proteins

2026-06-11 | Hepatic CREB Binding Protein/E1A Binding Protein p300 Maintain Bile Acid Homeostasis Through Histone Acetylation-Mediated Kruppel-Like Transcription Factor 10-Bile Salt Export Pump Axis.

Disruption of bile acid metabolism contributes to various liver diseases. Although CREB binding protein/E1A binding protein p300 are known regulators of hepatic metabolism, their role in bile acid homeostasis remains elusive. Metabolomic and transcriptomic analyses on liver-specific CREB binding protein/E1A binding protein p300 knockout mice were performed to assess bile acid metabolism. Cleavage Under Targets & Tagmentation sequencing and chromatin immunoprecipitation-quantitative polymerase chain reaction were used to investigate the epigenetic mechanisms underlying CREB binding protein/E1A binding protein p300-mediated bile acid regulation. A mouse model of cholestatic liver disease was established to validate the findings from the liver-specific CREB binding protein/E1A binding protein p300 double-knockout mice by administrating a 0.1% 3,5-diethoxycarbonyl-1,4-dihydrocollidine diet. Liver-specific CREB binding protein/E1A binding protein p300 deletion caused intrahepatic cholestasis due to impaired bile acid efflux, along with elevated plasma bile acid levels. Transcriptomic and metabolomic analyses revealed decreased expression of bile acid synthesis and transport genes, including Abcb11 (encoding BSEP, the bile salt export pump). Mechanistically, CBP/p300 promoted Abcb11 transcription via H3K27 acetylation. In addition, CREB binding protein/E1A binding protein p300 epigenetically regulated the transcription factor Kruppel-like transcription factor 10, which directly activated Abcb11 expression. Loss of CREB binding protein/E1A binding protein p300 reduced H3K27Ac enrichment at both Klf10 and Abcb11 promoters, leading to their downregulation. Overexpression of either Klf10 or Abcb11 in CREB binding protein/E1A binding protein p300-deficient hepatocytes partially rescued bile acid transport defects. Impaired Kruppel-like transcription factor 10-bile salt export pump regulatory axis was also observed in a 3,5-diethoxycarbonyl-1,4-dihydrocollidine-induced model of hepatic cholestasis. Our findings uncover a novel CREB binding protein/E1A binding protein p300-Kruppel-like transcription factor 10-bile salt export pump regulatory axis that orchestrates bile acid transport and highlight its therapeutic potential for cholestatic liver diseases.

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2026-04-09 | A Novel Broad pH-Adaptive Bile Salt Hydrolase from Nomascus concolor Fecal Microbial Metagenome Facilitates the Cholesterol-Lowering Ability of Escherichia coli Nissle 1917.

High serum cholesterol levels are among the key risk factors for atherosclerosis cardiovascular disease. The utilization of probiotics to lower cholesterol is a relatively safe and efficient therapy, and the bile salt hydrolase gene serves a key function in this process. We aim to identify novel bile salt hydrolase genes from the wild western black crested gibbon (Nomascus concolor) faecal metagenome. Additionally, we intend to develop a new generation of probiotics with cholesterol-lowering properties. Our study amplified and heterologously expressed novel bile salt hydrolases from the faecal metagenome of western black crested gibbons and investigated their enzymatic properties. The recombinant probiotic was constructed using Escherichia coli Nissle 1917 (EcN), and its physiological characteristics and cholesterol-lowering ability were evaluated. We screened uncharacterized bile salt hydrolase genes (NCbsh3 and NCbsh5) from Eubacterium and Roseburia. NCbsh3 exhibited broad pH adaptability and stability; its optimal pH range was 4–7, and the relative enzyme activity was maintained at 80% after 60 min at pH 3–9. The recombinant probiotic EcN/NCbsh3 was constructed, and its bile salt tolerance and cholesterol-lowering ability significantly increased (P < 0.05). These results indicated that NCbsh3 may adapt to the complex pH environment of the intestine and that the NCbsh3 gene is expected to increase the colonization capacity of the EcN strain in the gastrointestinal tract and reduce host serum cholesterol levels. EcN/NCbsh3 has favourable application potential and may contribute positively to the treatment of diseases caused by bile acid metabolism disorders.

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2026-03-29 | Activated hepatic stellate cells maintain hepatic bile acid homeostasis through paracrine FGF10/FGFR2 signaling.

Hepatic stellate cells (HSCs) are activated during cholestasis. However, beyond their role in liver fibrosis, whether HSCs influence bile acid (BA) synthesis and homeostasis remains largely unknown. This study investigates the role of activated HSCs (aHSCs) in the regulation of hepatic BA metabolism. Human liver tissues, human primary HSCs, and hepatocytes were utilized in this study. Fgf10 knock-in reporter mice, aHSC-specific Fgf10 knockout mice, and hepatocyte-specific Fgfr2 knockdown mice were generated. Mouse models of obstructive cholestasis and western diet-induced MASH were established. Recombinant FGF10 was administered to further validate the role of the FGF10/FGFR2 axis in regulating BA homeostasis and fibrosis. Bulk RNA sequencing (RNA-seq) and single-nucleus RNA-seq were performed to elucidate how aHSCs regulate BA metabolism via FGF10. FGF10 was upregulated in human fibrotic livers. The expression of FGF10 by aHSCs was increased in animal models of obstructive cholestasis and diet-induced MASH. Cholestasis-induced ductular reaction and tissue fibrosis were aggravated in livers of aHSC-specific Fgf10 knockout mice. RNA-seq and single-nucleus RNA-seq indicated that aHSCs regulate primary BA synthesis through FGF10-mediated CYP7A1 inhibition. Paracrine FGF10 induced the upregulation of FGFR2 in hepatocytes, resulting in the activation of PAK1/ERK/JNK signaling, thereby inhibiting CYP7A1 expression and the synthesis of primary BAs. In diet-induced MASH, FGF10 exerted no significant effect on steatosis or fibrosis but significantly influenced BA metabolism. Additionally, recombinant FGF10 treatment ameliorated cholestatic liver injury and reduced BA accumulation in mice, which was abrogated by Fgfr2 knockdown in hepatocytes. This study identifies a critical role for aHSCs in the regulation of hepatic BA homeostasis and demonstrates that the aHSC-FGF10/FGFR2 signaling axis may represent a potential therapeutic target for cholestatic liver disease. Cholestasis and metabolic dysfunction-associated steatohepatitis (MASH) are associated with the activation of hepatic stellate cells (HSCs), yet whether HSCs influence bile acid metabolism during these processes remains largely unknown. Here, we demonstrate that murine activated HSCs control CYP7A1 expression in hepatocytes via paracrine FGF10, which inhibits primary bile acid (BA) synthesis in obstructive cholestasis and MASH. Paracrine FGF10/FGFR2 signaling controlled hepatic primary BA synthesis and alleviated ductular reaction and liver injury in cholestatic mice, but showed no significant effect on steatosis and fibrosis during MASH progression. This study implies specific roles for activated HSCs and FGF10 in the maintenance of hepatic BA homeostasis.

