RESUMEN
Liver fibrosis is characterized by chronic inflammatory responses and progressive fibrous scar formation. Macrophages play a central role in the pathogenesis of hepatic fibrosis by reconstructing the immune microenvironment. Picroside II (PIC II), extracted from Picrorhizae Rhizoma, has demonstrated therapeutic potential for various liver damage. However, the mechanisms by which macrophage polarization initiates immune cascades and contributes to the development of liver fibrosis, and whether this process can be influenced by PIC II, remain unclear. In the current study, RNA sequencing and multiple molecular approaches were utilized to explore the underlying mechanisms of PIC II against liver fibrosis in multidrug-resistance protein 2 knockout (Mdr2-/-) mice. Our findings indicate that PIC II activates M1-polarized macrophages to recruit natural killer cells (NK cells), potentially via the CXCL16-CXCR6 axis. Additionally, PIC II promotes the apoptosis of activated hepatic stellate cells (aHSCs) and enhances the cytotoxic effects of NK cells, while also reducing the formation of neutrophil extracellular traps (NETs). Notably, the anti-hepatic fibrosis effects associated with PIC II were largely reversed by macrophage depletion in Mdr2-/- mice. Collectively, our research suggests that PIC II is a potential candidate for halting the progression of liver fibrosis.
Asunto(s)
Apoptosis , Cinamatos , Células Estrelladas Hepáticas , Glucósidos Iridoides , Cirrosis Hepática , Macrófagos , Animales , Masculino , Ratones , Apoptosis/efectos de los fármacos , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Miembro 4 de la Subfamilia B de Casete de Unión a ATP/genética , Cinamatos/farmacología , Células Estrelladas Hepáticas/efectos de los fármacos , Glucósidos Iridoides/farmacología , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Cirrosis Hepática/tratamiento farmacológico , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Previous research highlighted the involvement of the cannabinoid CB1 receptor in regulating the physiology of hepatocytes and hepatic stellate cells. The inhibition of the CB1 receptor via peripherally restricted CB1 receptor inverse agonist JD5037 has shown promise in inhibiting liver fibrosis in mice treated with CCl4. However, its efficacy in phospholipid transporter-deficiency-induced liver fibrosis remains uncertain. In this study, we investigated the effectiveness of JD5037 in Mdr2-/- mice. Mdr2 (Abcb4) is a mouse ortholog of the human MDR3 (ABCB4) gene encoding for the canalicular phospholipid transporter. Genetic disruption of the Mdr2 gene in mice causes a complete absence of phosphatidylcholine from bile, leading to liver injury and fibrosis. Mdr2-/- mice develop spontaneous fibrosis during growth. JD5037 was orally administered to the mice for four weeks starting at eight weeks of age. Liver fibrosis, bile acid levels, inflammation, and injury were assessed. Additionally, JD5037 was administered to three-week-old mice to evaluate its preventive effects on fibrosis development. Our findings corroborate previous observations regarding global CB1 receptor inverse agonists. Four weeks of JD5037 treatment in eight-week-old Mdr2-/- mice with established fibrosis led to reduced body weight gains. However, contrary to expectations, JD5037 significantly exacerbated liver injury, evidenced by elevated serum ALT and ALP levels and exacerbated liver histology. Notably, JD5037-treated Mdr2-/- mice exhibited significantly heightened serum bile acid levels. Furthermore, JD5037 treatment intensified liver fibrosis, increased fibrogenic gene expression, stimulated ductular reaction, and upregulated hepatic proinflammatory cytokines. Importantly, JD5037 failed to prevent liver fibrosis formation in three-week-old Mdr2-/- mice. In summary, our study reveals the exacerbating effect of JD5037 on liver fibrosis in genetically MDR2-deficient mice. These findings underscore the need for caution in the use of peripherally restricted CB1R inverse agonists for liver fibrosis treatment, particularly in cases of dysfunctional hepatic phospholipid transporter.
Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP , Miembro 4 de la Subfamilia B de Casete de Unión a ATP , Cirrosis Hepática , Receptor Cannabinoide CB1 , Animales , Ratones , Receptor Cannabinoide CB1/genética , Receptor Cannabinoide CB1/metabolismo , Receptor Cannabinoide CB1/agonistas , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP/deficiencia , Cirrosis Hepática/patología , Cirrosis Hepática/metabolismo , Cirrosis Hepática/inducido químicamente , Cirrosis Hepática/tratamiento farmacológico , Cirrosis Hepática/genética , Hígado/efectos de los fármacos , Hígado/patología , Hígado/metabolismo , Masculino , Ratones Noqueados , Ácidos y Sales Biliares/metabolismo , Agonismo Inverso de Drogas , Ratones Endogámicos C57BLRESUMEN
Primary sclerosing cholangitis (PSC) is an (auto)immune-mediated cholestatic liver disease with a yet unclear etiology. Increasing evidence points to an involvement of neutrophils in chronic liver inflammation and cirrhosis but also liver repair. Here, we investigate the role of the neutrophil extracellular trap (NET) component myeloperoxidase (MPO) and the therapeutic potential of DNase I and of neutrophil elastase (NE) inhibitor GW311616A on disease outcome in the multidrug resistance 2 knockout (Mdr2-/-) mouse, a PSC animal model. Initially, we observed the recruitment of MPO expressing cells and the formation of NETs in liver biopsies of PSC patients and in Mdr2-/- livers. Furthermore, sera of Mdr2-/- mice contained perinuclear anti-neutrophil cytoplasmic antibody (p-ANCA)-like reactivity similar to PSC patient sera. Also, hepatic NE activity was significantly higher in Mdr2-/- mice than in wild type littermates. Flow cytometry analyses revealed that during disease development a highly active neutrophil subpopulation established specifically in the liver of Mdr2-/- mice. However, absence of their MPO activity, as in MPO-deficient Mdr2-/- mice, showed no effect on hepatobiliary disease severity. In contrast, clearance of extracellular DNA by DNase I reduced the frequency of liver-resident neutrophils, plasmacytoid dendritic cells (pDCs) and CD103+ conventional DCs and decreased cholangiocyte injury. Combination of DNase I with a pDC-depleting antibody was additionally hepatocyte-protective. Most importantly, GW311616A, an orally bioavailable inhibitor of human NE, attenuated hepatobiliary injury in a TNFα-dependent manner and damped hyperproliferation of biliary epithelial cells. Further, hepatic immigration and activity of CD11b+ DCs as well as the secretion of IFNγ by hepatic CD4 and CD8 T cells were reduced. Our findings delineate neutrophils as important participants in the immune cell crosstalk that drives cholestatic liver disease and identify NET components as potential therapeutic targets.
