瘀证相关的粪便代谢物,涉及到的3个相关性最强的代谢通路,分别为缬氨酸、亮氨酸和异亮氨酸生物合成,泛酸和辅酶A生物合成,组氨酸代谢。另外,肠道菌群的属与粪便代谢物有强相关性。结论:16S rRNA的高通量基因测序技术与代谢组学技术的联用可用于评价寒凝血瘀证的病理机制。

Analysis of Intestinal Flora Diversity and Fecal Metabolic Characteristics in Rats with Cold Coagulation and Blood Stasis Syndrome

Objective:To explore the intestinal flora diversity and endogenous metabolites in fecal of rats with cold coagulation and blood stasis syndrome,and to investigate gut flora,metaboites and metabolic pathways closely related to cold coagulation and blood stasis syndrome. Method:The rats model of cold coagulation and blood stasis syndrome induced by ice-water bath was established,16S rRNA high-throughput gene sequencing coupled with UPLC-TOF-MS based on fecal metabonomics were used to analyze relativity between intestinal flora and endogenous biomarkers. Result:Based on Illumina MiSeq platform,V4 region was selected as the ideal variable regions.Compared with the blank group,it was found that Firmicutes was markedly up-regulated in model group(P<0.05) with Bacteroidetes decreased(P<0.01),there were 23 genus with significant differences.Based on fecal metabonomics technology,seven biomarkers were identified in model rats,of which 6 metabolites was markedly up-regulated except for docosapentaenoic acid.Three key metabolic pathways including histidine metabolism,biosynthesis of valine,leucine and isoleucine,biosynthesis of pantothenic acid and coenzyme A(CoA) were founded.There was a strong correlation between gut microbiota genera and fecal metabolites. Conclusion:It is suggested that 16S rRNA high-throughput gene sequencing combined with metabolomics can be further applied to assess pathogenesis of cold coagulation and blood stasis syndrome.