Elucidation of pericholangitis and periductal fibrosis in cholestatic liver diseases via extracellular vesicles released by polarized biliary epithelial cells.

Cholestatic liver diseases causes inflammation and fibrosis around bile ducts. However, the pathological mechanism has not been elucidated. Extracellular vesicles (EVs) are released from both the basolateral and apical sides of polarized biliary epithelial cells. We aimed to investigate the possibility that EVs released from the basolateral sides of biliary epithelial cells by bile acid stimulation induce inflammatory cells and fibrosis around bile ducts, and they may be involved in the pathogenesis of cholestatic liver disease. Human biliary epithelial cells (H69) were grown on cell culture inserts and stimulated with chenodeoxycholic acid + IFN-γ. Human THP-1-derived M1-macrophages, LX-2 cells, and KMST-6 cells were treated with the extracted basolateral EVs, and inflammatory cytokines and fibrosis markers were detected by RT-PCR. Highly expressed proteins from stimulated EVs were identified, and M1-macrophages, LX-2, KMST-6 were treated with these recombinant proteins. Stimulated EVs increased the expression of TNF, IL-1ß, and IL-6 in M1-macrophages, TGF-ß in LX-2 and KMST-6 compared with the corresponding expression levels in unstimulated EVs. Nucleophosmin, nucleolin, and midkine levels were increased in EVs from stimulated cells compared with protein expression in EVs from unstimulated cells. Leukocyte cell-derived chemotaxin-2 (LECT2) is highly expressed only in EVs from stimulated cells. Stimulation of M1-macrophages with recombinant nucleophosmin, nucleolin, and midkine significantly increased the expression of inflammatory cytokines. Stimulation of LX-2 and KMST-6 with recombinant LECT2 significantly increased the expression of fibrotic markers. These results suggest that basolateral EVs are related to the development of pericholangitis and periductal fibrosis in cholestatic liver diseases.NEW & NOTEWORTHY Our research elucidated that the composition of basolateral EVs from the biliary epithelial cells changed under bile acid exposure and the basolateral EVs contained the novel inflammation-inducing proteins NPM, NCL, and MK and the fibrosis-inducing protein LECT2. We report that these new results are possible to lead to the potential therapeutic target of cholestatic liver diseases in the future.

Melatonin improves cholestatic liver disease via the gut-liver axis.

Cholestatic liver disease is characterized by disturbances in the intestinal microbiota and excessive accumulation of toxic bile acids (BA) in the liver. Melatonin (MT) can improve liver diseases. However, the underlying mechanism remains unclear. This study aimed to explore the mechanism of MT on hepatic BA synthesis, liver injury, and fibrosis in 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-fed and Mdr2-/- mice. MT significantly improved hepatic injury and fibrosis with a significant decrease in hepatic BA accumulation in DDC-fed and Mdr2-/- mice. MT reprogramed gut microbiota and augmented fecal bile salt hydrolase activity, which was related to increasing intestinal BA deconjugation and fecal BA excretion in both DDC-fed and Mdr2-/- mice. MT significantly activated the intestinal farnesoid X receptor (FXR)/fibroblast growth factor 15 (FGF-15) axis and subsequently inhibited hepatic BA synthesis in DDC-fed and Mdr2-/- mice. MT failed to improve DDC-induced liver fibrosis and BA synthesis in antibiotic-treated mice. Furthermore, MT provided protection against DDC-induced liver injury and fibrosis in fecal microbiota transplantation mice. MT did not decrease liver injury and fibrosis in DDC-fed intestinal epithelial cell-specific FXR knockout mice, suggesting that the intestinal FXR mediated the anti-fibrosis effect of MT. In conclusion, MT ameliorates cholestatic liver diseases by remodeling gut microbiota and activating intestinal FXR/FGF-15 axis-mediated inhibition of hepatic BA synthesis and promotion of BA excretion in mice.

Protective and aggressive bacterial subsets and metabolites modify hepatobiliary inflammation and fibrosis in a murine model of PSC.

