摘要
目的:该研究旨在利用湿热瘀阻证溃疡性结肠炎(UC)患者的菌群人源化小鼠模型,以探讨乌梅丸加减对UC相关的肠道辅助性T细胞17(Th17)/调节性T细胞(Treg)免疫平衡及肠道菌群的调节作用,为UC的相关研究和治疗策略提供新的思路和依据。方法:选取24只雄性C57BL/6J小鼠作为实验对象,将小鼠随机分为正常组、模型组和乌梅丸加减组,每组8只。模型组和乌梅丸加减组首先给予含四联抗生素(万古霉素0.1 g·kg^(-1)、硫酸新霉素0.2 g·kg^(-1)、氨苄青霉素0.2 g·kg^(-1)、甲硝唑0.2 g·kg^(-1))的水溶液,持续21 d。抗生素清菌结束后,开始灌胃湿热瘀阻证UC患者粪便菌悬液,灌胃剂量0.2 mL·d^(-1),连续灌胃19 d,以此获得UC菌群人源化小鼠模型。治疗组每日灌胃乌梅丸加减药液(12.5 g·kg^(-1)),正常组和模型组灌胃等量无菌水,连续灌胃7 d。观察相关湿热症候,检测小鼠的结肠长度和脾指数,流式检测Th17、Treg细胞比例。应用16S rRNA高通量测序技术对肠道菌群进行基因测序,观察肠道菌群落的变化。结果:与正常组比较,模型组的小鼠结肠长度缩短(P<0.05),脾脏指数显著升高(P<0.01)。乌梅丸加减治疗小鼠较模型组的结肠长度显著增加(P<0.01),而脾脏指数明显降低(P<0.05)。与模型组比较,乌梅丸加减组结肠Th17、Th17/Treg显著下降(P<0.01),Treg比例升高(P<0.05)。肠道菌群方面,乌梅丸加减显著调节小鼠的物种数量及α与β多样性(P<0.05)。物种组成方面,乌梅丸加减显著增加UC菌群人源化小鼠的厚壁菌门、变形菌门、梭杆菌门、放线菌门、芽单胞菌门等丰度,同时上调了拟杆菌属等菌属和多形拟杆菌、脆弱拟杆菌等菌种的丰度。Spearman相关性分析显示,多形拟杆菌、脆弱拟杆菌等的相对丰度与Th17水平呈明显负相关(P<0.05)。此外,上述肠道菌群的改变可引起涉及糖酵解、氨基酸降解、无机营养素代谢、生物合成嘧啶脱氧核糖核苷酸、抗生素耐药性、多聚糖降解等14个通路功能的菌群基因功能的改变。结论:菌群人源化小鼠模型较好地模拟了湿热证UC患者以拟杆菌属减少为主要标志的菌群结构变化,乌梅丸加减可能通过富集多形拟杆菌、脆弱拟杆菌等肠道有益菌的丰度,通过细菌多糖利用位点促进以糖酵解为核心、氨基酸代谢、核苷酸合成等菌群代谢功能协同的肠道免疫调控,降低Th17/Treg细胞比例,从而对UC发挥保护效应。
Objective:To investigate the modulating effect of modified Wumeiwan(MWMW)on the ulcerative colitis(UC)-associated intestinal helper T cell 17(Th17)/regulatory T cell(Treg)balance and intestinal flora by using a human floraassociated model of UC patients with dampness-heat obstruction syndrome,thus providing a new idea for the UC-related research and therapeutic strategies.Methods:The 24 male C57BL/6J mice were randomized into normal control,model,and MWMW groups(n=8).Model and MWMW groups were first treated with an antibiotic cocktail(vancomycin,0.1 g·kg^(-1);neomycin sulfate,0.2 g·kg^(-1);ampicillin,0.2 g·kg^(-1);metronidazole,0.2 g·kg^(-1))for 21 days.At the end of antibiotic treatment,the gavage of fecal microbiota suspension from UC patients with dampness-heat obstruction syndrome was started at a dose of 0.2 mL·d^(-1)for 19 consecutive days,by which a human flora-associated model of UC was obtained.The MWMW group was administrated daily with MWMW liquid(12.5 g·kg^(-1)),while the normal control and model groups were administrated by gavage with an equal amount of sterile water for 7 consecutive days.The symptoms of dampness-heat obstruction were observed.The colon length and spleen index were measured and calculated,and the proportions of Th17 and Treg cells were detected by flow assay.The intestinal flora was analyzed by 16S rRNA high-throughput sequencing.Results:Compared with the normal control group,the model group showed shortened colon(P<0.05)and increased spleen index(P<0.01).Compared with the model group,the MWMW group showed prolonged colon(P<0.01)and decreased spleen index(P<0.05).After the intervention of MWMW,the Th17 proportion and Th17/Treg ratio in the colon decreased(P<0.01),and the proportion of Treg cells increased(P<0.05).The number of species and alpha and beta diversity of intestinal flora in mice were regulated by MWMW(P<0.05).In terms of intestinal flora composition,MWMW increased the relative abundance of several phyla(Firmicutes,Proteobacteria,Fusobacteriota,Actinobacteriota,and Gemmatimonadota),the genus Bacteroides,and two species(Bacteroides thetaiotaomicron and B.fragilis)in model mice.Moreover,Spearman's correlation analysis showed that the relative abundance of B.thetaiotaomicron and B.fragilis were negatively correlated with the Th17 level(P<0.05).In addition,the above changes in intestinal flora caused the changes in microbial genes involved in 14 pathways,such as glycolysis,amino acid degradation,inorganic nutrient metabolism,biosynthesis of pyrimidine deoxyribonucleotides,antibiotic resistance,and degradation of polysaccharides.Conclusions:The human floraassociated model successfully simulated the changes(marked by a decrease in the abundance of Bacteroides)of intestinal flora in UC patients with dampness-heat obstruction syndrome.MWMW can enrich the abundance of beneficial bacteria such as B.thetaiotaomicron and B.fragilis and promote the synergistic intestinal immune modulation with the metabolic functions centered on glycolysis,amino acid metabolism,and nucleotide synthesis through bacterial polysaccharide utilization sites to reduce the Th17/Treg ratio,thereby exerting a protective effect on UC.
作者
张崇豪
马沛广
刘华琛
苏佳龙
刘洁
李亚兰
徐贵川
李娜
彭桂英
ZHANG Chonghao;MA Peiguang;LIU Huachen;SU Jialong;LIU Jie;LI Yalan;XU Guichuan;LI Na;PENG Guiying(School of Life Sciences,Beijing University of Chinese Medicine,Beijing 100029,China)
出处
《中国实验方剂学杂志》
北大核心
2025年第20期86-93,共8页
Chinese Journal of Experimental Traditional Medical Formulae
基金
“十三五”国家重点研发计划项目(2018YFC1705405)
国家自然科学基金青年基金项目(82204888)
北京中医药大学基本科研业务费项目(2019-JYB-TD014,2023-JYB-JBQN-040)。
关键词
乌梅丸加减
溃疡性结肠炎
粪菌移植
菌群人源化
肠道菌群
modified Wumeiwan
ulcerative colitis
fecal microbiota transplantation
human-associated flora
intestinal flora
