Prolonged administration of a secretin receptor antagonist inhibits biliary senescence and liver fibrosis in Mdr2 -/- mice.

BACKGROUND AND AIMS: Secretin (SCT) and secretin receptor (SR, only expressed on cholangiocytes within the liver) play key roles in modulating liver phenotypes. Forkhead box A2 (FoxA2) is required for normal bile duct homeostasis by preventing the excess of cholangiocyte proliferation. Short-term administration of the SR antagonist (SCT 5-27) decreased ductular reaction and liver fibrosis in bile duct ligated and Mdr2 -/- [primary sclerosing cholangitis (PSC), model] mice. We aimed to evaluate the effectiveness and risks of long-term SCT 5-27 treatment in Mdr2 -/- mice. APPROACH AND RESULTS: In vivo studies were performed in male wild-type and Mdr2 -/- mice treated with saline or SCT 5-27 for 3 months and human samples from late-stage PSC patients and healthy controls. Compared with controls, biliary SCT/SR expression and SCT serum levels increased in Mdr2 -/- mice and late-stage PSC patients. There was a significant increase in ductular reaction, biliary senescence, liver inflammation, angiogenesis, fibrosis, biliary expression of TGF-ß1/VEGF-A axis, and biliary phosphorylation of protein kinase A and ERK1/2 in Mdr2 -/- mice. The biliary expression of miR-125b and FoxA2 decreased in Mdr2 -/- compared with wild-type mice, which was reversed by long-term SCT 5-27 treatment. In vitro , SCT 5-27 treatment of a human biliary PSC cell line decreased proliferation and senescence and SR/TGF-ß1/VEGF-A axis but increased the expression of miR-125b and FoxA2. Downregulation of FoxA2 prevented SCT 5-27-induced reduction in biliary damage, whereas overexpression of FoxA2 reduced proliferation and senescence in the human PSC cell line. CONCLUSIONS: Modulating the SCT/SR axis may be critical for managing PSC.

p16 INK4A drives nonalcoholic fatty liver disease phenotypes in high fat diet fed mice through biliary E2F1/FOXO1/IGF-1 signaling.

BACKGROUND AND AIMS: NAFLD is characterized by steatosis, hepatic inflammation, and fibrosis, which can develop into NASH. Patients with NAFLD/NASH have increased ductular reaction (DR) and biliary senescence. High fat/high cholesterol diet feeding increases biliary senescence, DR, and biliary insulin-like growth factor-1 (IGF-1) expression in mice. p16/IGF-1 converges with fork-head box transcription factor O1 (FOXO1) through E2F1. We evaluated p16 inhibition on NAFLD phenotypes and biliary E2F1/FOXO1/IGF-1 signaling. APPROACH AND RESULTS: 4-week wild-type (C57BL/6J) male mice were fed a control diet (CD) or high fat/high cholesterol diet and received either p16 or control Vivo Morpholino (VM) by tail vein injection 2× during the 16th week of feeding. We confirmed p16 knockdown and examined: (i) NAFLD phenotypes; (ii) DR and biliary senescence; (iii) serum metabolites; and (iv) biliary E2F1/FOXO1/IGF-1 signaling. Human normal, NAFLD, and NASH liver samples and isolated cholangiocytes treated with control or p16 VM were evaluated for p16/E2F1/FOXO1/IGF-1 signaling. p16 VM treatment reduced cholangiocyte and hepatocyte p16. In wild-type high fat/high cholesterol diet mice with control VM, there were increased (i) NAFLD phenotypes; (ii) DR and biliary senescence; (iii) serum metabolites; and (iv) biliary E2F1/FOXO1/IGF-1 signaling; however, p16 VM treatment reduced these parameters. Biliary E2F1/FOX-O1/IGF-1 signaling increased in human NAFLD/NASH but was blocked by p16 VM. In vitro , p16 VM reduced biliary E2f1 and Foxo1 transcription by inhibiting RNA pol II binding and E2F1 binding at the Foxo1 locus, respectively. Inhibition of E2F1 reduced biliary FOXO1 in vitro. CONCLUSION: Attenuating hepatic p16 expression may be a therapeutic approach for improving NAFLD/NASH phenotypes.