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2025-12-19 | Chemical proteomics reveals regulation of bile salt hydrolases via oxidative post-translational modifications.

The gut microbiome is the vast, diverse ecosystem of microorganisms that inhabits the human intestines and provides numerous essential functions for the host. One such key role is the metabolism of primary bile acids that are biosynthesized in the host liver into a plethora of secondary bile acids produced by gut bacteria. These metabolites serve as both antimicrobial and chemical signaling agents within the host. The critical microbial enzyme that plays a gatekeeping role in secondary bile acid metabolism is bile salt hydrolase (BSH), a cysteine hydrolase that is primarily known for its deconjugating and reconjugating activities on bile acid substrates. Despite the crucial nature of these biotransformations, regulation of BSH activity is not well understood. Here, we found that the catalytic cysteine 2 (Cys2) within the BSH active site exists in multiple sulfur oxidation states including sulfenic acid (Cys-SOH). Importantly, we show this reversible oxidative post-translational modification (oxPTM) ablates BSH catalytic activity. We have leveraged this discovery to develop a chemoproteomic platform featuring a sulfenic acid-reactive bile acid probe to profile BSH Cys2 oxPTMs throughout the gut microbiome. Our results reveal that though most gut microbiota-associated BSHs exist in the active Cys2-SH state, some are preferentially and reversibly inactivated in the Cys2-SOH state. This reversible oxidation of Cys2 may serve as a general mechanism to regulate BSH activity in vivo in response to a changing physiological environment.

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2025-06-12 | Liver-specific Nr1h4 deletion in mice with human-like bile acid composition causes severe liver injury.

The farnesoid X receptor, encoded by NR1H4, is crucial for bile acid, lipid, and glucose metabolism. NR1H4 mutations in humans cause a severe liver injury called progressive familial intrahepatic cholestasis 5. However, Nr1h4 deletion in mice did not cause severe liver damage at a young age, likely because of the higher levels of hydrophilic bile acids synthesized by the mouse-specific bile acid metabolic enzymes Cyp2a12 and Cyp2c70. We aimed to assess hepatic NR1H4 function by taking advantage of the recently established Cyp2a12/Cyp2c70 double-knockout (CYPDKO) mouse model, which has a human-like bile acid composition containing mainly hydrophobic bile acids. Liver-specific Nr1h4-deficient CYPDKO mice were established using an adeno-associated virus-derived genome-editing method. Nr1h4-deficient wild-type (WT) mice showed no significant changes in marker levels for serum liver injury. In contrast, Nr1h4-deficient CYPDKO mice showed an increase in the liver/body weight ratio and serum liver injury markers, suggesting that the combination of human-like bile acid composition and Nr1h4 deletion induces liver injury. Nr1h4 deletion increased total bile acid levels in the liver through the upregulation of bile acid metabolic genes and downregulation of bile acid transporters. Conversely, overexpression of a small heterodimer partner (Shp), a downstream gene of Nr1h4, suppresses liver injury induced by Nr1h4 deletion in CYPDKO mice. Overall, liver-specific Nr1h4 deficiency induced significant liver damage in mice with human-like bile acids, unlike in WT mice, validating its use as a new animal model for cholestatic liver disease. Therefore, Shp may be a potential target for the treatment of cholestasis.

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small molecules

2026-09-22 | Adult-onset deficiency of β-klotho in the liver reveals a novel role in lipid homeostasis.

β-klotho (KLB), the obligate co-receptor for fibroblast growth factor (FGF) 19 and 21 signalling, has emerged as a therapeutic target for metabolic disease. Current understanding of KLB function is based largely on germline knockout mouse models, where developmental abnormalities obscure its function in adult physiology. Here, we used somatic hepatocyte-specific gene editing in adult mice to define hepatic KLB function. Hepatocyte-specific KLB deficiency was induced by retro-orbital delivery of adeno-associated virus particles containing guide RNAs targeting Klb in adult male mice expressing Cas9 in the liver. Following transduction, mice were maintained on a 4-week chow diet or a 12-week high-fat diet, prior to comprehensive metabolic and biochemical phenotyping. Adult-onset hepatic KLB deficiency did not alter body weight or composition, indicating that reduced bodyweight in germline models reflects developmental effects. In line with previous studies, KLB deficiency resulted in derepressed bile acid synthesis, elevated faecal bile acid excretion, and a shift towards a more hydrophobic bile acid pool. Importantly, we also uncovered a role for hepatic KLB in regulating lipid homeostasis. Chow-fed KLB-deficient mice exhibited reduced circulating very-low-density lipoprotein levels (VLDL) with marked hepatic triglyceride accumulation, suggesting altered VLDL metabolism as a potential mechanism driving steatosis. Using an adult-onset liver-specific knockout model, we disentangled KLB function from developmental confounders and established KLB as a critical regulator of adult lipid and lipoprotein homeostasis. Our study redefines the physiological role of KLB in hepatic metabolism and has important implications for the interpretation of FGF19/KLB-directed therapies for metabolic diseases.

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2026-09-18 | Dietary flavonoids target the gut-liver axis for the management of cholestatic liver injury.

