ABSTRACT
-
Background/Aims
Cholestatic liver disease (CLD) is a pathological condition characterized by impaired bile formation, secretion, and excretion. However, the key pathophysiological mechanisms of CLD remain elusive, and therapeutic efficacy is unsatisfactory.
-
Methods
We administered berberine (BBR) or dihydroberberine (dhBBR) in bile duct ligation-, ANIT-, and mdr2-/- CLD mouse models to evaluate the anti-CLD effect. We conducted fecal microbiota transplantation to determine the role of gut microbiota in BBR’s effect. We conducted a randomized, controlled clinical trial to evaluate the effects of BBR in patients with CLD.
-
Results
Oral BBR alleviates cholestatic liver injury in multiple mouse models. Gut microbes can transform BBR into dhBBR, which suppresses 5-hydroxytryptamine (5-HT) production in gut enterochromaffin cells by antagonizing tryptophan hydroxylase 1 (TPH1) activity and downregulating Tph1 transcription. This further ameliorates CLD by interrupting the 5-HT/5-HTR axis. A clinical study validated that BBR improved blood biochemical indicators in patients with CLD and decreased 5-HT levels.
-
Conclusions
BBR is transformed by gut microbiota to ameliorate CLD via inhibiting 5-HT, suggesting potential novel strategies for further clinical use.
-
Keywords: Cholestatic liver disease; Berberine; Gut microbiota; Dihydroberberine; 5-HT
Study Highlights
• BBR reduced the progression of CLD in various mouse models and patients with cholestasis.
• dhBBR, produced by the bacterial metabolism of BBR, served as the primary substance to alleviate CLD.
• dhBBR reduces 5-HT by inhibiting TPH1 activity in gut enterochromaffin cells.
Graphical Abstract
INTRODUCTION
Cholestatic liver disease (CLD) is a complex condition characterized by impaired bile production, secretion, and excretion. This disrupts the normal flow of bile into the duodenum, resulting in its accumulation in the bloodstream [
1,
2]. Patients with CLD may not exhibit obvious symptoms in the early stages; however, elevated serum alkaline phosphatase (ALP) and gamma-glutamyl transferase (GGT) levels may be observed. Hyperbilirubinemia may occur as the disease progresses. In severe cases, it can lead to cirrhosis, liver failure, and even death. Various factors have been implicated, including bile secretion disorders in hepatocytes and biliary epithelial cells, inflammation and obstruction of intrahepatic bile ducts, immune-mediated cholangitis, genetic predisposition, and the influence of drugs and the environment [
3,
4]. However, its exact cause remains unclear. Currently, the FDA-approved medications for the treatment of CLD are ursodeoxycholic acid (UDCA) and obeticholic acid (OCA) [
5]. However, their efficacy is limited and patients are at a high risk of disease progression. Therefore, there is an urgent need for the development of novel therapeutic agents and strategies.
Berberine (BBR), an isoquinoline alkaloid isolated from the Chinese herb
Coptis chinensis and other
Berberis species, has a wide range of pharmacological properties. For centuries, it has been used to treat gastrointestinal infections and diarrhea, displaying notable antimicrobial properties [
6,
7]. The beneficial effect of BBR has been described in mouse models of liver disease and in patients with primary sclerosing cholangitis (PSC) and primary biliary cirrhosis (PBC) [
8,
9,
10]. However, current work overlooked BBR’s extremely low bioavailability, and low concentrations in plasma and liver after oral administration, suggesting an indirect mechanism of action in BBR’s protective effect [
11]. The gut microbiota, a crucial environmental regulator, plays a substantial role in various physiological and pathological processes and the development of multiple diseases [
12,
13]. BBR exerts anti-obesity, anti-diabetic, and anti-tumor effects, and improves metabolic syndrome through gut microbiota regulation [
12]. An important mechanism by which gut microbes affect host regulation is the synthesis of various metabolites, including trimethylamine, short-chain fatty acids, bile acids (BAs), and branched-chain amino acids [
14]. BBR directly interacts with the intestinal flora responsible for producing these metabolites, leading to improved outcomes in disorders, such as insulin resistance, metabolic disorders, and dextran sulfate sodium salt (DSS)-induced colitis [
12]. However, the mechanisms by which BBR and microbes are involved in CLD are poorly established.
Here, we showed that BBR could be converted to dihydroberberine (dhBBR) by the gut microbiota and dhBBR could suppress the production of 5-hydroxytryptamine (5-HT) by inhibiting the activity of the tryptophan hydroxylase 1 (TPH1) enzyme and downregulating Tph1 transcription in gut enterochromaffin cells (ECs), thus ameliorating CLD by blocking 5-HT/5-hydroxytryptamine receptor (5-HTR) signaling in the liver. Our clinical study on patients with CLD showed that BBR is a potential agent for the treatment of cholestatic liver injury.
MATERIAL AND METHODS
A complete list of reagents, antibodies, kits, and consumables used in this study is provided in
Supplementary Table 1
The study entitled “Clinical study of combined application of berberine hydrochloride tablets in the treatment of cholestatic liver disease” was approved by the Medical Ethics Committee of the Second Affiliated Hospital of Chongqing Army Medical University in China (Approval No. 2022-453-01) and registered with the Chinese Clinical Trial Registry (ChiCTR) in the WHO registration network (Registration No. ChiCTR2300068536). All volunteers provided written informed consent and clinical information prior to participating in the study. Peripheral blood samples were centrifuged at 2,000 g to obtain serum. Serum ALP, GGT, alkaline phosphatase (ALT), and aspartate aminotransferase (AST) levels were measured using an automated biochemical analyzer. Serum 5-HT levels were determined using liquid chromatography-mass spectrometry (LC-MS/MS).
Detailed baseline characteristics and clinical information of patients with CLD are provided in
Supplementary Table 2.
The animal protocol was approved by the Experimental Animal Welfare and Ethics Committee, and adhered to the Animal Ethics Statement (Approval Number: AMUWEC20210143). The C57BL/6J mice were purchased from Vital River (Beijing, China). Mdr2
-/- mice were obtained from Chengdu Pharmaceutical Company. Male mice (20–22 g, 6–8 weeks old) were housed in a specific pathogen-free environment with controlled conditions, experiencing a 12-h light/dark cycle at 21±1°C and 45±5% humidity. Mouse samples were collected as previously described [
15-
17]. Fresh fecal, liver, and intestinal samples from mice were collected and immediately frozen at –80°C until use. Peripheral blood samples from mice were centrifuged at 3,000 × g to obtain serum. Beckman automatic biochemical instruments were used to measure serum ALP, GGT, ALT, and AST levels. Serum 5-HT levels were determined using LC-MS/MS.
RESULTS
BBR ameliorates cholestatic liver injury in multiple mice models in a gut microbiota-dependent manner
To investigate the effects of BBR on CLD, we performed bile duct ligation (BDL) in C57BL/6J mice to induce cholestatic liver injury. BBR was orally administered for 7 days (
Fig. 1A). BDL led to a significant increase in hepatocyte necrosis (
Fig. 1B) as well as elevated levels of total BA (TBA), ALP, total bilirubin (TBIL), and direct bilirubin (DBIL) (
Fig. 1C). However, oral administration of BBR not only reversed hepatocyte necrosis (
Fig. 1B), but also decreased the elevated levels of TBA, ALP, TBIL, DBIL (
Fig. 1C), ALT, and AST (
Supplementary Fig. 1A,
1B). Additionally, we expanded our investigation to include another two models of CLD, the α-naphthylisothiocyanate (ANIT)-induced intrahepatic cholestasis and the mdr2
-/- mouse model [
18,
19]. In both models, we found significantly decreased hepatocyte necrosis and lower levels of TBA, ALP, TBIL, DBIL, ALT, and AST in BBR-treated mice than in mice treated with PBS (
Supplementary Fig. 1C–
1J,
Supplementary Fig. 2). Therefore, our study suggested that BBR exhibits a beneficial effect in ameliorating experimental CLD.