OBJECTIVE: Conflicting microbiota data exist for primary sclerosing cholangitis (PSC) and experimental models. GOAL: define the function of complex resident microbes and their association relevant to PSC patients by studying germ-free (GF) and antibiotic-treated specific pathogen-free (SPF) multidrug-resistant 2 deficient (mdr2-/- ) mice and microbial profiles in PSC patient cohorts. DESIGN: We measured weights, liver enzymes, RNA expression, histological, immunohistochemical and fibrotic biochemical parameters, faecal 16S rRNA gene profiling and metabolomic endpoints in gnotobiotic and antibiotic-treated SPF mdr2-/- mice and targeted metagenomic analysis in PSC patients. RESULTS: GF mdr2-/- mice had 100% mortality by 8 weeks with increasing hepatic bile acid (BA) accumulation and cholestasis. Early SPF autologous stool transplantation rescued liver-related mortality. Inhibition of ileal BA transport attenuated antibiotic-accelerated liver disease and decreased total serum and hepatic BAs. Depletion of vancomycin-sensitive microbiota exaggerated hepatobiliary disease. Vancomycin selectively decreased Lachnospiraceae and short-chain fatty acids (SCFAs) but expanded Enterococcus and Enterobacteriaceae. Antibiotics increased Enterococcus faecalis and Escherichia coli liver translocation. Colonisation of GF mdr2-/- mice with translocated E. faecalis and E. coli strains accelerated hepatobiliary inflammation and mortality. Lachnospiraceae colonisation of antibiotic pretreated mdr2-/- mice reduced liver fibrosis, inflammation and translocation of pathobionts, and SCFA-producing Lachnospiraceae and purified SCFA decreased fibrosis. Faecal Lachnospiraceae negatively associated, and E. faecalis/ Enterobacteriaceae positively associated, with PSC patients' clinical severity by Mayo risk scores. CONCLUSIONS: We identified novel functionally protective and detrimental resident bacterial species in mdr2-/- mice and PSC patients with associated clinical risk score. These insights may guide personalised targeted therapeutic interventions in PSC patients.

Sodium+/taurocholate cotransporting polypeptide as target therapy for liver fibrosis.

OBJECTIVE: Sodium+/ taurocholate cotransporting polypeptide (NTCP) is a membrane transporter affecting the enterohepatic circulation of bile acids (BAs). We aimed to evaluate NTCP's roles in humans and animal models of liver fibrosis (LF). DESIGN: Primary hepatic stellate cells (pHSCs) isolated from livers biopsies of patients with LF with different fibrosis grading were stained for NTCP. NTCP gene silencing, taurocholic acid (TCA), obeticholic acid (OCA), epigallocatechin gallate (EGCG) and HA-100 dihydrochloride (HA-100) were used as tools to modulate NTCP expression on human HSC line (LX2). BA trafficking/uptake were assessed extracellularly (LX2 culture medium) and intracellularly following treatment with/without NTCP neutralizing antibody. LF models of C57/BL6 mice of carbon tetrachloride (CCl4) and leptin-deficient (Ob/Ob) fed with high-fat diet (Ob/Ob HFD ) were evaluated for pHSCs-NTCP expressions, metabolic and LF profiles following intraperitoneal injections of NTCP neutralizing antibody. RESULTS: pHSCs from F3/F4-scored patients of LF exhibit threefold increased NTCP expressions compared with F0-scored patients (p<0.0001). Sorted-activated HSCs (LX2αSMA+) showed high expressions of NTCP and high TCA uptake in vitro and triggered a further increase in their activations. This phenomenon was inhibited with NTCP small interfering RNA and the NTCP neutralizing antibody. Sorted LX2NTCP+ (high alpha smooth muscle actin (αSMA)/high NTCP) cells showed high phosphorylated pathways of AKT/mTOR and protein kinase C (PKC) accompanied with a decrease in farnesoid X receptor expression. Moreover, LX2NTCP+ cells treated with EGCG, OCA and PKC inhibitor HA-100 significantly decreased NTCP and αSMA. NTCP neutralizing antibody inhibited NTCP (less TCA uptake); it attenuated LF in both CCl4 and Ob/Ob HFD animal models with ameliorated metabolic profile. CONCLUSION: NTCP expression is linearly correlated with fibrosis severity. Modulated BA trafficking could be an important step in LF pathogenesis. Antagonising BA uptake may suggest a therapeutic strategy for preventing disease progression.

