Effects of Zhenggan Huayu decoction (正肝化瘀方) combined with entecavir on gut microbiota in patients with chronic hepatitis B fibrosis

doi:10.19852/j.cnki.jtcm.20230208.002

Abstract

Abstract:

OBJECTIVE: To evaluate the effects of Zhenggan Huayu decoction (正肝化瘀方, ZGHY) combined with entecavir (ETV) on the gut microbiota in patients with chronic hepatitis B (CHB) fibrosis.

METHODS: A total of 59 CHB-related fibrosis patients were enrolled and treated with ZGHY combined with ETV (ZGHY + ETV) and ETV alone. Fecal samples were collected from patients at weeks 0, 12, and 24 after treatment and gut microbiota were analyzed by 16S rRNA gene sequencing.

RESULTS: Compared to the ETV group, microbiota diversity in the ZGHY + ETV group was increased after 24 weeks. Some potentially pathogenic bacteria, including Fusobacteriaceae spp., Acidaminococcus spp., and Erysipelotrichaceae spp. were reduced in the ZGHY + ETV group, while Eubacteriaceae spp., Lactococcus spp., and several other beneficial bacteria were increased.

CONCLUSION: Decreases in pathogenic bacteria and increases in probiotics were not always observed in the Traditional Chinese Medicine (TCM) group (e.g., Rothia was abundant). As an adjuvant TCM formulation for ETV, ZGHY had a positive role in the treatment of CHB patients.

Key words: hepatitis B, chronic, fibrosis, RNA, ribosomal, 16S, Zhenggan Huayu decoction, entecavir, gut microbiota

CHEN Wenlin, LIANG Fang, ZHANG Yuncheng, LV Jinzhen, YANG Daguo. Effects of Zhenggan Huayu decoction (正肝化瘀方) combined with entecavir on gut microbiota in patients with chronic hepatitis B fibrosis[J]. Journal of Traditional Chinese Medicine, 2023, 43(3): 559-567.

Figures/Tables 5

Table 1 Characteristics and clinical indices of participants

	Before treatment (0 week)			After treatment (24 weeks)
Item	ETV (n = 21)	TCM (n = 38)	P value	ETV (n = 21)	TCM (n = 38)	P value
Sex (F/M)	3/17	3/35	0.405	0.176	0.086	0.405
Age	40.45±7.35	41.70±9.29	0.456	40.45±7.35	41.70±9.29	0.456
ALT	86.31±105.55	241.84±808.09	0.697	25.32±9.48	26.93±12.22	0.563
AST	80.46±108.09	68.51±52.44	0.715	27.24±4.97	31.34±15.09	0.953
GGT	63.01±48.67	92.53±138.73	0.841	36.38±24.81	40.73±31.62	0.759
ALP	98.60±36.55	106.80±58.69	0.804	80.57±32.67	89.26±51.46	0.991
TBA	13.59±13.09	24.39±38.46	0.532	11.33±17.36	13.60±28.28	0.654
TP	73.59±6.55	72.82±5.75	0.637	71.79±8.05	74.48±5.25	0.328
TBIL	22.89±13.56	26.70±23.48	0.953	19.06±6.41	18.63±6.64	0.715
DBIL	6.01±5.98	8.59±12.24	0.741	3.45±1.14	3.97±2.80	0.878
IBIL	17.44±7.68	18.10±11.97	0.663	15.61±5.44	14.23±5.08	0.268
CHE	7485.12±2208.47	7555.21±2626.55	0.805	7815.45±1555.8	8081.00±2335.77	0.596
TG	1.28±0.71	1.37±0.69	0.340	1.16±0.41	1.56±1.21	0.396
TC	4.71±1.11	4.47±0.89	0.509	4.88±1.13	4.68±0.88	0.841
HDL-C	1.39±0.34	1.22±0.37	0.283	1.43±0.27	1.28±0.22	0.087
LDL-C	3.13±0.84	3.02±0.71	0.588	3.33±0.83	3.27±0.80	1.000
UREA	4.87±1.15	4.69±0.91	0.540	29.08±107.35	4.86±0.78	0.334
CR	72.57±13.14	70.72±8.75	0.741	75.46±9.86	73.99±11.98	0.841
UA	365.34±102.41	342.92±79.17	0.760	369.56±110.51	361.81±65.52	0.769
HA	182.92±237.93	202.16±272.55	0.800	87.81±88.53	60.15±41.98	0.266
PIIINP	12.13±10.30	11.51±9.62	0.888	8.92±5.80	6.63±2.14	0.490
IV-C	100.76±88.14	104.07±143.10	0.422	56.73±32.23	45.04±27.97	0.254
LN	2604.00±10286.62	119.10±67.09	0.746	113.61±75.35	93.10±38.79	0.526
RL	122.30±10.74	123.58±12.82	0.962	121.55±9.55	122.88±8.65	0.509
DPV	10.85±1.24	10.50±0.76	0.617	10.60±1.28	10.50±0.65	0.440
TS	41.15±5.72	37.38±6.42	0.041	36.55±5.01	35.50±5.33	0.594