To evaluate whether BBR exerts its effect directly in the liver, it was intraperitoneally administered. However, we did not observe improvements in the area of hepatocyte necrosis or serum levels of TBA, ALP, ALT, AST, TBIL, and DBIL in these experimental CLD models when compared with those in the control group (
Supplementary Figs. 3–
5), indicating that BBR may not directly target the liver to mitigate its effects. Oral administration of BBR is mainly found in the gut lumen with a low plasma concentration [
20,
21]. Therefore, we treated mice with an antibiotic cocktail (ABX) for 7 days to deplete the gut microbiota (
Fig. 1D,
Supplementary Fig. 6A) to investigate whether BBR’s beneficial effects on CLD depended on the gut microbiota. ABX treatment reduced the positive effects of BBR in mouse models of CLD (
Fig. 1E,
1F,
Supplementary Figs. 6,
7), indicating that BBR improves CLD in a gut microbiota–dependent manner.
To further explore the role of the gut microbiota in the potency of BBR in CLD, we performed fecal microbiota transplantation (FMT) in mice using fresh fecal samples from patients with CLD treated with oral BBR (FMT
CLD+BBR) or not (FMT
CLD) (
Fig. 2A). The FMT
CLD+BBR group showed significant improvements in hepatocyte necrosis (
Fig. 2B) and reduced serum levels of TBA and ALP (
Fig. 2C), ALT, AST, TBIL, and DBIL (
Supplementary Fig. 8A–
8D) compared with those in the FMT
CLD group. The fecal microbiota from mice orally treated with BBR (Group FMT
BDL+BBR,
Fig. 2D) also improved the hepatocyte necrosis area (
Fig. 2E), liver enzymes, and lipid profile in mice with cholestatic liver injury (
Fig. 2F,
Supplementary Fig. 8E–
8H).
To further investigate whether the efficacy of BBR stems from changes in the structure of the gut microbiota or from gut microbe-produced metabolites from the biotransformation of BBR, ABX-pretreated mice were randomized into five groups (
Fig. 3A): sham (sham operation receiving PBS); BDL (BDL mice receiving PBS); FMTBDL (BDL mice receiving microbiota from BDL donors); FMT
BDL+BBR (BDL mice receiving microbiota from BBR-treated BDL donors); and FMT
BDL+BBR+ABX (BDL mice receiving microbiota from BBR-treated BDL donors, additionally receiving ABX). In the FMT
BDL+BBR+ABX group, both donor and recipient microbiota were eliminated. We observed reduced cholestatic liver injury in the FMT
BDL+BBR and FMT
BDL+BBR+ABX groups (
Fig. 3B,
3C,
Supplementary Fig. 9A–
9E). Most importantly, there was no significant difference in the CLD parameters between the FMT
BDL+BBR and FMT
BDL+BBR+ABX groups (
Fig. 3B,
3C,
Supplementary Fig. 9A–
9E), suggesting that metabolites from BBR-treated mice might play a vital role in the anticholestatic effect. To further investigate the role of these metabolites, we collected fecal bacterial supernatants from BDL mice with or without BBR treatment using centrifugation and filtration (
Fig. 3D). These supernatants were then administered to BDL mice pretreated with ABX and untreated BDL mice. Results demonstrated that administration of the fecal bacterial supernatant significantly alleviated cholestatic liver injury in both non-ABX treated BDL mice (
Supplementary Fig. 9F–
9M) and ABX-pretreated BDL mice (
Fig. 3E,
3F;
Supplementary Fig. 9N,
9O). Taken together, these findings demonstrate that BBR exerts its effect of alleviating CLD through metabolites produced by gut microbes.
Gut microbiota plays a crucial role in the metabolism of pharmaceuticals, converting them into multiple metabolites [
22]. Gut microbes metabolize BBR into several compounds, including methylation and reduction product [
23]. One particular metabolite, dhBBR, is predominantly found in feces and is considered the primary metabolite produced by gut microbes [
24,
25]. The specific enzyme nitrosoreductase, which is expressed exclusively by gut microbes, reduced BBR to dhBBR (
Fig. 4A) [
24]. Composition analysis using 16S rRNA sequencing on fecal microbiota from BDL mice ± BBR treatment revealed the enrichment of nitroreductase-producing bacteria, such as
Bacteroides and
Bifidobacterium (
Fig. 4B). Using LC-MS/MS, we confirmed the presence of dhBBR in mouse and human feces following the oral administration of BBR (
Fig. 4C,
4D). To further investigate the potential role of dhBBR in CLD, we orally administered dhBBR to mice with BDL-induced cholestasis. dhBBR significantly reduced the area of hepatocyte necrosis (
Fig. 4E) and lowered serum levels of ALT, AST, TBA, ALP, TBIL, and DBIL (
Fig. 4F,
Supplementary Fig. 10A,
10B). Furthermore, we observed that the oral administration of dhBBR alleviated CLD, whereas BBR failed to improve the phenotype after ABX treatment in BDL mice (
Supplementary Fig. 10C–
10J), suggesting that dhBBR, produced by the bacterial metabolism of BBR, served as the fundamental substance to alleviate CLD.
Pharmacokinetic studies revealed that the plasma concentration of dhBBR is low when administered orally to mice [
25]. Notably, dhBBR showed a higher concentration in the intestinal tissue than in the liver after oral administration (
Fig. 4C). To explore how dhBBR improves cholestatic liver injury, liver samples from BDL mice with or without dhBBR administration were collected for untargeted metabolomic analysis. Partial least squares discriminant analysis showed robust alterations in metabolite composition in dhBBR-treated mice compared with that in PBS-treated mice (
Fig. 5A). Among the 620 metabolites identified (
Fig. 5B,
Supplementary Fig. 11A,
11B,
Supplementary Table 3), we focused on 5-HT, which was elevated in BDL mice compared to dhBBR-treated mice. Although 5-HT was not the highest-ranked metabolite by VIP score (
Supplementary Fig. 11A), we specifically highlight it due to its well-established biological significance in CLD [
26]. This change is further supported by our KEGG pathway enrichment analysis, which identified the tryptophan metabolism pathway––the primary pathway for 5-HT synthesis and degradation––as one of the most significantly altered metabolic pathways (
Supplementary Fig. 11C). Subsequently, LC-MS/MS quantification confirmed 5-HT levels were significantly decreased in the dhBBR-treated BDL mice compared to BDL mice (
Fig. 5C). Additionally, 5-HT levels were significantly increased in the serum, liver, and intestine of BDL mice compared with those in the sham group (
Fig. 5D). Correspondingly, hepatic expression of the 5-HT receptor subtypes 2a, 2b, and 2c (5-HTR
2a, 5-HTR
2b, and 5-HTR
2c) was upregulated in BDL mice (
Supplementary Fig. 12A). Exogenous administration of 5-HT exacerbated cholestasis in BDL mice, whereas the 5-HTR2a/2b/2c antagonists attenuated this effect (
Supplementary Fig. 12B–
12I). Co-administered BDL mice with 5-HT and 5-HTR2a/2b/2c antagonists demonstrated that antagonists mitigated the 5-HT-induced increase in the hepatocyte necrosis area and reduced the elevated serum levels of ALT, AST, TBA, ALP, and TBIL (
Supplementary Fig. 13A–
13G). These data establish a critical role for the 5-HT/5-HTR axis in CLD.
