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基于高通量测序分析凉血解毒方对HBV相关慢加急性肝衰竭患者肠道菌群的影响

侯艺鑫 张群 江宇泳 于浩 杨玉英 王宪波

引用本文:
Citation:

基于高通量测序分析凉血解毒方对HBV相关慢加急性肝衰竭患者肠道菌群的影响

DOI: 10.3969/j.issn.1001-5256.2022.06.013
基金项目: 

北京市科学技术委员会“首都临床特色应用研究”资助项目 (Z181100001718052)

北京市医院管理中心“青苗”计划 (QMl220201802)

北京市中医药科技发展资金项目 (Qn-2020-25)

国家中医药管理局重大疑难疾病中西医临床协作试点项目 (2018-6-4)

国家中医药管理局区域中医诊疗中心 (2019-3-18)

伦理学声明:本研究方案于2018年10月29日经由北京地坛医院伦理委员会审批,批号:2018第(036)-01号,所纳入患者均签署知情同意书。
利益冲突声明:本研究不存在研究者、伦理委员会成员、受试者监护人以及与公开研究成果有关的利益冲突。
作者贡献声明:侯艺鑫、张群负责课题设计,资料分析,撰写论文;江宇泳、于浩、杨玉英参与收集数据,修改论文;王宪波负责拟定写作思路,指导撰写文章并最后定稿。
详细信息
    通信作者:

    王宪波, wangxianbo638@163.com

Effect of Liangxue Jiedu decoction on intestinal flora in patients with hepatitis B virus-related acute-on-chronic liver failure: An analysis based on high-throughput sequencing

Research funding: 

"Capital Featured Clinical Application Research Projects" of Beijing Science and Technology Committee (Z181100001718052);

Green Seed Plan of Beijing Hospitals Authority (QMl220201802);

Fund for Beijing Science & Technology Development of TCM (Qn-2020-25);

Pilot Project of Clinical Cooperation Between Traditional Chinese Medicine and Western Medicine for Major and Difficult Diseases, State Administration of Traditional Chinese Medicine (2018-6-4);

Regional Traditional Chinese Medicine Diagnosis and Treatment Center, State Administration of Traditional Chinese Medicine (2019-3-18)

More Information
  • 摘要:   目的  观察凉血解毒方对HBV相关慢加急性肝衰竭(HBV-ACLF)患者肠道菌群的影响。  方法  选取2018年10月—2019年10月于北京地坛医院住院诊断为HBV-ACLF的患者,招募同期正常健康人群作为对照(HP组),通过高通量测序的方法筛选出HBV-ACLF患者与正常健康人群的菌群多样性和菌种的差异,从门、属水平筛选出两组的差异菌;利用体外模拟发酵实验,HBV-ACLF患者给予不同浓度(0、10%、50%、100%)凉血解毒方培养基培养24 h,从属分类水平比较分析HBV-ACLF用药组、HBV-ACLF未用药组及HP组菌群变化。正态分布的计量资料两组间比较采用t检验;非正态分布的计量资料两组间比较采用Mann-Whitney U检验。  结果  共纳入HBV-ACLF患者10例,其中HBV-ACLF用药组5例,未用药组5例;HP组15例。HBV-ACLF未用药组较HP组肠道菌群多样性及丰度下降。在门分类水平,HP组每个样本均以拟杆菌门和厚壁菌门为主;HBV-ACLF未用药组拟杆菌门明显减少,梭杆菌门、变形菌门、纤维杆菌门增多。在属分类水平上,HBV-ACLF未用药组瘤胃菌、布劳特菌属、真杆菌属较HP组明显减少,而副杆菌属、乳酸杆菌、梭杆菌、链球菌较HP组增多。体外模拟发酵实验结果显示,HBV-ACLF用药组瘤胃菌、毛螺菌属、拟杆菌、氏菌属相对丰度值较HBV-ACLF未用药组显著升高,而梭杆菌、变形菌较未用药组显著减少(P值均<0.05)。  结论  凉血解毒方能够调节肠道菌群紊乱,恢复肠道菌群多样性,增加优势菌,减少致病菌,这可能是其治疗HBV-ACLF重要的作用机制之一。

     

  • 图  1  Alpha多样性分析结果

    注:a,Chao指数;b,Shannon指数;c,Simpson指数。

    Figure  1.  Alpha results of the diversity analysis

    图  2  物种累积曲线

    Figure  2.  Species accumulation curve

    图  3  门水平菌群比较

    注:a,不同门分类在每个病例中所占的比例;b,HBV-ACLF未用药组和HP组3种门分类的比较。Actinobacteris,放线杆菌门;Epsilonbacteraeota,表皮菌门;Tenericutes,马鞭菌门;Gemmatimonadetes,芽单孢菌门;Cynobacteria,嗜肺杆菌门;Acidobacteria,酸杆菌门;Nitrospirae,硝基绣线菌门;Spirochaetes,螺旋体菌门;Deferribacteres,脱铁杆菌门;Laescibacteria,不动杆菌门;Fibrobacteres,纤维杆菌门;Fusobacterio,梭杆菌门;Proteobacteria,变形菌门。