Asunto(s)
Miembro 4 de la Subfamilia B de Casete de Unión a ATP , Colangitis Esclerosante , Modelos Animales de Enfermedad , Trampas Extracelulares , Ratones Noqueados , Neutrófilos , Animales , Trampas Extracelulares/inmunología , Trampas Extracelulares/metabolismo , Ratones , Humanos , Colangitis Esclerosante/inmunología , Neutrófilos/inmunología , Neutrófilos/metabolismo , Colestasis/inmunología , Colestasis/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/deficiencia , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Hígado/patología , Hígado/inmunología , Hígado/metabolismo , Peroxidasa/metabolismo , Peroxidasa/inmunología , Desoxirribonucleasa I/metabolismo , Elastasa de Leucocito/metabolismo , Elastasa de Leucocito/antagonistas & inhibidores , Masculino , FemeninoRESUMEN
OBJECTIVES: Biallelic variants in the adenosine triphosphate binding cassette subfamily B member 4 (ABCB4) gene which encodes the multidrug resistance 3 protein (MDR3) leads to progressive familiar intrahepatic cholestasis type 3. However, monoallelic variants are increasingly recognized as contributing to liver disease in adults. Our aim was to describe the clinical characteristics of MDR3 heterozygous variants in a large cohort of infants and children with cholestatic liver disease. METHODS: The clinical and genotypic data on pediatric patients seen at King's College Hospital, London, between 2004 and 2022 and found to harbour heterozygous variants in ABCB4 were reviewed. RESULTS: Ninety-two patients amongst 1568 tested were identified with a monoallelic variant (5.9%). The most common presenting problem was conjugated hyperbilirubinemia (n = 46; 50%) followed by cholelithiasis (n = 12; 13%) and cholestatic hepatitis (n = 10; 11%). The median values of liver biochemistry at presentation were: GGT 105 IU/L and total bilirubin 86 µmol/L. Thirty-two genetic variants were identified including 22 missense (69%), 4 deletions (13%), 5 splice site (16%) and 1 termination (3%). At a median follow up of 1 year there was resolution of liver disease. CONCLUSIONS: Rare variants in ABCB4 were found amongst infants and children with cholestatic liver disease. The presenting problems were variable and abnormalities tended to normalize over time. Those with severe mutations could develop liver disease later in life when exposed to further insult and should be counseled appropriately.
Asunto(s)
Miembro 4 de la Subfamilia B de Casete de Unión a ATP , Colestasis Intrahepática , Colestasis , Adulto , Niño , Humanos , Lactante , Colestasis/genética , Colestasis Intrahepática/genética , Colestasis Intrahepática/metabolismo , Heterocigoto , Mutación , Miembro 4 de la Subfamilia B de Casete de Unión a ATP/genéticaRESUMEN
OBJECTIVE: Multidrug resistance protein 2 (MRP2) is a bottleneck in bilirubin excretion. Its loss is sufficient to induce hyperbilirubinaemia, a prevailing characteristic of acute liver failure (ALF) that is closely associated with clinical outcome. This study scrutinises the transcriptional regulation of MRP2 under different pathophysiological conditions. DESIGN: Hepatic MRP2, farnesoid X receptor (FXR) and Forkhead box A2 (FOXA2) expression and clinicopathologic associations were examined by immunohistochemistry in 14 patients with cirrhosis and 22 patients with ALF. MRP2 regulatory mechanisms were investigated in primary hepatocytes, Fxr -/- mice and lipopolysaccharide (LPS)-treated mice. RESULTS: Physiologically, homeostatic MRP2 transcription is mediated by the nuclear receptor FXR/retinoid X receptor complex. Fxr-/- mice lack apical MRP2 expression and rapidly progress into hyperbilirubinaemia. In patients with ALF, hepatic FXR expression is undetectable, however, patients without infection maintain apical MRP2 expression and do not suffer from hyperbilirubinaemia. These patients express FOXA2 in hepatocytes. FOXA2 upregulates MRP2 transcription through binding to its promoter. Physiologically, nuclear FOXA2 translocation is inhibited by insulin. In ALF, high levels of glucagon and tumour necrosis factor α induce FOXA2 expression and nuclear translocation in hepatocytes. Impressively, ALF patients with sepsis express low levels of FOXA2, lose MRP2 expression and develop severe hyperbilirubinaemia. In this case, LPS inhibits FXR expression, induces FOXA2 nuclear exclusion and thus abrogates the compensatory MRP2 upregulation. In both Fxr -/- and LPS-treated mice, ectopic FOXA2 expression restored apical MRP2 expression and normalised serum bilirubin levels. CONCLUSION: FOXA2 replaces FXR to maintain MRP2 expression in ALF without sepsis. Ectopic FOXA2 expression to maintain MRP2 represents a potential strategy to prevent hyperbilirubinaemia in septic ALF.