To investigate whether dhBBR alleviates CLD by reducing 5-HT levels, we measured 5-HT concentrations in mouse models using mass spectrometry. The oral administration of dhBBR significantly reduced serum 5-HT levels in BDL mice (
Fig. 5C). BBR treatment also decreased 5-HT concentrations in the serum, intestine, and liver (
Fig. 5D). Additionally, tail vein administration of 5-HT to dhBBR-treated mice reversed the anticholestatic effects of dhBBR (
Fig. 5E,
5F,
Supplementary Fig. 13H,
13I). Our data indicated that dhBBR exerts beneficial effects by reducing 5-HT levels in CLD.
It is well known that 5-HT is mainly produced by ECs [
27]. To investigate the gut-dependent mechanism dhBBR, we administered dhBBR intraperitoneally in BDL mice. Results showed dhBBR failed to exert a significant therapeutic effect in BDL mice (
Supplementary Fig. 14), supporting its gut-dependent mechanism. To determine whether dhBBR could reduce 5-HT levels in EC cells, we constructed mouse intestinal organoids containing ECs (
Supplementary Fig. 15A). DhBBR incubation significantly inhibited the production of 5-HT in the organoids, whereas BBR did not (
Supplementary Fig. 15B,
15C). To confirm that the gut is the primary source of 5-HT, we measured 5-HT levels in the portal vein of BDL mice (
Fig. 6A, left). Portal 5-HT concentrations were significantly higher than those in the peripheral circulation. Additionally, the increase in portal 5-HT induced by BDL was notably higher than that in the periphery. BBR treatment decreased portal 5-HT in BDL mice, which was significantly greater than the reduction in the periphery (
Fig. 6A, right). Immunofluorescence of colonic tissues showed increased ChgA
+-5HT
+ double positive cell numbers in BDL mice, both of which were reduced by treatment with BBR (
Fig. 6B).
To elucidate the underlying mechanism, we explored the role of TPH1, a critical enzyme in the conversion of L-tryptophan (L-Trp) to 5-HT, which is predominantly expressed in gut EC cells [
28]. qPCR analysis indicated a significant increase in TPH1 mRNA expression in the BDL mice colon, which was decreased by BBR treatment (
Supplementary Fig. 15D). The molecular docking results showed that dhBBR binds to TPH1 by interacting with multiple amino acids in the TPH1 protein pocket (
Supplementary Fig. 15E). We further confirmed that dhBBR inhibits TPH1 activity in a dose-dependent manner, with an IC
50 value of 0.69 mM (
Supplementary Fig. 15F). Microscale thermophoresis (MST) results further supported the interactions of dhBBR and TPH1, with a K
d value of 7.16 μM (
Supplementary Fig. 15G). To further explore the mode of interaction between dhBBR and TPH1, we incubated TPH1 enzyme with L-Trp and dhBBR. We observed a partial inhibition of TPH1 activity in the presence of 50 μM L-Trp and 0.5 mM dhBBR (
Supplementary Fig. 15H), indicating that dhBBR is a competitive inhibitor against L-Trp toward TPH1. Next, we performed a competitive binding assay using MST. LX-1031, a selective TPH1 inhibitor that occupies the L-Trp-binding pocket [
29], binds to TPH1 with a K
d value of 0.27 μM. DhBBR at 200 μM enhanced the K
d value of LX1031 to 1.2 mM (
Supplementary Fig. 15G), suggesting that dhBBR may exert its inhibitory effect by partially competing for binding to TPH1 substrates. We found that dhBBR significantly reduced the levels of 5-HTP, which are produced by the conversion of tryptophan by TPH1 (
Supplementary Fig. 15I). When LX-1031 blocked TPH1 in BDL mice, dhBBR did not provide additional relief in cholestasis liver injury, as evidenced by the hepatocyte necrosis area (
Fig. 6C) and serum levels of TBA, ALP, and TBIL (
Fig. 6D), confirming the anticholestatic efficacy of dhBBR through TPH1 inhibition. Our data demonstrate that dhBBR inhibits 5-HT production in gut EC by targeting TPH1 and alleviating cholestatic liver injury.
To evaluate the efficacy of BBR in patients with CLD, we conducted a randomized controlled clinical trial. Thirty-three (33) patients were enrolled and randomly divided into control (n=15) and treatment (n=18) groups. The control group received regular UDCA medication, whereas the treatment group received BBR hydrochloride tablets orally three times a day for 15 days in addition to UDCA medication (
Fig. 7A). After the 15-day intervention, patients in the treatment group had better outcomes than those in the control group in terms of mean changes in critical liver enzyme levels and lipid profiles. The GGT, ALP, ALT, and AST levels were significantly lower in the treatment group (
Fig. 7B) despite no initial differences between the two groups. Furthermore, the recovery rates of the GGT, ALP, and ALT levels in the treatment group were higher than those in the control group (
Fig. 7C). Moreover, the serum concentrations of 5-HT were reduced in the treatment group compared with those in the control group, and the recovery rate of 5-HT was higher in the treatment group (
Fig. 7D,
7E). These findings suggest that BBR promotes recovery in patients with CLD.
DISCUSSION
Owing to the limited efficacy of the current drugs for CLD treatment, new therapeutic agents and strategies are urgently needed. In the present study, BBR alleviated CLD progression in multiple mouse models and patients with cholestasis. BBR is metabolized to dhBBR by gut microbes, and dhBBR further reduces 5-HT production in gut EC cells by inhibiting TPH1 activity. Our clinical study showed that oral treatment with BBR for half a month improved the recovery rates of GGT, ALP, and ALT, accompanied by a reduction in 5-HT levels, identifying BBR as a potential drug for treating CLD by inhibiting 5-HT production in the gut.
Due to extensive structural conjugation, BBR exhibits low water solubility and poor bioavailability [
30,
31]. BBR modulates various diseases primarily via interaction with the gut microbiota, either by altering microbial composition or being metabolized into active derivatives [
23,
32,
33]. A key metabolite, dhBBR, generated via microbial nitroreductase, was reported to reduce BH₂ to BH₄ to promote L-DOPA biosynthesis, which can alleviate Parkinsonian symptoms [
34]. Nitroreductase is widespread among gut microbiota, such as
Bacteroides,
Enterococcus,
Staphylococcus aureus,
Enterococcus faecium,
Lactobacillus casei and
Lactobacillus acidophilus [
24,
33]. Supporting the clinical relevance, patients with PSC exhibit fecal dysbiosis enriched in nitroreductase-producing taxa like
Bacteroides and
Enterococcus compared to healthy controls [
35]. Composition analysis revealed the enrichment of nitroreductase-producing bacteria, particularly
Bacteroides and
Bifidobacterium, in both groups, with
Bacteroides abundance showing a significant increase after BBR treatment (
Fig. 4B). This microbial variability may underlie individual differences in BBR response, which warrants further investigation.
We further investigated the mechanism by which dhBBR improves CLD and found that dhBBR did not directly exert its effect in the liver. However, this effect was dependent on gut administration. Untargeted metabolomics and LC-MS/MS analyses revealed that 5-HT levels significantly decreased in the intestine, serum, and liver after dhBBR treatment, implying that 5-HT might be a target of dhBBR. A previous study revealed hepatic 5-HTR2a/2b/2c upregulation exacerbates cholestatic injury in BDL/mdr2
−/− models. Their activation promotes cytokine release, oxidative stress, mitochondrial dysfunction, and caspase-3-mediated apoptosis via MAPK, STAT3, ROS, and PKA pathways. These pathways drive oxidative stress and apoptosis in cholestatic liver injury, elucidating the mechanism by which BBR exerts hepatoprotective effects through the reduction of intestinal 5-HT levels [
26]. Our findings contextualize prior work linking enterohepatic 5-HT to biliary injury, positioning this axis as an emerging therapeutic target in cholestasis.