Effect of CFTR correctors on the traffic and the function of intracellularly retained ABCB4 variants.

BACKGROUND & AIM: ABCB4 is expressed at the canalicular membrane of hepatocytes. This ATP-binding cassette (ABC) transporter is responsible for the secretion of phosphatidylcholine into bile canaliculi. Missense genetic variations of ABCB4 are correlated with several rare cholestatic liver diseases, the most severe being progressive familial intrahepatic cholestasis type 3 (PFIC3). In a repurposing strategy to correct intracellularly retained ABCB4 variants, we tested 16 compounds previously validated as cystic fibrosis transmembrane conductance regulator (CFTR) correctors. METHODS: The maturation, intracellular localization and activity of intracellularly retained ABCB4 variants were analyzed in cell models after treatment with CFTR correctors. In addition, in silico molecular docking calculations were performed to test the potential interaction of CFTR correctors with ABCB4. RESULTS: We observed that the correctors C10, C13, and C17, as well as the combinations of C3 + C18 and C4 + C18, allowed the rescue of maturation and canalicular localization of four distinct traffic-defective ABCB4 variants. However, such treatments did not permit a rescue of the phosphatidylcholine secretion activity of these defective variants and were also inhibitory of the activity of wild type ABCB4. In silico molecular docking analyses suggest that these CFTR correctors might directly interact with transmembrane domains and/or ATP-binding sites of the transporter. CONCLUSION: Our results illustrate the uncoupling between the traffic and the activity of ABCB4 because the same molecules can rescue the traffic of defective variants while they inhibit the secretion activity of the transporter. We expect that this study will help to design new pharmacological tools with potential clinical interest.

Functional rescue of an ABCB11 mutant by ivacaftor: A new targeted pharmacotherapy approach in bile salt export pump deficiency.

BACKGROUND & AIM: The canalicular bile salt export pump (BSEP/ABCB11) of hepatocytes is the main adenosine triphosphate (ATP)-binding cassette (ABC) transporter responsible for bile acid secretion. Mutations in ABCB11 cause several cholestatic diseases, including progressive familial intrahepatic cholestasis type 2 (PFIC2) often lethal in absence of liver transplantation. We investigated in vitro the effect and potential rescue of a BSEP mutation by ivacaftor, a clinically approved cystic fibrosis transmembrane conductance regulator (CFTR/ABCC7) potentiator. METHODS: The p.T463I mutation, identified in a PFIC2 patient and located in a highly conserved ABC transporter motif, was studied by 3D structure modelling. The mutation was reproduced in a plasmid encoding a rat Bsep-green fluorescent protein. After transfection, mutant expression was studied in Can 10 cells. Taurocholate transport activity and ivacaftor effect were studied in Madin-Darby canine kidney (MDCK) clones co-expressing the rat sodium-taurocholate co-transporting polypeptide (Ntcp/Slc10A1). RESULTS: As the wild-type protein, BsepT463I was normally targeted to the canalicular membrane of Can 10 cells. As predicted by 3D structure modelling, taurocholate transport activity was dramatically low in MDCK clones expressing BsepT463I . Ivacaftor treatment increased by 1.7-fold taurocholate transport activity of BsepT463I (P < .0001), reaching 95% of Bsepwt activity. These data suggest that the p.T463I mutation impairs ATP-binding, resulting in Bsep dysfunction that can be rescued by ivacaftor. CONCLUSION: These results provide experimental evidence of ivacaftor therapeutic potential for selected patients with PFIC2 caused by ABCB11 missense mutations affecting BSEP function. This could represent a significant step forward for the care of patients with BSEP deficiency.

Effect of scheduled endoscopic dilatation of dominant strictures on outcome in patients with primary sclerosing cholangitis.

