兰州大学机构库 >生命科学学院
党参多糖调节结肠炎小鼠肠道菌群影响皂苷代谢的机制
Alternative TitleMetabolism of Codonopsis pilosula saponins by coexisting polysaccharides alleviates gut microbial dysbiosis with acute colitis mice
景亚萍
Thesis Advisor张春江
2018-05-10
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name硕士
Keyword党参多糖 党参皂苷 急性结肠炎 肠道菌群 高通量测序 代谢
Abstract

炎症性肠病 (IBD) 一般由溃疡性结肠炎 (UC) 和克罗恩病 (CD) 两类组成,是基因、代谢、免疫等多种因素共同介导引发的自身免疫性疾病。恢复紊乱的肠道菌群是防治IBD的新型靶点。传统中医药为代表的补充及替代疗法(CAM)是临床防治IBD的主要方法。党参作为临床补中益气的经典中药,其代表性组分党参多糖和党参皂苷的药理活性广泛,具有抗炎、调节免疫等作用,且党参多糖可在肠道菌群的作用下,分解为小分子物质进而被宿主吸收发挥药效作用。但到目前为止,党参多糖在IBD的防治中并未得到充分利用。加之,在长期的临床应用中,党参主要是以水煎剂的形式口服给药;因中药材组成的复杂性及其主要化学组分的协同作用尚不清楚,其对寻找疾病治疗过程中发挥主要药效作用的组分、追踪有效组分之间的互作机制带来了困难。故深入研究党参有效成分之间的互作关系,及在IBD中发挥药效时的互作机制具有重要意义。
本项目首先利用低聚果糖、党参多糖和党参皂苷分别在健康小鼠中预防21天后,采用3 % 的葡聚糖硫酸钠(DSS)诱导7天(从第22天给药到第28天截止)建立急性结肠炎小鼠模型。通过评估实验小鼠体重变化、结肠长度、疾病病理指数(DAI)、组织病理切片和髓过氧化物酶(MPO)活性等指标变化,比较三种药物对结肠炎宏观病理症状的缓解情况。采用Illumina MiSeq 250测序平台,基于细菌16SrRNA基因 V3-V4可变区,探究了药物对结肠炎小鼠肠道菌群的调节,明确各药物干预组中的优势菌属。气相色谱(GC)测定粪便中短链脂肪酸(SCFAs)的含量,追踪与产SCFAs相关菌属的变化。酶联免疫法(ELISA)检测结肠炎小鼠中参与调节TH17/Treg平衡的促炎及抗炎细胞因子的分泌。实验结束前,于第29天,各实验组灌胃给予300 mg/kg的党参皂苷一次,收集血液及盲肠样本;应用UPLC-QTOF-MS技术结合PKsolver软件分析药物防治后,党参皂苷中暴露量最高的单体皂苷在血清及盲肠中的吸收情况;阐明党参多糖通过改变肠道菌群作用于皂苷代谢、进而影响其发挥疾病治疗作用的机制。
结果表明:低聚果糖、党参多糖和党参皂苷干预防治均可不同程度改善DSS诱导的结肠炎小鼠的宏观病理状况;下调与Th17/Treg平衡相关促炎细胞因子:白细胞介素 [IL] -17A,IL-17F,IL-6,IL-22和肿瘤坏死因子 [TNF]-α的分泌,上调抗炎因子IL-10和转化生长因子 [TGF]-β的表达。16SrRNA高通量测序结果显示:三种药物防治可不同程度改善结肠炎小鼠肠道菌群的结构和多样性。其中,低聚果糖效果最优,但与党参多糖之间并无显著差异。同时,党参多糖通过选择性刺激三种重要益生菌属Bifidobacterium spp., Akkermansia spp和Lactobacillus spp的生长,下调有害菌属Desulfovibio spp., Alistipes spp 和 Helicobacter spp 的丰度,改善了结肠炎小鼠肠道菌群的紊乱。GC结果显示:低聚果糖、党参多糖和党参皂苷不仅增加了结肠炎小鼠肠道内SCFAs的含量,而且通过选择性富集具有促SCFAs产生的Blautia,Prevotellaceae UCG-001,Oscillibacter和Quinella菌属的生长,改善了SCFAs的系统和局部能量供应情况。此外,研究发现:三种药物防治后,通过缓解结肠炎小鼠肠道菌群的失调,提高了党参皂苷中暴露量最高的单体化合物Cs-415.2在结肠炎小鼠体内的吸收率,其中,低聚果糖作用最优,党参多糖次之。综上所述:党参多糖具有良好的益生元特性,在急性结肠炎的防治过程中显示出良好的应用前景。
本研究一方面证实了党参多糖的潜在益生元效应,另一方面也揭示了党参中共存的多糖通过恢复结肠炎小鼠肠道菌群的紊乱进而影响皂苷代谢的机制。为以党参多糖作为益生元开发新型微生态制剂奠定基础,同时也为发现中药药效物质提供理论依据。