The 5-HT is synthesized from L-Trp by TPH, which has two isoforms: peripheral TPH1 and neuronal TPH2 [
36,
37]. Our data indicated BBR downregulates
Tph1 transcription, potentially via Rfx6 or HDAC1 modulation [
38,
39], a mechanism requiring further investigation. We further observed dhBBR partially reversed L-Trp-induced TPH1 activation and significantly decreased the K
d of the TPH1 inhibitor LX-1031, implying distinct binding modes: while L-Trp binds via both hydrophobic and polar interactions near the catalytic iron, dhBBR primarily interacts hydrophobically with Pro268 and His272, lacking polar contacts. Although dhBBR may affect dopamine production via tyrosine hydroxylase [
34], our convergent evidence from computational docking, biochemical enzymatic assays, cellular experiments, and in vivo rescue studies supports its primary action as a potent TPH1 inhibitor under the conditions tested.
TPH1 activity has been reported to be enhanced by PKA phosphorylation [
40]. In the pathological state of cholestasis (BDL), dysregulated BAs potently drive sustained activation of PKA/cAMP signaling [
41,
42], which maintains TPH1 in a highly phosphorylated, hyperactive state, where dhBBR potently inhibits it and reduces 5-HT. Under physiological conditions (Sham), where BA homeostasis is preserved, TPH1 likely operates at a basal, low-phosphorylation state with inherently lower activity. Inhibiting this low-activity enzyme with dhBBR would therefore yield a minimal effect on 5-HT production. The precise mechanism underlying the changes in TPH1 phosphorylation during cholestasis represents an important direction for our future research.
Current treatments for CLD (UDCA and OCA), exhibit limited clinical efficacy and poor prognosis [
3]. Our clinical trial showed BBR significantly improved recovery rates of GGT, ALP, and ALT, and reduced 5-HT levels, consistent with previous studies on
Berberis vulgaris in PSC and PBC [
43]. Although both UDCA and BBR reduce serum 5-HT, their mechanisms differ: UDCA enhances BA excretion and ameliorates liver injury, indirectly reducing 5-HT, whereas BBR directly inhibits intestinal TPH1, suppressing 5-HT biosynthesis at its source. Their complementary mechanisms lead to superior efficacy in combination therapy (>65% reduction). Meanwhile, dhBBR remains in early-stage clinical investigation, with ongoing studies focused on bioavailability and metabolism, warranting further preclinical and clinical evaluation for CLD applications.
In conclusion, our study highlights the therapeutic effects of BBR in CLD, which was achieved through the biotransformation of BBR to dhBBR by the gut microbiota. DhBBR inhibits TPH1 activity in ECs and downregulates Tph1 transcription, reducing 5-HT synthesis, and inhibiting 5-HT/5-HTR signaling to alleviate CLD. BBR-related 5-HT reduction and its therapeutic effects have been validated in patients with CLD, providing a potential novel strategy for clinical intervention. Our study uncovers a novel role of gut microbiota in enhancing the effects of low-bioavailability drugs, offering valuable insights for therapeutic strategies.
FOOTNOTES
-
Authors’ contributions
Shiming Yang conceptualized the study. Dianji Tu, Cheng Lu, Junfeng Guo, Qiao Chen, Xin Li, Yingjie Wang, Lulu Cheng, Hongfei Jiang, Jinchen Jian, Yusong Ge, Zhanjie Hou, Xiaojie Feng, Yunxuan Feng, Jianchun Zhou, Yuanyuan Lei, Lei Ran, Yuanyuan Zhou, Zhengguo Xu, Hua Diao, and Jiyin Zhou performed the experiments and analyzed the data. Junfeng Guo and Qiao Chen conducted extensive additional experiments and data analysis during the revision process. Cheng Lu, Dianji Tu, Bo Tang, and Shiming Yang wrote the manuscript. Bo Tang and Shiming Yang designed the study. All authors edited the manuscript and approved the final manuscript.
-
Acknowledgements
This work was supported by the National Natural Science Foundation of China (No. 82370586 to Bo Tang), and Key Program of National Natural Science Foundation of China (No. 82030020 to Shiming Yang.). We acknowledge Dr. Shuang Feng for guidance and technical assistance in the ultrasound-guided localization of the mouse portal vein for blood sampling.
-
Conflicts of Interest
The authors have no conflicts to disclose.
SUPPLEMENTARY MATERIAL
Supplementary material is available at Clinical and Molecular Hepatology website (
http://www.e-cmh.org).
Supplementary Figure 1.
BBR alleviates CLD progression in the BDL and ANIT-induced intrahepatic cholestasis mouse model. (A, B) Serum ALT and AST levels from BDL and sham mice gavaged with BBR or not. (C–J) C57BL/6J mice were administered ANIT (50 mg/kg/day) intraperitoneally for 14 days, and BBR (20 mg/kg/day) or PBS was gavaged on the last 5 days of the 14 days of ANIT-dosing. n=6 mice per group. (C) H&E staining of liver sections. Scale bar, 200 μm. (D) Histopathological score of the liver sections. (E–J) Serum ALT, AST, TBA, ALP, TBIL, and DBIL levels. Data were represented as mean±SEM. P-values were calculated by a two-sided Mann–Whitney test depending on the sample distribution type. ALP, alkaline phosphatase; ALT, alanine aminotransferase; ANIT, α-naphthylisothiocyanate; AST, aspartate aminotransferase; BBR, berberine; BDL, bile duct ligation; CLD, cholestatic liver disease; DBIL, direct bilirubin; PBS, phosphate-buffered saline; TBA, total bile acid; TBIL, total bilirubin.
Supplementary Figure 2.
BBR alleviates CLD progression in the mdr2-/- mouse model. (A–H) Wild type (control) or mdr2-/- mice were gavaged with PBS or BBR (20 mg/kg/day) for 5 days. n=6 mice per group. (A) H&E staining of liver sections. Scale bar, 200 μm. (B) Histopathological score of the liver sections. (C–H) Serum AST, ALT, TBA, ALP, TBIL, and DBIL levels. Data were represented as mean±SEM. P-values were calculated by a two-sided Mann–Whitney test depending on the sample distribution type. ALP, alkaline phophatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BBR, berberine; CLD, cholestatic liver disease; DBIL, direct bilirubin; PBS, phosphate-buffered saline; TBA, total bile acid; TBIL, total bilirubin.
Supplementary Figure 3.
Intraperitoneal injections of BBR did not ameliorate CLD in the BDL mouse model. (A–H) C57BL/6J mice were subjected to BDL or sham operation, followed by intraperitoneal injections of PBS or BBR (20 mg/kg/day) for 7 days. n=6 mice per group. (A) H&E staining of liver sections. Scale bar, 200 μm. (B) Histopathological score of the liver sections. (C–H) Serum TBA, ALP, ALT, AST, TBIL, and DBIL levels. Data were represented as mean±SEM. P-values were calculated by a two-sided Mann–Whitney test depending on the sample distribution type. ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BBR, berberine; BDL, bile duct ligation; CLD, cholestatic liver disease; DBIL, direct bilirubin; PBS, phosphate-buffered saline; TBA, total bile acid; TBIL, total bilirubin.
Supplementary Figure 4.