Asunto(s)
Factor Nuclear 3-beta del Hepatocito , Fallo Hepático Agudo , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Animales , Ratones , Bilirrubina , Factor Nuclear 3-beta del Hepatocito/metabolismo , Hepatocitos/metabolismo , Hiperbilirrubinemia/metabolismo , Hiperbilirrubinemia/patología , Lipopolisacáridos/metabolismo , Hígado/metabolismo , Fallo Hepático Agudo/metabolismo , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos/metabolismo , Miembro 4 de la Subfamilia B de Casete de Unión a ATPRESUMEN
Bile salt export pump (Bsep) (Abcb11)-/- mice are protected from acquired cholestatic injury due to metabolic preconditioning with a hydrophilic bile acid (BA) pool with formation of tetrahydroxylated bile acids (THBAs). We aimed to explore whether loss of Bsep and subsequent elevation of THBA levels may have immunomodulatory effects, thus improving liver injury in the multidrug resistance protein 2 (Mdr2) (Abcb4)-/- mouse. Cholestatic liver injury in Mdr2-/- Bsep-/- double knockout (DKO), Mdr2-/- , Bsep-/- , and wild-type mice was studied for comparison. Mdr2-/- mice were treated with a THBA (3α,6α,7α,12α-Tetrahydroxycholanoic acid). RNA/protein expression of inflammatory/fibrotic markers were investigated. Serum BA-profiling was assessed by ultra-performance liquid chromatography tandem mass spectrometry. Hepatic immune cell profile was quantified by flow cytometric analysis (FACS). In vitro, the THBA effect on chenodeoxycholic acid (CDCA)-induced inflammatory signaling in hepatocyte and cholangiocytes as well as lipopolysaccharide (LPS)/interferon-γ (IFN-γ)-induced macrophage activation was analyzed. In contrast to Mdr2-/- , DKO mice showed no features of sclerosing cholangitis. Sixty-seven percent of serum BAs in DKO mice were polyhydroxylated (mostly THBAs), whereas Mdr2-/- mice did not have these BAs. Compared with Mdr2-/- , DKO animals were protected from hepatic inflammation/fibrosis. THBA feeding in Mdr2-/- mice improved liver injury. FACS analysis in DKO and Mdr2-/- THBA-fed mice showed changes of the hepatic immune cell profile towards an anti-inflammatory pattern. Early growth response 1 (EGR1) protein expression was reduced in DKO and in Mdr2-/- THBA-fed mice compared with Mdr2-/- control mice. In vitro, THBA-reduced CDCA induced EGR1 protein and mRNA expression of inflammatory markers in hepatocytes and cholangiocytes. LPS/IFN-γ-induced macrophage activation was ameliorated by THBA. THBAs repress EGR1-related key pro-inflammatory pathways. Conclusion: THBA and their downstream targets may represent a potential treatment strategy for cholestatic liver diseases.
Asunto(s)
Ácidos y Sales Biliares , Colangitis Esclerosante , Colestasis , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Animales , Ácidos y Sales Biliares/química , Ácidos y Sales Biliares/farmacología , Conductos Biliares/patología , Colangitis Esclerosante/genética , Colestasis/complicaciones , Colestasis/genética , Modelos Animales de Enfermedad , Inmunomodulación/efectos de los fármacos , Interferón gamma , Lipopolisacáridos/farmacología , Cirrosis Hepática/genética , Ratones , Ratones Noqueados , Miembro 4 de la Subfamilia B de Casete de Unión a ATPRESUMEN
Clinical studies have revealed that the ABCB4 gene encodes the phospholipid transporter on the canalicular membrane of hepatocytes, and its mutations and variants are the genetic basis of low phospholipid-associated cholelithiasis (LPAC), a rare type of gallstone disease caused by a single-gene mutation or variation. The main features of LPAC include a reduction or deficiency of phospholipids in bile, symptomatic cholelithiasis at <40 years of age, intrahepatic sludge and microlithiasis, mild chronic cholestasis, a high cholesterol/phospholipid ratio in bile, and recurrence of biliary symptoms after cholecystectomy. Needle-like cholesterol crystals, putatively "anhydrous" cholesterol crystallization at low phospholipid concentrations in model and native bile, are characterized in ABCB4 knockout mice, a unique animal model for LPAC. Gallbladder bile with only trace amounts of phospholipids in these mice is supersaturated with cholesterol, with lipid composition plotting in the left two-phase zone of the ternary phase diagram, consistent with "anhydrous" cholesterol crystallization. In this review, we summarize the molecular biology and physiological functions of ABCB4 and comprehensively discuss the latest advances in the genetic analysis of ABCB4 mutations and variations and their roles in the pathogenesis and pathophysiology of LPAC in humans, based on the results from clinical studies and mouse experiments. To date, approximately 158 distinct LPAC-causing ABCB4 mutations and variants in humans have been reported in the literature, indicating that it is a monogenic risk factor for LPAC. The elucidation of the ABCB4 function in the liver, the identification of ABCB4 mutations and variants in LPAC patients, and the exploration of gene therapy for ABCB4 deficiency in animal models can help us to better understand the cellular, molecular, and genetic mechanisms underlying the onset of the disease, and will pave the way for early diagnosis and prevention of susceptible subjects and effective intervention for LPAC in patients.
Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP , Colelitiasis , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Animales , Colelitiasis/diagnóstico , Colelitiasis/genética , Colesterol , Pruebas Genéticas , Humanos , Ratones , Mutación , Fosfolípidos , Miembro 4 de la Subfamilia B de Casete de Unión a ATPRESUMEN
Fibroblast growth factor 1 (FGF1) belongs to a family of growth factors involved in cellular growth and division. MicroRNA 16 (miR-16) is a regulator of gene expression, which is dysregulated during liver injury and insult. However, the role of FGF1 in the progression of biliary proliferation, senescence, fibrosis, inflammation, angiogenesis, and its potential interaction with miR-16, are unknown. In vivo studies were performed in male bile duct-ligated (BDL, 12-week-old) mice, multidrug resistance 2 knockout (Mdr2-/-) mice (10-week-old), and their corresponding controls, treated with recombinant human FGF1 (rhFGF1), fibroblast growth factor receptor (FGFR) antagonist (AZD4547), or anti-FGF1 monoclonal antibody (mAb). In vitro, the human cholangiocyte cell line (H69) and human hepatic stellate cells (HSCs) were used to determine the expression of proliferation, fibrosis, angiogenesis, and inflammatory genes following rhFGF1 treatment. PSC patient and control livers were used to evaluate FGF1 and miR-16 expression. Intrahepatic bile duct mass (IBDM), along with hepatic fibrosis and inflammation, increased in BDL mice treated with rhFGF1, with a corresponding decrease in miR-16, while treatment with AZD4547 or anti-FGF1 mAb decreased hepatic fibrosis, IBDM, and inflammation in BDL and Mdr2-/- mice. In vitro, H69 and HSCs treated with rhFGF1 had increased expression of proliferation, fibrosis, and inflammatory markers. PSC samples also showed increased FGF1 and FGFRs with corresponding decreases in miR-16 compared with healthy controls. Conclusion: Our study demonstrates that suppression of FGF1 and miR-16 signaling decreases the presence of hepatic fibrosis, biliary proliferation, inflammation, senescence, and angiogenesis. Targeting the FGF1 and miR-16 axis may provide therapeutic options in treating cholangiopathies such as PSC.
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Colangitis Esclerosante , MicroARNs , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Animales , Colangitis Esclerosante/tratamiento farmacológico , Modelos Animales de Enfermedad , Factor 1 de Crecimiento de Fibroblastos/genética , Fibrosis , Humanos , Inflamación , Cirrosis Hepática/tratamiento farmacológico , Masculino , Ratones , Ratones Noqueados , MicroARNs/genética , Miembro 4 de la Subfamilia B de Casete de Unión a ATPRESUMEN
BACKGROUND AND OBJECTIVE: Acyclovir is effective in treating herpes simplex virus infections of the central nervous system. The purpose of this study was to investigate the interactions between acyclovir and the efflux pumps P-glycoprotein (P-gp), breast cancer resistance protein (Bcrp), multidrug resistance protein 2 (Mrp2), and organic anion transporter 3 (Oat3) at the blood-brain barrier (BBB). METHODS: Acyclovir concentrations in the blood and brain were evaluated by microdialysis and high-performance liquid chromatography. Acyclovir pharmacokinetic parameters, including the area under the unbound blood concentration-time curve (AUCu,blood), the area under the unbound brain concentration-time curve (AUCu,brain), and the ratio of AUCu,brain to AUCu,blood (Kp.uu.brain), were evaluated in the presence and absence of elacridar (P-gp/Bcrp inhibitor, 7.5 mg/kg), tariquidar (P-gp/Bcrp inhibitor, 7.5 mg/kg), MK571 (Mrp2 inhibitor, 7.5 mg/kg), cyclosporine (P-gp/Bcrp/Mrp2 inhibitor, 25 mg/kg), and probenecid (Oat3 inhibitor, 50 mg/kg). RESULTS: The average AUCu,blood, AUCu,brain, and Kp.uu.brain in rats who received acyclovir (25 mg/kg, intravenous) alone were 1377.7 min · µg/ml, 435.4 min · µg/ml, and 31.6%, respectively. Probenecid drastically increased the AUCu,blood of acyclovir 1.73-fold, whereas coadministration with elacridar, tariquidar, MK571, and cyclosporine did not alter the blood pharmacokinetic parameters of acyclovir. Elacridar, tariquidar, MK571, cyclosporine, and probenecid significantly increased the AUCu,brain of acyclovir 1.51-, 1.54-, 1.47-, 1.95-, and 2.34-fold, respectively. Additionally, the Kp.uu.brain of acyclovir markedly increased 1.48-, 1.63-, 1.39-, 1.90-, and 1.35-fold following elacridar, tariquidar, MK571, cyclosporine, and probenecid administration, respectively. CONCLUSION: The present study demonstrated that P-gp, Bcrp, Mrp2, and Oat3 inhibition increased the penetration of acyclovir across the BBB, supporting the hypothesis that these efflux pumps restrict the distribution of acyclovir in the brain.