Secretin alleviates biliary and liver injury during late-stage primary biliary cholangitis via restoration of secretory processes.

BACKGROUND & AIMS: Primary biliary cholangitis (PBC) is characterised by ductopenia, ductular reaction, impairment of anion exchanger 2 (AE2) and the 'bicarbonate umbrella'. Ductulo-canalicular junction (DCJ) derangement is hypothesised to promote PBC progression. The secretin (Sct)/secretin receptor (SR) axis regulates cystic fibrosis transmembrane receptor (CFTR) and AE2, thus promoting choleresis. We evaluated the role of Sct/SR signalling on biliary secretory processes and subsequent injury in a late-stage PBC mouse model and human samples. METHODS: At 32 weeks of age, female and male wild-type and dominant-negative transforming growth factor beta receptor II (late-stage PBC model) mice were treated with Sct for 1 or 8 weeks. Bulk RNA-sequencing was performed in isolated cholangiocytes from mouse models. RESULTS: Biliary Sct/SR/CFTR/AE2 expression and bile bicarbonate levels were reduced in late-stage PBC mouse models and human samples. Sct treatment decreased bile duct loss, ductular reaction, inflammation, and fibrosis in late-stage PBC models. Sct reduced hepatic bile acid levels, modified bile acid composition, and restored the DCJ and 'bicarbonate umbrella'. RNA-sequencing identified that Sct promoted mature epithelial marker expression, specifically anterior grade protein 2 (Agr2). Late-stage PBC models and human samples exhibited reduced biliary mucin 1 levels, which were enhanced by Sct treatment. CONCLUSION: Loss of Sct/SR signalling in late-stage PBC results in a faulty 'bicarbonate umbrella' and reduced Agr2-mediated mucin production. Sct restores cholangiocyte secretory processes and DCJ formation through enhanced mature cholangiocyte phenotypes and bile duct growth. Sct treatment may be beneficial for individuals with late-stage PBC. IMPACT AND IMPLICATIONS: Secretin (Sct) regulates biliary proliferation and bicarbonate secretion in cholangiocytes via its receptor, SR, and in mouse models and human samples of late-stage primary biliary cholangitis (PBC), the Sct/SR axis is blunted along with loss of the protective 'bicarbonate umbrella'. We found that both short- and long-term Sct treatment ameliorated ductular reaction, immune cell influx, and liver fibrosis in late-stage PBC mouse models. Importantly, Sct treatment promoted bicarbonate and mucin secretion and hepatic bile acid efflux, thus reducing cholestatic and toxic bile acid-associated injury in late-stage PBC mouse models. Our work perpetuates the hypothesis that PBC pathogenesis hinges on secretory defects, and restoration of secretory processes that promote the 'bicarbonate umbrella' may be important for amelioration of PBC-associated damage.

Loss of apical sodium bile acid transporter alters bile acid circulation and reduces biliary damage in cholangitis.