Table 1 Characteristics and clinical indices of participants

	Before treatment (0 week)			After treatment (24 weeks)
Item	ETV (n = 21)	TCM (n = 38)	P value	ETV (n = 21)	TCM (n = 38)	P value
Sex (F/M)	3/17	3/35	0.405	0.176	0.086	0.405
Age	40.45±7.35	41.70±9.29	0.456	40.45±7.35	41.70±9.29	0.456
ALT	86.31±105.55	241.84±808.09	0.697	25.32±9.48	26.93±12.22	0.563
AST	80.46±108.09	68.51±52.44	0.715	27.24±4.97	31.34±15.09	0.953
GGT	63.01±48.67	92.53±138.73	0.841	36.38±24.81	40.73±31.62	0.759
ALP	98.60±36.55	106.80±58.69	0.804	80.57±32.67	89.26±51.46	0.991
TBA	13.59±13.09	24.39±38.46	0.532	11.33±17.36	13.60±28.28	0.654
TP	73.59±6.55	72.82±5.75	0.637	71.79±8.05	74.48±5.25	0.328
TBIL	22.89±13.56	26.70±23.48	0.953	19.06±6.41	18.63±6.64	0.715
DBIL	6.01±5.98	8.59±12.24	0.741	3.45±1.14	3.97±2.80	0.878
IBIL	17.44±7.68	18.10±11.97	0.663	15.61±5.44	14.23±5.08	0.268
CHE	7485.12±2208.47	7555.21±2626.55	0.805	7815.45±1555.8	8081.00±2335.77	0.596
TG	1.28±0.71	1.37±0.69	0.340	1.16±0.41	1.56±1.21	0.396
TC	4.71±1.11	4.47±0.89	0.509	4.88±1.13	4.68±0.88	0.841
HDL-C	1.39±0.34	1.22±0.37	0.283	1.43±0.27	1.28±0.22	0.087
LDL-C	3.13±0.84	3.02±0.71	0.588	3.33±0.83	3.27±0.80	1.000
UREA	4.87±1.15	4.69±0.91	0.540	29.08±107.35	4.86±0.78	0.334
CR	72.57±13.14	70.72±8.75	0.741	75.46±9.86	73.99±11.98	0.841
UA	365.34±102.41	342.92±79.17	0.760	369.56±110.51	361.81±65.52	0.769
HA	182.92±237.93	202.16±272.55	0.800	87.81±88.53	60.15±41.98	0.266
PIIINP	12.13±10.30	11.51±9.62	0.888	8.92±5.80	6.63±2.14	0.490
IV-C	100.76±88.14	104.07±143.10	0.422	56.73±32.23	45.04±27.97	0.254
LN	2604.00±10286.62	119.10±67.09	0.746	113.61±75.35	93.10±38.79	0.526
RL	122.30±10.74	123.58±12.82	0.962	121.55±9.55	122.88±8.65	0.509
DPV	10.85±1.24	10.50±0.76	0.617	10.60±1.28	10.50±0.65	0.440
TS	41.15±5.72	37.38±6.42	0.041	36.55±5.01	35.50±5.33	0.594

