术苦芩总多糖对湿热泄泻仔猪肠道菌群和免疫功能的影响

doi:10.11843/j.issn.0366-6964.2022.03.024

摘要/Abstract

摘要： 旨在探究术苦芩总多糖(ZKQPs)对湿热泄泻仔猪肠道菌群和免疫功能的影响。通过HPLC分析ZKQPs的单糖组成,体外抑菌试验研究ZKQPs体外对常见肠道致病菌的直接影响,同时建立仔猪腹泻模型,用白头翁散(PP)及3种剂量的ZKQPs (25、50和75 mg·kg^-1)治疗腹泻仔猪,治疗结束后,收集小肠各段组织、盲肠内容物,对菌落进行鉴定与计数;通过16S rDNA高通量测序分析肠道菌群的变化,测定小肠免疫相关细胞因子mRNA表达。结果显示:ZKQPs一级结构主要由鼠李糖、葡萄糖、半乳糖和木糖构成。在体外试验中,大肠杆菌、沙门菌、猪源产气荚膜梭菌肠道常见有害菌对ZKQPs敏感,并在一定范围内呈现浓度依赖性。动物试验中,ZKQPs有效促进了腹泻仔猪肠道乳酸杆菌的生长,对致病大肠杆菌生长有较强的抑制作用,其中,50 mg·kg^-1ZKQPs影响最为显著。16S rDNA高通量测序结果表明,ZKQPs调节了腹泻仔猪菌群Alpha多样性,对肠道菌群在纲和属水平上有显著影响,腹泻仔猪芽胞杆菌纲、乳酸杆菌属相对丰度显著下降,梭菌纲相对丰度显著上升;ZKQPs治疗后芽胞杆菌纲、乳酸杆菌属相对丰度均显著上升且高于正常值,梭菌纲相对丰度极显著下降,显著改善了腹泻仔猪肠道菌群结构。仔猪腹泻时,小肠各段Th1细胞因子IL-2 mRNA水平上升(P<0.01),Th2细胞因子IL-4、IL-5 mRNA水平均下降;ZKQPs治疗后,腹泻仔猪小肠各段IL-2 mRNA水平下降,IL-4、IL-5 mRNA水平普遍上升,且不同细胞因子mRNA水平在小肠不同位置变化有所差异,50和75 mg·kg^-1ZKQPs治疗后对其影响最为显著,综合分析50 mg·kg^-1ZKQPs治疗腹泻仔猪对细胞因子mRNA水平影响最大。综上表明,ZKQPs在体外对常见肠道致病菌生长具有良好的抑制作用,可以改变腹泻仔猪肠道微生物群的丰富度和多样性,提高乳酸杆菌属的相对丰度,并改善菌群结构,同时调节肠道免疫反应,有效增强了仔猪抗腹泻能力。

关键词: 术苦芩总多糖, 腹泻仔猪, 肠道菌群, 免疫

Abstract: This study was carried out to investigate the effect of polysaccharides from ZhuKuQin (ZKQPs) on the intestinal flora and immune function of piglets with dampness-heat diarrhea. We analyzed the monosaccharide composition of ZKQPs by HPLC, and studied the direct effects of ZKQPs on common intestinal pathogens in vitro antibacterial experiments. At the same time, we establish a piglet diarrhea model, and treated with Pulsatilla powder and three doses of ZKQPs (25, 50 and 75 mg·kg^-1). After the treatment, the small intestine tissues and the contents of the cecum were collected. The cecal colony was identified and counted; the changes of intestinal flora were analyzed by 16S rDNA high-throughput sequencing, and the mRNA expression of small intestinal immune-related cytokines was determined. The results showed that the primary structure of ZKQPs is mainly composed of rhamnose, glucose, galactose and xylose. In vitro tests, E. coli, Salmonella, and Clostridium wischii were sensitive to ZKQPs and showed a concentration dependence within a certain range. In animal experiments, ZKQPs effectively promoted the growth of intestinal Lactobacilli in piglets with diarrhea, and had a strong inhibitory effect on the growth of pathogenic E. coli. Among them, 50 mg·kg^-1ZKQPs had the most significant effect. The results of 16S rDNA high-throughput sequencing showed that ZKQPs regulate the Alpha diversity of diarrhea piglets, and had a significant impact on the intestinal flora at the class and genera level. The relative abundance of Bacillus and Lactobacillus decreased significantly and the relative abundance of bacteria increased significantly in diarrhea piglets; after ZKQPs treatment, the relative abundances of Bacillus and Lactobacillus increased significantly and were higher than normal, and the relative abundance of Clostridia significantly decreased, which significantly improved the intestinal flora structure of diarrhea piglets. In piglets with diarrhea, the level of Th1 cytokine IL-2 mRNA in each segment of the small intestine increased (P<0.01), while the Th2 cytokine IL-4 and IL-5 mRNA levels decreased; after treatment with ZKQPs, the levels of IL-2 mRNA in the small intestine decreased, the levels of IL-4 and IL-5 mRNA generally increased, and the mRNA levels of different cytokines vary in different locations in the small intestine. ZKQPs 50 and 75 mg·kg^-1 have the most significant effects on them, comprehensive analysis ZKQPs 50 mg·kg^-1 treatment of diarrhea piglets has the greatest impact on the level of cytokine mRNA. This study shows that ZKQPs have a good inhibitory effect on the growth of intestinal pathogens in vitro, can change the abundance and diversity of the intestinal microbiota of diarrhea piglets, increase the relative abundance of Lactobacillus and improve the structure of the flora. At the same time, ZKQPs regulate the intestinal immune response and effectively enhance the anti-diarrhea ability of piglets.

Key words: polysaccharides from Zhukuqin, diarrhea piglets, intestinal flora, immune

中图分类号:

S853.74

陶未来, 刘佳, 刘琼丹, 郝永峰, 胡宇, 朱兆荣, 刘娟. 术苦芩总多糖对湿热泄泻仔猪肠道菌群和免疫功能的影响[J]. 畜牧兽医学报, 2022, 53(3): 913-924.

TAO Weilai, LIU Jia, LIU Qiongdan, HAO Yongfeng, HU Yu, ZHU Zhaorong, LIU Juan. Effects of Total Polysaccharides from Zhukuqin on Intestinal Flora and Immune Function in Piglets with Dampness-heat Diarrhea[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(3): 913-924.

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