高通量测序分析附子、红参不同比例配伍对大鼠肠道菌群的影响

Effect of Aconiti Lateralis Radix Praeparata and Ginseng Radix Et Rhizoma Rubra in different compounding ratio on gut microbiota in SD rats evaluated by high-throughput sequencing

目的初步研究附子、红参不同比例配伍对大鼠肠道菌群的影响。方法红参、附子分别采用8倍体积的70%乙醇加热回流提取2次,每次1 h,制备醇提物。将15只SD大鼠随机分成A、B、C 3组,每组5只,A组为附子醇提物单独给药组,给药量为0.415 g/kg,B、C组分别为附子、红参配伍比例1∶1(红参醇提物1.17 g/kg)和1∶2(红参醇提物2.34 g/kg)给药组,每天ig给药1次,连续给药7 d。给药结束后,于第8天收集大鼠粪便,提取粪便中细菌的DNA,对粪便菌群的16Sr-RNA的V4区进行扩增,利用Miseq高通量测序平台进行基因序列的测定,利用Uparse software对序列进行分析,将相似性在97%以上的序列进行归并,生成分类操作单元(OTU);利用QIIME软件计算样品的菌群丰度指数(Ace、Chao1)、菌群多样性指数(Simpson、Shannon)。结果 A组和B组的OTU数量较C组显著增多(P<0.05);A组和B组Ace、Chao1指数为1 393、1 368和1 085、1 057,C组Ace和Chao1指数为889和884,A组和B组较C组显著增多(P<0.05、0.01);厚壁菌门、拟杆菌们和变形菌门均为3组样品中的优势菌门;在属的水平上,C组中乳酸杆菌属和拟杆菌属含量较A组和B组增加。结论红参与附子不同比例配伍可对大鼠肠道菌群产生显著的影响,随红参比例的增加,可能在一定程度上改善肠道的微生态环境。

Objective To analyze the effect of AconitiLateralis Radix Praeparata (ALRP) and Red Ginseng in different compounding ratios on the intestinal microbiota of rats by using Miseq high-throughput sequencing technology.Methods Red Ginseng and ALRP were extracted by refluxing with 70% ethanol of eight times volume for two times, 1 h each time, to prepare ethanol extracts. Fifteen SD rats were randomly divided into A, B and C group, which were treated with ALRP ethanol extracts alone, ALRP and Red Ginseng in compounding ratio of 1:1 (Red Ginseng ethanol extracts 1.17 g/kg) and 1:2 (Red Ginseng ethanol extracts 2.34 g/kg) respectively, ig administrated once daily for 7 d. The fecal samples were collected on the 8th to extract bacterial DNA. The fecal bacteria composition was investigated by Miseq high-throughput sequencing of the V4 region of the 16Sr-RNA gene. Uparse Software was used to analyze the sequences, and the sequences with 97% similarity were merged to generate OTU, and QⅡME Software was used to calculate the bacterial abundance index (Ace and Chao1) and bacterial diversity index (Simpson and Shannon). Results The number of OTUs in group A and group B was significantly higher than that in group C (P<0.05); the Ace and C hao1 indices in group A and group B were 1393 and 1368, 1085 and 1057, and the Ace and Chao1 indices in group C were 889 and 884, while the A and B groups were significantly higher than that in group C (P<0.05 and 0.01); Fimicutes, Bacteroides and Proteobacteria were the dominant phylum in the three groups; at the genus level, Lactobacillus and Bacteroides in group C were the dominant phylum. Conclusion ALRP and Red Ginseng in different compounding ratio have significant effect on gut microbiota. With the increase of the proportion of Red Ginseng, the intestinal microecological environment may be improved to a certain extent.