Figure 1 Effects of ETV and TCM on the gut microbiota of patients A: rarefaction curve analysis of ASVs in each group of samples. The number of identified ASVs in each group was calculated based on a randomly selected specific number of samples with 100 replacements. Quartiles numbers are plotted by a line segment; B, C: differences in gut microbiota diversity between ETV and TCM groups at different treatment periods. The significance level in the Wilcoxon signed-rank test was marked as a numerical P-value; D: the dbRDA analysis based on microbiota composition showed separations between groups. The R2 and P values generated by PERMANOVA analysis are shown in the lower-left quadrant of the plot; E: dbRDA analysis showed separations at different treatment periods within ETV and TCM groups, and R2 as well as P values generated by PERMANOVA analysis are shown at the top of the image; F: PERMANOVA analysis showed differences between treatment periods. Patients in the TCM group were treated with ZGHY 1 potion/d (oral administration in two doses) and ETV 0.5 mg/d, and the ETV group were treated with ETV 0.5 mg/d. All patients were treated for a total of 24 weeks, fecal samples were collected and analyzed in the 12 and 24 weeks, respectively. ETV: entecavir; TCM: Traditional Chinese Medicine (FZHY+ETV group); CON: control (ETV group); ZGHY: Zhenggan Huayu decoction; ASVs: amplicon sequence variants; dbRDA: distance-based redundancy analysis; PERMANOVA: permutational multivariate analysis of variance. Significant differences in TCM group: 12 w vs 24 w, aP<0.05; 0+12 w vs 24 w, aP<0.05. n = 38.

Figure 2 Microbiota composition in patients’ gut A: the composition of phyla levels in different groups. Only the top 10 members in relative abundance are shown; B: species differences at phylum levels between groups; C: the composition of family levels in different groups. Only the top 10 members in relative abundance are shown; D: species differences at family levels between groups. Patients in the TCM group were treated with ZGHY 1 potion/d (oral administration in two doses) and ETV 0.5 mg/d, and the ETV group were treated with ETV 0.5 mg/d. All patients were treated for a total of 24 weeks, fecal samples were collected and analyzed in the 12 and 24 weeks, respectively. ETV: entecavir; ZGHY: Zhenggan Huayu decoction; TCM: Traditional Chinese Medicine (FZHY + ETV group); CON: control (ETV group).

Figure 3 Heat maps show correlations between feature genera and clinical indicators Spearman correlation test showed that: * q<0.05; + q<0.01. A correlation coefficient>0.6 was considered as a positive correlation, and<-0.6 as a negative correlation. ALP: alkaline phosphatase; ALT: alanine aminotransferase; AST: aspartate aminotransferase; CHE: cholinesterase; CR: creatinine; DBIL: direct bilirubin; GGT: γ-glutamyl transferase; HA: hyaluronic acid; HDL-C: high-density lipoprotein cholesterol; IBIL: indirect bilirubin; IV-C: Type IV collagen; LDL-C: low-density lipoprotein cholesterol; LN: laminin; PIIINP: type III procollagen peptide; TBA: total bile acid; TBIL: total bilirubin; TC: total cholesterol; TG: triglyceride; TP: total protein; UA: uric acid; UREA: urea; RL: right lobe of liver oblique diameter; DPV: portal vein diameter; TS: spleen thickness.

Figure 4 Effects of ETV and TCM on gut microbiota functions A: rarefaction curve analysis of KOs in each group of samples. The number of annotated KO genes in each group was calculated based on a randomly selected specific number of samples with 100 replacements. Quartile numbers were plotted by a line segment. B: boxplot showing the Shannon index of gut microbiota function for patients in ETV and TCM groups at different treatment periods, revealing microbiota function diversity in patients’ gut. C: fold change of gut microbiota function between patients in TCM and ETV groups. Only functional items with significant differences (P<0.05) are shown, TCM group: n = 38, ETV group, n = 21. Patients in the TCM group were treated with ZGHY 1 potion/d (oral administration in two doses) and ETV 0.5 mg/d, and the ETV group were treated with ETV 0.5 mg/d. All patients were treated for a total of 24 weeks, fecal samples were collected and analyzed in the 12 and 24 weeks, respectively. ETV: entecavir; TCM: Traditional Chinese Medicine (FZHY + ETV group); CON: control (ETV group); KO: Kyoto Encyclopedia of Genes and Genomes Orthology; ZGHY: Zhenggan Huayu decoction.