Asunto(s)
Aciclovir , Barrera Hematoencefálica , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Animales , Transporte Biológico/fisiología , Barrera Hematoencefálica/metabolismo , Encéfalo/metabolismo , Proteínas de Neoplasias , Ratas , Miembro 4 de la Subfamilia B de Casete de Unión a ATPRESUMEN
Clinical trials of olanzapine combined with fluoxetine (Olanzapine/Fluoxetine Combination, OFC) in the treatment of refractory depression have shown significant efficacy, but the drug-drug interaction (DDI) between them remains unclear. In this report, the pharmacokinetic interaction between olanzapine and fluoxetine was studied in wild-type (WT) and Mdr1a/b gene knockout (KO) rats. By analyzing the pharmacokinetics and tissue distribution of olanzapine in single dose and combination, the potential DDI mediated by P-gp was explored. The results showed that in WT rats, the combination of fluoxetine increased the peak concentration (Cmax, 44.1 ± 5.1 ng/mL in the combination group vs 9.0 ± 1.5 ng/mL in the monotherapy group) and the exposure (AUC0-t, 235.8 ± 22.7 h × ng/mL in the combination group vs 47.5 ± 8.4 h × ng/mL in monotherapy group) of olanzapine, and decreased the clearance (CL, 8119.0 ± 677.9 mL/h/kg in the combination group vs 49,469.0 ± 10,306.0 mL/h/kg in monotherapy group). At the same time, fluoxetine significantly increased the in vivo exposure of olanzapine in brain, liver, kidney and ileum of WT rats, indicating the occurrence of DDI. The same phenomenon was observed in Caco-2 cells in vitro as well. However, in KO rats, there was no significant difference in pharmacokinetic parameters between the monotherapy group and the combination group. In conclusion, P-gp plays an important role in the pharmacokinetic interaction between olanzapine and fluoxetine in rats. This study may provide a reference for the clinical safety of olanzapine combined with fluoxetine.
Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Antidepresivos de Segunda Generación/farmacocinética , Antipsicóticos/farmacocinética , Fluoxetina/farmacocinética , Olanzapina/farmacocinética , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Administración Oral , Animales , Antidepresivos de Segunda Generación/administración & dosificación , Antipsicóticos/administración & dosificación , Células CACO-2 , Interacciones Farmacológicas , Fluoxetina/administración & dosificación , Humanos , Masculino , Olanzapina/administración & dosificación , Ratas Sprague-Dawley , Ratas Transgénicas , Distribución Tisular , Miembro 4 de la Subfamilia B de Casete de Unión a ATPRESUMEN
Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease of unknown aetiology for which there are no approved therapeutic options. Patients with PSC display changes in gut microbiota and in bile acid (BA) composition; however, the contribution of these alterations to disease pathogenesis remains controversial. Here we identify a role for microbiota-dependent changes in BA synthesis that modulates PSC pathophysiology. In a genetic mouse model of PSC, we show that loss of microbiota-mediated negative feedback control of BA synthesis results in increased hepatic BA concentrations, disruption of bile duct barrier function and, consequently, fatal liver injury. We further show that these changes are dependent on decreased BA signalling to the farnesoid X receptor, which modulates the activity of the rate-limiting enzyme in BA synthesis, CYP7A1. Moreover, patients with advanced stages of PSC show suppressed BA synthesis as measured by serum C4 levels, which is associated with poor disease prognosis. Our preclinical data highlight the microbiota-dependent dynamics of BA metabolism in cholestatic liver disease, which could be important for future therapies targeting BA and gut microbiome interactions, and identify C4 as a potential biomarker to functionally stratify patients with PSC and predict disease outcomes.
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Ácidos y Sales Biliares/metabolismo , Colestasis/metabolismo , Microbioma Gastrointestinal , Receptores Citoplasmáticos y Nucleares/metabolismo , Transducción de Señal , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Animales , Antibacterianos/administración & dosificación , Colangitis Esclerosante/metabolismo , Colangitis Esclerosante/patología , Humanos , Hígado/metabolismo , Ratones , Pronóstico , Miembro 4 de la Subfamilia B de Casete de Unión a ATPRESUMEN
BACKGROUND AND AIMS: Fibroblast growth factor (FGF) 21 has recently been shown to play a potential role in bile acid metabolism. We aimed to investigate the FGF21 response in an ethanol-induced acute-on-chronic liver injury (ACLI) model in Abcb4-/- mice with deficiency of the hepatobiliary phospholipid transporter. METHODS: Total RNA was extracted from wild-type (WT, C57BL/6J) and Abcb4-/- (KO) mice, which were either fed a control diet (WT-Cont and KO-Cont groups; n = 28/group) or ethanol diet, followed by an acute ethanol binge (WT-EtOH and KO-EtOH groups; n = 28/group). A total of 58 human subjects were recruited into the study, including patients with alcohol-associated liver disease (AALD; n = 31) and healthy controls (n = 27). The hepatic and ileal expressions of genes involved in bile acid metabolism, plasma FGF levels, and bile acid and its precursors 7α- and 27-hydroxycholesterol (7α- and 27-OHC) concentrations were determined. Primary mouse hepatocytes were isolated for cell culture experiments. RESULTS: Alcohol feeding significantly induced plasma FGF21 and decreased hepatic Cyp7a1 levels. Hepatic expression levels of Fibroblast growth factor receptor 1 (Fgfr1), Fgfr4, Farnesoid X-activated receptor (Fxr), and Small heterodimer partner (Shp) and plasma FGF15/FGF19 levels did not differ with alcohol challenge. Exogenous FGF21 treatment suppressed Cyp7a1 in a dose-dependent manner in vitro. AALD patients showed markedly higher FGF21 and lower 7α-OHC plasma levels while FGF19 did not differ. CONCLUSIONS: The simultaneous upregulation of FGF21 and downregulation of Cyp7a1 expressions upon chronic plus binge alcohol feeding together with the invariant plasma FGF15 and hepatic Shp and Fxr levels suggest the presence of a direct regulatory mechanism of FGF21 on bile acid homeostasis through inhibition of CYP7A1 by an FGF15-independent pathway in this ACLI model. Lay Summary: Alcohol challenge results in the upregulation of FGF21 and repression of Cyp7a1 expressions while circulating FGF15 and hepatic Shp and Fxr levels remain constant both in healthy and pre-injured livers, suggesting the presence of an alternative FGF15-independent regulatory mechanism of FGF21 on bile acid homeostasis through the inhibition of Cyp7a1.
