J Neurogastroenterol Motil 2023; 29(4): 520-531  https://doi.org/10.5056/jnm22182
Distinct Effects of Non-absorbed Agents Rifaximin and Berberine on the Microbiota-Gut-Brain Axis in Dysbiosis-induced Visceral Hypersensitivity in Rats
Jindong Zhang, Cunzheng Zhang, Tao Zhang, Lu Zhang, and Liping Duan*
Department of Gastroenterology, Peking University Third Hospital, Beijing, China
Correspondence to: *Liping Duan, MD
Department of Gastroenterology, Peking University Third Hospital, No. 49 North Garden Road, Haidian District, Beijing, China
Tel: +86-10-8280-6003, Fax: +86-10-8280-1250, E-mail: duanlp@bjmu.edu.cn
Received: October 26, 2022; Revised: January 21, 2023; Accepted: February 12, 2023; Published online: October 30, 2023
© The Korean Society of Neurogastroenterology and Motility. All rights reserved.

cc This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Irritable bowel syndrome (IBS) is accepted as a disorder of gut-brain interactions. Berberine and rifaximin are non-absorbed antibiotics and have been confirmed effective for IBS treatment, but there is still lack of direct comparison of their effects. This study aims to compare the effect of the 2 drugs on the alteration of gut-brain axis caused by gut microbiota from IBS patients.
Germ-free rats received fecal microbiota transplantation from screened IBS patients and healthy controls. After 14 days’ colonization, rats were administrated orally with berberine, rifaximin or vehicle respectively for the next 14 days. The visceral sensitivity was evaluated, fecal microbiota profiled and microbial short chain fatty acids were determined. Immunofluorescence staining and morphological analysis were performed to evaluate microglial activation.
Visceral hypersensitivity induced by IBS–fecal microbiota transplantation was relieved by berberine and rifaximin, and berberine increased sucrose preference rate. Microbial α-diversity were reduced by both drugs. Compared with rifaximin, berberine significantly changed microbial structure and enriched Lachnoclostridium. Furthermore, berberine but not rifaximin significantly increased fecal concentrations of acetate and propionate acids. Berberine restored the morphological alterations of microglia induced by dysbiosis, which may be associated with its effect on the expression of microbial gene pathways involved in peptidoglycan biosynthesis. Rifaximin affected neither the numbers of activated microglial cells nor the microglial morphological alterations.
Berberine enriched Lachnoclostridium, reduced the expression of peptidoglycan biosynthesis genes and increased acetate and propionate. The absence of these actions of rifaximin may explain the different effects of the drugs on microbiota-gut-brain axis.
Keywords: Berberine; Microbiota-gut-brain axis; Microglia; Rifaximin

Irritable bowel syndrome (IBS) is the most common functional gastrointestinal disorder in clinical practice, which is defined as disorder of gut-brain interactions based on the Rome IV criteria.1 This chronic heterogeneous disorder affects 5% to 10% of the general population worldwide.2 Due to the recurrent abdominal pain associated with altered stool form or frequency, patients with IBS often have a poor quality of life and work productivity.3 Traditionally, the possible pathophysiological mechanisms of IBS include psychosocial distress, mild mucosal inflammation, colonic barrier impairment, alteration of gastrointestinal sensitivity and motility.4 Accordingly, the commonly used treatment for IBS consists of antispasmodics, antidiarrheals, antidepressants, and psychological/behavioral therapies.

In recent years, emerging evidence indicates that IBS patients have a condition of dysbiosis compared with healthy people.5 Our previous studies have demonstrated that both intestinal microbial composition and gene functions of IBS patients differed significantly from healthy controls.6,7 Thus, the concept of microbiota-gut-brain has been widely accepted by an increasing number of researchers in this field. These conclusions promote the application of treatment targeting microbiota for IBS, including probiotics, prebiotics, antibiotics, and fecal microbiota transplantation. Among them, antibiotics such as neomycin, rifaximin and systemic antibiotics, have higher quality of evidence than other methods, as they have been proved effective in randomized controlled trials.8,9

Rifaximin is a nonsystemic, broad-spectrum antibiotic that locally acts on the gut. Treatment with rifaximin for 2 weeks significantly relieved IBS symptoms in patients who had IBS without constipation.9 In vivo experiment showed that rifaximin prevented stress-induced visceral hypersensitivity and altered gut microbiota.10 Berberine, the major active compound in the Chinese herb Coptis chinensis (Huanglian), is also a non-absorbed agent with antibacterial properties. Besides the bacterial diarrhea, berberine has been shown to be effective in alleviating both abdominal and psychological symptoms in IBS patients.11 Treatment with berberine significantly modulated microbial composition in different clinical trials and rodent experiments.12-14 Interestingly, although rifaximin and berberine have a similar therapeutic effect and characteristic of modulating gut microbiota, there is no direct comparison of their effect on the microbiota-gut-brain axis.