Primary sclerosing cholangitis (PSC) is characterized by increased ductular reaction (DR), liver fibrosis, hepatic total bile acid (TBA) levels, and mast cell (MC) infiltration. Apical sodium BA transporter (ASBT) expression increases in cholestasis, and ileal inhibition reduces PSC phenotypes. FVB/NJ and multidrug-resistant 2 knockout (Mdr2-/-) mice were treated with control or ASBT Vivo-Morpholino (VM). We measured 1) ASBT expression and MC presence in liver/ileum; 2) liver damage/DR; 3) hepatic fibrosis/inflammation; 4) biliary inflammation/histamine serum content; and 5) gut barrier integrity/hepatic bacterial translocation. TBA/BA composition was measured in cholangiocyte/hepatocyte supernatants, intestine, liver, serum, and feces. Shotgun analysis was performed to ascertain microbiome changes. In vitro, cholangiocytes were treated with BAs ± ASBT VM, and histamine content and farnesoid X receptor (FXR) signaling were determined. Treated cholangiocytes were cocultured with MCs, and FXR signaling, inflammation, and MC activation were measured. Human patients were evaluated for ASBT/MC expression and histamine/TBA content in bile. Control patient- and PSC patient-derived three-dimensional (3-D) organoids were generated; ASBT, chymase, histamine, and fibroblast growth factor-19 (FGF19) were evaluated. ASBT VM in Mdr2-/- mice decreased 1) biliary ASBT expression, 2) PSC phenotypes, 3) hepatic TBA, and 4) gut barrier integrity compared with control. We found alterations between wild-type (WT) and Mdr2-/- mouse microbiome, and ASBT/MC and bile histamine content increased in cholestatic patients. BA-stimulated cholangiocytes increased MC activation/FXR signaling via ASBT, and human PSC-derived 3-D organoids secrete histamine/FGF19. Inhibition of hepatic ASBT ameliorates cholestatic phenotypes by reducing cholehepatic BA signaling, biliary inflammation, and histamine levels. ASBT regulation of hepatic BA signaling offers a therapeutic avenue for PSC.NEW & NOTEWORTHY We evaluated knockdown of the apical sodium bile acid transporter (ASBT) using Vivo-Morpholino in Mdr2KO mice. ASBT inhibition decreases primary sclerosing cholangitis (PSC) pathogenesis by reducing hepatic mast cell infiltration, altering bile acid species/cholehepatic shunt, and regulating gut inflammation/dysbiosis. Since a large cohort of PSC patients present with IBD, this study is clinically important. We validated findings in human PSC and PSC-IBD along with studies in novel human 3-D organoids formed from human PSC livers.

The Functional Roles of Immune Cells in Primary Liver Cancer.

Primary liver cancer includes hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA). Incidence of liver cancer has been increasing in recent years, and the 5-year survival is <20%. HCC and CCA are often accompanied with a dense stroma coupled with infiltrated immune cells, which is referred to as the tumor microenvironment. Populations of specific immune cells, such as high density of CD163+ macrophages and low density of CD8+ T cells, are associated with prognosis and survival rates in both HCC and CCA. Immune cells in the tumor microenvironment can be a therapeutic target for liver cancer treatments. Previous studies have introduced immunotherapy using immune checkpoint inhibitors, pulsed dendritic cells, or transduced T cells, to enhance cytotoxicity of immune cells and inhibit tumor growth. This review summarizes current understanding of the roles of immune cells in primary liver cancer covering HCC and CCA.

Melatonin receptor 1A, but not 1B, knockout decreases biliary damage and liver fibrosis during cholestatic liver injury.

BACKGROUND AND AIMS: Melatonin reduces biliary damage and liver fibrosis in cholestatic models by interaction with melatonin receptors 1A (MT1) and 1B (MT2). MT1 and MT2 can form heterodimers and homodimers, but MT1 and MT2 can heterodimerize with the orphan receptor G protein-coupled receptor 50 (GPR50). MT1/GPR50 dimerization blocks melatonin binding, but MT2/GPR50 dimerization does not affect melatonin binding. GPR50 can dimerize with TGFß receptor type I (TGFßRI) to activate this receptor. We aimed to determine the differential roles of MT1 and MT2 during cholestasis. APPROACH AND RESULTS: Wild-type (WT), MT1 knockout (KO), MT2KO, and MT1/MT2 double KO (DKO) mice underwent sham or bile duct ligation (BDL); these mice were also treated with melatonin. BDL WT and multidrug resistance 2 KO (Mdr2-/- ) mice received mismatch, MT1, or MT2 Vivo-Morpholino. Biliary expression of MT1 and GPR50 increases in cholestatic rodents and human primary sclerosing cholangitis (PSC) samples. Loss of MT1 in BDL and Mdr2-/- mice ameliorated biliary and liver damage, whereas these parameters were enhanced following loss of MT2 and in DKO mice. Interestingly, melatonin treatment alleviated BDL-induced biliary and liver injury in BDL WT and BDL MT2KO mice but not in BDL MT1KO or BDL DKO mice, demonstrating melatonin's interaction with MT1. Loss of MT2 or DKO mice exhibited enhanced GPR50/TGFßR1 signaling, which was reduced by loss of MT1. CONCLUSIONS: Melatonin ameliorates liver phenotypes through MT1, whereas down-regulation of MT2 promotes liver damage through GPR50/TGFßR1 activation. Blocking GPR50/TGFßR1 binding through modulation of melatonin signaling may be a therapeutic approach for PSC.