Cholestatic liver injury (CLI) is a progressive liver disorder characterized by impaired bile acid (BA) synthesis, transport, and excretion. This dysfunction leads to the intrahepatic accumulation of toxic BAs, which in turn triggers hepatic oxidative stress, inflammation, and subsequent fibrogenesis. Through enterohepatic circulation, the gut and liver form an integrated gut-liver axis that regulates BA metabolism. Gut microbiota and nuclear receptor farnesoid X receptor (FXR) are tightly involved in modulating this axis and play a critical role in maintaining BA homeostasis. Disruption of the gut-liver axis under cholestatic conditions contributes to gut dysbiosis, BA imbalance, impaired intestinal barrier integrity, hepatic inflammation, and fibrotic progression. Flavonoids, a major class of polyphenols present in various foods, possess microbial-dependent biotransformation and gut enrichment characteristics. Through coordinated modulation of gut microbiota, BA metabolism, and host signaling pathways, flavonoids display promising protective effects against multiple pathological outcomes associated with CLI. This review summarizes the pathological mechanisms of CLI and the molecular interactions through which dietary flavonoids modulate the gut microbiota-BA-FXR axis. It further discusses the dietary exposure, bioavailability, and translational relevance of flavonoids by comparing human dietary intake and systemic exposure with preclinical doses, while evaluating safety considerations and available clinical trial evidence. These insights provide a framework for evaluating the nutritional relevance of flavonoids and their potential as functional food ingredients for the prevention and management of CLI.

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2026-09-12 | The hypoxia–gut microbiota–bile acid axis: mechanistic crosstalk and therapeutic opportunities

Hypoxia is a central feature of a wide range of pathological conditions; however, the metabolic regulatory networks underlying hypoxic adaptation remain incompletely understood. In recent years, accumulating evidence suggests that bile acids, traditionally regarded as metabolic end products, also function as signaling molecules and may participate in hypoxia-related regulation. Nevertheless, this association is largely indirect and context-dependent. In this context, the gut microbiota has emerged as a key intermediary. It is highly sensitive to fluctuations in oxygen tension and plays a dominant role in shaping bile acid composition, including the generation of secondary and microbially conjugated bile acids. Collectively, these observations raise the possibility that hypoxic signaling, microbial dynamics, and bile acid metabolism are functionally interconnected within an integrated regulatory network that remains to be fully defined. In this review, we propose a hypoxia–microbiota–bile acid axis as one of a central regulatory hub in hypoxic adaptation. Hypoxia-inducible factors regulate bile acid synthesis and transport while reshaping gut microbial composition, thereby altering the bile acid pool. In turn, bile acids feed back on hypoxia signaling, modulating oxidative stress, inflammation, and metabolic homeostasis. Microbiota-derived bile acids further amplify these effects through receptor-mediated pathways. Notably, these interactions are highly tissue- and context-dependent, leading to both protective and detrimental outcomes. Within this framework, we further delineate how host HIF signaling may selectively enrich MCBA-competent bacterial taxa and activate their biosynthetic machinery at the host–microbial interface, thereby connecting hypoxic sensing to a structurally distinct class of microbial metabolites. Elucidating this network may advance our understanding of hypoxia-related diseases and uncover therapeutic opportunities targeting the microbiota–bile acid interface. Oxygen shortage (also called hypoxia) is common in many diseases, such as heart problems, stroke, and chronic inflammation. When oxygen levels drop, the body must adjust how it produces energy and maintains balance. One important but often overlooked part of this response involves the gut, where trillions of microbes help regulate many processes that keep us healthy. These gut microbes play a key role in transforming bile acids—natural substances made by the liver that not only help digest food but also act as signals controlling metabolism, immunity, and stress responses. Recent research shows that low oxygen levels can reshape the gut microbial community, which in turn changes how bile acids are modified. This creates a close link between oxygen levels, gut microbes, and bile acid activity. In this review, we explain how this interconnected system works and how it influences the body’s response to stress. Some bile acids produced by microbes can help protect tissues, reduce inflammation, and support energy balance. However, under certain conditions, they may also contribute to cell damage and disease. Overall, this system acts like a double-edged sword. A better understanding of these interactions may lead to new treatments for diseases linked to low oxygen, metabolic disorders, and inflammation by targeting gut microbes or bile acid pathways.

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2026-09-04 | Saxifraga umbellulata Attenuates Cholestatic Liver Injury and Modulates Farnesoid X Receptor-Related Bile Acid Homeostasis.

Disrupted bile acid homeostasis represents a central feature of cholestatic liver injury, for which effective therapeutic options remain limited. Saxifraga umbellulata Hook. f. et Thoms. (SU) has traditionally been used in Tibetan medicine to treat hepatobiliary disorders, yet the pharmacological basis of SU remains unclear. Therefore, this study aimed to evaluate the effects of SU on cholestatic liver injury and explore the regulatory mechanisms underlying these effects. A mouse model of cholestatic liver injury was established by administering α-naphthyl isothiocyanate (ANIT). The effects of SU were evaluated using serum biochemical measurements, liver histopathology, and targeted profiling of hepatic bile acids. Protein expression associated with farnesoid X receptor (FXR) signaling was assessed by Western blotting in liver and ileal tissues. Representative triterpenoids in the SU extract were further examined using molecular docking and HepG2 cell experiments. SU administration reduced serum markers of hepatocellular injury and cholestasis and lessened hepatic necrosis and inflammatory cell infiltration in ANIT-treated mice. SU also shifted the altered hepatic bile acid profile toward a more balanced state, including partial correction of the disturbed proportions of primary and secondary bile acids. These metabolic changes were accompanied by reduced hepatic CYP7A1 expression and increased expression of BSEP and MRP2. In addition, SU increased ileal FXR and FGF15 expression and hepatic FGFR4 expression, a pattern consistent with coordinated regulation of the intestinal FXR-FGF15/hepatic FGFR4 axis. Meanwhile, molecular docking and HepG2 cell experiments suggested that cucurbitacin IIb and saxifragic acid may contribute to the observed FXR-related responses. SU mitigated ANIT-induced cholestatic liver injury and promoted partial recovery of hepatic bile acid homeostasis. The protective effects of SU were associated with coordinated changes in hepatic and intestinal FXR-related signaling and with reduced inflammatory injury. These results provide experimental evidence supporting the traditional use of SU for hepatobiliary disorders and highlight cucurbitacin IIb and saxifragic acid as potential bioactive triterpenoids.