Therefore, in this study, we investigate the effect of rifaximin and berberine on dysbiosis-induced visceral hypersensitivity, the microbial and metabolites alterations and microglial activation, aiming to compare the influence of the 2 drugs on the microbiota-gut-brain axis.

Materials and Methods

Animals and Drugs

Six-week old male germ-free (GF) Sprague-Dawley rats were kept under sterile conditions at the Department of Laboratory Animal Science, Peking University Health Science Center in Beijing, China. All animals were housed on a 12-hour light/dark cycle at a constant temperature (23 ± 2℃) and humidity (63 ± 2%), and were provided autoclaved food and water ad libitum. All protocols were approved by the Laboratory Animal Welfare Ethics branch of the Biomedical Ethics Committee of Peking University (Approval No. LA2016230).

After 7 days of acclimatization, GF rats were assigned into 4 groups. Group 1 (GH group) received fecal microbiota transplantation (FMT) from a healthy donor, whereas groups 2 (GI group, GF rats receiving FMT from IBS patients), 3, and 4 received FMT from a patient with diarrhea-predominant IBS (IBS-D). After humanized for 14 days, groups 1 and 2 underwent vehicle gavage, while group 3 (GIBBR group, GI rats receiving berberine treatment) and 4 (GIR group, GI rats receiving rifaximin treatment) were orally administrated with berberine and rifaximin, respectively. All treatments lasted for 14 days. Berberine and rifaximin were both purchased from Shanghai Shifeng Biotechnology Ltd, Shanghai, China, and they were dissolved in sterile water with the dosage of 200 mg/kg/day (berberine) and 150 mg/kg/day (rifaximin) respectively. On the 14th day, the visceral sensitivity was assessed by colorectal distension tests, and on the next day, all rats were sacrificed and samples collected.

Fecal Microbiota Transplantation

The stool sample were collected from a treatment-naïve IBS-D patients and a healthy control (Supplementary Table 1). The stool preparation was performed as described in our previous study.15 Briefly, 600 mg frozen stool was resolved in 30 mL sterile phosphate buffered saline-20% glycerol (volume/volume) mixed solution. The GF rats were humanized by oral gavage with 2 mL suspension and inoculation of the bedding.

Colorectal Distension

Visceral hypersensitivity was assessed by colorectal distension (CRD) tests. Briefly, a flexible balloon made of a polyethylene plastic bag (4-5 cm) was inserted into the distal colon and inflated by 20, 40, 60, and 80 mmHg, and each distension lasted for 20 seconds, with a 4-minute interstimulus interval. Abdominal withdrawal score (AWR) was scored using a previously described scale.16 Five series of CRD tests were conducted in different pressure orders.

Sucrose Preference Test

In the sucrose preference test (SPT), rats were habituated to 1% sucrose solution or pure water for 48 hours. After removing the 2 bottles for 6 hours, each rat was free to get pure and sucrose water for 1 hour. The sucrose preference rate was calculated as (consumption of sucrose/total consumption of both kind of water) × 100%.

Hematoxylin and Eosin Staining

To evaluate the histological morphology, formalin-fixed distal colon tissues were cut into 5 μm sections and then stained with hematoxylin and eosin. Images were scanned with a Nano Zoomer scanning device (Hamamatsu, Shizuoka, Japan).

Microbiota Sequencing and Analysis

Fecal pellets were collected after 14 days of treatment and stored at –80°C. An OMEGA-soil DNA Kit (Omega Bio-Tek, Norcross, GA, USA) was used to extract microbial DNA. The V1-V3 regions of the 16S ribosomal RNA gene were amplified by polymerase chain reaction with primers (338 forward primer: 5’-ACTCCTACGGGAGGCAGCAG-3’; 806 reverse primer: 5’-GGACTACHVGGGTWTCTAAT-3’). The obtained amplicons were then sequenced on the Illumina MiSeq platform (Illumina, San Diego, CA, USA). Operational taxonomic units were clustered with a 97% similarity cutoff value, and the sequences were classified taxonomically by searching the SILVA reference database. Predictions of the microbial functions were performed using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt).

Determination of Fecal Short-chain Fatty Acids

As previously reported,15 fresh feces were weighted and mixed with aqueous acetonitrile (100 mg/1 mL), and after centrifuged and derivatized by mixing the supernatant with 3-nitrophenylhydrazine and N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide·HCl-6% pyridine solution, 10 μL of the solution were used to determine the short-chain fatty acids (SCFAs) concentration on a mass spectrometer (Thermo Fisher, Waltham, MA, USA). The data are calculated and expressed as SCFA (mg) per gram feces.

