解毒化瘀通腑方对乙型肝炎肝硬化肝胆湿热证患者肠道菌群的影响

刘江凯; 牛亚蒙; 李素领; 马庆亮; 张建文; 张雅儒; 李冰倩

doi:10.3969/j.issn.1001-5256.2022.04.016

解毒化瘀通腑方对乙型肝炎肝硬化肝胆湿热证患者肠道菌群的影响

DOI: 10.3969/j.issn.1001-5256.2022.04.016

1.
河南中医药大学第一附属医院脾胃肝胆科, 郑州 450000
2.
河南中医药大学第一临床医学院, 郑州 450000

基金项目:

国家自然科学基金联合基金 (U1504825);

河南省高等学校重点科研项目计划 (20A360014);

河南省中医药拔尖人才培养项目 (Yuwei Zhongyi Han [2021] No.15)

伦理学声明：本研究方案于2019年12月9日经河南中医药大学第一附属医院伦理委员会审批，批号：2019HL-171，所有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：刘江凯对研究的思路或设计有关键贡献；李素领、牛亚蒙参与病例资料收集；刘江凯、牛亚蒙、马庆亮参与研究数据的获取分析及解释；张建文、张雅儒、李冰倩参与数据汇总及指标检测。

详细信息

通信作者:
刘江凯, xmlc001@126.com

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出版历程
- 收稿日期: 2021-08-25
- 录用日期: 2021-11-05
- 出版日期: 2022-04-20

Effect of Jiedu Huayu Tongfu prescription on intestinal flora in patients with hepatitis B cirrhosis with liver-gallbladder damp-heat syndrome

1.
Spleen, Stomach and Hepatobiliary Department, The First Affiliated Hospital of Henan University of Chinese Medicine, Zhengzhou 450000, China
2.
The First Clinical Medical College of Henan University of Chinese medicine, Zhengzhou 450000, China

Research funding:

Joint Fund of National Natural Science Foundation of China (U1504825);

Key Scientific Research Project Plan of Colleges and Universities in Henan Province (20A360014);

Henan Provincial Top Talent Training Program of Traditional Chinese Medicine (Yuwei Zhongyi Han [2021] No.15)

More Information

Corresponding author: LIU Jiangkai, xmlc001@126.com (ORCID: 0000-0002-1529-5089)