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Subfamilia B de Transportador de Casetes de Unión a ATP/fisiología , Insuficiencia Hepática Crónica Agudizada/patología , Ácidos y Sales Biliares/metabolismo , Colesterol 7-alfa-Hidroxilasa/metabolismo , Factores de Crecimiento de Fibroblastos/metabolismo , Hepatocitos/patología , Receptores Citoplasmáticos y Nucleares/metabolismo , Insuficiencia Hepática Crónica Agudizada/metabolismo , Animales , Estudios de Casos y Controles , Colesterol 7-alfa-Hidroxilasa/genética , Femenino , Factores de Crecimiento de Fibroblastos/genética , Hepatocitos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores Citoplasmáticos y Nucleares/genética , Miembro 4 de la Subfamilia B de Casete de Unión a ATPRESUMEN
Hepatocellular carcinoma (HCC) typically develops on a background of chronic hepatitis for which the proinflammatory cytokine IL6 is conventionally considered a crucial driving factor. Paradoxically, IL6 also acts as a hepatoprotective factor in chronic liver injury. Here we used the multidrug-resistant gene 2 knockout (Mdr2-/-) mouse model to elucidate potential roles of IL6 in chronic hepatitis-associated liver cancer. Long-term analysis of three separate IL6/Stat3 signaling-deficient Mdr2-/- strains revealed aggravated liver injury with increased dysplastic nodule formation and significantly accelerated tumorigenesis in all strains. Tumorigenesis in the IL6/Stat3-perturbed models was strongly associated with enhanced macrophage accumulation and hepatosteatosis, phenotypes of nonalcoholic steatohepatitis (NASH), as well as with significant reductions in senescence and the senescence-associated secretory phenotype (SASP) accompanied by increased hepatocyte proliferation. These findings reveal a crucial suppressive role for IL6/Stat3 signaling in chronic hepatitis-associated hepatocarcinogenesis by impeding protumorigenic NASH-associated phenotypes and by reinforcing the antitumorigenic effects of the SASP. SIGNIFICANCE: These findings describe a context-dependent role of IL6 signaling in hepatocarcinogenesis and predict that increased IL6-neutralizing sgp130 levels in some patients with NASH may herald early HCC development.See related commentary by Huynh and Ernst, p. 4671.
Asunto(s)
Transformación Celular Neoplásica/metabolismo , Senescencia Celular , Hígado Graso/etiología , Hígado Graso/metabolismo , Interleucina-6/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Animales , Biomarcadores , Transformación Celular Neoplásica/genética , Senescencia Celular/genética , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Hígado Graso/patología , Femenino , Inmunohistoquímica , Interleucina-6/genética , Neoplasias Hepáticas/etiología , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Ratones , Ratones Noqueados , Ratones Transgénicos , Miembro 4 de la Subfamilia B de Casete de Unión a ATPRESUMEN
Polysaccharides have hypoglycemic activity and pea protein has high nutritional value. The purified pea glycoprotein PGP2 has been shown to inhibit the activity of α-glucosidase and α-amylase in previous studies. To study the mechanism of PGP2-induced blood glucose lowering in vivo, this paper established a diabetic mouse model by intraperitoneal injection of STZ and high-fat diet, and evaluated the blood-glucose-lowering activity of the pea component PGP2 at different doses. The results showed that intragastric administration of PGP2 could effectively reduce diabetic weight loss and polyphagia symptoms, reduce fasting blood glucose levels in mice, and improve oral glucose tolerance levels in mice. PGP2 could promote insulin secretion and had a protective effect on mouse organs. After intragastric administration of PGP2 in mice, the serum levels of total cholesterol, triglycerides and low-density lipoprotein decreased. PGP2 up-regulated the gene expression of insulin receptor substrates IRS-1 and IRS-2 in liver tissues, thereby reducing insulin resistance. Based on the above experimental results, PGP2 had good hypoglycemic activity and was expected to be developed as a natural medicine for the treatment of type II diabetes.
Asunto(s)
Diabetes Mellitus Tipo 2/metabolismo , Glicoproteínas/farmacología , Hipoglucemiantes/farmacología , Pisum sativum/química , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Animales , Glucemia/metabolismo , Diabetes Mellitus Experimental/tratamiento farmacológico , Dieta Alta en Grasa/efectos adversos , Prueba de Tolerancia a la Glucosa , Glicoproteínas/uso terapéutico , Hipoglucemiantes/uso terapéutico , Insulina/sangre , Proteínas Sustrato del Receptor de Insulina/metabolismo , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos ICR , Triglicéridos/sangre , Miembro 4 de la Subfamilia B de Casete de Unión a ATPRESUMEN
BACKGROUND: Emerging evidence implicates the gut microbiome in liver inflammation and hepatocellular carcinoma (HCC) development. We aimed to characterize the temporal evolution of gut dysbiosis, in relation to the phenotype of systemic and hepatic inflammatory responses leading to HCC development. In the present study, Mdr2 -/- mice were used as a model of inflammation-based HCC. Gut microbiome composition and function, in addition to serum LPS, serum cytokines/chemokines and intrahepatic inflammatory genes were measured throughout the course of liver injury until HCC development. RESULTS: Early stages of liver injury, inflammation and cirrhosis, were characterized by dysbiosis. Microbiome functional pathways pertaining to gut barrier dysfunction were enriched during the initial phase of liver inflammation and cirrhosis, whilst those supporting lipopolysaccharide (LPS) biosynthesis increased as cirrhosis and HCC ensued. In parallel, serum LPS progressively increased during the course of liver injury, corresponding to a shift towards a systemic Th1/Th17 proinflammatory phenotype. Alongside, the intrahepatic inflammatory gene profile transitioned from a proinflammatory phenotype in the initial phases of liver injury to an immunosuppressed one in HCC. In established HCC, a switch in microbiome function from carbohydrate to amino acid metabolism occurred. CONCLUSION: In Mdr2 -/- mice, dysbiosis precedes HCC development, with temporal evolution of microbiome function to support gut barrier dysfunction, LPS biosynthesis, and redirection of energy source utilization. A corresponding shift in systemic and intrahepatic inflammatory responses occurred supporting HCC development. These findings support the notion that gut based therapeutic interventions could be beneficial early in the course of liver disease to halt HCC development.
