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

doi:10.3969/j.issn.1001-5256.2022.06.013

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

首都医科大学附属北京地坛医院中西医结合中心, 北京 100015

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

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出版历程
- 收稿日期: 2021-06-04
- 录用日期: 2021-07-08
- 出版日期: 2022-06-20

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

Center of Integrative Medicine, Beijing Ditan Hospital, Capital Medical University, Beijing 100015, China

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

Corresponding author: WANG Xianbo, wangxianbo638@163.com (ORCID:0000-0002-3593-5741)

摘要

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。
- 乙型肝炎病毒 /
- 慢加急性肝功能衰竭 /
- 中西医结合疗法 /
- 胃肠道微生物组
Abstract: Objective To investigate the effect of Liangxue Jiedu decoction on intestinal flora in patients with hepatitis B virus-related acute-on-chronic liver failure (HBV-ACLF). Methods The patients who were hospitalized and diagnosed with HBV-ACLF in Beijing Ditan Hospital from October 2018 to October 2019 were enrolled, and healthy individuals were enrolled as HP group. High-throughput sequencing was used to screen for the differences in bacterial diversity and species between HBV-ACLF patients and healthy individuals, and differentially expressed bacteria between the two groups were screened out at the phylum and genus levels. With the help of in vitro simulated fermentation experiment, fecal samples were collected from the patients with HBV-ACLF and were then cultured in the medium containing different concentrations of Liangxue Jiedu decoction (0, 10%, 50%, and 100%) for 24 hours, and the changes in intestinal flora were analyzed and compared between the HBV-ACLF treatment group, the HBV-ACLF non-treatment group, and the HP group at the genus level. The t-test was used for comparison of normally distributed continuous data between two groups, and the Mann-Whitney U test was used for comparison of non-normally distributed continuous data between two groups. Results A total of 10 HBV-ACLF patients were enrolled, with 5 in the HBV-ACLF treatment group and 5 in the HBV-ACLF non-treatment group, and there were 15 individuals in the HP group. Compared with the HP group, the HBV-ACLF non-treatment group had significant reductions in the diversity and abundance of intestinal flora. At the phylum level, Bacteroidetes and Firmicutes were mainly observed in the samples of the HP group, while the HBV-ACLF non-treatment group had a significant reduction in Bacteroidetes and significant increases in Fusobacteria, Proteobacteria, and Fibrobacteres. At the genus level, compared with the HP group, the HBV-ACLF non-treatment group had significant reductions in Ruminococcus, Blautia, and Eubacterium and significant increases in Parabacteroides, Lactobacillus, Fusobacterium, and Streptococcus. The in vitro fermentation experiment showed that compared with the HBV-ACLF non-treatment group, the HBV-ACLF treatment group had significant increases in Ruminococcus, Lachnospira, Bacteroides, and Genusgenus and significant reductions in Fusobacterium and Proteobacteria (all P < 0.05). Conclusion Liangxue Jiedu decoction can regulate intestinal flora disturbance, restore the diversity of intestinal flora, increase dominant bacteria, and reduce pathogenic bacteria, which may be one of its important mechanisms of action in the treatment of HBV-ACLF.
- Hepatitis B Virus /
- Acute-On-Chronic Liver Failure /
- TCM WM THERAPY /
- Gastrointestinal Microbiome

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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

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图 2 物种累积曲线

Figure 2. Species accumulation curve

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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

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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

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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

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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

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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

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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

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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)

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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。

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表 4 各个水平菌群比较

Table 4. Comparison of microflora at different levels

方法	OTU	门	纲	目	科	属	种
t检验	142	3	6	13	20	39	10
Wilcoxon检验	263	2	6	13	34	77	22

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