Immunofluorescence Staining

The Immunofluorescence staining and reconstruction of microglial cells was performed according to previously reported.15 Briefly, animals were anesthetized with pentobarbital and the specimens of brains and spinal cords were collected and fixed in paraformaldehyde. The brains and spinal cord were cut into 35-μm parasagittal and cross-sectional sections respectively, and were incubated with anti-Iba-1 primary antibody (1:200; Wako, Osaka, Japan) overnight at 4°C, and finally incubated with a secondary anti-rabbit antibody (1:500; Invitrogen, Carlsbad, CA, USA). Given the relationships between amygdala, prefrontal cortex, hippocampus, and L6-S1 dorsal spinal cord with visceral hypersensitivity,17,18 they were chosen to count the number of Iba-1-positive cells and to evaluate the microglial cell morphology parameters using IMARIS (Bitplane, Victoria, Australia). For the analysis of microglial cell morphology, we performed semiautomated reconstruction of microglial cell bodies and processes, and quantified the area, volume, total process length, number of branch points, number of segments, and number of terminal points. In addition, automated Sholl analysis was also performed on each cell using concentric spheres with radii that increased by 1 μm per step, and the number of intersections at each concentric sphere was analyzed.

Statistical Methods

Abdominal withdrawal reflex scores were analyzed using two-way ANOVA with CRD pressure and treatment as factors with Fisher’s least significant difference post hoc tests. The microbial data were analyzed by Wilcoxon tests. The data for SCFA contents were analyzed using one-way ANOVA followed with Fisher’s least significant difference post hoc tests. The results are expressed as the means ± standard errors of the means. Differences between groups were considered significant if P < 0.05. Statistical analyses were performed with SPSS version 20.0 (IBM Corp, Armonk, NY, USA).


Berberine and Rifaximin Alleviated Irritable Bowel Syndrome–Fecal Microbiota Transfer-induced Visceral Hypersensitivity in Germ-free Rats

The experiment design was shown in Figure 1A. Germ-free rats received FMT from ether healthy or IBS donors. In the CRD test, GF rats colonized with IBS-derived microbiota for 2 weeks showed significantly increased AWR scores at 20, 40, and 60 mmHg, compared with those receiving FMT from the healthy control. Rats in the GIBBR and GIR groups had significantly lowered AWR scores at 20 mmHg and 40 mmHg, and berberine also decreased AWR score at 60 mmHg (Fig. 1B). There were no obvious inflammatory cell infiltration and mucosal damage of the colon observed under the microscope in each group, and all colon scored 0 (Fig. 1C, data not shown). In the SPT, the sucrose preference rate in the berberine-treated group was significantly higher than the GI group, while this effect was not observed in the rifaximin-treated rats (Fig. 1D).

Figure 1. Effect of berberine and rifaximin on visceral hypersensitivity. (A) Design of the experiment. (B) Abdominal withdrawal reflex scores in response to colorectal distension. (C) Hematoxylin and eosin staining for distal colonic tissue. (D) Sucrose preference rate reflecting depressive behavior. GF, germ-free; GH, GF rats receiving fecal microbiota transplantation (FMT) from healthy control, n = 5; GI, GF rats receiving FMT from irritable bowel syndrome (IBS) patients, n = 8; GIBBR, GI rats receiving berberine treatment, n = 8; GIR, GI rats receiving rifaximin treatment, n = 7; SPT, sucrose preference test; CRD, colorectal distension. *P < 0.05, **P < 0.01, ****P < 0.0001.

Berberine and Rifaximin Had Different Effect on Microbial Structure

The rarefaction curve became smooth with the increase of number of reads sampled, which demonstrated that the number of sequencing data were enough for analysis (Fig. 2A). Chao1 and Shannon diversity indexes were significantly higher in GI rats, and berberine and rifaximin decreased the microbial richness and diversity, consistent with the antibacterial effects of the 2 drugs (Fig. 2B and 2C). Principle coordinate analysis showed significant difference of overall structure between GH and GI groups. Interestingly, berberine changed the structure towards GH group, while the microbial structure in rifaximin-treated group remained similar as GI group (Fig. 2D). As shown in Figure 2E, more than 90% of the obtained operational taxonomic units were assigned to Bacteroidetes and Firmicutes at the phylum level. The relative abundance of Firmicutes in the GI and rifaximin-treated group were higher than the other 2 groups.

Figure 2. Comparison of microbial diversity and structure altered by berberine and rifaximin. (A) The rarefaction curve showing the depth of sequencing data. (B) Chao1 and (C) shannon diversity indexes. (D) Principal coordinate analysis showing microbial structure. (E) Bacterial composition at the phylum level. GH, germ-free (GF) rats receiving fecal microbiota transplantation (FMT) from healthy control, n = 5; GI, GF rats receiving FMT from IBS patients, n = 8; GIBBR, GI rats receiving berberine treatment, n = 8; GIR, GI rats receiving rifaximin treatment, n = 7; OTU, observable taxonomic unit. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Berberine and Rifaximin Enriched Different Bacterial Taxa

Taxonomic analysis identified differentially enriched genera and species among the 4 groups. Particularly, at the genus level, the abundance of Akkermansia, Eisenbergiella, and Lachnoclostridium were significantly lower in the GI group, and neither berberine nor rifaximin reversed the decreased relative abundance of Akkermansia and Eisenbergiella in IBS-derived microbiota (Fig. 3A and 3B). Intriguingly, the 2 drugs have opposite effects on several SCFAs-producing genera, including Lachnoclostridium and Faecalibacterium. Lachnoclostridium was enriched by berberine but depleted by rifaximin, while Faecalibacterium was enriched by rifaximin but depleted by berberine (Fig. 3C and 3D). The interaction network analysis showed that Lachnoclostridium was negatively correlated with Faecalibacterium (Fig. 3E).