Asunto(s)
Carcinoma Hepatocelular/complicaciones , Carcinoma Hepatocelular/microbiología , Disbiosis/complicaciones , Disbiosis/inmunología , Neoplasias Hepáticas/complicaciones , Neoplasias Hepáticas/microbiología , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Animales , Carcinoma Hepatocelular/inmunología , Modelos Animales de Enfermedad , Disbiosis/microbiología , Microbioma Gastrointestinal/fisiología , Inflamación/complicaciones , Inflamación/microbiología , Neoplasias Hepáticas/inmunología , Ratones , Tiempo , Miembro 4 de la Subfamilia B de Casete de Unión a ATPRESUMEN
BACKGROUND: Dermatophytes showing reduced sensitivity to antifungal agents have emerged in several countries. One terbinafine resistant strain of Trichophyton rubrum, TIMM20092, also showed reduced sensitivity to itraconazole (ITC) and voriconazole (VRC). The expression of two genes (TruMDR2 and TruMDR3) encoding multidrug transporters of the ABC family was found to be highly up-regulated in this strain. Deletion of TruMDR3 in TIMM20092 abolished its resistance to VRC but only slightly reduced its resistance to ITC. OBJECTIVES: We examined the potential of T rubrum to develop resistance to ITC by analysing the mechanism of ITC resistance in TIMM20092. METHODS: The deletion of TruMDR2 by gene replacement was performed in TIMM20092 and one TruMDR3-lacking mutant (∆TruMDR3) previously generated from TIMM20092. TruMDR2 single and TruMDR2/TruMDR3 double mutants (∆TruMDR2 and ∆TruMDR2/3) were successfully obtained, respectively. RESULTS: The suppression of TruMDR2 was shown to abolish resistance to ITC in TIMM20092 and the TruMDR3-lacking mutant, strongly suggesting that TruMDR2 is a major contributor to ITC resistance in TIMM20092. CONCLUSIONS: Our study highlights the possible role of the ABC transporter TruMDR2 in ITC resistance of T. rubrum.
Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Antifúngicos/farmacología , Arthrodermataceae/efectos de los fármacos , Arthrodermataceae/genética , Farmacorresistencia Fúngica/genética , Itraconazol/farmacología , Humanos , Pruebas de Sensibilidad Microbiana , Miembro 4 de la Subfamilia B de Casete de Unión a ATPRESUMEN
Bile salts, such as cholate, glycocholate, taurocholate, and glycochenodeoxycholate, are taken up from the portal blood into hepatocytes via transporters, such as the Na+-taurocholate-cotransporting polypeptide (NTCP) and organic anion-transporting polypeptides (OATPs). These bile salts are later secreted into bile across the canalicular membrane, which is facilitated by the bile salt export pump (BSEP). Apart from bile salt transport, some of these proteins (e.g., OATPs) are also key transporters for drug uptake into hepatocytes. In vivo studies of transporter function in patients by using tracer compounds have emerged as an important diagnostic tool to complement classic liver parameter measurements by determining dynamic liver function both for diagnosis and monitoring progression or improvement of liver diseases. Such approaches include use of radioactively labeled bile salts (e.g., for positron emission tomography) and fluorescent bile salt derivatives or dyes (e.g., indocyanine green). To expand the list of liver function markers, we synthesized fluorescent derivatives of cholic and chenodeoxycholic acid by conjugating small organic dyes to the bile acid side chain. These novel fluorescent probes were able to block substrate transport in a concentration-dependent manner of NTCP, OATP1B1, OATP1B3, OATP2B1, BSEP, and intestinal apical sodium-dependent bile salt transporter (ASBT). Whereas the fluorescent bile acid derivatives themselves were transported across the membrane by OATP1B1, OATP1B3, and OATP2B1, they were not transport substrates for NTCP, ASBT, BSEP, and multidrug resistance-related protein 2. Accordingly, these novel fluorescent bile acid probes can potentially be used as imaging agents to monitor the function of OATPs. SIGNIFICANCE STATEMENT: Synthetic modification of common bile acids by attachment of small organic fluorescent dyes to the bile acid side chain resulted in bright, fluorescent probes that interact with hepatic and intestinal organic anion [organic anion-transporting polypeptide (OATP) 1B1, OATP1B3, OATP2B1], bile salt uptake (Na+-taurocholate-cotransporting polypeptide, apical sodium-dependent bile salt transporter), and bile salt efflux (bile salt export pump, multidrug resistance-related protein 2) transporters. Although the fluorescent bile salt derivatives are taken up into cells via the OATPs, the efflux transporters do not transport any of them but one.