Mast Cells Promote Nonalcoholic Fatty Liver Disease Phenotypes and Microvesicular Steatosis in Mice Fed a Western Diet.

BACKGROUND AND AIMS: Nonalcoholic fatty liver disease (NAFLD) is simple steatosis but can develop into nonalcoholic steatohepatitis (NASH), characterized by liver inflammation, fibrosis, and microvesicular steatosis. Mast cells (MCs) infiltrate the liver during cholestasis and promote ductular reaction (DR), biliary senescence, and liver fibrosis. We aimed to determine the effects of MC depletion during NAFLD/NASH. APPROACH AND RESULTS: Wild-type (WT) and KitW-sh (MC-deficient) mice were fed a control diet (CD) or a Western diet (WD) for 16 weeks; select WT and KitW-sh WD mice received tail vein injections of MCs 2 times per week for 2 weeks prior to sacrifice. Human samples were collected from normal, NAFLD, or NASH mice. Cholangiocytes from WT WD mice and human NASH have increased insulin-like growth factor 1 expression that promotes MC migration/activation. Enhanced MC presence was noted in WT WD mice and human NASH, along with increased DR. WT WD mice had significantly increased steatosis, DR/biliary senescence, inflammation, liver fibrosis, and angiogenesis compared to WT CD mice, which was significantly reduced in KitW-sh WD mice. Loss of MCs prominently reduced microvesicular steatosis in zone 1 hepatocytes. MC injection promoted WD-induced biliary and liver damage and specifically up-regulated microvesicular steatosis in zone 1 hepatocytes. Aldehyde dehydrogenase 1 family, member A3 (ALDH1A3) expression is reduced in WT WD mice and human NASH but increased in KitW-sh WD mice. MicroRNA 144-3 prime (miR-144-3p) expression was increased in WT WD mice and human NASH but reduced in KitW-sh WD mice and was found to target ALDH1A3. CONCLUSIONS: MCs promote WD-induced biliary and liver damage and may promote microvesicular steatosis development during NAFLD progression to NASH through miR-144-3p/ALDH1A3 signaling. Inhibition of MC activation may be a therapeutic option for NAFLD/NASH treatment.

Inhibition of Secretin/Secretin Receptor Axis Ameliorates NAFLD Phenotypes.