Figure 3. Comparison of changes of selected genera in the berberine- and rifaximin-treated groups. (A-D) The relative abundance of representative genera. (E) Correlation network of genus-genus interactions. GH, germ-free (GF) rats receiving fecal microbiota transplantation (FMT) from healthy control, n = 5; GI, GF rats receiving FMT from irritable bowel syndrome patients, n = 8; GIBBR, GI rats receiving berberine treatment, n = 8; GIR, GI rats receiving rifaximin treatment, n = 7. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Berberine Promoted More Short-chain Fatty Acids Production Than Rifaximin

The major metabolites SCFAs were determined to investigate their role in the effect of the 2 drugs. The concentration of formate acid was significantly lower in rifaximin-treated group than the other 3 groups (Fig. 4A). We observed that berberine increased fecal concentration of acetate, propionate and total SCFAs by 94.6%, 52.3%, and 56.1%, respectively, compared with GI rats. On the contrary, fecal acetate, propionate and total SCFAs decreased by 61.1%, 52.6%, and 59.3% in the rifaximin-treated group, respectively (Fig. 4B, 4C, and 4F). The butyrate acid was not influenced by any of the 2 drugs (Fig. 4D). In addition, treatment with berberine and rifaximin reduced the concentration of valerate acid to a level comparable to that of GH group (Fig. 4E). The correlation analysis showed positive relationship between the abundance of Lachnoclostridium and the concentration of acetate and propionate. The abundance of Lachnoclostridium and Bacteroides were negatively correlated with the concentration of valerate.

Figure 4. Effect of berberine and rifaximin on fecal concentrations of short chain fatty acids (SCFAs). (A-F) The fecal concentration of the 6 SCFAs. (G) Heat map of the Spearman’s rank correlation coefficient between the 6 SCFAs and selected genera. GH, germ-free (GF) rats receiving fecal microbiota transplantation (FMT) from healthy control, n = 5; GI, GF rats receiving FMT from irritable bowel syndrome patients, n = 8; GIBBR, GI rats receiving berberine treatment, n = 8; GIR, GI rats receiving rifaximin treatment, n = 7. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Berberine and Rifaximin Had Different Effect on Microbial Gene Functions

Kyoto Encyclopedia of Genes and Genomes pathway analysis of the microbial gene function showed increased cluster distance between berberine-treated and rifaximin-treated group (Fig. 5A). Post hoc analysis demonstrated berberine reduced the expression of pathways involved in peptidoglycan (PGN) biosynthesis, pantothenate and coenzyme A biosynthesis and lysine biosynthesis (Fig. 5B). Of these, we observed peptidoglycan biosynthesis associated pathways were decreased in berberine-treated group, but remained unchanged in rifaximin group (Fig. 5C). Furthermore, the relative abundance of Lachnoclostridium was negatively correlated with the proportion of sequences of PGN biosynthesis, while Faecalibacterium was positively with number of genes involved in PGN biosynthesis pathways (Fig. 5D and 5E).

Figure 5. Effect of berberine and rifaximin on microbial gene functions. (A) Principal coordinate analysis of the enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. (B) Comparison of significantly enriched KEGG pathways between the berberine- and rifaximin-treated groups. (C) Enrichment of pathways involved in peptidoglycan biosynthesis. (D-E) Correlation between the relative abundance of Lachnoclostridium and Faecalibacterium and the proportion of sequences of peptidoglycan biosynthesis. GH, germ-free (GF) rats receiving fecal microbiota transplantation (FMT) from healthy control, n = 5; GI, GF rats receiving FMT from irritable bowel syndrome patients, n = 8; GIBBR, GI rats receiving berberine treatment, n = 8; GIR, GI rats receiving rifaximin treatment, n = 7; PGN, peptidoglycan. ***P < 0.001, ****P < 0.0001.

Berberine but not Rifaximin Reversed Alleviated Irritable Bowel Syndrome–Fecal Microbiota Transfer-induced Microglial Activation

Figure 6 showed the different effects of berberine and rifaximin on activated microglia in the spinal cords. Although fecal microbiota from IBS patients did not influence the numbers of activated microglial cells (Fig. 6B), it induced amebic-like alteration of microglia compared with GH rats (Fig. 6C-G). Both drugs did not affect the numbers of activated microglial cells (Fig. 6B), however, berberine significantly reversed the microglial morphological alterations in the central nervous system (CNS), as reflected by the parameters of dendrite and branch, while the morphology of microglia of rifaximin-treated rats remained similar as GI rats (Fig. 6C-G). Furthermore, the microglial morphological parameters in amygdala, hippocampus, prefrontal cortex were also analyzed, and there was no obvious difference between GIBBR and GIR groups (Supplementary Tables 2 and 3).