Asunto(s)
Transportadores de Anión Orgánico , Subfamilia B de Transportador de Casetes de Unión a ATP , Ácidos y Sales Biliares , Hepatocitos , Miembro 4 de la Subfamilia B de Casete de Unión a ATPRESUMEN
The transporter multidrug resistance protein 2 (MRP2) can transport some tobacco carcinogens and plays an important role in the transport of mediators related to pulmonary inflammatory diseases. However, it is not fully understood whether the pulmonary inflammation caused by cigarette smoke extract (CSE) and lipopolysaccharide (LPS) is related to the regulation of MRP2. In this study, CSE and LPS were used alone and in combination as stimuli to induce pulmonary inflammation. In addition, the establishment of a pulmonary inflammation model was verified by animal experiments in vivo. We found that compared with those in the control group, the expression of MRP2 protein was downregulated and the expression of inflammatory cytokines was upregulated in pulmonary inflammation in the CSE group and the CSE combined with LPS group. However, there was almost no change in the expression of MRP2 stimulated by LPS alone. Our results show that CSE and CSE combined with LPS downregulate the expression of MRP2 under inflammatory conditions, while LPS has almost no effect on the expression of MRP2 under inflammatory conditions. The in vivo experimental results of CSE combined with LPS were consistent with the cellular results of CSE combined with LPS, which provides a model and basis for other studies of the role of MRP2 in pulmonary inflammation.
Asunto(s)
Neumonía , Subfamilia B de Transportador de Casetes de Unión a ATP , Animales , Regulación hacia Abajo , Inflamación/inducido químicamente , Lipopolisacáridos/toxicidad , Neumonía/inducido químicamente , Humo/efectos adversos , Fumar , Nicotiana , Miembro 4 de la Subfamilia B de Casete de Unión a ATPRESUMEN
BACKGROUND & AIMS: Cholangiopathies are chronic liver diseases in which damaged cholangiocytes trigger a proinflammatory and profibrotic reaction. The nuclear vitamin D receptor (VDR) is highly expressed in cholangiocytes and exerts immune-regulatory functions in these cells. In the present study, we examined the protective function of VDR and other vitamin D signaling pathways in chronic cholangiopathy and cholangiocytes. METHODS: Vdr was invalidated in Abcb4 knockout mice, a widely used animal model of chronic cholangiopathy. The impact of vitamin D signaling on cholangiopathy features was examined in vivo and in cholangiocytes (primary and cell lines). RESULTS: Cholangiopathy features (i.e, cholestasis, ductular reaction and fibrosis) were aggravated in Vdr;Abcb4 double knockout mice compared to the Abcb4 simple knockout, and associated with an overexpression of proinflammatory factors. The proinflammatory phenotype of cholangiocytes was also exacerbated following VDR silencing in vitro. The expression of proinflammatory factors and the severity of cholangiopathy were reduced in the double knockout mice treated with the vitamin D analog calcipotriol or with vitamin D. In vitro, the inflammatory response to TNFα was significantly reduced by calcipotriol in biliary cells silenced for VDR, and this effect was abolished by co-silencing the plasma membrane receptor of vitamin D, protein disulfide-isomerase A3 (PDIA3). CONCLUSIONS: Our results demonstrate an anti-inflammatory role of VDR signaling in cholangiocytes and cholangiopathy. They also provide evidence for PDIA3-mediated anti-inflammatory effects of vitamin D and vitamin D analog in these settings.
Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Colestasis/genética , Receptores de Calcitriol/genética , Vitamina D/metabolismo , Animales , Colestasis/tratamiento farmacológico , Colestasis/metabolismo , Colestasis/patología , Fibrosis , Eliminación de Gen , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de Calcitriol/metabolismo , Transducción de Señal/efectos de los fármacos , Vitamina D/uso terapéutico , Vitaminas/metabolismo , Vitaminas/uso terapéutico , Miembro 4 de la Subfamilia B de Casete de Unión a ATPRESUMEN
BACKGROUND & AIMS: Transforming growth factor ß (TGFß) upregulates cholangiocyte-derived signals that activate myofibroblasts and promote fibrosis. Using epigenomic and transcriptomic approaches, we sought to distinguish the epigenetic activation mechanisms downstream of TGFß that mediate transcription of fibrogenic signals. METHODS: Chromatin immunoprecipitation (ChIP)-seq and RNA-seq were performed to assess histone modifications and transcriptional changes following TGFß stimulation. Histone modifications and acetyltransferase occupancy were confirmed using ChIP assays. Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) was used to investigate changes in chromatin accessibility. Cholangiocyte cell lines and primary cholangiocytes were used for in vitro studies. Mdr2-/- and 3,5-diethoxycarboncyl-1,4-dihydrocollidine (DDC)-fed mice were used as animal models. RESULTS: TGFß stimulation caused widespread changes in histone 3 lysine 27 acetylation (H3K27ac), and was associated with global TGFß-mediated transcription. In contrast, H3K9ac was gained in a smaller group of chromatin sites and was associated with fibrosis pathways. These pathways included overexpression of hepatic stellate cell (HSC) activators such as fibronectin 1 (FN1) and SERPINE1. The promoters of these genes showed H3K9ac enrichment following TGFß. Of the acetyltransferases responsible for H3K9ac, cholangiocytes predominantly express Lysine Acetyltransferases 2A (KAT2A). Small interfering RNA knockdown of KAT2A or H3K9ac inhibition prevented the TGFß-mediated increase in FN1 and SERPINE1. SMAD3 ChIP-seq and ATAC-seq suggested that TGFß-mediated H3K9ac occurs through SMAD signaling, which was confirmed using colocalization and genetic knockdown studies. Pharmacologic inhibition or cholangiocyte-selective deletion of Kat2a was protective in mouse models of biliary fibrosis. CONCLUSIONS: Cholangiocyte expression of HSC-activating signals occurs through SMAD-dependent, KAT2A-mediated, H3K9ac, and can be targeted to prevent biliary fibrosis.