BACKGROUND AND AIMS: Human NAFLD is characterized at early stages by hepatic steatosis, which may progress to NASH when the liver displays microvesicular steatosis, lobular inflammation, and pericellular fibrosis. The secretin (SCT)/secretin receptor (SCTR) axis promotes biliary senescence and liver fibrosis in cholestatic models through down-regulation of miR-125b signaling. We aim to evaluate the effect of disrupting biliary SCT/SCTR/miR-125b signaling on hepatic steatosis, biliary senescence, and liver fibrosis in NAFLD/NASH. APPROACH AND RESULTS: In vivo, 4-week-old male wild-type, Sct-/- and Sctr-/- mice were fed a control diet or high-fat diet (HFD) for 16 weeks. The expression of SCT/SCTR/miR-125b axis was measured in human NAFLD/NASH liver samples and HFD mouse livers by immunohistochemistry and quantitative PCR. Biliary/hepatocyte senescence, ductular reaction, and liver angiogenesis were evaluated in mouse liver and human NAFLD/NASH liver samples. miR-125b target lipogenesis genes in hepatocytes were screened and validated by custom RT2 Profiler PCR array and luciferase assay. Biliary SCT/SCTR expression was increased in human NAFLD/NASH samples and in livers of HFD mice, whereas the expression of miR-125b was decreased. Biliary/hepatocyte senescence, ductular reaction, and liver angiogenesis were observed in human NAFLD/NASH samples as well as HFD mice, which were decreased in Sct-/- and Sctr-/- HFD mice. Elovl1 is a lipogenesis gene targeted by miR-125b, and its expression was also decreased in HFD mouse hepatocytes following Sct or Sctr knockout. Bile acid profile in fecal samples have the greatest changes between wild-type mice and Sct-/- /Sctr-/- mice. CONCLUSION: The biliary SCT/SCTR/miR-125b axis promotes liver steatosis by up-regulating lipid biosynthesis gene Elovl1. Targeting the biliary SCT/SCTR/miR-125b axis may be key for ameliorating phenotypes of human NAFLD/NASH.

Kupffer Cells: Inflammation Pathways and Cell-Cell Interactions in Alcohol-Associated Liver Disease.

Chronic alcohol consumption is linked to the development of alcohol-associated liver disease (ALD). This disease is characterized by a clinical spectrum ranging from steatosis to hepatocellular carcinoma. Several cell types are involved in ALD progression, including hepatic macrophages. Kupffer cells (KCs) are the resident macrophages of the liver involved in the progression of ALD by activating pathways that lead to the production of cytokines and chemokines. In addition, KCs are involved in the production of reactive oxygen species. Reactive oxygen species are linked to the induction of oxidative stress and inflammation in the liver. These events are activated by the bacterial endotoxin, lipopolysaccharide, that is released from the gastrointestinal tract through the portal vein to the liver. Lipopolysaccharide is recognized by receptors on KCs that are responsible for triggering several pathways that activate proinflammatory cytokines involved in alcohol-induced liver injury. In addition, KCs activate hepatic stellate cells that are involved in liver fibrosis. Novel strategies to treat ALD aim at targeting Kupffer cells. These interventions modulate Kupffer cell activation or macrophage polarization. Evidence from mouse models and early clinical studies in patients with ALD injury supports the notion that pathogenic macrophage subsets can be successfully translated into novel treatment options for patients with this disease.

Knockout of the Tachykinin Receptor 1 in the Mdr2-/- (Abcb4-/-) Mouse Model of Primary Sclerosing Cholangitis Reduces Biliary Damage and Liver Fibrosis.

Activation of the substance P (SP)/neurokinin 1 receptor (NK1R) axis triggers biliary damage/senescence and liver fibrosis in bile duct ligated and Mdr2-/- (alias Abcb4-/-) mice through enhanced transforming growth factor-ß1 (TGF-ß1) biliary secretion. Recent evidence indicates a role for miR-31 (MIR31) in TGF-ß1-induced liver fibrosis. We aimed to define the role of the SP/NK1R/TGF-ß1/miR-31 axis in regulating biliary proliferation and liver fibrosis during cholestasis. Thus, we generated a novel model with double knockout of Mdr2-/- and NK1R-/ (alias Tacr1-/-) to further address the role of the SP/NK1R axis during chronic cholestasis. In vivo studies were performed in the following 12-week-old male mice: (i) NK1R-/-; (ii) Mdr2-/-; and (iii) NK1R-/-/Mdr2-/- (Tacr1-/-/Abcb4-/-) and their corresponding wild-type controls. Liver tissues and cholangiocytes were collected, and liver damage, changes in biliary mass/senescence, and inflammation as well as liver fibrosis were evaluated by both immunohistochemistry in liver sections and real-time PCR. miR-31 expression was measured by real-time PCR in isolated cholangiocytes. Decreased ductular reaction, liver fibrosis, biliary senescence, and biliary inflammation were observed in NK1R-/-/Mdr2-/- mice compared with Mdr2-/- mice. Elevated expression of miR-31 was observed in Mdr2-/- mice, which was reduced in NK1R-/-/Mdr2-/- mice. Targeting the SP/NK1R and/or miR-31 may be a potential approach in treating human cholangiopathies, including primary sclerosing cholangitis.