Intraperitoneal injection of BBR did not ameliorate CLD in the ANIT-induced intrahepatic cholestasis mouse model. (A–H) C57BL/6J mice were administered ANIT (50 mg/kg/day) intraperitoneally for 14 days, and BBR (20 mg/kg/day) or PBS was intraperitoneally injected on the last 5 days of the 14 days of ANIT-dosing. n=6 mice per group. (A) H&E staining of liver sections. Scale bar, 200 μm. (B) Histopathological score of the liver sections. (C–H) Serum ALT, AST, TBA, ALP, TBIL, and DBIL levels. Data were represented as mean±SEM. P-values were calculated by a two-sided Mann–Whitney test depending on the sample distribution type. ALP, alkaline phosphatase; ALT, alanine aminotransferase; ANIT, α-naphthylisothiocyanate; AST, aspartate aminotransferase; BBR, berberine; CLD, cholestatic liver disease; DBIL, direct bilirubin; NS, not significant; PBS, phosphate-buffered saline; TBA, total bile acid; TBIL, total bilirubin.
Supplementary Figure 5.
Intraperitoneal injection of BBR did not ameliorate CLD in the mdr2-/- mouse model. (A–H) Wild type (control) or mdr2-/- mice were treated with PBS or BBR (20 mg/kg/day) intraperitoneally for 5 days. n = 6 mice per group. (A) H&E staining of liver sections. Scale bar, 200 μm. (B) Histopathological score of the liver sections. (C–(H) Serum ALT, AST, TBA, ALP, TBIL, and DBIL levels. Data were represented as mean±SEM. P-values were calculated by a two-sided Mann–Whitney test or unpaired, two-sided t-test, depending on the sample distribution type. ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BBR, berberine; CLD, cholestatic liver disease; DBIL, direct bilirubin; NS, not significant; PBS, phosphate-buffered saline; TBA, total bile acid; TBIL, total bilirubin.
Supplementary Figure 6.
ABX attenuated the beneficial effects of BBR in the BDL and ANIT-induced intrahepatic cholestasis mouse model. (A) qPCR analysis for gut microbiota depletion, n=8. (B, C) Serum ALT and AST levels from BDL mice pretreated with ABX, followed by gavage with BBR or not. (D–K) C57BL/6J mice were pretreated with ABX cocktail for 7 days, then received ANIT intraperitoneally for 14 days, and BBR (20 mg/kg/day) was gavaged on the last 5 of the 14 days of ANIT-dosing. n=6 mice per group. (D) H&E staining of liver sections. Scale bar, 200 μm. (E) Histopathological score of the liver sections. (F–K) Serum ALT, AST, TBA, ALP, TBIL, and DBIL levels. Data were represented as mean±SEM. P-values were calculated by a two-sided Mann–Whitney test depending on the sample distribution type. ABX, antibiotic cocktail; ALP, alkaline phosphatase; ALT, alanine aminotransferase; ANIT, α-naphthylisothiocyanate; AST, aspartate aminotransferase; BBR, berberine; BDL, bile duct ligation; DBIL, direct bilirubin; TBA, total bile acid; TBIL, total bilirubin.
Supplementary Figure 7.
ABX attenuated the beneficial effects of BBR in mdr2-/- mouse model. (A–H): Wild type (control) or mdr2-/- mice were pretreated with ABX cocktail for 7 days, then received BBR (20 mg/kg/day) or PBS orally for 5 days. n=6 mice per group. (A) H&E staining of liver sections. Scale bar, 200 μm. (B) Histopathological score of the liver sections. (C–H) Serum ALT, AST, TBA, ALP, TBIL, and DBIL levels. Data were represented as mean±SEM. P-values were calculated by a two-sided Mann–Whitney test depending on the sample distribution type. ABX, antibiotic cocktail; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BBR, berberine; DBIL, direct bilirubin; NS, not significant; PBS, phosphate-buffered saline; TBA, total bile acid; TBIL, total bilirubin.
Supplementary Figure 8.
BBR’s ameliorating effects on CLD are dependent on gut microbiota. (A–D) Serum AST, ALT, TBIL, and DBIL levels from mice in sham/BDL/ABX/FMTCLD/FMTCLD+BBR group. (E–H) Serum AST, ALT, TBIL, and DBIL levels from mice in sham/BDL/ABX/FMTBDL/FMTBDL+BBR group. Data were represented as mean±SEM. P-values were calculated by a two-sided Mann–Whitney test depending on the sample distribution type. ABX, antibiotic cocktail; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BBR, berberine; BDL, bile duct ligation; CLD, cholestatic liver disease; DBIL, direct bilirubin; FMT, fecal microbiota transplantation; TBIL, total bilirubin.
Supplementary Figure 9.
Metabolites produced from BBR by gut microbiota alleviate CLD. (A–E) Serum TBA, ALT, AST, TBIL, and DBIL levels from mice in sham/BDL/FMTBDL/FMTBDL+BBR/FMTBDL+BBR+ABX group. (F–M) C57BL/6J mice were subjected to BDL or sham operation, then gavaged with PBS or fecal metabolites from BDL mice treated with BBR. n=6 mice per group. (F) H&E staining of liver sections. Scale bar, 200 μm. (G) Histopathological score of the liver sections. (H–M) Serum ALT, AST, TBA, ALP, TBIL, and DBIL levels. (N, O) Serum ALT and AST from BDL mice gavaged with fecal metabolites. Data were represented as mean±SEM. P-values were calculated by a two-sided Mann–Whitney test depending on the sample distribution type. ALT, alanine aminotransferase; AST, aspartate aminotransferase; BBR, berberine; CLD, cholestatic liver disease; BDL, bile duct ligation; DBIL, direct bilirubin; FMT, fecal microbiota transplantation; phosphate-buffered saline; TBA, total bile acid; TBIL, total bilirubin.
Supplementary Figure 10.
dhBBR alleviates CLD in BDL mice pretreated with ABX. (A, B) Serum ALT and AST levels from mice subjected to BDL or sham operation gavaged with dhBBR. (C–J) C57BL/6J mice were pretreated with ABX cocktail for 7 days, then subjected to BDL or sham operation, followed by receiving dhBBR (20 mg/kg/day) or BBR (20 mg/kg/day) orally for 7 days. n=6 mice per group. (C) H&E staining of liver sections. Scale bar, 200 μm. (D) Histopathological score of the liver sections. (E–J) Serum TBA, ALP, ALT, AST, TBIL, and DBIL levels. Data were represented as mean±SEM. P-values were calculated by a two-sided Mann–Whitney test depending on the sample distribution type. ABX, antibiotic cocktail; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BDL, bile duct ligation; CLD, cholestatic liver disease; DBIL, direct bilirubin; dhBBR, dihydroberberine; PBS, phosphate-buffered saline; TBA, total bile acid; TBIL, total bilirubin.
Supplementary Figure 11.
Untargeted metabolomics analysis of liver tissues from BDL and BDL+dhBBR mice. (A) Stem plot showing differentially abundant metabolites between dhBBR+BDL and BDL groups. (B) Cluster heatmap of differential metabolites between dhBBR+BDL and BDL groups. (C) KEGG pathway network analysis of altered metabolic pathways in dhBBR+BDL versus BDL groups. BDL, bile duct ligation; dhBBR, dihydroberberine; KEGG, Kyoto Encyclopedia of Genes and Genomes.
Supplementary Figure 12.