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2026-09-03 | Bile Acid Metabolism as a Unifying Readout for Diet, Microbiome Function, and Gastrointestinal Disease.

Bile acid biology has advanced through significant conceptual shifts. Once understood primarily as biological detergents, bile acids are now recognized as signaling molecules and, more recently, as a chemically diverse set of host- and microbe-derived metabolites. The 2020 discovery of microbially conjugated bile acids (MCBAs) and the expansion of the recognized catalog from approximately 20 species to more than 200 mark a new phase in this trajectory. Clinical translation has not kept up. Direct farnesoid X receptor (FXR) agonism failed twice in trials for non-alcoholic steatohepatitis, later received a serious liver injury safety communication from the U.S. Food and Drug Administration, and was voluntarily withdrawn from the United States market. The standard of care for bile acid diarrhea and post-cholecystectomy diarrhea still relies largely on chemical binding with drugs introduced in the 1960s and 1970s. This Perspective argues that the gap between bile acid biology and bile acid medicine persists in part because the field has not been organized around the bile acid pool as a shared measurable output. Researchers studying dietary modulators of the gut microbiome have worked in separate communities around fiber, fermented foods, polyphenols, protein, and dietary fat. Each of these inputs can shape bile acid metabolism, yet many intervention studies do not measure it. The most immediately implementable bile acid-targeted strategy is specified dietary intervention designed with measurable bile acid outcomes and evaluated with the precision of pharmacological therapy. Four recommendations follow: intervention studies should routinely measure bile acid outcomes; clinicians should test bile acid metabolism in conditions involving dysregulation; regulators should develop a framework for multicomponent dietary therapies; and researchers should build infrastructure for population-scale longitudinal monitoring.

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cell therapies

2026-04-20 | A 3D In Vitro Model of the Human Hepatobiliary Junction.

Bile flow is an essential feature of the liver, and disruption of this process contributes to a range of liver pathologies. Efficient bile transport requires coordinated organization between hepatocytes and cholangiocytes at the hepatobiliary junction, a structure that remains poorly captured in existing in vitro models of liver disease. Here, we present a 3D multicellular spheroid-based model of the human hepatobiliary junction. Building on advances in organoid and spheroid engineering, we co-aggregate human hepatocytes and intrahepatic cholangiocytes, supported by murine fibroblasts, into adult hepatobiliary organoids (aHBOs). aHBOs directionally transport bile from hepatocyte canaliculi to cholangiocyte-lined ductule-like structures, visualized through a high-throughput imaging assay. Hepatobiliary junction formation and bile flow dynamics are quantified over time using a fluorescent bile acid analog and AI-assisted image analysis. When subjected to hypoxia-reoxygenation, aHBOs exhibit disrupted bile transport and distinct cell-type-specific responses, enabling interrogation of hepatocyte and cholangiocyte vulnerability to transplant-associated biliary hypoxia. Our findings suggest a reversible reduction in hepatocyte canalicular function under hypoxia, followed by selective cholangiocyte death upon reoxygenation, potentially contributing to biliary dysfunction after ischemic injury. This human-derived, scalable platform provides a phenotypically relevant model for dissecting mechanisms of biliary dysfunction and discovering therapeutics for hypoxic liver injury and cholestatic diseases.

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2026-03-18 | Probiotic Lactobacillus rhamnosus mitigates PBC-like features in Mcpip1-deficient mice via modulation of gut-liver crosstalk.

Primary biliary cholangitis (PBC) is a chronic autoimmune liver disease characterized by progressive biliary destruction and cholestasis. Current therapies, including ursodeoxycholic acid (UDCA), exhibit limited efficacy in advanced disease. In this study, we investigate the therapeutic potential of microbial intervention using Lactobacillus rhamnosus (Lbr) in the Mcpip1fl/flAlbCre knockout mouse model of PBC, which we described previously. Knockout mice develop human PBC-like features such as bile acid dysregulation, autoantibodies, cholangiocyte hyperplasia and fibrosis. Six-week-old Mcpip1fl/fl (wild-type) and Mcpip1fl/flAlbCre (knockout) mice were treated with Lactobacillus rhamnosus supplementation, UDCA (15 mg/kg/day), UDCA + Lbr, and UDCA + OCA (obeticholic acid, 10 mg/kg/day) for six weeks. Treatment response was characterized by liver and gut pathology, serum biomarkers, transcriptomic profiles, and microbiome composition. Treatment of Mcpip1fl/flAlbCre animals with Lbr decreased serum bile acids and reduced pathological cholangiocyte dysplasia in the liver, decreased leukocyte infiltration and fibrosis. RNAseq of liver tissue revealed enrichment of humoral immune responses and T cell activation pathways in knockouts, all of which were significantly attenuated by Lbr monotherapy. Gut pathology marked by increased intraepithelial lymphocyte infiltration and mucosal hypertrophy, was also normalized upon Lbr administration. Finally, probiotic treatment modulated the microbiome by increasing the Firmicutes/Bacteroidetes ratio and enriching butyrate-producing Lachnospiraceae. Administration of UDCA and UDCA+OCA had less pronounced effects: only decreased serum bile acids was detected in both groups. Probiotic intervention with Lbr represents a feasible strategy to attenuate fibrotic progression in a mouse model of autoimmune cholestatic disease by modulation of the gut-microbiome-immune crosstalk.

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2025-11-03 | Microbiome and gut-liver interactions: From mechanisms to therapies.

The gut-liver axis represents a bidirectional and dynamic communication system between the gastrointestinal tract and liver, critically modulated by gut microbiota, bile acids, immune responses, and metabolic pathways. Disruption of this finely tuned axis contributes to the pathogenesis of several liver diseases, including alcohol-associated hepatitis, metabolic dysfunction-associated steatotic liver disease, cirrhosis, hepatic encephalopathy, and cholangiopathies like primary biliary cholangitis and primary sclerosing cholangitis. Dysbiosis, marked by reduced microbial diversity and dominance of pathogenic species, alters bile acid metabolism, increases gut permeability, and fuels hepatic inflammation. In cholangiopathies, the gut microbiome modulates immune dysregulation and fibrosis through complex microbial-host interactions. Emerging therapies targeting the microbiota, such as fecal microbiota transplantation, antibiotics (e.g., rifaximin, vancomycin), bile acid modulators, and probiotics, show promise in restoring microbial equilibrium, improving liver biochemistry, and reducing disease progression. Precision medicine strategies integrating genomics, metabolomics, and microbiomics offer a tailored approach for therapy and prognosis. This review highlights the central role of the gut-liver axis in liver diseases and the potential of microbiome-based interventions to shift management from symptomatic relief toward disease modification and personalized hepatology, underscoring a new frontier in liver disease therapeutics.

