基于16S rDNA扩增子测序与流式细胞仪分析葛根改善免疫与肠道微生物的关系

Based on 16S rDNA Amplicon Sequencing and Flow Cytometry, the Relationship between Pueraria Improving Immunity and Intestinal Microorganism Was Analyzed

本文基于16S rDNA扩增子测序与流式细胞仪深入探索葛根改善免疫的机制,并分析肠道微生物与免疫的关系。葛根灌胃四周后,取血,测定血清中TNF-α、IFN-γ、IL-6,运用流式细胞仪测定血清中T、B淋巴细胞数量,16S rDNA扩增子测序粪便中肠道微生物菌群变化。结果显示,与空白对照组比较,各剂量葛根皆降低炎性因子,改善机体免疫状态,其中以低剂量最为显著(P < 0.05)。流式细胞仪结果显示,各剂量组葛根提取物都显著增加血清中T、B淋巴细胞数量(P < 0.05),增长幅度以低剂量最为显著(P < 0.01)。16S rDNA扩增子测序结果显示,葛根提取物可以特异性增高与免疫相关肠道微生物的丰度,如梭状芽孢杆菌、罗氏杆菌、狄氏副拟杆菌、肠球菌等,以狄氏副拟杆菌最为显著。Alpha多样性指数组间差异分析提示葛根能够提高肠道微生物的丰度,提高微生物分布均匀性。PCoA分析提示葛根能较显著改变肠道内微生物的菌种丰度与组成结构。因此,葛根能够提高免疫功能,降低炎性因子,增加T、B淋巴细胞数量,并可能通过特异性改变肠道微生物,如狄氏副拟杆菌,增加肠道微生物多样性等途径发挥免疫调节作用。

The aim of this study was to explore the immune improvement mechanism of Pueraria based on 16S rDNA amplicon sequencing and flow cytometry, and to analyze the relationship between intestinal microbes and immunity, measuring the serum levels of TNF-α, IFN-γ and IL-6 w, determining the number of T and B lymphocytes in serum by flow cytometry. The changes of intestinal microflora in feces were sequenced by amplicon of 16S rDNA. The results showed that compared with the blank control group, all the doses of pueraria decreased inflammatory factors and improved the immune state of the body, and the low dose was the most significant (P < 0.05). Flow cytometry results showed that the number of T and B lymphocytes in serum of pueraria root extract in each dose group was significantly increased (P < 0.05), and the in-crease rate was most significant in low dose group (P < 0.01). 16S rDNA amplified sequencing re-sults showed that pueraria root extract could specifically increase the abundance of im-mune-related intestinal microorganisms, such as Clostridium difficile, Roche, Parabacteroides dieldii, enterococcus, etc. The difference analysis of Alpha diversity index between groups suggested that pueraria root could improve the abundance and distribution uniformity of intestinal microorgan-isms. PCoA analysis indicated that pueraria could significantly change the species abundance and composition of intestinal microorganisms. Therefore, pueraria root can improve immune function, reduce inflammatory factors, increase the number of T and B lymphocytes, besides, it may play an immunomodulatory role by changing the intestinal microflora, such as parabens Diels, and in-creasing the diversity of intestinal microflora.