    Figure  3.  Phyla level flora ratio

    图  4  属水平菌群比较

    注:Eubacterium coprostanoligenes,粪甾烷内酯真杆菌属;Eubacterium hallii-group,真杆菌属;Agathobacter,阴沟杆菌属;Agathobacter Blautla,布劳特菌属;Fusobacterium,梭杆菌属;Lactobacllus,乳酸杆菌;Parabacteroides,副杆菌属;Ruminococcus,瘤胃菌;Streptococcus链球菌属;uncultured-bacterium, 未增养菌。

    Figure  4.  Comparison of microflora at genus level

    图  5  聚类分析结果

    注:a,整体趋势;b,聚类分析。

    Figure  5.  Results of cluster analysis

    图  6  不同浓度凉血解毒方对肠道菌群Alpha多样性的影响

    Figure  6.  Effect of different concentrations of Liangxue Jiedu decoction on Alpha diversity of intestinal flora

    图  7  凉血解毒方对菌属的影响

    注:a、c、e、g、i、k,HP组与HBV-ACLF未用药组瘤胃菌(Ruminococus)、毛螺菌属(Lachnospira)、拟杆菌(Bacteroidetes)、氏菌属(Genusgenus)、梭杆菌(Fusobacterio)、变形菌(Proteobacteria)菌群变化;b、d、f、h、j、l,HBV-ACLF不同凉血解毒方含药浓度瘤胃菌(Ruminococus)、毛螺菌属(Lachnospira)、拟杆菌(Bacteroidetes)、氏菌属(Genusgenus)、梭杆菌(Fusobacterio)、变形菌(Proteobacteria)菌群变化。

    Figure  7.  Effect of Liangxue Jiedu Fang on fungi

    表  1  含有不同凉血解毒方浓度的培养基配方

    Table  1.   Culture medium containing different concentrations of Liangxue Jiedu formula

    含药浓度 YCFA培养基 凉血解毒方溶液
    0 10 mL 0 mL
    10% 9 mL 1 mL
    50% 5 mL 5 mL
    100% 0 mL 10 mL
    下载: 导出CSV

    表  2  两组研究对象的一般资料

    Table  2.   General information of the two groups of subjects

    指标 HP组
    (n=15)
    HBV-ACLF组
    (n=10)
    年龄(岁) 41.3±13.2 42.3±10.2
    BMI(kg/m2) 20.3±6.2 21.0±7.8
    Child-Pugh分级(例)
      A级 0
      B级 6
      C级 4
    MELD评分 24.7±3.5
    TBil(μmol/L) 293.5±103.5
    Alb(g/L) 29.5±4.9
    PTA(%) 25.0(20.6~30.7)
    INR 2.2(1.9~2.8)
    下载: 导出CSV

    表  3  每个样本信息表

    Table  3.   Information sheet for each sample

    Sample_ID clean_tags valid_tags valid_percent valid mean
    length
    OTU_counts
    LF.1 41 033 33 826 82.44% 420.39 424
    LF.2 41 802 37 043 88.62% 410.31 436
    LF.3 40 602 36 443 89.76% 412.07 477
    LF.4 39 174 33 063 84.40% 415.78 529
    LF.5 40 065 35 353 88.24% 411.78 395
    LF.6 40 416 36 302 89.82% 424.90 393
    LF.7 39 940 34 481 86.33% 423.41 389
    LF.8 41 002 35 022 85.42% 415.13 411
    LF.9 39 690 35 844 90.31% 410.98 356
    LF.10 40 678 35 815 88.05% 416.40 446
    H.1 38 761 34 957 90.19% 425.93 315
    H.2 40 329 34 492 85.53% 427.77 385
    H.3 38 753 33 801 87.22% 429.53 357
    H.4 38 848 34 017 87.56% 426.16 300
    H.5 38 462 35 138 91.36% 432.39 405
    H.6 38 194 32 350 84.70% 431.75 355
    H.7 39 211 34 487 87.95% 410.10 439
    H.8 41 783 35 965 86.08% 409.92 442
    H.9 38 162 32 184 84.34% 413.61 449
    H.10 40 125 36 203 90.23% 416.68 481
    H.11 38 234 33 521 87.67% 414.28 524
    H.12 38 990 34 198 87.71% 419.20 453
    H.13 39 733 34 258 86.22% 416.84 451
    H.14 40 022 33 981 84.91% 413.43 447
    H.15 38 009 32 751 86.17% 413.66 536
    注:Sample_ID,病例编码。
    下载: 导出CSV

    表  4  各个水平菌群比较

    Table  4.   Comparison of microflora at different levels

    方法 OTU
    t检验 142 3 6 13 20 39 10
    Wilcoxon检验 263 2 6 13 34 77 22
    下载: 导出CSV
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  • 收稿日期:  2021-06-04
  • 录用日期:  2021-07-08
  • 出版日期:  2022-06-20
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