OBJECTIVE: Scheduled endoscopic dilatation of dominant strictures (DS) in primary sclerosing cholangitis (PSC) might improve outcome relative to endoscopic treatment on demand, but evidence is limited. Since randomisation is difficult in clinical practice, we present a large retrospective study comparing scheduled versus on-demand endoscopic retrograde cholangiopancreatography (ERCP) based on patient preferences. DESIGN: Between 1987 and 2017, all new patients with PSC had been offered scheduled ERCP with dilatation of a DS if diagnosed; the latter was repeated at defined intervals until morphological resolution, independent of clinical symptoms (treatment group). Patients who refused participation were clinically evaluated annually and received endoscopic treatment only on demand (control group). The primary clinical endpoint was transplantation-free survival. Secondary outcomes were overall survival, bacterial cholangitis episodes, hepatic decompensation of liver cirrhosis and endoscopy-related adverse events. RESULTS: The final study included 286 patients, 133 (46.5%) receiving scheduled ERCP and 153 (53.5%) receiving on-demand ERCP. After a mean follow-up of 9.9 years, the rate of transplantation-free survival was higher in patients receiving scheduled ERCP (51% vs 29.3%; p<0.001), as was transplantation-free survival time (median: 17.9 vs 15.2 years; log-rank: p=0.008). However, the benefit of scheduled ERCP was significant only in patients with the initial (17.1%) or later (45.5%) diagnosis of a DS (17.8 vs 11.1 years; log-rank: p<0.001). IBD (p=0.03), DS (p=0.006), higher Mayo Risk Score (p=0.02) and non-adherence to scheduled endoscopy (p=0.005) were independently associated with transplantation-free survival. CONCLUSION: In our large retrospective study, regular ERCP with endoscopic balloon dilatation significantly benefits patients with PSC with DS, diagnosed both at initial presentation and during surveillance, even if asymptomatic. Further studies have to find out how to best identify stricture patients non-invasively.

Intestinal dysbiosis augments liver disease progression via NLRP3 in a murine model of primary sclerosing cholangitis.

OBJECTIVE: There is a striking association between human cholestatic liver disease (CLD) and inflammatory bowel disease. However, the functional implications for intestinal microbiota and inflammasome-mediated innate immune response in CLD remain elusive. Here we investigated the functional role of gut-liver crosstalk for CLD in the murine Mdr2 knockout (Mdr2-/-) model resembling human primary sclerosing cholangitis (PSC). DESIGN: Male Mdr2-/-, Mdr2-/- crossed with hepatocyte-specific deletion of caspase-8 (Mdr2-/- /Casp8∆hepa) and wild-type (WT) control mice were housed for 8 or 52 weeks, respectively, to characterise the impact of Mdr2 deletion on liver and gut including bile acid and microbiota profiling. To block caspase activation, a pan-caspase inhibitor (IDN-7314) was administered. Finally, the functional role of Mdr2-/- -associated intestinal dysbiosis was studied by microbiota transfer experiments. RESULTS: Mdr2-/- mice displayed an unfavourable intestinal microbiota signature and pronounced NLRP3 inflammasome activation within the gut-liver axis. Intestinal dysbiosis in Mdr2-/- mice prompted intestinal barrier dysfunction and increased bacterial translocation amplifying the hepatic NLRP3-mediated innate immune response. Transfer of Mdr2-/- microbiota into healthy WT control mice induced significant liver injury in recipient mice, highlighting the causal role of intestinal dysbiosis for disease progression. Strikingly, IDN-7314 dampened inflammasome activation, ameliorated liver injury, reversed serum bile acid profile and cholestasis-associated microbiota signature. CONCLUSIONS: MDR2-associated cholestasis triggers intestinal dysbiosis. In turn, translocation of endotoxin into the portal vein and subsequent NLRP3 inflammasome activation contribute to higher liver injury. This process does not essentially depend on caspase-8 in hepatocytes, but can be blocked by IDN-7314.

ACOX2 deficiency: An inborn error of bile acid synthesis identified in an adolescent with persistent hypertransaminasemia.