Figure 6. Effect of berberine and rifaximin on microglial activation in spinal cords. (A) Representative images of Iba-1-stained microglia in the dorsal lumbar spinal cord. (B) Number of Iba-1-positive cells in the dorsal lumbar spinal cord. (C-G) Morphological parameters of microglia. GH, germ-free (GF) rats receiving fecal microbiota transplantation (FMT) from healthy control; GI, GF rats receiving FMT from irritable bowel syndrome patients; GIBBR, GI rats receiving berberine treatment; GIR, GI rats receiving rifaximin treatment. n = 4/group. *P < 0.05, **P < 0.01.

In the present study, we compared the effect of berberine and rifaximin on the microbiota-gut-brain axis. Both drugs were effective on alleviating visceral hypersensitivity, while berberine increased sucrose preference rate. For microbiota analysis, we found that berberine could reverse the microbial structural alteration induced by IBS-derived microbiota, while rifaximin did not show this action. Furthermore, the bacterial taxa enriched and the key metabolites SCFAs altered by the 2 drugs were also different. We also demonstrated that berberine but not rifaximin reversed IBS-derived microbiota induced amoebic like alteration, and this may be associated with the decreased gene pathways expression of peptidoglycan biosynthesis (Fig. 7).

Figure 7. Distinct effect of rifaximin and berberine on the microbiota-gut-brain axis in dysbiosis-induced visceral hypersensitivity. IBS, irritable bowel syndrome; PGN, peptidoglycan; CNS, central nervous system.

Previous studies have shown that fecal microbiota from IBS patients could result in visceral hypersensitivity and behavior abnormalities in germfree rodent animals.19,20 To investigate the effect of the drugs on microbiota under an ideal situation, we also established the IBS model by FMT method. The stool used was from our previous study, which showed significant difference in microbial structure and function.6,7 The increased AWR scores in CRD test in GI rats proved the successful establishment of IBS model.

Multiple randomized controlled trials (RCTs) have provided evidence that rifaximin treatment for 10 days or 14 days was more efficacious than placebo for IBS symptom improvement.21 In a randomized double-blind placebo-controlled clinical trial enrolling 132 IBS-D patients, berberine hydrochloride administration for 8 weeks significantly reduced both abdominal and psychological symptoms in IBS-D patients.11 Several rodent experiments also verified the efficacy of rifaximin and berberine. In Xu et al10 study, treatment with rifaximin was found effective to prevent intestinal abnormalities and visceral hyperalgesia induced by chronic psychological stress (water avoidance stress and repeat restraint stress). In another study, post-infectious IBS model was established by Trichinella spiralis infection, and 7 days oral gavage of rifaximin alleviated visceral hypersensitivity, intestinal inflammation and barrier disruption.22 Consistent with these results, our data also suggested alleviation of visceral hypersensitivity by berberine and rifaximin.

Our results also showed that berberine increased the sucrose preference rate more than rifaximin in the sucrose preference test. As we know, berberine is an oral hypoglycemic agent with anti-dyslipidemia and anti-obesity activities, so the higher sucrose preference may be related with metabolic change in the rats. Meanwhile, we suppose this may indicate that berberine had better effect on improving psychological status, as reflected by higher weight gain rate in the berberine-treated rats than other groups (data not shown).

The modulating effect of gut microbiota by rifaximin and berberine have been reported respectively. In a RCT comparing the alteration of gut microbiota in the rifaximin and placebo group, IBS-D patients receiving rifaximin treatment for 10 weeks showed short-lived decreased richness at 2 weeks, but no differences of Shannon diversity and evenness were observed between the 2 groups.23 Another study also found that 14 days treatment with rifaximin decreased the richness of the microbial community.24 However, Li et al25 study showed that the microbial diversity was not influenced by rifaximin in IBS patients. Berberine was also verified to reduce the community richness in the RCTs investigating its effectiveness on metabolic diseases.12,26 In our study, rifaximin and berberine showed the same trend on the richness and diversity of microbiota community. This phenomenon was in accord with their broad antibacterial attribute.