Modulation of the Tryptophan Hydroxylase 1/Monoamine Oxidase-A/5-Hydroxytryptamine/5-Hydroxytryptamine Receptor 2A/2B/2C Axis Regulates Biliary Proliferation and Liver Fibrosis During Cholestasis.

BACKGROUND AND AIMS: Serotonin (5HT) is a neuroendocrine hormone synthetized in the central nervous system (CNS) as well as enterochromaffin cells of the gastrointestinal tract. Tryptophan hydroxylase (TPH1) and monoamine oxidase (MAO-A) are the key enzymes for the synthesis and catabolism of 5HT, respectively. Previous studies demonstrated that 5-hydroxytryptamine receptor (5HTR)1A/1B receptor agonists inhibit biliary hyperplasia in bile-duct ligated (BDL) rats, whereas 5HTR2B receptor antagonists attenuate liver fibrosis (LF) in mice. Our aim was to evaluate the role of 5HTR2A/2B/2C agonists/antagonists in cholestatic models. APPROACH AND RESULTS: While in vivo studies were performed in BDL rats and the multidrug resistance gene 2 knockout (Mdr2-/- ) mouse model of PSC, in vitro studies were performed in cell lines of cholangiocytes and hepatic stellate cells (HSCs). 5HTR2A/2B/2C and MAO-A/TPH1 are expressed in cholangiocytes and HSCs from BDL rats and Mdr2-/- - mice. Ductular reaction, LF, as well as the mRNA expression of proinflammatory genes increased in normal, BDL rats, and Mdr2-/- - mice following treatment 5HTR2A/2B/2C agonists, but decreased when BDL rats and Mdr2-/- mice were treated with 5HTR2A/2B/2C antagonists compared to BDL rats and Mdr2-/- mice, respectively. 5HT levels increase in Mdr2-/- mice and in PSC human patients compared to their controls and decrease in serum of Mdr2-/- mice treated with 5HTR2A/2B/2C antagonists compared to untreated Mdr2-/- mice. In vitro, cell lines of murine cholangiocytes and human HSCs express 5HTR2A/2B/2C and MAO-A/TPH1; treatment of these cell lines with 5HTR2A/2B/2C antagonists or TPH1 inhibitor decreased 5HT levels as well as expression of fibrosis and inflammation genes compared to controls. CONCLUSIONS: Modulation of the TPH1/MAO-A/5HT/5HTR2A/2B/2C axis may represent a therapeutic approach for management of cholangiopathies, including PSC.

Correction to: Knockout of α-calcitonin gene-related peptide attenuates cholestatic liver injury by differentially regulating cellular senescence of hepatic stellate cells and cholangiocytes.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Amelioration of Ductular Reaction by Stem Cell Derived Extracellular Vesicles in MDR2 Knockout Mice via Lethal-7 microRNA.