Other Abstract

Inflammatory bowel disease (IBD) mainly includes Ulcerative Colitis (UC) and Crohn's Disease (CD). It is an autoimmune disease caused by genes, metabolism, immunity and other factors. Recovering disorder of gut microbiota is a new research target for the treatment of IBD. Herbal Medicine (HM) as the representative of the supplement and alternative therapy is the key method in the clinical treatment of IBD. Codonopsis pilosula (Franch) Nannf (CPN) serves as a classic HM for replenishing qi. Its representative components, Codonopsis pilosula polysaccharides (Cp) and Codonopsis pilosula saponins (Cs), have a wide range of pharmacological activities, such as anti-inflammatory and immuno-modulate effect. In addition, Cp can be broken down into small molecules under the action of gut microbiota and absorbed by the host to exert its pharmacodynamic effect. But so far, Cp has not been fully utilized in the treatment of IBD. In long-term clinical application, CPN is mainly prepared by decoction and administered orally. Because the complexity of the composition of HM and the synergistic effects of their major chemical components are not yet clear. So it is difficult to find the components that play an effective role in the treatment of disease, and to track the interaction mechanism between the active ingredients. Therefore, it is of great significance to conduct in-depth research on the interactions between the active ingredients of CPN and its interaction mechanism in IBD.
In this project, the mouse model of acute colitis was established with 3% dextran sodium sulfate (DSS) induction for 7 days (from the 22nd day of administration to the 28th day) after Fro, Cp and Cs were gastric administration with healthy mouse models in 21 days. By evaluating the changes of body weight, colon length, disease pathology index, colon tissue sections, and myeloperoxidase (MPO) in colitis mice, the effects of the three drugs on the macroscopic pathological symptoms of colitis were compared. Using Illumina MiSeq 250 sequence, based on the 16S V3-V4 variable region of bacteria, the regulation of gut microbiota structure and diversity by different drugs in colitis mice was explored, and the dominant genus in each group after intervention with each drug were analyzed. Gas chromatography (GC) was used to determine the content of short-chain fatty acids (SCFAs) in feces, and the changes associated with SCFAs-associated genus were searched. ELISA was used to detect the expression of pro-inflammatory and anti-inflammatory cytokines associated with Th17/Treg balance. At the end of the experiment, each experimental group was given 300 mg/kg of Cs once on the 29th day to collect blood and cecal samples; using UPLC-QTOF-MS technology combined with PKsolver software to analyze the absorption of the highest exposure for monomeric compounds in Cs after the administration of three drugs for prevention. Elucidation that Cp could be used as prebiotics to regulate gut microbiota, which affect the mechanism of Cs metabolism.
The results showed Fro, Cp and Cs improved the macroscopic pathological status of mice with DSS-induced colitis. Down-regulated the secretion of pro-inflammatory cytokines: interleukin [IL]-17A, IL-17F, IL-6, IL-22, TNF-α, up-regulated the expression of IL-10 and TGF-β in Th17/Treg balance, which has the anti-inflammatory effect. 16SrRNA sequence results showed that the three drugs regulated the structure and diversity of gut mocrobiota in mice with colitis to varying degrees. At the same time, Cp selectively stimulated the growth of three important probiotics, Bifidobacterium spp., Akkermansia spp and Lactobacillus spp., reduced the abundance of Desulfovibio spp., Alistipes spp., Helicobacter spp., and restored the gut microbiota in colitis mice. Among them, Fro had the best effect, but there was no significant difference from the Cp group. GC results showed that Fro, Cp and Cs increased the content of SCFAs. Through selective enrichment of Blautia, Prevotellaceae UCG-001, Oscillibacter and Quinella were reported to promote the production of SCFAs, improved the systemic and local energy supply of SCFAs. In addition, the study found after prevention of three drugs for 21 days, by adjusting the imbalance of gut microbiota in mice with colitis, the absorption of the most exposed monomeric compound Cs-415.2 of Cs in colitis mice was enhanced. At the same time, among which, Fro had the best effect, followed by Cp. In summary: Cp had the good prebiotic properties and shown good application prospects in the prevention and treatment of acute colitis.
On the one hand, this study confirmed the potential prebiotic effect of Cp. On the other hand, it also revealed the Cp in CPN could affect the metabolic mechanism of Cs in the serum by restoring the disorder of gut microbiota in mice with colitis. To lay the foundation for the development of new micro-ecological preparation by using Cp as a prebiotic. In addition, it also provided a theoretical basis for the discovery of effective substances in HM.

URL查看原文
Language中文
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/221568
Collection生命科学学院
Recommended Citation
GB/T 7714
景亚萍. 党参多糖调节结肠炎小鼠肠道菌群影响皂苷代谢的机制[D]. 兰州. 兰州大学,2018.
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