摘要

摘要: 目的研究解毒化瘀通腑方对乙型肝炎肝硬化肝胆湿热证患者肠道稳态的调节作用及对内毒素、炎性因子和细胞免疫功能的影响。方法纳入2019年6月至2021年1月在河南中医药大学第一附属医院就诊，符合诊断及纳入标准的72例患者为研究对象，随机分为观察组和对照组，每组各36例。其治疗组脱落2例，剔除2例，完成32例；对照组脱落2例，剔除1例，完成33例。两组均给予抗病毒、保肝等基础治疗，观察组加服解毒化瘀通腑颗粒，对照组给予双歧杆菌四联活菌片口服，疗程为4周。运用16S rDNA测序技术对两组粪便菌群进行基因测序，同时检测治疗前后肝功能(ALT、AST、TBil、Alb)、内毒素(ET)、TNFα、IL-6水平以及T淋巴细胞亚群(CD3⁺T、CD4⁺T、CD8⁺T、CD4⁺/CD8⁺)的变化。计量资料服从正态分布及方差齐性时，组内比较采用配对t检验，2组间比较用独立样本t检验，不服从正态分布时用Wilcoxon秩和检验；计数资料组间比较采用χ²检验。结果观察组临床总有效率为87.5%，对照组总有效率为60.6%，观察组总有效率明显高于对照组(χ²=-2.299，P=0.022)。两组治疗后ALT、AST、TBil均降低，Alb水平升高(P值均＜0.05)，与对照组相比，观察组TBil水平降低更显著(Z=-2.165，P=0.030)。两组患者治疗后CD3⁺T、CD4⁺T、CD4⁺/CD8⁺水平均明显改善, 且观察组较对照组更显著(Z值分别为-2.146、-2.940、3.157，P值分别为0.032、0.003、0.002)。两组患者治疗后炎性因子TNFα、IL-6与ET水平均明显下降，且观察组优于对照组(Z值分别为-2.139、-1.982、-2.062，P值分别为0.032、0.048、0.043)。两组治疗后OTU数量均较前上升。肠道菌群结构丰度在门水平上，观察组治疗后厚壁菌门丰度的上升和拟杆菌门丰度的下降较治疗前更显著(Z值分别为-3.181、-2.215，P值分别为0.001、0.027)；与对照组相比，观察组厚壁菌门、蓝细菌门丰度的升高和拟杆菌门、梭菌门、ε-变形菌门的降低更明显(P值均＜0.05)；在属水平上，观察组双歧杆菌属较治疗前升高更明显(Z=-2.045，P=0.041)。Alpha多样性分析表明，观察组Chao1、Ace指数较治疗前升高更加显著(t值分别为-4.263、-3.328，P值分别为0.001、0.005)，其中Ace指数较对照组上升更明显(t=2.292，P=0.030)。Beta多样性分析表明，各组菌群构成相似，无明显统计学差异(P值均＞0.05)。结论在病因及基础治疗的基础上联合解毒化瘀通腑方治疗能够缓解乙型肝炎肝硬化肝胆湿热证患者临床症状，减轻肝损伤，改善细胞免疫功能。解毒化瘀通腑方可能通过增加厚壁菌门、乳杆菌属、双歧杆菌属等有益菌的丰度和减少拟杆菌门、梭菌门等致病菌的丰度来改善患者肠道菌群失调，其对肝脏和免疫功能的进一步改善作用可能与调整肠道微生态有关。
- 乙型肝炎 /
- 肝硬化 /
- 肠道菌群 /
- 肝胆湿热证 /
- 解毒化瘀通腑方
Abstract: Objective To investigate the regulatory effect of Jiedu Huayu Tongfu prescription on intestinal homeostasis in patients with hepatitis B cirrhosis with liver-gallbladder damp-heat syndrome, as well as its effect on endotoxin, inflammatory factors, and cellular immune function. Methods A total of 72 patients who attended The First Affiliated Hospital of Henan University of Chinese Medicine from June 2019 to January 2021 and met the diagnostic and inclusion criteria were enrolled as subjects and then randomly divided into observation group and control group, with 36 patients in each group. In the treatment group, 2 patients were lost to follow-up, 2 patients were excluded, and 32 patients completed the study; in the control group, 2 patients were lost to follow-up, 1 patient was excluded, and 33 patients completed the study. In addition to the basic treatment including antiviral therapy and liver-protecting treatment, the patients in the observation group were given Jiedu Huayu Tongfu granules, and those in the control group were given oral administration of Bifidobacterium tetravaccine tablets; the course of treatment was 4 weeks for both groups. The 16S rDNA sequencing technique was used for sequencing of fecal flora, and the two groups were measured in terms of the changes in liver function [alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBil), and albumin (Alb)], endotoxin (ET), levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), and T lymphocyte subsets (CD3⁺T, CD4⁺T, CD8⁺T, and CD4⁺/CD8⁺) after treatment. For normally distributed continuous data with homogeneity of variance, the paired t-test was used for comparison within each group, and the independent samples t-test was used for comparison between two groups; the Wilcoxon rank-sum test was used for comparison of non-normally distributed continuous data. The chi-square test was used for comparison of categorical data. Results The observation group had a significantly higher overall response rate than the control group (87.5% vs 60.6%, χ²=-2.299, P=0.022). After treatment, both groups had significant reductions in ALT, AST, and TBil and a significant increase in Alb (all P < 0.05), and compared with the control group, the observation group had a significantly greater reduction in TBil (Z=-2.165, P=0.030). After treatment, both groups had significant improvements in the levels of CD3⁺T, CD4⁺T, and CD4⁺/CD8⁺, and the observation group had significantly greater improvements than the control group (Z=-2.146, -2.940, and 3.157, P=0.032, 0.003, and 0.002). After treatment, both groups had significant reductions in the levels of TNF-α, IL-6, and ET, and the observation group had significantly greater reductions than the control group (Z=-2.139, -1.982, and -2.062, P=0.032, 0.048, and 0.043). Both groups had an increase in the number of operational taxonomic units after treatment. As for the abundance of intestinal flora at the phylum level, the observation group had a significant increase in the abundance of Firmicutes and a significant reduction in the abundance of Bacteroidetes after treatment (Z=-3.181 and -2.215, P=0.001 and 0.027); compared with the control group, the observation group had significantly greater increases in the abundance of Firmicutes and Cyanobacteria and significantly greater reductions in the abundance of Bacteroidetes, Cercozoa, and ε-Proteobacteria (allP < 0.05). At the genus level, the observation group had a significant increase in the abundance of Bifidobacterium after treatment (Z=-2.045, P=0.041). The alpha-diversity analysis showed that the observation group had significant increases in Chao1 and Ace indices after treatment (t=-4.263 and -3.328, P=0.001 and 0.005) and a significantly greater increase in Ace index than the control group (t=2.292, P=0.030). The beta-diversity analysis showed that the two groups had a similar composition of flora without significant difference (all P > 0.05). Conclusion Jiedu Huayu Tongfu prescription, in combination with etiological and basic treatments, can alleviate clinical symptoms, reduce liver injury, and improve cellular immune function in patients with hepatitis B cirrhosis with liver-gallbladder damp-heat syndrome. Jiedu Huayu Tongfu prescription can improve the imbalance of intestinal flora by increasing the abundance of the probiotic bacteria such as Firmicutes, Lactobacillus, and Bifidobacterium and the pathogenic bacteria such as Bacteroidetes and Cercozoa, and its effect in further improving liver and immune function may be associated with the regulation of intestinal microecology.
- Hepatitis B /
- Liver Cirrhosis /
- Gut Microbiota /
- Damp Heat Syndrome Of Liver And Gallbladder /
- Jiedu Huayu Tongfu Recipe