References 44

[1]	Nguyen MH, Wong G, Gane E, Kao JH, Dusheiko G. Hepatitis B virus: advances in prevention, diagnosis, and therapy. Clin Microbiol Rev 2020; 33: e00046-19.
[2]	Suhail M, Abdel-Hafiz H, Ali A, Fatima K, et al. Potential mechanisms of hepatitis B virus induced liver injury. World J Gastroenterol 2014; 20: 12462-72. DOI URL
[3]	Tang LSY, Covert E, Wilson E, Kottilil S. Chronic hepatitis B Infection: a review. JAMA 2018; 319: 1802-13. DOI PMID
[4]	Seto WK, Lo YR, Pawlotsky JM, Yuen MF. Chronic hepatitis B virus infection. Lancet 2018; 392: 2313-24. DOI URL
[5]	Sarin SK, Kumar M, Lau GK, et al. Asian-Pacific clinical practice guidelines on the management of hepatitis B: a 2015 update. Hepatol Int 2016; 10: 1-98. DOI PMID
[6]	Zhou WC, Zhang QB, Qiao L. Pathogenesis of liver cirrhosis. World J Gastroenterol 2014; 20: 7312-24. DOI URL
[7]	Calvaruso V, Craxì A. Fibrosis in chronic viral hepatitis. Best Pract Res Clin Gastroenterol 2011; 25: 219-30. DOI PMID
[8]	Wang T, Jin W, Huang Q, et al. Clinical efficacy and safety of eight Traditional Chinese Medicine combined with entecavir in the treatment of chronic hepatitis B liver fibrosis in adults: a network Meta-analysis. Evid Based Complement Alternat Med: eCAM 2020; 2020: 7603410.
[9]	Choi WM, Yip TC, Lim YS, Wong GL, Kim WR. Methodological challenges of performing Meta-analyses to compare the risk of hepatocellular carcinoma between chronic hepatitis B treatments. J Hepatol 2022; 76: 186-94. DOI URL
[10]	Lange CM, Bojunga J, Hofmann WP, et al. Severe lactic acidosis during treatment of chronic hepatitis B with entecavir in patients with impaired liver function. Hepatology 2009; 50: 2001-6. DOI PMID
[11]	Zhang L, Wang G, Hou W, Li P, Dulin A, Bonkovsky HL. Contemporary clinical research of Traditional Chinese Medicines for chronic hepatitis B in China: an analytical review. Hepatology 2010; 51: 690-8. DOI PMID
[12]	Hu J, Teng J, Wang W, et al. Clinical efficacy and safety of Traditional Chinese Medicine Xiaoyao San in insomnia combined with anxiety. Medicine 2021; 100: e27608.
[13]	Zhang DQ, Mu YP, Xu Y, Chen JM, Liu P, Liu W. Research progress in Chinese Medicine preparations for promoting blood circulation and removing blood stasis for cirrhotic patients with portal vein thrombosis following splenectomy. Chin J Integr Med 2020; 28: 855-863. DOI
[14]	Huang C, Shen D, Sun S, et al. Effect of Fufang Biejia Ruangan Tablet on lowering biochemical and virological parameters of hepatic fibrosis in patients with chronic hepatitis B: protocol for a systematic review and Meta-analysis of randomized controlled trials and cohort studies. Medicine 2019; 98: e15297.
[15]	Wang T, Zhou X, Liu H, et al. Fuzheng Huayu capsule as an adjuvant treatment for HBV-related cirrhosis: a systematic review and Meta-analysis. Phytother Res 2018; 32: 757-68. DOI PMID
[16]	Li X, Zhou D, Chi X, et al. Entecavir combining Chinese herbal medicine for HBeAg-positive chronic hepatitis B patients: a randomized, controlled trial. Hepatol Int 2020; 14:985-96. DOI PMID
[17]	Li X, Zhang L, Qiu M, et al. Chinese herbal medicine combined with entecavir to reduce the off-therapy recurrence risk in HBeAg-positive chronic hepatitis B patients: a multicener, double-blind, randomized controlled trial in China. Trials 2020; 21: 708. DOI PMID
[18]	Jones RM, Neish AS. Gut microbiota in intestinal and liver disease. Annu Rev Pathol 2021; 16: 251-75. DOI PMID
[19]	Wang X, Chen L, Wang H, Cai W, Xie Q. Modulation of bile acid profile by gut microbiota in chronic hepatitis B. J Cell Mol Med 2020; 24: 2573-81. DOI PMID
[20]	Bajaj JS. Alcohol, liver disease and the gut microbiota. Nat Rev Gastroenterol Hepatol 2019; 16: 235-46. DOI PMID
[21]	Fan Y, Pedersen O. Gut microbiota in human metabolic health and disease. Nat Rev Microbiol 2021; 19: 55-71. DOI
[22]	Yang R, Xu Y, Dai Z, Lin X, Wang H. The immunologic role of gut microbiota in patients with chronic HBV infection. J Immunol Res 2018; 2018: 2361963.
