红芪多糖-1-1的分离纯化及其调节抗生素诱导小鼠肠道菌群失调的最佳剂量分析

旨在筛选红芪多糖-1-1（Radix Hedysari polysaccharide-1-1，RHPS-1-1）调节小鼠肠道菌群失调的最佳剂量。将红芪粗多糖分别用DEAE-52和Sephadex G-100进行分离纯化得单一多糖RHPS-1-1，采用高效阴离子交换色谱法及甲基化和红外光谱法鉴定其单糖组成与结构。将C57BL/6小鼠90只随机分为正常对照组、模型组、自愈组和不同剂量RHPS-1-1（12.5、25、50、100、200和400 mg·kg^-1）给药组，每组10只。通过连续14 d灌服抗生素鸡尾酒（氨苄西林、新霉素、甲硝唑、万古霉素）的方法建立小鼠肠道菌群失调模型，造模结束后，模型组小鼠处死采样，各给药组灌服不同剂量RHPS-1-1进行治疗，正常对照组与自愈组给予等量生理盐水，连续14 d。应用16S rDNA高通量测序法分析各组小鼠盲肠内容物的肠道菌群变化特征，并观测正常对照组、自愈组和25 mg·kg^-1 RHPS-1-1组主要脏器的器官指数和组织病理变化。结果表明，经纯化得到单一多糖RHPS-1-1，重均分子量为19.420 ku，主要由葡萄糖组成，主链连接方式为1，4-D-Glcp，并具有植物多糖的特征吸收峰。灌服抗生素鸡尾酒使小鼠出现了严重的肠道菌群失调，拟杆菌门、厚壁菌门和放线菌门等优势菌丰度极度减少，变形菌门相对增多，且不能自然恢复；经RHPS-1-1给药后肠道菌群的多样性和丰富度发生改变，25 mg·kg^-1 RHPS-1-1治疗组Chaol指数显著升高（P<0.05），PCA和UPGMA聚类分析结果均显示，25 mg·kg^-1的RHPS-1-1给药组与正常对照组肠道菌群结构最接近，有害菌Muribaculaceae_unclassified相对丰度降低，有益菌Ruminococcaceae_UCG-014和Clostridiales_unclassified相对丰度增加。自愈组相比正常对照组仅肝脏指数显著减小（P<0.05），经25 mg·kg^-1的RHPS-1-1治疗后显著升高（P<0.05）。但正常对照组、自愈组及25 mg·kg^-1的RHPS-1-1治疗组的心、肝、脾、肺、肾和脑均无明显病理变化。结果证明，所获RHPS-1-1是一种分子量约为19.420 ku的植物葡聚糖，治疗剂量为25 mg·kg^-1时，促进抗生素所致小鼠肠道菌群失调恢复的效果最佳，且可改善肝脏指数下降，本研究为红芪多糖调节肠道菌群的临床应用提供依据。

Isolation and Purification of Radix Hedysari Polysaccharide-1-1 and Analysis of Its Optimal Dosage for Regulating Intestinal Flora Imbalance Induced by Antibiotics in Mice

The aim of this study was to screen the best dosage of Radix Hedysari polysaccharide-1-1 (RHPS-1-1) regulating intestinal flora imbalance in mice. The single polysaccharide RHPS-1-1 was isolated and purified from the crude RHPS by DEAE-52 and Sephadex G-100, respectively. The monosaccharide composition of RHPS-1-1 was determined by high-performance anion-exchange chromatography (HPAEC), and the structure of the polysaccharide was identified by methylation and infrared spectroscopy. Ninety C57BL/6 mice were randomly divided into normal control group, model group, self-healing group, and different doses of RHPS-1-1 groups (12.5, 25, 50, 100, 200 and 400 mg·kg^-1) with 10 mice in each group. The intestinal flora imbalance mouse model was established by intragastric administration of antibiotic cocktail (ampicillin, neomycin, metronidazole and vancomycin) for 14 consecutive days. At the end of model establishing, the mice in model group were killed and sampled. Each drug administration group was gavaged with different doses of RHPS-1-1, and normal control and self-healing groups were gavaged with equal volume of normal saline for 14 consecutive days. Finally, 16S rDNA high-throughput sequencing method was used to analyze the changes of intestinal flora of cecal contents in each group, and the organ indices and histopathological changes of major organs in normal control, self-healing and 25 mg·kg^-1 RHPS-1-1 groups were calculated and observed. The results showed that the purified RHPS-1-1 was a single polysaccharide with a weight average molecular weight of 19.420 ku. RHPS-1-1 was mainly composed of glucose, the main chain of which was connected by 1,4-D-Glcp, and had a special absorption peak of plant polysaccharides. Gavaging with antibiotic cocktail caused a serious intestinal flora imbalance in mice. The abundance of Bacteroidetes, Firmicutes and Actinomycetes were extremely reduced, while Proteobacteria were relatively increased. And this alteration could not recover naturally. The diversity and richness of intestinal flora were changed after RHPS-1-1 administration, and the Chao 1 index of 25 mg·kg^-1 RHPS-1-1 group was significantly increased (P<0.05). The principal components analysis (PCA) and unweighted pair-group method with arithmetic averages (UPGMA) showed that the intestinal flora structure of 25 mg·kg^-1 RHPS-1-1 group was the closest to that of the normal control group. The abundance of harmful bacteria Muribaculaceae_unclassified was decreased, while the abundance of beneficial bacteria Ruminococcaceae_UCG-014 and Clostridiales_unclassified were increased. Compared with normal control group, liver index in self-healing group was significantly decreased (P<0.05), and significantly increased after 25 mg·kg^-1 RHPS-1-1 treatment (P<0.05). However, there were no obvious pathological changes in heart, liver, spleen, lung, kidney and brain in normal control group, self-healing group and 25 mg·kg^-1 RHPS-1-1 treatment group. The results demonstrated that RHPS-1-1 was a plant glucan with a molecular weight of 19.420 ku. At the dose of 25 mg·kg^-1, RHPS-1-1 had the best effect on promoting the recovery of intestinal flora disorder caused by antibiotics in mice, and could improve the decline of liver index. The present study provided evidences for the clinical application of RHPS regulating intestinal flora.