BACKGROUND & AIMS: Acyl-CoA oxidase (ACOX2) is involved in the shortening of C27 cholesterol derivatives to generate C24 bile acids. Inborn errors affecting the rest of peroxisomal enzymes involved in bile acid biosynthesis have been described. Here we aimed at investigating the case of an adolescent boy with persistent hypertransaminasemia of unknown origin and suspected dysfunction in bile acid metabolism. METHODS: Serum and urine samples were taken from the patient, his sister and parents and underwent HPLC-MS/MS and HPLC-TOF analyses. Coding exons in genes of interest were amplified by high-fidelity PCR and sequenced. Wild-type or mutated (mutACOX2) variants were overexpressed in human hepatoblastoma HepG2 cells to determine ACOX2 enzymatic activity, expression and subcellular location. RESULTS: The patient's serum and urine showed negligible amounts of C24 bile acids, but augmented levels of C27 intermediates, mainly tauroconjugated trihydroxycholestanoic acid (THCA). Genetic analysis of enzymes potentially involved revealed a homozygous missense mutation (c.673C>T; R225W) in ACOX2. His only sister was also homozygous for this mutation and exhibited similar alterations in bile acid profiles. Both parents were heterozygous and presented normal C24 and C27 bile acid levels. Immunofluorescence studies showed similar protein size and peroxisomal localization for both normal and mutated variants. THCA biotransformation into cholic acid was enhanced in cells overexpressing ACOX2, but not in those overexpressing mutACOX2. Both cell types showed similar sensitivity to oxidative stress caused by C24 bile acids. In contrast, THCA-induced oxidative stress and cell death were reduced by overexpressing ACOX2, but not mutACOX2. CONCLUSION: ACOX2 deficiency, a condition characterized by accumulation of toxic C27 bile acid intermediates, is a novel cause of isolated persistent hypertransaminasemia. LAY SUMMARY: Elevation of serum transaminases is a biochemical sign of liver damage due to multiplicity of causes (viruses, toxins, autoimmunity, metabolic disorders). In rare cases the origin of this alteration remains unknown. We have identified by the first time in a young patient and his only sister a familiar genetic defect of an enzyme called ACOX2, which participates in the transformation of cholesterol into bile acids as a cause of increased serum transaminases in the absence of any other symptomatology. This treatable condition should be considered in the diagnosis of those patients where the cause of elevated transaminases remains obscure.

Stratification of hepatocellular carcinoma risk in primary biliary cirrhosis: a multicentre international study.

OBJECTIVE: Hepatocellular carcinoma (HCC) is an infrequent yet critical event in primary biliary cirrhosis (PBC); however, predictive tools remain ill-defined. Our objective was to identify candidate risk factors for HCC development in patients with PBC. DESIGN: Risk factor analysis was performed in over 15 centres from North America and Europe spanning >40 years observation period using Cox proportional hazards assumptions, logistic regression, and Kaplan-Meier estimates. RESULTS: Of 4565 patients with PBC 123 developed HCC, yielding an incidence rate (IR) of 3.4 cases/1000 patient-years. HCC was significantly more common in men (p<0.0001), and on univariate analysis factors at PBC diagnosis associated with future HCC development were male sex (unadjusted HR 2.91, p<0.0001), elevated serum aspartate transaminase (HR 1.24, p<0.0001), advanced disease (HR 2.72, p=0.022), thrombocytopenia (HR 1.65, p<0.0001), and hepatic decompensation (HR 9.89, p<0.0001). As such, non-treatment with ursodeoxycholic acid itself was not associated with cancer development; however, 12-month stratification by biochemical non-response (Paris-I criteria) associated significantly with future risk of HCC (HR 4.52, p<0.0001; IR 6.6 vs 1.4, p<0.0001). Non-response predicted future risk in patients with early stage disease (IR 4.7 vs 1.2, p=0.005), advanced disease (HR 2.79, p=0.02; IR 11.2 vs 4.4, p=0.033), and when restricting the analysis to only male patients (HR 4.44, p<0.001; IR 18.2 vs 5.4, p<0.001). On multivariable analysis biochemical non-response remained the most significant factor predictive of future HCC risk (adjusted HR 3.44, p<0.0001). CONCLUSIONS: This uniquely powered, internationally representative cohort robustly demonstrates that 12-month biochemical non-response is associated with increased future risk of developing HCC in PBC. Such risk stratification is relevant to patient care and development of new therapies.

Pleiotrophin regulates the ductular reaction by controlling the migration of cells in liver progenitor niches.