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图 1 组间Venn图

注: ZYQ为观察组治疗前; ZYH为观察组治疗后; XYQ为对照组治疗前; XYH为对照组治疗后。

Figure 1. Venn diagram between groups

下载: 全尺寸图片幻灯片

图 2 肠道菌群结构丰度柱状图

注: a, 门水平上的差异; b, 属水平上的差异。ZYQ为观察组治疗前, ZYH为观察组治疗后, XYQ为对照组治疗前, XYH为对照组治疗后。

Figure 2. Structural abundance histogram of intestinal flora

下载: 全尺寸图片幻灯片

图 3 Alpha多样性分析图

注: ZYQ为观察组治疗前; ZYH为观察组治疗后; XYQ为对照组治疗前; XYH为对照组治疗后。

Figure 3. Alpha diversity analysis

下载: 全尺寸图片幻灯片

图 4 肠道菌群结构PCoA、NMDS分析图

注: a, PCoA分析图; b, NMDS分析图。ZYQ为观察组治疗前, ZYH为观察组治疗后, XYQ为对照组治疗前, XYH为对照组治疗后。

Figure 4. Analysis of intestinal flora structure PCoA and NMDS

下载: 全尺寸图片幻灯片

表 1 两组中医症状疗效比较

Table 1. Comparison of curative effects of TCM symptoms between the two groups

组别	例数	显效[例(%)]	有效[例(%)]	无效[例(%)]	总有效率(%)
观察组	32	7(21.9)	21(65.6)	4(12.5)	87.5
对照组	33	4(12.1)	16(48.5)	13(39.4)	60.6