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2025-10-20 | Microbial changes resulting from VSG attenuate MASLD by modulating bile acid metabolism and the intestinal FXR-FGF19 axis.

Vertical sleeve gastrectomy (VSG) is a highly effective intervention for metabolic dysfunction-associated steatotic liver disease (MASLD) and is associated with significant alterations in the gut microbiota. However, the precise mechanisms underlying its metabolic benefits remain poorly understood. In this study, we revealed that VSG mitigates MASLD by reshaping gut microbiota-mediated bile acid metabolism. Through integrated 16S rRNA sequencing, targeted metabolomics, and functional validation experiments, we demonstrated that VSG markedly enhances bile salt hydrolase (BSH) activity within the gut microbiota, resulting in elevated levels of unconjugated bile acids. These unconjugated bile acids serve as potent agonists for the intestinal farnesoid X receptor (FXR), thereby activating the intestinal FXR-fibroblast growth factor 19 signaling pathway. This activation leads to significant improvements in metabolic health, including enhanced glucose regulation and attenuated hepatic lipid accumulation. Fecal microbiota transplantation (FMT) from VSG-treated rats replicated these metabolic improvements, whereas antibiotic treatment abolished these beneficial effects, highlighting the indispensable role of the gut microbiota in mediating the anti-MASLD effects of VSG. Importantly, inhibition of intestinal FXR signaling negated the metabolic benefits of FMT, further emphasizing the critical role of the gut microbiota-BSH-FXR axis. Our findings reveal a novel mechanism by which VSG alleviates MASLD through gut microbiota-dependent activation of intestinal FXR, offering new perspectives for microbiome-targeted therapeutic strategies in MASLD. Fecal transplantation from bariatric surgery patients and mice to germ-free mice has shown that the gut microbiota may contribute to metabolic benefits after bariatric surgery. However, the mechanisms by which the gut microbiota contributes to metabolic benefits after bariatric surgery require further investigation. To address this gap, we investigated the effects of the vertical sleeve gastrectomy (VSG) gut microbiota on metabolic dysfunction-associated steatotic liver disease (MASLD) in vivo and elucidated its underlying mechanisms. Our study demonstrated that VSG significantly improved the gut microbiota, especially by increasing bile salt hydrolase (BSH) activity, in MASLD rats. Increased BSH activity significantly increased the proportion of FXR-agonistic bile acids and further activated the intestinal FXR-FGF19 axis, thereby improving MASLD. These findings explored the key roles and mechanisms of the gut microbiota in the metabolic benefits of VSG, offering new microbiome-based treatment strategies.

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2025-09-02 | Fecal microbiota transplantation improves bile acid malabsorption in patients with inflammatory bowel disease: results of microbiota and metabolites from two cohort studies.

Bile acid malabsorption (BAM) or bile acid diarrhea (BAD) complicates more than 30% of Crohn's disease (CD), yet no non-invasive biomarker reliably identifies patients who will benefit from fecal microbiota transplantation (FMT). We investigated whether serum 7α-hydroxy-4-cholesten-3-one (C4), a hepatic bile-acid synthesis precursor, can predict BAM and FMT response in inflammatory bowel disease (IBD). We included 106 pairs of IBD patients treated with FMT from two longitudinal cohorts of prospective trials and 24 matched healthy individuals to identify a multi-omics analysis of microbiota-metabolism and evaluate real-world effectiveness of FMT. Fecal and serum samples before and after FMT along with medical information were collected and detected through 16S rRNA amplicon sequencing and untargeted liquid chromatography mass spectrometry. Mice models were used to preliminarily verify the exacerbation of colitis through administration of primary BAs and treated by FMT. Patients in BAM group tended to achieve sustained higher and stable clinical response (66.67% vs. 49.41%) and remission (52.38% vs. 40.00%) than non-BAM group at 3 months after FMT, along with a significantly decrease of C4 (P < 0.001), improvement of obvious abdominal pain and diarrhea, which was especially obvious in CD patients with ileal resection and ileal /ileocolonic type. Random forest classifiers predicted BAM in IBD patients with 18 or top 4 differential OTUs, showing an area under the curve of 0.92 and 0.83, respectively. Furthermore, results from primary bile acid-induced colitis mice models reinforced these findings. Serum C4 and a minimal gut microbiota may identify IBD patients with BAM who are most likely to achieve durable remission after FMT. These translatable biomarkers can guide precision use of microbiota-directed therapy. ClinicalTrials.gov: NCT01790061 and NCT01793831.

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antibodies

2025-10-20 | Innate immunity of bile and cholangiocytes in primary biliary cholangitis.