OBJECTIVE: The ductular reaction (DR) involves mobilisation of reactive-appearing duct-like cells (RDC) along canals of Hering, and myofibroblastic (MF) differentiation of hepatic stellate cells (HSC) in the space of Disse. Perivascular cells in stem cell niches produce pleiotrophin (PTN) to inactivate the PTN receptor, protein tyrosine phosphatase receptor zeta-1 (PTPRZ1), thereby augmenting phosphoprotein-dependent signalling. We hypothesised that the DR is regulated by PTN/PTPRZ1 signalling. DESIGN: PTN-GFP, PTN-knockout (KO), PTPRZ1-KO, and wild type (WT) mice were examined before and after bile duct ligation (BDL) for PTN, PTPRZ1 and the DR. RDC and HSC from WT, PTN-KO, and PTPRZ1-KO mice were also treated with PTN to determine effects on downstream signaling phosphoproteins, gene expression, growth, and migration. Liver biopsies from patients with DRs were also interrogated. RESULTS: Although quiescent HSC and RDC lines expressed PTN and PTPRZ1 mRNAs, neither PTN nor PTPRZ1 protein was demonstrated in healthy liver. BDL induced PTN in MF-HSC and increased PTPRZ1 in MF-HSC and RDC. In WT mice, BDL triggered a DR characterised by periportal accumulation of collagen, RDC and MF-HSC. All aspects of this DR were increased in PTN-KO mice and suppressed in PTPRZ1-KO mice. In vitro studies revealed PTN-dependent accumulation of phosphoproteins that control cell-cell adhesion and migration, with resultant inhibition of cell migration. PTPRZ1-positive cells were prominent in the DRs of patients with ductal plate defects and adult cholestatic diseases. CONCLUSIONS: PTN, and its receptor, PTPRZ1, regulate the DR to liver injury by controlling the migration of resident cells in adult liver progenitor niches.

Functional analysis of ABCB4 mutations relates clinical outcomes of progressive familial intrahepatic cholestasis type 3 to the degree of MDR3 floppase activity.

OBJECTIVE: Progressive familial intrahepatic cholestasis type 3 (PFIC3) is a potentially lethal autosomal recessive liver disease associated with mutations in ABCB4, the gene encoding the canalicular translocator of phosphatidylcholine MDR3. While some affected children benefit from ursodeoxycholic acid (UDCA) therapy, others evolve to end-stage liver disease. We aimed to evaluate whether these different outcomes are related to the impact of ABCB4 mutations. DESIGN: Six children with PFIC3 were investigated by sequencing of ABCB4 exons and flanking intron-exon boundaries and by immunohistochemistry. ABCB4 missense mutations were phenotyped in vitro by assessing their effects on MDR3 expression, subcellular localisation, and phosphatidylcholine-translocating activity. The resulting data were contrasted with the clinical outcomes. RESULTS: Eight distinct ABCB4 mutations were identified: one nonsense, one splicing and six missense mutations, four of which (G68R, T201M, P479L, D459H) affected MDR3 expression level. G68R and D459H also led to retention of the protein in endoplasmic reticulum. Phosphatidylcholine efflux assays indicated that T201M, P479L, S978P and E1118K mutations impaired MDR3 activity to variable degrees. Three children with mutations that caused a total loss of MDR3 expression/function manifested progressive liver disease refractory to UDCA treatment. This was also the case in a patient carrying two different mutations that, in combination, resulted in a 90% reduction in total MDR3 activity. A favourable response to UDCA was achieved in two patients with estimated MDR3 activities of 50% and 33%, respectively. CONCLUSIONS: These data provide experimental evidence of the correlation between the degree of MDR3 floppase activity and the clinical outcomes of PFIC3.

Jnk1 in murine hepatic stellate cells is a crucial mediator of liver fibrogenesis.