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表 2 两组肝功能比较

Table 2. Comparison of liver function between the two groups

组别	例数	ALT(U/L)				AST(U/L)
组别	例数	治疗前	治疗后	Z值	P值	治疗前	治疗后	Z值	P值
观察组	32	44.45(36.78~66.38)	32.50(27.45~44.08)	7.007	< 0.001	48.85(33.15~63.33)	37.40(29.93~42.53)	-4.507	< 0.001
对照组	33	47.50(33.75~65.30)	33.80(24.80~49.55)	5.599	< 0.001	46.20(33.40~65.00)	35.10(26.85~50.25)	-4.083	< 0.001
Z值		-0.046	-0.387			-0.092	-0.013
P值		0.963	0.699			0.927	0.990

组别	例数	TBil(μmol/L)				Alb(g/L)
组别	例数	治疗前	治疗后	Z值	P值	治疗前	治疗后	Z值	P值
观察组	32	20.08(17.25~39.75)	15.80(12.85~21.23)	-4.506	< 0.001	35.50(30.35~41.83)	38.25(34.60~43.78)	-2.940	0.006
对照组	33	25.20(16.30~38.45)	23.70(15.10~33.00)	-2.207	0.027	35.20(29.95~40.05)	36.20(33.50~40.00)	-2.472	0.019
Z值		-0.020	-2.165			0.326	1.760
P值		0.984	0.030			0.745	0.083

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表 3 两组T淋巴细胞亚群水平比较

Table 3. Comparison of T lymphocyte subsets between the two groups

组别	例数	CD3⁺T(%)				CD4⁺T(%)
组别	例数	治疗前	治疗后	Z值	P值	治疗前	治疗后	Z值	P值
观察组	32	61.10(49.73~68.80)	71.55(67.60~74.25)	-5.189	< 0.001	32.05(24.48~40.28)	43.20(38.70~46.15)	-5.321	< 0.001
对照组	33	61.70(48.90~68.70)	68.20(60.35~71.10)	-6.745	< 0.001	33.70(25.85~39.95)	38.00(32.85~41.45)	-2.537	0.011
Z值		-0.066	-2.146			-0.217	-2.940
P值		0.948	0.032			0.829	0.003

组别	例数	CD8⁺T(%)				CD4⁺/CD8⁺
组别	例数	治疗前	治疗后	Z值	P值	治疗前	治疗后	Z值	P值
观察组	32	26.40(22.00~36.75)	25.55(20.65~28.43)	2.565	0.015	1.27(0.92~1.52)	1.69(1.55~1.93)	-6.983	< 0.001
对照组	33	29.70(24.25~36.25)	27.70(23.50~30.90)	1.788	0.083	1.05(0.72~1.59)	1.43(1.06~1.80)	-2.381	0.023
Z值		-0.513	-1.887			-0.741	3.157
P值		0.610	0.064			0.458	0.002

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表 4 两组治疗前后炎症指标比较

Table 4. Comparison of inflammatory indexes between the two groups

组别	例数	IL-6(pg/mL)		TNFα(pg/mL)		ET(pg/mL)
组别	例数	治疗前	治疗后	治疗前	治疗后	治疗前	治疗后
观察组	32	5.04(4.05~6.00)	2.73(1.77~3.16)	57.59(42.37~67.27)	26.94(16.20~42.59)	9.28(7.49~11.41)	6.18(4.00~8.20)
对照组	33	4.99(4.27~6.18)	3.05(2.51~3.90)	58.38(32.30~71.68)	48.18(15.69~65.41)	10.05(8.59~12.16)	8.12(5.48~9.90)
Z值		-0.518	-2.139	-0.039	-1.982	-0.688	-2.062
P值		0.604	0.032	0.969	0.048	0.494	0.043

下载: 导出CSV

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