5-HT exacerbates cholestasis in BDL mice, while antagonists of 5-HTR2a/2b/2c attenuate these effects. (A) Immunohistochemical labeling of sham and BDL in liver sections. Scale bar, 200 μm. (B–I): C57BL/6J mice were subjected to sham operation or BDL, then received 5-HT (50 mg/kg/day), 5-HTR antagonist (Spiperone hydrochloride, 5-HTR2a antagonist, 100 nmol/kg/day; SB204741, 5-HTR2b antagonist, 100 nmol/kg/day; N-desmethylclozapine, 5-HTR2c antagonist, 100 nmol/kg/day) via tail intravenous injection for 7 days. n=6 mice per group. (B) H&E staining of liver sections. Scale bar, 200 μm. (C) Histopathological score of the liver sections. (D–I) Serum ALT, AST, TBA, ALP, TBIL and DBIL levels. Data were represented as mean±SEM. P-values were calculated by a two-sided Mann–Whitney test depending on the sample distribution type. ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BDL, bile duct ligation; DBIL, direct bilirubin; TBA, total bile acid; TBIL, total bilirubin; 5-HT, 5-hydroxy-tryptamine; 5-HTR, 5-hydroxytryptamine receptor.
Supplementary Figure 13.
5-HT promoting CLD progression via 5-HTR. (A–G) C57BL/6J mice were subjected to sham operation or BDL, then received 5-HT (50 mg/kg/day)+5-HTR antagonist (spiperone hydrochloride, 5-HTR2a antagonist, 100 nmol/kg/day; SB204741, 5-HTR antagonist, 100 nmol/kg/day; N-desmethylclozapine, 5-HTR2c antagonist, 100 nmol/kg/day) via tail intravenous injection for 7 days. n=6 mice per group. (A) H&E staining of liver sections. Scale bar, 200 μm. (B) Histopathological score of the liver sections. (C–G) Serum ALT, AST, TBA, ALP, and TBIL levels. (H, I) Serum ALT and AST of mice treated by dbBBR, 5-HT, or dhBBR plus 5-HT. n=6 mice per group. Data were represented as mean±SEM. P-values were calculated by two-sided Mann–Whitney test or unpaired, two-sided t-test depending on the sample distribution type. ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BDL, bile duct ligation; CLD, cholestatic liver disease; dhBBR, dihydroberberine; TBA, total bile acid; TBIL, total bilirubin; 5-HT, 5-hydroxytryptamine; 5-HTR, 5-hydroxytryptamine receptor.
Supplementary Figure 14.
Intraperitoneal administration of dhBBR failed to attenuate disease progression in BDL mice. (A–H) C57BL/6J mice underwent sham surgery or BDL, then were subsequently treated with intraperitoneal injections of dhBBR at a dose of 20 mg/kg/day for 5 days. n=6 mice per group. (A) H&E staining of liver sections. Scale bar, 200 μm. (B) Histopathological score of the liver sections. (C–H) Serum ALT, AST, TBA, ALP, TBIL and DBIL levels. Data were represented as mean±SEM. P-values were calculated by a two-sided Mann–Whitney test depending on the sample distribution type. ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BDL, bile duct ligation; DBIL, direct bilirubin; dhBBR, dihydroberberine; TBA, total bile acid; TBIL, total bilirubin.
Supplementary Figure 15.
dhBBR decreased 5-HT production through inhibiting gut TPH1. (A) Mouse intestinal organoids were developed for 7 days post-seeding and stained with chromogranin A (ChgA, green). Scale bar, 50 μm. (B) Immunofluorescent staining and relative fluorescence of ChgA (green) and 5-HT (red) of mouse intestinal organoids by incubating with dhBBR (20 μg/mL). Scale bar, 50 μm. n=3 or 4 independent incubations. (C) Level of 5-HT in the supernatant of gut organoids by incubating with BBR (20 μg/mL) or dhBBR (20 μg/mL) (n=3–4 independent incubations). (D) qPCR analysis of TPH1 in the mouse colon (n=8). (E) Molecular docking of dhBBR with TPH1 (PDB ID: 3HF6). (F) Concentration-dependent curve of dhBBR inhibiting TPH1 activity. (G) Microscale thermophoresis assay for dhBBR and LX1031 against TPH1. (H) The inhibition effect of dhBBR (0.5 mM) in L-Trp (50 μM)-induced activation of TPH1 under the enzymatic reaction system of TPH1 in vitro (n=3 independent incubations). (I) 5-HTP concentration in the intestine of BDL mice treated with BBR or PBS. n=6 mice per group. Data were represented as mean±SEM. P-values were calculated by two-sided Mann–Whitney test or unpaired depending on the sample distribution type. BBR, berberine; dhBBR, dihydroberberine; L-Trp, L-tryptophan; PBS, phosphate-buffered saline; TPH1, tryptophan hydroxylase 1; 5-HT, 5-hydroxytryptamine.
Figure 1.Berberine (BBR) alleviates cholestatic liver disease progression in multiple models and is associated with gut microbiota. (A) Experimental scheme for (B, C). n=6 mice per group. (B) H&E staining of liver sections and histopathological score of the liver sections. Scale bar, 200 μm. (C) Serum TBA, ALP, TBIL, and DBIL levels. (D) Experimental scheme for (E, F). n=6 mice per group. C57BL/6J mice were first treated with ABX for 7 days, then subjected to BDL or sham operation, followed by administration of BBR (20 mg/kg/day) or ABX plus BBR (20 mg/kg/day) for another 7 days. (E) H&E staining of liver sections and histopathological score of the liver sections. Scale bar, 200 μm. (F) Serum TBA, ALP, TBIL, and DBIL levels. Data were represented as mean±SEM. P-values were calculated by a two-sided Mann-Whitney test depending on the sample distribution type. ABX, antibiotic cocktail; ALP, alkaline phosphatase; BDL, bile duct ligation; DBIL, direct bilirubin; NS, not significant; PBS, phosphate-buffered saline; SPF, specific pathogen-free; TBA, total bile acid; TBIL, total bilirubin.
Figure 2.BBR’s ameliorating effects on CLD are dependent on gut microbiota. (A) Experimental scheme for (B, C). (B) H&E staining of liver sections and histopathological score of the liver sections. Scale bar, 200 μm. (C) Serum TBA and ALP levels. (D) Experimental scheme for (E, F). C57BL/6J mice were subjected to BDL or sham operation, then received fecal microbiota from BDL mice gavaged with BBR or not. n=6 mice per group. (E) H&E staining of liver sections and histopathological score of the liver sections. Scale bar, 200 μm. (F) Serum TBA and ALP levels. Data were represented as mean±SEM. P-values were calculated by a two-sided Mann–Whitney test depending on the sample distribution type. ABX, antibiotic cocktail; ALP, alkaline phosphatase; BBR, berberine; BDL, bile duct ligation; CLD, cholestatic liver disease; FMT, fecal microbiota transplantation; PBS, phosphate-buffered saline; SPF, specific pathogen-free; TBA, total bile acid.
Figure 3.Metabolites produced from BBR by gut microbiota alleviate CLD. (A) Experimental scheme for (B, C). n=6 mice per group. (B) H&E staining of liver sections and histopathological score of the liver sections. Scale bar, 200 μm. (C) Serum TBA and ALP levels. (D) Workflow for isolation of the fecal metabolites derived from BDL mice gavaged with BBR. Feces from BDL mice were collected and resuspended, followed by centrifugation and filtration to obtain the metabolites. (E, F) C57BL/6J mice were pretreated with ABX for 7 days, followed by BDL or sham operation. The mice then received daily oral gavage of either PBS or fecal metabolites derived from BDL mice treated with BBR. n=6 mice per group. (E) H&E staining of liver sections and histopathological score of the liver sections. Scale bar, 200 μm. (F) Serum TBA, ALP, TBIL, and DBIL levels. Data were represented as mean±SEM. P-values were calculated by a two-sided Mann-Whitney test depending on the sample distribution type. ABX, antibiotic cocktail; ALP, alkaline phosphatase; BBR, berberine; BDL, bile duct ligation; CLD, cholestatic liver disease; DBIL, direct bilirubin; FMT, fecal microbiota transplantation; PBS, phosphate-buffered saline; SPF, specific pathogen-free; TBA, total bile acid; TBIL, total bilirubin.
