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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.

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 Kit W-sh (MC-deficient) mice were fed a control diet (CD) or a Western diet (WD) for 16 weeks; select WT and Kit W-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 insulinlike 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 Kit W-sh WD mice. Loss of MCs prominently reduced microvesicular steatosis in zone 1 hepatocytes. MC injection promoted WDinduced 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 Kit W-sh WD mice. MicroRNA 144-3 prime (miR-144-3p) expression was increased in WT WD mice and human NASH but reduced in Kit W-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. (Hepatology 2021;74:164-182). N onalcoholic fatty liver disease (NAFLD) can develop into nonalcoholic steatohepatitis (NASH). (1) High mortality rates are seen in patients with NAFLD, and NASH is the third most common indication for liver transplantation in

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.

Biliary senescence and hepatic fibrosis are hallmarks of cholangiopathies including primary sclerosing cholangitis (PSC). Senescent cholangiocytes display senescence-associated secretory phenotypes (SASPs, e. g., TGF-β1) that further increase biliary senescence (by an autocrine loop) and trigger liver fibrosis by paracrine mechanisms. To determine the effect of p16 inhibition and role of the TGF-β1/miR-34a/SIRT1 axis in biliary damage and liver fibrosis in the Mdr2-/- mouse model of PSC. We treated (i) in vivo male wild-type (WT) and Mdr2-/- mice with p16 Vivo-Morpholino or controls before measuring biliary mass (IBDM) and senescence, biliary SASPs levels and liver fibrosis; and (ii) in vitro intrahepatic murine cholangiocyte lines (IMCLs) with siRNA against p16 before measuring the mRNA expression of proliferation, senescence and fibrosis markers. p16 and miR-34a increased but SIRT1 decreased in Mdr2-/- mice and PSC human liver samples compared to controls. P16 immunoreactivity and biliary senescence and SASPs levels increased in Mdr2-/- mice but decreased in Mdr2-/- mice treated with p16 Vivo-Morpholino. The increase in IBDM and hepatic fibrosis (observed in Mdr2-/- mice) returned to normal values in Mdr2-/- mice treated with p16 Vivo-Morpholino. TGF-β1 immunoreactivity and biliary SASPs levels were higher in Mdr2-/- compared to those of WT mice, but returned to normal values in Mdr2-/- mice treated with p16 Vivo-Morpholino. The expression of fibrosis/senescence markers decreased in cholangiocytes from Mdr2-/- mice treated with p16 Vivo-Morpholino (compared to Mdr2-/- mice) and in IMCLs (after p16 silencing) compared to controls. Modulation of the TGF-β1/miR-34a/SIRT1 axis may be important in the management of PSC phenotype.