Primary Biliary Cholangitis (PBC) is a chronic autoimmune liver disease characterized by immune-mediated destruction of intrahepatic bile ducts. This review synthesizes current knowledge on the critical role of innate immunity, specifically involving cholangiocytes, bile components, and associated immune cells. Cholangiocytes function not only as passive targets but also as active immunomodulators through mechanisms including Toll-like receptor (TLR) signaling, antigen presentation, and immune cell recruitment. Dysregulated bile acid signaling via receptors like TGR5 disrupts immune homeostasis, while apoptosis of biliary epithelial cells releases antigens (e.g., PDC-E2), triggering aberrant innate and adaptive immune responses. Innate lymphoid cells (ILCs), natural killer (NK) cells, and macrophages exhibit altered frequencies and functions in PBC, driving chronic inflammation and fibrosis through cytokine cascades (e.g., IL-17, IFNγ) and interactions within the gut-liver axis. Furthermore, biliary microbiota dysbiosis exacerbates disease by promoting bacterial translocation, modifying bile acid metabolism, and activating innate immune pathways. Current clinical management with ursodeoxycholic acid (UDCA) and obeticholic acid (OCA) primarily addresses cholestasis. However, the immunomodulatory effects of these agents remain constrained. Targeted therapeutic strategies addressing innate immune pathways-exemplified by RIPK2 (Receptor Interacting Serine/Threonine Kinase 2) inhibition, IL-1 blockade(Canakinumab), and T cell immunoglobulin mucin domain-containing protein 3 (TIM-3) modulation-alongside cell-based interventions such as mesenchymal stem cell therapy, demonstrate considerable therapeutic potential. Advancing these modalities necessitates multidisciplinary integration to facilitate clinical translation. Additionally, Prognostic indices like the neutrophil-to-lymphocyte ratio (NLR) and monocyte-to-lymphocyte ratio (MLR) reflect systemic inflammation and correlate with disease progression. Achieving therapeutic precision requires deeper elucidation of the gut-biliary-immune axis, trained immunity mechanisms, and cholangiocyte senescence, paving the way for targeted interventions in PBC. Establishing a comprehensive treatment burden assessment system is imperative to facilitate the transition from investigational platforms to clinical care.

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2024-02-14 | Management and outcomes after liver transplantation for progressive familial intrahepatic cholestasis: A systematic review and meta-analysis.

Progressive familial intrahepatic cholestasis (PFIC) is a heterogeneous rare congenital cholestatic liver disease. Disease progression might necessitate liver transplantation (LT). The aim of this study was to describe the outcome of PFIC1-4 patients after LT. Electronic databases were searched to identify studies on PFIC and LT. Patients were categorized according to PFIC type, genotype, graft type, age at LT, time of follow-up, and complications and treatment during follow-up. Seventy-nine studies with 507 patients met inclusion criteria; most patients were classified as PFIC1-3. The median age at LT was 50 months. The overall 5-year patient survival was 98.5%. PFIC1 patients with diarrhea after LT were at significant risk of developing graft steatosis ( p < 0.0001). Meta-analysis showed an efficacy of 100% [95% CI: 73.9%-100%] for surgical biliary diversion to ameliorate steatosis and 94.9% [95% CI: 53.7%-100%] to improve diarrhea (n = 8). PFIC2 patients with bile salt export pump (BSEP)2 or BSEP3-genotype were at significant risk of developing antibody-induced BSEP deficiency (AIBD) ( p < 0.0001), which was reported in 16.2% of patients at a median of 36.5 months after LT. Meta-analysis showed an efficacy of 81.1% [95% CI: 47.5%-100%] for rituximab-based treatment regimens to improve AIBD (n = 18). HCC was detected in 3.6% of PFIC2 and 13.8% of PFIC4 patients at LT. Fifty percent of PFIC1 patients develop diarrhea and steatosis after LT. Biliary diversion can protect the graft from injury. PFIC2 patients with BSEP2 and BSEP3 genotypes are at significant risk of developing AIBD, and rituximab-based treatment regimens effectively improve AIBD. PFIC3 patients have no PFIC-specific complications following LT.

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2021-04-19 | Imbalance of the CD226/TIGIT Immune Checkpoint Is Involved in the Pathogenesis of Primary Biliary Cholangitis.

The relationship between the cluster of differentiation 226 (CD226)/T cell Ig and ITIM domain (TIGIT) immune checkpoint and primary biliary cholangitis (PBC) pathogenesis is unknown. Herein, PBC patients (n = 42) showed significantly higher proportions of peripheral CD8+ T and CD4+ T cells expressing either CD226 or TIGIT than disease (n = 25) and healthy (n = 30) controls. The percentage of CD8+TIGIT+ T cell was negatively associated with total bilirubin, direct bilirubin, total bile acid, γ-glutamyl transpeptidase, and alkaline phosphatase, but positively correlated with platelet count; alkaline phosphatase was positively associated with the frequency of CD8+CD226+ T cell; and the CD226/TIGIT ratio of CD8+ T cell was positively associated with total bilirubin, direct bilirubin, total bile acid, γ-glutamyl transpeptidase, alkaline phosphatase, and aspartate aminotransferase to platelet ratio, but negatively correlated with albumin and platelet count. The effector function of CD8+CD226+ T cells was more robust than the CD8+CD226- counterparts. CD226 blockade reduced CD107a+, IFN-γ+, and TNF-α+ proportions among CD8+CD226+ T cells, inhibiting CD8+ T cell proliferation. In conclusion, CD226/TIGIT immune checkpoint imbalance is involved in the pathogenesis of PBC. The CD226/TIGIT ratio of CD8+ T cell is a potential biomarker for evaluating the disease status and the prognosis of PBC patients. Moreover, CD8+CD226+ T cells represent a possible therapeutic target for PBC, and blocking CD226 could inhibit the activity of this cell subset in vitro.

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2020-07-20 | Therapeutic trials of biologics in primary biliary cholangitis: An open label study of abatacept and review of the literature.

Primary biliary cholangitis (PBC) is a classic autoimmune disease in which humoral, cytotoxic, and innate immune responses have been implicated with the specific targeting of a mitochondrial antigen. The mainstay of treatment remains the bile acid ursodeoxycholic acid (UDCA). Corticosteroids may have some benefits, but to date, clinical trials of biologics targeting B cells and IL-12/23 have not shown any efficacy. Because activated T cells target the intrahepatic bile ducts in PBC and pre-clinical models suggested that blocking CD80/CD86 with CTLA-4 Ig might have therapeutic benefit in PBC, we performed an open-label trial to determine if CTLA-4 Ig (abatacept) is safe and potentially efficacious in PBC patients with an incomplete response to UDCA. PBC patients with an alkaline phosphatase (ALP) > 1.67 × the upper limit of normal after 6 months on UDCA treatment or who were intolerant of UDCA received abatacept 125 mg s.q. weekly for 24 weeks. The co-primary endpoint was ALP normalization or a >40% reduction from baseline. Among 16 subjects enrolled and who received at least 1 dose of abatacept, 1 (6.3%) met the co-primary endpoint. Absolute and percent changes in ALP [median (95% CI)] were +2.8 U/L (-90.9-96.6) and -0.28% (-21.1-15.5), respectively. No significant changes were observed in ALP, ALT, total bilirubin, albumin, immunoglobulins, or liver stiffness. Abatacept treatment decreased several non-terminally differentiated CD4+ but not CD8+ T cell populations, including decreases in CD4+ CCR5+ (p = 0.02) and CD4+ PD1+ (p = 0.03) lymphocytes. In contrast there were increases in CD4+ CCR7+ lymphocytes (p = 0.034). Treatment emergent adverse events occurred in 4 subjects. Abatacept was well tolerated in this population of PBC patients but like other biologics in PBC was ineffective in achieving biochemical responses associated with improved clinical outcomes.