Our data also revealed that rifaximin and berberine showed different effects on the microbial structure. Beyond our expectation, rifaximin did not affect the structure of IBS-derived microbiota. Previous studies have provided similar evidence as ours. An in vitro experiment using continuous culture colonic model system investigated the effect of rifaximin on the intestinal microbiota from patients suffering colonic active Crohn’s disease, and found that the overall composition remained stable during rifaximin treatment.27 The in vivo rodent experiment also found no alteration of microbial structure by rifaximin in post-infectious IBS rats.22 However, there were studies showing significant changes of microbial community in rifaximin-treated patients24 or stressed animals.10 These divergent results may be explained by different baseline microbial features of IBS patients, as rifaximin had better modulating effect on fecal microbiota in IBS patients with gut dysbiosis compared with those without.25

The analysis of bacterial abundance demonstrated that the taxa enriched by rifaximin and berberine were inconsistent. The Lachnoclostridium was enriched in berberine-treated group, which has been discussed in our previous study.15 Briefly, Lachnoclostridium was found lower in abundance in colitis mice and supplementary of Lachnoclostridium could protect against experimental colitis. Interestingly, neither rifaximin nor berberine enriched the Akkermansia and Bifidobacterium, which was in contrast with previous studies.13,25 This suggested us that different baseline state of microbial community influenced the regulating effect of the drugs.

Compared with rifaximin, berberine significantly reduced the expression of pathways involved in peptidoglycan biosynthesis. PGN is a macromolecule that is a cell wall component of both Gram-positive and Gram-negative bacteria. PGN and its fragments can act on Toll-like receptors, nucleotide-binding oligomerization domain (NOD)-like receptors, and other PGN recognition proteins, thus causing a proinflammatory effect.28 Recent studies found that PGN can cross the impaired gut epithelium, entered into blood, and eventually reached the brain. This demonstrates that PGN is associated with acute or chronic neuroinflammation, which is reflected by activated microglia. Indeed, Muramyl dipeptide, a widely studied signature motif of PGN, is a ligand of NOD2 and the nucleotide-binding domain and leucine-rich repeat protein-3 inflammasome in human microglia.29 This may partially explain the effect of berberine on CNS microglia and depressive behavior.

SCFAs are key metabolites produced by gut microbiota. Compared with GI rats, the concentration of acetate, propionate acids and total SCFAs were significantly increased by berberine, but not changed by rifaximin. This phenomenon was also observed in 1 previous clinical trial, in which rifaximin did not significantly alter total or individual SCFAs including acetate, propionate and butyrate in stool in IBS patients.24 SCFAs were reported to contribute to intestinal homeostasis and the regulation of energy metabolism, and increasing evidence confirmed the important role of SCFAs beyond the gut, for instance the microbiota-gut-brain axis.30 They were shown to be able to cross the blood-brain barrier.31 Perhaps this is the reason that the CNS microglia showed different state in the berberine and rifaximin rats. The microglia were brain resident macrophages which are activated in response to stress, injury and pathogens.32 Several animal experiments have demonstrated that microglia in the spinal cord and hippocampus play key roles in visceral hypersensitivity.33-35 For instance, intrathecal injection of agonist of microglia (fractalkine) could enhance visceral nociception, while an inhibitor of microglia minocycline inhibits visceral hypersensitivity.33 Erny et al36 first found that the gut microbiota and its metabolites short chain fatty acids (acetate, propionate, and butyrate) control the maturation and functions of microglia. Our data suggested the possibility that acetate and propionate increased by berberine could reverse the activated morphology of CNS microglia.

Berberine is the major pharmacological component of the Chinese herb Coptis chinensis (Huang-Lian). Besides treating bacterial diarrhea, it has been found effective in the treatment of colorectal adenoma recurrence, metabolic disorders, and neuropsychiatric disorders.13,37,38 In 1 study investigating the role of berberine in regulating microbiota-gut-brain axis in Parkinson’s disease, the results demonstrated that berberine was an agonist of tyrosine hydroxylase in Enterococcus and could lead to the production of levodopa in the gut, thus ameliorate Parkinson’s disease manifestation in mice.38

The limitation of this study is that we used AWR rather than electromyographic recording for measuring visceral hypersensitivity. We did not implant the electrodes to perform electromyographic recording because we hoped to guarantee the sterile environment and avoid contamination to the germ-free rats. Meanwhile, during the CRD test, 2 researchers evaluated the AWR scores independently in a blind manner, using uniform standards to reduce the subjectivity.

In conclusion, this study showed that both rifaximin and berberine can alleviate visceral hypersensitivity induced by dysbiosis. However, they have different effects on microbial diversity, structure and taxa. The alteration of fecal SCFAs and peptidoglycan biosynthesis pathways by the 2 drugs may result in divergent state of microglia. Further studies are needed to reveal the mechanisms of the 2 drugs to regulate the microbiota-gut-brain axis.


Part of the results was selected for a poster presentation (ranking top 10%) during Digestive Disease Week 2020.

Financial support:

This work was supported by National Key R&D Program of China (No. 2019YFA0905600) and National Natural Science Foundation of China (No.82000510, 82170557).

Supplementary Materials

Note: To access the supplementary tables mentioned in this article, visit the online version of Journal of Neurogastroenterology and Motility at http://www.jnmjournal.org/, and at https://doi.org/10.5056/jnm22182.