Figure 4.Gut microbiota transformed BBR into dhBBR to attenuate the progression of CLD. (A) Schematic representation of BBR metabolized into dhBBR by gut microbial nitroreductase. (B) Gut microbiota composition and diversity in BDL mice with or without BBR treatment. (C) dhBBR concentration determined by LC-MS/MS in the feces, intestine, and liver from BDL mice gavaged with BBR. (D) Extracted ion chromatograms of dhBBR in human feces by LC-MS/MS. (E, F) C57BL/6J mice were subjected to BDL or sham operation, then administered with dhBBR or not. n=6 mice per group. (E) H&E staining of liver sections and histopathological score of the liver sections. Scale bar, 200 μm. (F) Serum TBA, ALP, TBIL, and DBIL levels. Data were represented as mean±SEM. P-values were calculated by a two-sided Mann–Whitney test depending on the sample distribution type. ALP, alkaline phosphatase; BBR, berberine; BDL, bile duct ligation; CLD, cholestatic liver disease; DBIL, direct bilirubin; dhBBR, dihydroberberine; LC-MS/MS, liquid chromatography-mass spectrometry; TBA, total bile acid; TBIL, total bilirubin.
Figure 5.dhBBR alleviates CLD progression in a 5-HT/5-HTR dependent way. (A) Principal component analysis for liver metabolites from BDL mice gavaged with dhBBR or not. n=6 mice per group. (B) Volcano plot for serum metabolites from BDL mice gavaged with dhBBR or not. Data were compared using the Wilcoxon rank-sum test. (C) 5-HT concentration in serum from BDL/sham mice gavaged with dhBBR or not. n=6 mice per group. (D) 5-HT concentration in liver, intestine, and serum from BDL/sham mice gavaged with BBR or not. n=12 mice per group. (E, F) C57BL/6J mice were subjected to BDL or sham operation, then received dhBBR (20 mg/kg/day, p.o.) or dhBBR (20 mg/kg/day, p.o.) plus 5-HT (50 mg/kg/day, I.V.). n=6 mice per group. (E) H&E staining of liver sections and histopathological score of the liver sections. Scale bar, 200 μm. (F) Serum TBA, ALP, TBIL, and DBIL levels. Data were represented as mean±SEM. P-values were calculated by a two-sided Mann–Whitney test depending on the sample distribution type. ALP, alkaline phosphatase; BDL, bile duct ligation; CLD, cholestatic liver disease; DBIL, direct bilirubin; dhBBR, dihydroberberine; I.V., intravenous; PBS, phosphate-buffered saline; p.o., per os; TBA, total bile acid; TBIL, total bilirubin; 5-HT, 5-hydroxytryptamine; 5-HTR, 5-hydroxytryptamine receptor.
Figure 6.dhBBR decreased 5-HT production through inhibiting gut TPH1. (A) Ultrasound-guided localization and blood sampling from the portal vein in mice (left). Comparison of 5-HT levels and their reduction ratio by BBR in portal (n=6) versus peripheral (n=12) blood across sham, BDL, and BDL+BBR groups (right). (B) Immunofluorescence staining of ChgA+ (green) and 5-HT+ (red) double-positive cells (yellow overlap, indicated by white arrows) in colon sections from sham, BDL, and BDL+BBR mice. Scale bar, 50 μm. n=6. (C, D) C57BL/6J mice were subjected to BDL or sham operation, followed by 7-day treatment with dhBBR (20 mg/kg/day, p.o.), LX-1031 (45 mg/kg/day, p.o.), or their combination. n=6 mice per group. (C) H&E staining of liver sections and histopathological score of the liver sections. Scale bar, 200 μm. (D) Serum TBA, ALP, TBIL, and DBIL levels. Data were represented as mean±SEM. P-values were calculated by two-sided Mann–Whitney test or unpaired depending on the sample distribution type. ALP, alkaline phosphatase; BBR, berberine; BDL, bile duct ligation; DBIL, direct bilirubin; dhBBR, dihydroberberine; p.o., per os; TBA, total bile acid; TBIL, total bilirubin; TPH1, tryptophan hydroxylase 1; 5-HT, 5-hydroxytryptamine.
Figure 7.The combination of UDCA and BBR improves the clinical indicators in CLD patients. (A) Study design and workflow summary. (B) Serum GGT, ALP, ALT, and AST levels from CLD patients administered with UDCA or UDCA plus BBR. (C) Recovery rates of GGT, ALP, ALT, and AST levels from CLD patients administered with UDCA or UDCA plus BBR. (D) Serum 5-HT concentration from CLD patients administered with UDCA or UDCA plus BBR. (E) Recovery rates of 5-HT concentrations from CLD patients administered with UDCA or UDCA plus BBR. Data were represented as mean±SEM. P-values were calculated by a two-sided t-test depending on the sample distribution type. UDCA, ursodeoxycholic acid; BBR, berberine; CLD, cholestatic liver disease; GGT, gamma-glutamyl transferase; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; 5-HT, 5-hydroxytryptamine.
Abbreviations
aspartate aminotransferase
cyclic adenosine monophosphate
cholestatic liver disease
fecal microbiota transplantation
gamma-glutamyl transferase
liquid chromatography-mass spectrometry
multidrug resistance protein
microscale thermophoresis
primary biliary cirrhosis
primary sclerosing cholangitis
5-hydroxytryptamine receptor
REFERENCES
- 1. Ibrahim SH, Kamath BM, Loomes KM, Karpen SJ. Cholestatic liver diseases of genetic etiology: advances and controversies. Hepatology 2022;75:1627-1646.
- 2. Trauner M, Fuchs CD. Novel therapeutic targets for cholestatic and fatty liver disease. Gut 2022;71:194-209.
- 3. Hasegawa S, Yoneda M, Kurita Y, Nogami A, Honda Y, Hosono K, et al. Cholestatic liver disease: current treatment strategies and new therapeutic agents. Drugs 2021;81:1181-1192.
- 4. Mayo MJ. Mechanisms and molecules: What are the treatment targets for primary biliary cholangitis? Hepatology 2022;76:518-531.
- 5. Schattenberg JM, Pares A, Kowdley KV, Heneghan MA, Caldwell S, Pratt D, et al. A randomized placebo-controlled trial of elafibranor in patients with primary biliary cholangitis and incomplete response to UDCA. J Hepatol 2021;74:1344-1354.
- 6. Habtemariam S. Berberine pharmacology and the gut microbiota: a hidden therapeutic link. Pharmacol Res 2020;155:104722.
- 7. Feng X, Sureda A, Jafari S, Memariani Z, Tewari D, Annunziata G, et al. Berberine in cardiovascular and metabolic diseases: from mechanisms to therapeutics. Theranostics 2019;9:1923-1951.
- 8. Koperska A, Wesołek A, Moszak M, Szulińska M. Berberine in non-alcoholic fatty liver disease-a review. Nutrients 2022;14:3459.
- 9. Wang Y, Zhao D, Su L, Tai YL, Way GW, Zeng J, et al. Therapeutic potential of berberine in attenuating cholestatic liver injury: insights from a PSC mouse model. Cell Biosci 2024;14:14.
- 10. Naghibi Z, Rakhshandeh H, Jarahi L, Hosseini SM, Yousefi M. Evaluation of the effects of additional therapy with Berberis vulgaris oxymel in patients with refractory primary sclerosing cholangitis and primary biliary cholangitis: a quasi-experimental study. Avicenna J Phytomed 2021;11:154-167.