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2019-09-20 | Lysyl oxidase-like protein 2 (LOXL2) modulates barrier function in cholangiocytes in cholestasis.

The lysyl oxidase-like protein 2 (LOXL2) promotes stabilization of the extracellular matrix, chemotaxis, cell growth and cell mobility. We aimed to (i) identify stimuli of LOXL2 in cholangiopathies, (ii) characterize the effects of LOXL2 on biliary epithelial cells' (BECs) barrier function, (iii) compare LOXL2 expression in primary sclerosing cholangitis (PSC), primary biliary cholangitis, and disease controls, and (iv) to determine LOXL2 expression and its cellular sources in four mouse models of cholangiopathies. Cultured murine BECs were challenged with well-known triggers of cellular senescence, hypoxia, phospholipid-deficient Abcb4-/- mouse bile and chenodeoxycholic acid and investigated for LOXL2, SNAIL1 and E-cadherin expression and transepithelial electrical resistance with and without LOX-inhibition. In vivo, LOXL2 expression was studied in PSC livers, and controls and mouse models. We compared LOXL2 serum levels in patients with PSC, secondary SC, primary biliary cholangitis, and controls. Cellular senescence, hypoxia, Abcb4-/- bile and chenodeoxycholic acid induced LOXL2 and SNAIL1 expression, repressed E-cadherin expression, and significantly reduced transepithelial electrical resistance in BECs. Notably, all of the pathological changes could be recovered via pharmacological LOX-inhibition. Mouse models showed induced LOXL2 expression in the portal region and in association with ductular reaction. LOXL2 serum levels were significantly elevated in patients with cholangiopathies. In PSC, LOXL2 expression was located to characteristic periductal onion skin-type fibrosis, ductular reaction, Kupffer cells, and fibrotic septa. Importantly, in PSC, LOXL2 overexpression was paralleled by E-cadherin loss in BECs from medium-sized bile ducts. Reactive BECs produce LOXL2, resulting in increased tight junction permeability, which can be ameliorated by pharmacological LOX-inhibition in vitro. Reactive BECs, portal myofibroblasts, and Kupffer cells are the main sources of LOXL2 in cholangiopathies. In this study, we investigate the role of lysyl oxidase-like protein 2 (LOXL2), an enzyme pivotal in the development of organ fibrosis, in the pathogenesis of cholangiopathies (diseases of bile ducts), such as primary sclerosing cholangitis. We found LOXL2 to be expressed in association with bile duct epithelial injury and uncovered mechanisms for its upregulation and the subsequent effects in vitro and in vivo. Our findings support testing of anti-LOXL2 treatment strategies for patients with primary sclerosing cholangitis.

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2026-09-22 | Myelin-Inspired 3D PEGylation of Framework Nucleic Acids Enables Simultaneous Long Circulation and Rapid Renal Clearance.

The rational design of nanomedicines is fundamentally constrained by a physicochemical paradox at the bio-nano interface: strategies to prolong circulation inevitably suppress clearance, often resulting in long-term accumulation and systemic toxicity. To address this, we propose spatial conformation engineering as an additional design dimension to decouple these opposing processes. Inspired by the compact, multilamellar architecture of the myelin sheath, we program poly(ethylene glycol) (PEG) chains into a three-dimensional matrix on a DNA tetrahedral framework. Unlike conventional linear or planar PEGylation, this 3D conformation yields a dense yet compact interfacial topology, enabling effective dynamic steric shielding (reducing protein adsorption by >60%) while maintaining a small hydrodynamic diameter (7.2 nm). This design strategy achieves a two-fold extension of blood circulation half-life while promoting a dominant renal elimination pathway (>90% clearance within 24 h). As a proof-of-concept, we functionalize the platform with a bile acid aptamer to construct a "patrolling" detoxification system capable of continuous sequestration and removal of blood-borne toxins in a murine hypercholanemia model. Our work establishes spatial conformation as a programmable parameter for regulating nanoparticle biodistribution and clearance, thereby shifting the design paradigm from compositional adjustment to architectural control. This architectural control opens avenues for targeted delivery, imaging, and immunotherapy.

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2026-08-11 | Liver-directed gene therapy results in amelioration of progressive familial intrahepatic cholestasis type 2 in mice.

Progressive familial intrahepatic cholestasis type 2 (PFIC2) is a rare disease affecting the ABCB11 gene, encoding the bile salt export pump (BSEP). BSEP dysfunction impairs bile acid (BA) secretion, causing hepatic damage and leading to pruritus, cholestasis, hepatomegaly, and often fibrosis and end-stage hepatic disease. Treatments include ileal BA transporter inhibitors, surgical bile diversion, or ultimately, liver transplantation. Our aim was to develop a PFIC2 gene therapy approach based on the restoration of BSEP hepatocyte expression. We designed several expression cassettes containing the human ABCB11 gene downstream of either a liver-specific constitutive promoter or a BA-inducible promoter. After in vitro screening, the AAV vectors with the best expression cassette for each promoter were tested in a PFIC2 mouse model. The AAV vector containing the constitutive promoter, named VTX-802, showed higher BSEP expression, resulting in better restoration of BA secretion 3 and 7 weeks post-treatment. We then performed a dose-range-finding study of VTX-802 in 5-week-old female PFIC2 mice in which the therapeutic efficacy was monitored until 5 months of age. Treated mice showed a sustained dose-dependent improvement in serum transaminase levels. These mice also exhibited significant, but partial, correction of hepatomegaly, and increased BA levels in bile and small intestine, indicating partial restoration of normal BA secretion. VTX-802 restores hepatic BSEP expression and partially corrects disease phenotype in PFIC2 mice. To our knowledge, VTX-802 is the first gene therapy approach that could potentially benefit PFIC2 patients.