Conflicts of interest


Author contributions

Jindong Zhang conducted the animal experiments and sample analyses, and wrote the draft of the manuscript; Cunzheng Zhang, Tao Zhang, and Lu Zhang performed the experiments and data interpretation; and Liping Duan designed and supervised the experiments, and performed critical revisions of the manuscript.

  1. Drossman DA, Hasler WL. Rome IV-functional GI disorders: disorders of gut-brain interaction. Gastroenterology 2016;150:1257-1261.
    Pubmed CrossRef
  2. Sperber AD, Dumitrascu D, Fukudo S, et al. The global prevalence of IBS in adults remains elusive due to the heterogeneity of studies: a rome foundation working team literature review. Gut 2017;66:1075-1082.
    Pubmed CrossRef
  3. Ford AC, Lacy BE, Talley NJ. Irritable bowel syndrome. N Engl J Med 2017;376:2566-2578.
    Pubmed CrossRef
  4. Ford AC, Sperber AD, Corsetti M, Camilleri M. Irritable bowel syndrome. Lancet 2020;396:1675-1688.
    Pubmed CrossRef
  5. Pittayanon R, Lau JT, Yuan Y, et al. Gut microbiota in patients with irritable bowel syndrome-a systematic review. Gastroenterology 2019;157:97-108.
    Pubmed CrossRef
  6. Liu Y, Zhang L, Wang X, et al. Similar fecal microbiota signatures in patients with diarrhea-predominant irritable bowel syndrome and patients with depression. Clin Gastroenterol Hepatol 2016;14:1602-1611, e5.
    Pubmed CrossRef
  7. Xu C, Jia Q, Zhang L, et al. Multiomics study of gut bacteria and host metabolism in irritable bowel syndrome and depression patients. Front Cell Infect Microbiol 2020;10:580980.
    Pubmed KoreaMed CrossRef
  8. Pimentel M, Chow EJ, Lin HC. Normalization of lactulose breath testing correlates with symptom improvement in irritable bowel syndrome. a double-blind, randomized, placebo-controlled study. Am J Gastroenterol 2003;98:412-419.
    Pubmed CrossRef
  9. Pimentel M, Lembo A, Chey WD, et al. Rifaximin therapy for patients with irritable bowel syndrome without constipation. N Engl J Med 2011;364:22-32.
    Pubmed CrossRef
  10. Xu D, Gao J, Gillilland M rd, et al. Rifaximin alters intestinal bacteria and prevents stress-induced gut inflammation and visceral hyperalgesia in rats. Gastroenterology 2014;146:484-496, e4.
    Pubmed KoreaMed CrossRef
  11. Chen C, Tao C, Liu Z, et al. A randomized clinical trial of berberine hydrochloride in patients with diarrhea-predominant irritable bowel syndrome. Phytother Res 2015;29:1822-1827.
    Pubmed CrossRef
  12. Zhang Y, Gu Y, Ren H, et al. Gut microbiome-related effects of berberine and probiotics on type 2 diabetes (the PREMOTE study). Nat Commun 2020;11:5015.
    Pubmed KoreaMed CrossRef
  13. Li S, Wang N, Tan HY, et al. Modulation of gut microbiota mediates berberine-induced expansion of immuno-suppressive cells to against alcoholic liver disease. Clin Transl Med 2020;10:e112.
    Pubmed KoreaMed CrossRef
  14. Fang Y, Zhang J, Zhu S, et al. Berberine ameliorates ovariectomy-induced anxiety-like behaviors by enrichment in equol generating gut microbiota. Pharmacol Res 2021;165:105439.
    Pubmed CrossRef
  15. Zhang JD, Liu J, Zhu SW, et al. Berberine alleviates visceral hypersensitivity in rats by altering gut microbiome and suppressing spinal microglial activation. Acta Pharmacol Sin 2021;42:1821-1833.
    Pubmed KoreaMed CrossRef
  16. Al-Chaer ED, Kawasaki M, Pasricha PJ. A new model of chronic visceral hypersensitivity in adult rats induced by colon irritation during postnatal development. Gastroenterology 2000;119:1276-1285.
    Pubmed CrossRef
  17. Seminowicz DA, Labus JS, Bueller JA, et al. Regional gray matter density changes in brains of patients with irritable bowel syndrome. Gastroenterology 2010;139:48-57, e2.
    Pubmed KoreaMed CrossRef
  18. Wouters MM, Van Wanrooy S, Casteels C, et al. Altered brain activation to colorectal distention in visceral hypersensitive maternal-separated rats. Neurogastroenterol Motil 2012;24:678-685, e297.
    Pubmed CrossRef
  19. Crouzet L, Gaultier E, Del'Homme C, et al. The hypersensitivity to colonic distension of IBS patients can be transferred to rats through their fecal microbiota. Neurogastroenterol Motil 2013;25:e272-e282.