- 11. Kowdley KV, Forman L, Eksteen B, Gunn N, Sundaram V, Landis C, et al. A randomized, dose-finding, proof-of-concept study of berberine ursodeoxycholate in patients with primary sclerosing cholangitis. Am J Gastroenterol 2022;117:1805-1815.
- 12. Wang Z, Zhao Y. Gut microbiota derived metabolites in cardiovascular health and disease. Protein Cell 2018;9:416-431.
- 13. Agus A, Clément K, Sokol H. Gut microbiota-derived metabolites as central regulators in metabolic disorders. Gut 2021;70:1174-1182.
- 14. Cheng H, Liu J, Tan Y, Feng W, Peng C. Interactions between gut microbiota and berberine, a necessary procedure to understand the mechanisms of berberine. J Pharm Anal 2022;12:541-555.
- 15. Lei Y, Tang L, Chen Q, Wu L, He W, Tu D, et al. Disulfiram ameliorates nonalcoholic steatohepatitis by modulating the gut microbiota and bile acid metabolism. Nat Commun 2022;13:6862.
- 16. Luo Y, Liu C, Luo Y, Zhang X, Li J, Hu C, et al. Thiostrepton alleviates experimental colitis by promoting RORγt ubiquitination and modulating dysbiosis. Cell Mol Immunol 2023;20:1352-1366.
- 17. Tang B, Li S, Li X, He J, Zhou A, Wu L, et al. Cholecystectomy-related gut microbiota dysbiosis exacerbates colorectal tumorigenesis. Nat Commun 2025;16:7638.
- 18. Liu Y, Binz J, Numerick MJ, Dennis S, Luo G, Desai B, et al. Hepatoprotection by the farnesoid X receptor agonist GW4064 in rat models of intra- and extrahepatic cholestasis. J Clin Invest 2003;112:1678-1687.
- 19. Zhang J, Lyu Z, Li B, You Z, Cui N, Li Y, et al. P4HA2 induces hepatic ductular reaction and biliary fibrosis in chronic cholestatic liver diseases. Hepatology 2023;78:10-25.
- 20. Chen W, Miao YQ, Fan DJ, Yang SS, Lin X, Meng LK, et al. Bioavailability study of berberine and the enhancing effects of TPGS on intestinal absorption in rats. AAPS PharmSciTech 2011;12:705-711.
- 21. Liu YT, Hao HP, Xie HG, Lai L, Wang Q, Liu CX, et al. Extensive intestinal first-pass elimination and predominant hepatic distribution of berberine explain its low plasma levels in rats. Drug Metab Dispos 2010;38:1779-1784.
- 22. Koppel N, Maini Rekdal V, Balskus EP. Chemical transformation of xenobiotics by the human gut microbiota. Science 2017;356:eaag2770.
- 23. Wang K, Feng X, Chai L, Cao S, Qiu F. The metabolism of berberine and its contribution to the pharmacological effects. Drug Metab Rev 2017;49:139-157.
- 24. Feng R, Shou JW, Zhao ZX, He CY, Ma C, Huang M, et al. Transforming berberine into its intestine-absorbable form by the gut microbiota. Sci Rep 2015;5:12155.
- 25. Feng R, Zhao ZX, Ma SR, Guo F, Wang Y, Jiang JD. Gut microbiota-regulated pharmacokinetics of berberine and active metabolites in beagle dogs after oral administration. Front Pharmacol 2018;9:214.
- 26. Kyritsi K, Chen L, O’Brien A, Francis H, Hein TW, Venter J, et al. Modulation of the tryptophan hydroxylase 1/monoamine oxidase-a/5-hydroxytryptamine/5-hydroxytryptamine receptor 2A/2B/2C axis regulates biliary proliferation and liver fibrosis during cholestasis. Hepatology 2020;71:990-1008. Erratum in: Hepatology 2023;78:E86.
- 27. Yano JM, Yu K, Donaldson GP, Shastri GG, Ann P, Ma L, et al. Indigenous bacteria from the gut microbiota regulate host serotonin biosynthesis. Cell 2015;161:264-276. Erratum in: Cell 2015;163:258.
- 28. Gershon MD, Tack J. The serotonin signaling system: from basic understanding to drug development for functional GI disorders. Gastroenterology 2007;132:397-414.
- 29. Camilleri M. LX-1031, a tryptophan 5-hydroxylase inhibitor, and its potential in chronic diarrhea associated with increased serotonin. Neurogastroenterol Motil 2011;23:193-200.
- 30. Cheng M, Liu R, Wu Y, Gu P, Zheng L, Liu Y, et al. LC-MS/MS determination and urinary excretion study of seven alkaloids in healthy Chinese volunteers after oral administration of Shuanghua Baihe tablets. J Pharm Biomed Anal 2016;118:89-95.
- 31. Liu CS, Zheng YR, Zhang YF, Long XY. Research progress on berberine with a special focus on its oral bioavailability. Fitoterapia 2016;109:274-282.
- 32. Wang Y, Shou JW, Li XY, Zhao ZX, Fu J, He CY, et al. Berberine-induced bioactive metabolites of the gut microbiota improve energy metabolism. Metabolism 2017;70:72-84.
- 33. Wang Y, Tong Q, Shou JW, Zhao ZX, Li XY, Zhang XF, et al. Gut microbiota-mediated personalized treatment of hyperlipidemia using berberine. Theranostics 2017;7:2443-2451.
- 34. Wang Y, Tong Q, Ma SR, Zhao ZX, Pan LB, Cong L, 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.
- 35. Sabino J, Vieira-Silva S, Machiels K, Joossens M, Falony G, Ballet V, et al. Primary sclerosing cholangitis is characterised by intestinal dysbiosis independent from IBD. Gut 2016;65:1681-1689.
- 36. Zhang X, Beaulieu JM, Sotnikova TD, Gainetdinov RR, Caron MG. Tryptophan hydroxylase-2 controls brain serotonin synthesis. Science 2004;305:217.
- 37. Patel PD, Pontrello C, Burke S. Robust and tissue-specific expression of TPH2 versus TPH1 in rat raphe and pineal gland. Biol Psychiatry 2004;55:428-433.
- 38. Zhang Y, Wang S, Zhang L, Zhou F, Zhu K, Zhu Q, et al. Protein acetylation derepresses serotonin synthesis to potentiate pancreatic beta-cell function through HDAC1-PKA-Tph1 signaling. Theranostics 2020;10:7351-7368.
- 39. Piccand J, Vagne C, Blot F, Meunier A, Beucher A, Strasser P, et al. Rfx6 promotes the differentiation of peptide-secreting enteroendocrine cells while repressing genetic programs controlling serotonin production. Mol Metab 2019;29:24-39.
- 40. Waløen K, Kleppe R, Martinez A, Haavik J. Tyrosine and tryptophan hydroxylases as therapeutic targets in human disease. Expert Opin Ther Targets 2017;21:167-180.
- 41. Dawson PA, Karpen SJ. Bile acids reach out to the spinal cord: new insights to the pathogenesis of itch and analgesia in cholestatic liver disease. Hepatology 2014;59:1638-1641.
- 42. Lieu T, Jayaweera G, Bunnett NW. GPBA: a GPCR for bile acids and an emerging therapeutic target for disorders of digestion and sensation. Br J Pharmacol 2014;171:1156-1166.
- 43. Naghibi Z, Rakhshandeh H, Jarahi L, Hosseini SM, Yousefi M. Evaluation of the effects of additional therapy with Berberis vulgaris oxymel in patients with refractory primary sclerosing cholangitis and primary biliary cholangitis: a quasi-experimental study. Avicenna J Phytomed 2021;11:154-167.
, Shiming Yang1,2