OBJECTIVE: The c-Jun N-terminal kinase-1 (Jnk1) gene has been shown to be involved in liver fibrosis. Here, we aimed to investigate the molecular mechanism and define the cell type involved in mediating the Jnk1-dependent effect on liver fibrogenesis. DESIGN: Jnk1(f/f) wildtype (WT), Jnk1(-/-) and Jnk1(Δhepa) (hepatocyte-specific deletion of Jnk1) mice were subjected to (i) bile duct ligation (BDL) and (ii) CCl4-induced liver fibrosis. Additionally, we performed bone marrow transplantations (BMT), isolated primary hepatic stellate cells (HSCs), studied their activation in vitro and investigated human diseased liver samples. RESULTS: Phosphorylated Jnk was expressed in myofibroblasts, epithelial and inflammatory cells during the progression of fibrogenesis in humans and mice. In mice, liver transaminases, alkaline phosphatase, bilirubin and liver histology revealed reduced injury in Jnk1(-/-) compared with WT and Jnk1(Δhepa) mice correlating with lower hepatocyte cell death and proliferation. Consequently, parameters of liver fibrosis such as Sirius red staining and collagen IA1 and α-smooth muscle actin expression were downregulated in Jnk1(-/-) compared with WT and Jnk1(Δhepa) livers, 4 weeks after CCl4 or BDL. BMT experiments excluded bone marrow-derived cells from having a major impact on the Jnk1-dependent effect on fibrogenesis, while primary HSCs from Jnk1(-/-) livers showed reduced transdifferentiation and extracellular matrix production. Moreover, Jnk1 ablation caused a reduced lifespan and poor differentiation of HSCs into matrix-producing myofibroblasts. CONCLUSIONS: Jnk1 in HSCs, but not in hepatocytes, significantly contribute to liver fibrosis development, identifying Jnk1 in HSCs as a profibrotic kinase and a promising cell-directed target for liver fibrosis.

Isoastragaloside I attenuates cholestatic liver diseases by ameliorating liver injury, regulating bile acid metabolism and restoring intestinal barrier.

ETHNOPHARMACOLOGICAL RELEVANCE: Cholestatic liver diseases (CLD) are liver disorders resulting from abnormal bile formation, secretion, and excretion from various causes. Due to the lack of suitable and safe medications, liver transplantation is the ultimate treatment for CLD patients. Isoastragaloside I (IAS I) is one of the main saponin found in Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao or Astragalus membranaceus (Fisch.) Bge, which has been demonstrated to obviously alleviate CLD. Nevertheless, the IAS I's specific anti-CLD mechanism remains undecipherable. AIM OF THE STUDY: This study's purpose was to elucidate the protective consequence of IAS I on 0.1% 3, 5-diethoxycarbonyl-1,4-dihydroxychollidine (DDC) diet-induced CLD mice, and to reveal its potential mechanism. MATERIALS AND METHODS: In this study, mice with CLD that had been fed a 0.1% DDC diet were distributed two doses of IAS I (20 mg/kg, 50 mg/kg). The effects of IAS I on CLD models were investigated by assessing blood biochemistry, liver histology, and Hyp concentrations. We investigated markers of liver fibrosis and ductular reaction using immunohistochemistry, Western blot, and qRT-PCR. Liver inflammation indicators, arachidonic acid (ARA), and ω-3 fatty acid (FA) metabolites were also analyzed. Quantitative determination of 39 bile acids (BAs) in different organs employing UHPLC-Q-Exactive Orbitrap HRMS technology. Additionally, the H&E and Western blot analysis were used to evaluate differences in intestinal barrier function in DDC-induced mice before and after administering IAS I. RESULTS: After treatment with IAS I, serum biochemical indicators and liver hydroxyproline (Hyp) increased in a dose-dependent manner in CLD mice. The IAS I group showed significant improvement in indicators of liver fibrosis and ductular response, including as α-smooth muscle actin (α-SMA) and cytokeratin 19 (CK19), and transforming growth factor-ß (TGF-ß)/Smads signaling pathway. And inflammatory factors: F4/80, tumor necrosis factor-α (TNF-α), Interleukin-1ß (IL-1ß), ARA and ω-3 FA metabolites showed significant improvement following IAS I treatment. Moreover, IAS I significantly ameliorated liver tau-BAs levels, particularly TCA, THCA, THDCA, TCDCA, and TDCA contents, which were associated with enhanced expression of hepatic farnesoid X receptor (FXR), small heterodimer partner (SHP), cholesterol 7α-hydroxylase (Cyp7a1), and bile-salt export pump (BSEP). Furthermore, IAS I significantly improved pathological changes and protein expression related to intestinal barrier function, including zonula occludens protein 1 (ZO-1), Muc2, and Occludin. CONCLUSIONS: IAS I alleviated cholestatic liver injury, relieved inflammation, improved the altered tau-BAs metabolism and restored intestinal barrier function to protect against DDC-induced cholestatic liver diseases.