    Pubmed CrossRef
  20. De Palma G, Lynch MD, Lu J, et al. Transplantation of fecal microbiota from patients with irritable bowel syndrome alters gut function and behavior in recipient mice. Sci Transl Med 2017;9:eaaf6397.
  21. Pimentel M. Review article: potential mechanisms of action of rifaximin in the management of irritable bowel syndrome with diarrhoea. Aliment Pharmacol Ther 2016;43(suppl 1):37-49.
    Pubmed CrossRef
  22. Jin Y, Ren X, Li G, et al. Beneficial effects of rifaximin in post-infectious irritable bowel syndrome mouse model beyond gut microbiota. J Gastroenterol Hepatol 2018;33:443-452.
    Pubmed CrossRef
  23. Pimentel M, Fodor AA, Golden P, Bortey E, Forbes WP. Mo1268 characterization of stool microbiota in subjects with IBS-D receiving repeat treatments with rifaximin in the TARGET 3 study. Gastroenterology 2015;148:S-655.
  24. Acosta A, Camilleri M, Shin A, et al. Effects of rifaximin on transit, permeability, fecal microbiome, and organic acid excretion in irritable bowel syndrome. Clin Transl Gastroenterol 2016;7:e173.
    Pubmed KoreaMed CrossRef
  25. Li Y, Hong G, Yang M, et al. Fecal bacteria can predict the efficacy of rifaximin in patients with diarrhea-predominant irritable bowel syndrome. Pharmacol Res 2020;159:104936.
    Pubmed CrossRef
  26. Ming J, Yu X, Xu X, et al. Effectiveness and safety of Bifidobacterium and berberine in human hyperglycemia and their regulatory effect on the gut microbiota: a multi-center, double-blind, randomized, parallel-controlled study. Genome Med 2021;13:125.
    Pubmed KoreaMed CrossRef
  27. Maccaferri S, Vitali B, Klinder A, et al. Rifaximin modulates the colonic microbiota of patients with crohn's disease: an in vitro approach using a continuous culture colonic model system. J Antimicrob Chemother 2010;65:2556-2565.
    Pubmed CrossRef
  28. Laman JD, t' Hart BA, Power C, Dziarski R. Bacterial peptidoglycan as a driver of chronic brain inflammation. Trends Mol Med 2020;26:670-682.
    Pubmed CrossRef
  29. Ramaswamy V, Walsh JG, Sinclair DB, et al. Inflammasome induction in rasmussen's encephalitis: cortical and associated white matter pathogenesis. J Neuroinflammation 2013;10:152.
    Pubmed KoreaMed CrossRef
  30. Dalile B, Van Oudenhove L, Vervliet B, Verbeke K. The role of short-chain fatty acids in microbiota-gut-brain communication. Nat Rev Gastroenterol Hepatol 2019;16:461-478.
    Pubmed CrossRef
  31. Frost G, Sleeth ML, Sahuri-Arisoylu M, et al. The short-chain fatty acid acetate reduces appetite via a central homeostatic mechanism. Nat Commun 2014;5:3611.
    Pubmed KoreaMed CrossRef
  32. Wolf SA, Boddeke HW, Kettenmann H. Microglia in physiology and disease. Annu Rev Physiol 2017;79:619-643.
    Pubmed CrossRef
  33. Saab CY, Wang J, Gu C, Garner KN, Al-Chaer ED. Microglia: a newly discovered role in visceral hypersensitivity?. Neuron Glia Biol 2006;2:271-277.
    Pubmed KoreaMed CrossRef
  34. Bradesi S, Svensson CI, Steinauer J, Pothoulakis C, Yaksh TL, Mayer EA. Role of spinal microglia in visceral hyperalgesia and NK1R up-regulation in a rat model of chronic stress. Gastroenterology 2009;136:1339-1348, e1-e2.
    Pubmed KoreaMed CrossRef
  35. Zhang G, Yu L, Chen ZY, et al. Activation of corticotropin-releasing factor neurons and microglia in paraventricular nucleus precipitates visceral hypersensitivity induced by colorectal distension in rats. Brain Behav Immun 2016;55:93-104.
    Pubmed CrossRef
  36. Erny D, Hrabě de Angelis AL, Jaitin D, et al. Host microbiota constantly control maturation and function of microglia in the CNS. Nat Neurosci 2015;18:965-977.
    Pubmed KoreaMed CrossRef
  37. Chen YX, Gao QY, Zou TH, et al. Berberine versus placebo for the prevention of recurrence of colorectal adenoma: a multicentre, double-blinded, randomised controlled study. Lancet Gastroenterol Hepatol 2020;5:267-275.
    Pubmed CrossRef
  38. Wang Y, Tong Q, Ma SR, et al. Oral berberine improves brain dopa/dopamine levels to ameliorate parkinson's disease by regulating gut microbiota. Signal Transduct Target Ther 2021;6:77.
    Pubmed KoreaMed CrossRef

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