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2025-06-25 | FXR overexpression alleviates cholestasis via NLRC4 inflammasome suppression and bile acid homeostasis regulation.

Cholestasis, a major driver of liver disease progression, is characterized by toxic bile acid accumulation due to impaired bile flow, potentially leading to hepatic fibrosis, cirrhosis, and hepatocellular carcinoma. To date, the pathogenesis of cholestasis has remained incompletely understood. In the present study, we investigated the role of farnesoid X receptor (FXR) in modulating the NLR family CARD domain-containing protein 4 (NLRC4) inflammasome activity using in vitro (AML-12 hepatocytes) and in vivo (C57BL/6 mice) models of lithocholic acid (LCA)-induced cholestasis. LCA suppressed FXR expression, downregulated bile acid transporters (Bsep, Mrp2, Ntcp), and elevated serum biomarkers of liver injury. Through restored hepatic function by FXR lentiviral vectors, FXR overexpression reduced bile acid accumulation and mitigated inflammation and oxidative stress. In addition, FXR overexpression suppressed bile acid synthesis enzymes (Cyp7a1, Cyp8b1) by upregulating Shp and Fgf15, while enhancing detoxification through Ugt1a1 and Sult2a1. Interestingly, molecular docking analysis and Co-IP experiments demonstrated a direct interaction between FXR and NLRC4. Furthermore, FXR overexpression significantly inhibited NLRC4 inflammasome activation and decreased the expression of NLRC4, caspase-1, IL-1β, and IL-18, thereby attenuating inflammation and oxidative stress. Conversely, FXR knockdown reversed these effects. In addition, to delineate the contribution of NLRC4 inflammasome activation to IL-18 and IL-1β elevation, NLRC4-targeting siRNA-mediated knockdown and NLRC4-encoding plasmid-driven overexpression strategies were systematically employed. Furthermore, DCFH-DA staining was adopted to visualize reactive oxygen species (ROS). In conclusion, for the first time, we found that FXR overexpression alleviates LCA-induced cholestasis by regulating bile acid metabolism and inhibiting NLRC4 inflammasome activation, providing a novel therapeutic strategy for drug development targeting the FXR-NLRC4 axis.

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2025-05-19 | Tetrahedral framework nucleic acids ameliorate cholestatic liver disease by activating Wnt/β-catenin signaling and promoting ERK1/2 phosphorylation.

Cholestatic liver disease (CLD) is characterized by disruptions in bile formation, secretion and excretion, leading to progressive liver injury, inflammation and fibrosis. Effective treatments to halt or reverse the progression of CLD remain limited. The Wnt/β-catenin signaling pathway has been implicated in the regulation of bile acid homeostasis and liver regeneration, playing a complex role in CLD pathophysiology. Tetrahedral framework nucleic acids (TFNAs), a class of anti-inflammatory and antioxidant DNA nanomaterials, have shown potential in promoting mammalian cell proliferation through activation of cell cycle and proliferation-related signaling pathways. However, their therapeutic potential in CLD has not been fully explored. In this study, we investigated the effects of TFNAs in an α-naphthyl isothiocyanate (ANIT)-induced mouse model of CLD. TFNAs demonstrated the ability to enter hepatocytes, where they activated the Wnt/β-catenin signaling pathway and enhanced ERK1/2 phosphorylation. These molecular changes resulted in significant improvements in liver injury markers, bile acid metabolism and liver regeneration. Complementary in vitro experiments revealed that TFNAs reduced hepatocyte apoptosis and oxidative stress, while promoting cell viability and proliferation. Histological analysis confirmed that TFNAs treatment mitigated liver necrosis, reduced ductular reactions and decreased neutrophil infiltration, highlighting their anti-inflammatory and tissue-protective effects. These findings provide compelling evidence that TFNAs can ameliorate CLD by modulating key signaling pathways involved in hepatocyte survival, regeneration and bile acid homeostasis. Collectively, our findings highlight the therapeutic potential of TFNAs as a novel treatment for CLD and paves the way for further exploration of nanomaterials in liver disease therapy.

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2025-02-15 | Liver specific transgenic expression of CYP7B1 attenuates early western diet-induced MASLD progression.

Effect of liver specific oxysterol 7α-hydroxylase (CYP7B1) overexpression on the Western diet (WD)-induced metabolic dysfunction-associated steatotic liver disease (MASLD) progression was studied in mice. Among various hepatic genes impacted during MASLD development, CYP7B1 is consistently suppressed in multiple MASLD mouse models and in human MASLD cohorts. CYP7B1 enzyme suppression leads to accumulations of bioactive oxysterols such as (25R)26-hydroxycholesterol (26HC) and 25-hydroxycholesterol (25HC). We challenged liver specific CYP7B1 transgenic (CYP7B1hep.tg) overexpressing mice with ad libitum WD feeding. Unlike their WT counterparts, WD-fed CYP7B1hep.tg mice developed no significant hepatotoxicity as evidenced by liver histology, lipid quantifications, and serum biomarker analyses. Hepatic 26HC and 25HC levels were maintained at the basal levels. The comparative gene expression/lipidomic analyses between WT and CYP7B1hep.tg mice revealed that chronically accumulated 26HC initiates LXR/PPAR-mediated hepatic fatty acid uptake and lipogenesis which surpasses fatty acid metabolism and export; compromising metabolic functions. In addition, major pathways related to oxidative stress, inflammation, and immune system including retinol metabolism, arachidonic acid metabolism, and linoleic acid metabolism were significantly impacted in the WD-fed WT mice. All pathways were unaltered in CYP7B1hep.tg mice liver. Furthermore, the nucleus of WT mouse liver but not of CYP7B1hep.tg mouse liver accumulated 26HC and 25HC in response to WD. These data strongly suggested that these two oxysterols are specifically important in nuclear transcriptional regulation for the described cytotoxic pathways. In conclusion, this study represents a "proof-of-concept" that maintaining normal mitochondrial cholesterol metabolism with hepatic CYP7B1 expression prevents oxysterol-driven liver toxicity; thus attenuating MASLD progression.

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