[23]	Zeng Y, Chen S, Fu Y, et al. Gut microbiota dysbiosis in patients with hepatitis B virus-induced chronic liver disease covering chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. J Viral Hepat 2020; 27: 143-55. DOI URL
[24]	Lv LJ, Li SH, Li SC, et al. Early-onset preeclampsia is associated with gut microbial alterations in antepartum and postpartum women. Front Cell Infect Microbiol 2019; 9: 224. DOI URL
[25]	Bolyen E, Rideout JR, Dillon MR, et al. Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. Nat Biotechnol 2019; 37: 852-7. DOI PMID
[26]	Quast C, Pruesse E, Yilmaz P, et al. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res 2013; 41: D590-6.
[27]	Douglas GM, Maffei VJ, Zaneveld JR, et al. PICRUSt2 for prediction of metagenome functions. Nat Biotechnol 2020; 38: 685-8. DOI PMID
[28]	Ren Z, Li A, Jiang J, et al. Gut microbiome analysis as a tool towards targeted non-invasive biomarkers for early hepatocellular carcinoma. Gut 2019; 68: 1014-23. DOI PMID
[29]	Li X, Wu S, Du Y, Yang L, Li Y, Hong B. Entecavir therapy reverses gut microbiota dysbiosis induced by hepatitis B virus infection in a mouse model. Int J Antimicrob Agents 2020; 56: 106000.
[30]	Compare D, Coccoli P, Rocco A, et al. Gut--liver axis: the impact of gut microbiota on non alcoholic fatty liver disease. Nutr Metab Cardiovasc Dis 2012; 22: 471-6. DOI PMID
[31]	Wan MLY, El-Nezami H. Targeting gut microbiota in hepatocellular carcinoma: probiotics as a novel therapy. Hepatobiliary Surg Nutr 2018; 7: 11-20. DOI URL
[32]	Gevers D, Kugathasan S, Denson LA, et al. The treatment-naive microbiome in new-onset Crohn's disease. Cell Host Microbe 2014; 15: 382-92. DOI PMID
[33]	Bajaj JS, Heuman DM, Hylemon PB, et al. Altered profile of human gut microbiome is associated with cirrhosis and its complications. J Hepatol 2014; 60: 940-7. DOI PMID
[34]	Shek D, Chen D, Read SA, Ahlenstiel G. Examining the gut-liver axis in liver cancer using organoid models. Cancer Lett 2021; 510: 48-58. DOI PMID
[35]	Roberti MP, Yonekura S, Duong CPM, et al. Chemotherapy-induced ileal crypt apoptosis and the ileal microbiome shape immunosurveillance and prognosis of proximal colon cancer. Nat Med 2020; 26: 919-31. DOI PMID
[36]	Kaakoush NO. Insights into the role of erysipelotrichaceae in the human host. Front Cell Infect Microbiol 2015; 5: 84.
[37]	Lurie Y, Webb M, Cytter-Kuint R, Shteingart S, Lederkremer GZ. Non-invasive diagnosis of liver fibrosis and cirrhosis. World J Gastroenterol 2015; 21: 11567-83. DOI URL
[38]	Qin F, Wu H, Li X, Han J. Correlation between changes in gut flora and serum inflammatory factors in children with noninfectious diarrhea. J Int Med Res 2020; 48: 300060519896154.
[39]	Zhu L, Baker SS, Gill C, et al. Characterization of gut microbiomes in nonalcoholic steatohepatitis (NASH) patients: a connection between endogenous alcohol and NASH. Hepatology 2013; 57: 601-9. DOI PMID
[40]	Maccioni L, Gao B, Leclercq S, et al. Intestinal permeability, microbial translocation, changes in duodenal and fecal microbiota, and their associations with alcoholic liver disease progression in humans. Gut microbes 2020; 12: 1782157. DOI URL
[41]	Wang B, Zhang SQ, Dong JL, et al. Ambient temperature structures the gut microbiota of zebrafish to impact the response to radioactive pollution. Environ Pollut 2021; 293: 118539. DOI URL
[42]	Martin-Gallausiaux C, Marinelli L, Blottière HM, Larraufie P, Lapaque N. SCFA: mechanisms and functional importance in the gut. Proc Nutr Soc 2021; 80: 37-49. DOI URL
[43]	Louis P, Flint HJ. Diversity, metabolism and microbial ecology of butyrate-producing bacteria from the human large intestine. FEMS Microbiol Lett 2009; 294: 1-8. DOI PMID
[44]	Suez J, Zmora N, Zilberman-Schapira G, et al. Post-antibiotic gut mucosal microbiome reconstitution is impaired by probiotics and improved by autologous FMT. Cell 2018; 174: 1406-23. e16. DOI PMID

Home

Online First

Author Center

Reviewer Center

Issues

Announcement

About Us