Cholangiopathies are diseases that affect cholangiocytes, the cells lining the biliary tract. Liver stem cells (LSCs) are able to differentiate into all cells of the liver and possibly influence the surrounding liver tissue by secretion of signaling molecules. One way in which cells can interact is through secretion of extracellular vesicles (EVs), which are small membrane-bound vesicles that contain proteins, microRNAs (miRNAs), and cytokines. We evaluated the contents of liver stem cell-derived EVs (LSCEVs), compared their miRNA contents to those of EVs isolated from hepatocytes, and evaluated the downstream targets of these miRNAs. We finally evaluated the crosstalk among LSCs, cholangiocytes, and human hepatic stellate cells (HSCs). We showed that LSCEVs were able to reduce ductular reaction and biliary fibrosis in multidrug resistance protein 2 (MDR2)-/- mice. Additionally, we showed that cholangiocyte growth was reduced and HSCs were deactivated in LSCEV-treated mice. Evaluation of LSCEV contents compared with EVs derived from hepatocytes showed a large increase in the miRNA, lethal-7 (let-7). Further evaluation of let-7 in MDR2-/- mice and human primary sclerosing cholangitis samples showed reduced levels of let-7 compared with controls. In liver tissues and isolated cholangiocytes, downstream targets of let-7 (identified by ingenuity pathway analysis), Lin28a (Lin28 homolog A), Lin28b (Lin28 homolog B), IL-13 (interleukin 13), NR1H4 (nuclear receptor subfamily 1 group H member 4) and NF-κB (nuclear factor kappa B), are elevated in MDR2-/- mice, but treatment with LSCEVs reduced levels of these mediators of ductular reaction and biliary fibrosis through the inhibition of NF-κB and IL-13 signaling pathways. Evaluation of crosstalk using cholangiocyte supernatants from LSCEV-treated cells on cultured HSCs showed that HSCs had reduced levels of fibrosis and increased senescence. Conclusion: Our studies indicate that LSCEVs could be a possible treatment for cholangiopathies or could be used for target validation for future therapies.

Knockout of α-calcitonin gene-related peptide attenuates cholestatic liver injury by differentially regulating cellular senescence of hepatic stellate cells and cholangiocytes.

α-Calcitonin gene-related peptide (α-CGRP) is a 37-amino acid neuropeptide involved in several pathophysiological processes. α-CGRP is involved in the regulation of cholangiocyte proliferation during cholestasis. In this study, we aimed to evaluate if α-CGRP regulates bile duct ligation (BDL)-induced liver fibrosis by using a α-CGRP knockout (α-CGRP-/-) mouse model. α-CGRP-/- and wild-type (WT) mice were subjected to sham surgery or BDL for 7 days. Then, liver fibrosis and cellular senescence as well as the expression of kinase such as p38 and C-Jun N-terminal protein kinase (JNK) in mitogen-activated protein kinases (MAPK) signaling pathway were evaluated in total liver, together with measurement of cellular senescence in cholangiocytes or hepatic stellate cells (HSCs). There was enhanced hepatic expression of Calca (coding α-CGRP) and the CGRP receptor components (CRLR, RAMP-1 and RCP) in BDL and in both WT α-CGRP-/- and BDL α-CGRP-/- mice, respectively. Moreover, there was increased CGRP serum levels and hepatic mRNA expression of CALCA and CGRP receptor components in late-stage PSC samples compared to healthy control samples. Depletion of α-CGRP reduced liver injury and fibrosis in BDL mice that was associated with enhanced cellular senescence of hepatic stellate cells and reduced senescence of cholangiocytes as well as decreased activation of p38 and JNK MAPK signaling pathway. Cholangiocyte supernatant from BDL α-CGRP-/- mice inhibited the activation and increased cellular senescence of cultured human HSCs (HHSCs) compared to HHSCs stimulated with BDL cholangiocyte supernatant. Taken together, endogenous α-CGRP promoted BDL-induced cholestatic liver fibrosis through differential changes in senescence of HSCs and cholangiocytes and activation of p38 and JNK signaling. Modulation of α-CGRP/CGRP receptor signaling may be key for the management of biliary senescence and liver fibrosis in cholangiopathies.

Possible application of melatonin treatment in human diseases of the biliary tract.