Pruritus in primary biliary cholangitis is under-recorded in patient medical records.

OBJECTIVE: Cholestatic pruritus in primary biliary cholangitis (PBC) reduces patients' health-related quality of life (HRQoL). Despite this, existing research suggests that pruritus is under-recorded in patients' health records. This study assessed the extent to which pruritus was recorded in medical records of patients with PBC as compared with patient-reported pruritus, and whether patients reporting mild itch were less likely to have pruritus recorded. We also evaluated clinico-demographic characteristics and HRQoL of patients with medical record-documented and patient-reported pruritus. DESIGN: This cross-sectional study used clinical information abstracted from medical records, together with patient-reported (PBC-40) data from patients with PBC in the USA enrolled in the PicnicHealth cohort. Medical record-documented pruritus was classified as 'recent' (at, or within 12 months prior to, enrolment) or 'ever' (at, or any point prior to, enrolment). Patient-reported pruritus (4-week recall) was assessed using the first PBC-40 questionnaire completed on/after enrolment; pruritus severity was classified by itch domain score (any severity: ≥1; clinically significant itch: ≥7). Patient clinico-demographic characteristics and PBC-40 domain scores were described in patients with medical record-documented and patient-reported pruritus; overlap between groups was evaluated. Descriptive statistics were reported. RESULTS: Pruritus of any severity was self-reported by 200/225 (88.9%) patients enrolled; however, only 88/225 (39.1%) had recent medical record-documented pruritus. Clinically significant pruritus was self-reported by 120/225 (53.3%) patients; of these, 64/120 (53.3%) had recent medical record-documented pruritus. Patients reporting clinically significant pruritus appeared to have higher mean scores across PBC-40 domains (indicating reduced HRQoL), versus patients with no/mild patient-reported pruritus or medical-record documented pruritus. CONCLUSION: Compared with patient-reported measures, pruritus in PBC is under-recorded in medical records and is associated with lower HRQoL. Research based only on medical records underestimates the true burden of pruritus, meaning physicians may be unaware of the extent and impact of pruritus, leading to potential undertreatment.

Causal associations between gut microbiota and Cholestatic liver diseases: a Mendelian randomization study.

Background: The etiological factors of Cholestatic Liver Diseases especially primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC) are not fully illustrated. It has been reported in previous observational studies that gut microbiota are associated with cholestatic liver diseases. However, there is uncertainty regarding the causality of this association. By using Mendelian randomization, this study aimed to examine the causal impact of gut microbiota on cholestatic liver diseases. Methods: From large-scale genome-wide association studies, genetic instruments for each gut microbiota taxa as well as primary biliary cholangitis and primary sclerosing cholangitis were developed. Subsequently, we conducted a two-sample Mendelian randomization analysis, supplemented by multiple post hoc sensitivity analyses. Additionally, we performed reverse MR analyses to investigate the possibility of the reverse causal association. Result: This two-sample MR study indicated that the order Bacillales, family Peptostreptococcaceae, family Ruminococcaceae, genus Anaerotruncu was associated with a decreased risk of developing PBC, and that order Selenomonadales, family Bifidobacteriaceae may be factors that increase the risk of PBC. On the other hand, we also identified order Selenomonadales, family Rhodospirillaceae, and genus RuminococcaceaeUCG013 were positively associated with PSC. The order Actinomycetales, family Actinomycetaceae, genus Actinomyces, genus Alloprevotella, genus Barnesiella, and genus Peptococcus were found negative associations with the risk of PSC. The reverse MR analysis demonstrated no statistically significant relationship between PBC, PSC and these specific gut microbial taxa. Conclusion: Our findings offered novel evidence that the abundance of particular bacteria contributes to the risk of PBC and PSC, which may contribute to more effective approaches to PBC and PSC therapy and prevention.