Melatonin was discovered in 1958 by Aaron Lerner. Its name comes from the ability of melatonin to change the shape of amphibian melanophores from stellate to roundish. Starting from the 1980s, the role of melatonin in the regulation of mammalian circadian and seasonal clocks has been elucidated. Presently, several other effects have been identified in different organs. For example, the beneficial effects of melatonin in models of liver damage have been described. This review gives first a general background on experimental and clinical data on the use of melatonin in liver damage. The second part of the review focuses on the findings related to the role of melatonin in biliary functions, suggesting a possible use of melatonin therapy in human diseases of the biliary tree.

Cellular Interactions and Crosstalk Facilitating Biliary Fibrosis in Cholestasis.

Biliary fibrosis is seen in cholangiopathies, including primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC). In PBC and PSC, biliary fibrosis is associated with worse outcomes and histologic scores. Within the liver, both hepatic stellate cells (HSCs) and portal fibroblasts (PFs) contribute to biliary fibrosis, but their roles can differ. PFs reside near the bile ducts and may be the first responders to biliary damage, whereas HSCs may be recruited later and initiate bridging fibrosis. Indeed, different models of biliary fibrosis can activate PFs and HSCs to varying degrees. The portal niche can be composed of cholangiocytes, HSCs, PFs, endothelial cells, and various immune cells, and interactions between these cell types drive biliary fibrosis. In this review, we discuss the mechanisms of biliary fibrosis and the roles of PFs and HSCs in this process. We will also evaluate cellular interactions and mechanisms that contribute to biliary fibrosis in different models and highlight future perspectives and potential therapeutics.

Endoplasmic reticulum stress: A possible connection between intestinal inflammation and neurodegenerative disorders.

BACKGROUND: Different studies have shown the key role of endoplasmic reticulum (ER) stress in autoimmune and chronic inflammatory disorders, as well as in neurodegenerative diseases. ER stress leads to the formation of misfolded proteins which affect the secretion of different cell types that are crucial for the intestinal homeostasis. PURPOSE: In this review, we discuss the role of ER stress and its involvement in the development of inflammatory bowel diseases, chronic conditions that can cause severe damage of the gastrointestinal tract, focusing on the alteration of Paneth cells and goblet cells (the principal secretory phenotypes of the intestinal epithelial cells). ER stress is also discussed in the context of neurodegenerative diseases, in which protein misfolding represents the signature mechanism. ER stress in the bowel and consequent accumulation of misfolded proteins might represent a bridge between bowel inflammation and neurodegeneration along the gut-to-brain axis, affecting intestinal epithelial homeostasis and the equilibrium of the commensal microbiota. Targeting intestinal ER stress could foster future studies for designing new biomarkers and new therapeutic approaches for neurodegenerative disorders.

Gallstone and Gallbladder Disease: Biliary Tract and Cholangiopathies.

Cholestatic liver diseases are named primarily due to the blockage of bile flow and buildup of bile acids in the liver. Cholestasis can occur in cholangiopathies, fatty liver diseases, and during COVID-19 infection. Most literature evaluates damage occurring to the intrahepatic biliary tree during cholestasis; however, there may be associations between liver damage and gallbladder damage. Gallbladder damage can manifest as acute or chronic inflammation, perforation, polyps, cancer, and most commonly gallstones. Considering the gallbladder is an extension of the intrahepatic biliary network, and both tissues are lined by biliary epithelial cells that share common mechanisms and properties, it is worth further evaluation to understand the association between bile duct and gallbladder damage. In this comprehensive article, we discuss background information of the biliary tree and gallbladder, from function, damage, and therapeutic approaches. We then discuss published findings that identify gallbladder disorders in various liver diseases. Lastly, we provide the clinical aspect of gallbladder disorders in liver diseases and ways to enhance diagnostic and therapeutic approaches for congruent diagnosis. © 2023 American Physiological Society. Compr Physiol 13:4909-4943, 2023.