Cholangiocyte-Derived Exosomal Long Noncoding RNA H19 Promotes Hepatic Stellate Cell Activation and Cholestatic Liver Fibrosis.

Activation of hepatic stellate cells (HSCs) represents the primary driving force to promote the progression of chronic cholestatic liver diseases. We previously reported that cholangiocyte-derived exosomal long noncoding RNA-H19 (lncRNA-H19) plays a critical role in promoting cholestatic liver injury. However, it remains unclear whether cholangiocyte-derived lncRNA-H19 regulates HSC activation, which is the major focus of this study. Both bile duct ligation (BDL) and Mdr2 knockout (Mdr2-/- ) mouse models were used. Wild-type and H19maternalΔExon1/+ (H19KO) mice were subjected to BDL. Mdr2-/- H19maternalΔExon1/+ (DKO) mice were generated. Exosomes isolated from cultured mouse and human cholangiocytes or mouse serum were used for in vivo transplantation and in vitro studies. Fluorescence-labeled exosomes and flow cytometry were used to monitor exosome uptake by hepatic cells. Collagen gel contraction and bromodeoxyuridine assays were used to determine the effect of exosomal-H19 on HSC activation and proliferation. Mouse and human primary sclerosing cholangitis (PSC)/primary biliary cholangitis (PBC) liver samples were analyzed by real-time PCR, western blot analysis, histology, and immunohistochemistry. The results demonstrated that hepatic H19 level was closely correlated with the severity of liver fibrosis in both mouse models and human patients with PSC and PBC. H19 deficiency significantly protected mice from liver fibrosis in BDL and Mdr2-/- mice. Transplanted cholangiocyte-derived H19-enriched exosomes were rapidly and preferentially taken up by HSCs and HSC-derived fibroblasts, and promoted liver fibrosis in BDL-H19KO mice and DKO mice. H19-enriched exosomes enhanced transdifferentiation of cultured mouse primary HSCs and promoted proliferation and matrix formation in HSC-derived fibroblasts. Conclusion: Cholangiocyte-derived exosomal H19 plays a critical role in the progression of cholestatic liver fibrosis by promoting HSC differentiation and activation and represents a potential diagnostic biomarker and therapeutic target for cholangiopathies.

C/EBP homologous protein-induced loss of intestinal epithelial stemness contributes to bile duct ligation-induced cholestatic liver injury in mice.

Impaired intestinal barrier function promotes the progression of various liver diseases, including cholestatic liver diseases. The close association of primary sclerosing cholangitis (PSC) with inflammatory bowel disease highlights the importance of the gut-liver axis. It has been reported that bile duct ligation (BDL)-induced liver fibrosis is significantly reduced in C/EBP homologous protein knockout (CHOP-/- ) mice. However, the underlying mechanisms remain unclear. In the current study, we demonstrate that BDL induces striking and acute hepatic endoplasmic reticulum (ER) stress responses after 1 day, which return to normal after 3 days. No significant hepatocyte apoptosis is detected 7-14 days following BDL. However, the inflammatory response is significantly increased after 7 days, which is similar to what we found in human PSC liver samples. BDL-induced loss of stemness in intestinal stem cells (ISCs), disruption of intestinal barrier function, bacterial translocation, activation of hepatic inflammation, M2 macrophage polarization and liver fibrosis are significantly reduced in CHOP-/- mice. In addition, intestinal organoids derived from CHOP-/- mice contain more and longer crypt structures than those from wild-type (WT) mice, which is consistent with the upregulation of stem cell markers (leucine-rich repeat-containing G-protein-coupled receptor 5, olfactomedin 4, and SRY [sex determining region Y]-box 9) and in vivo findings that CHOP-/- mice have longer villi and crypts as compared to WT mice. Similarly, mRNA levels of CD14, interleukin-1ß, tumor necrosis factor-alpha, and monocyte chemotactic protein-1 are increased and stem cell proliferation is suppressed in the duodenum of patients with cirrhosis. CONCLUSION: Activation of ER stress and subsequent loss of stemness of ISCs plays a critical role in BDL-induced systemic inflammation and cholestatic liver injury. Modulation of the ER stress response represents a potential therapeutic strategy for cholestatic liver diseases as well as other inflammatory diseases. (Hepatology 2018;67:1441-1457).

Continued Alcohol Misuse in Human Cirrhosis is Associated with an Impaired Gut-Liver Axis.

BACKGROUND: Cirrhosis and alcohol can independently affect the gut-liver axis with systemic inflammation. However, their concurrent impact in humans is unclear. METHODS: Our aim was to determine the effect of continued alcohol misuse on the gut-liver axis in cirrhotic patients. Age- and MELD-balanced cirrhotic patients who were currently drinking (Alc) or abstinent (NAlc) and healthy controls underwent serum and stool collection. A subset underwent upper endoscopy and colonoscopy for biopsies and duodenal fluid collection. The groups were compared regarding (i) inflammation/intestinal barrier: systemic tumor necrosis factor levels, intestinal inflammatory cytokine (duodenum, ileum, sigmoid), and ileal antimicrobial peptide expression; (ii) microbiota composition: 16SrRNA sequencing of duodenal, ileal, and colonic mucosal and fecal microbiota; and (iii) microbial functionality: duodenal fluid and fecal bile acid (BA) profile (conjugation and dehydroxylation status), intestinal BA transporter (ASBT, FXR, FGF-19, SHP) expression, and stool metabolomics using gas chromatography/mass spectrometry. RESULTS: Alc patients demonstrated a significant duodenal, ileal, and colonic mucosal and fecal dysbiosis, compared to NAlc and controls with lower autochthonous bacterial taxa. BA profile skewed toward a potentially toxic profile (higher secondary and glycine-conjugated BAs) in duodenal fluid and stool in Alc patients. Duodenal fluid demonstrated conjugated secondary BAs only in the Alc group. There was a greater expression of all ileal BA transporters in Alc patients. This group also showed higher endotoxemia, systemic and ileal inflammatory expression, and lower amino acid and bioenergetic-associated metabolites, without change in antimicrobial peptide expression. CONCLUSIONS: Despite cirrhosis, continued alcohol misuse predisposes patients to widespread dysbiosis with alterations in microbial functionality such as a toxic BA profile, which can lead to intestinal and systemic inflammation.

Therapeutic potential of berberine in attenuating cholestatic liver injury: insights from a PSC mouse model.

BACKGROUND AND AIMS: Primary sclerosing cholangitis (PSC) is a chronic liver disease characterized by progressive biliary inflammation and bile duct injury. Berberine (BBR) is a bioactive isoquinoline alkaloid found in various herbs and has multiple beneficial effects on metabolic and inflammatory diseases, including liver diseases. This study aimed to examine the therapeutic effect of BBR on cholestatic liver injury in a PSC mouse model (Mdr2-/- mice) and elucidate the underlying mechanisms. METHODS: Mdr2-/-mice (12-14 weeks old, both sexes) received either BBR (50 mg/kg) or control solution daily for eight weeks via oral gavage. Histological and serum biochemical analyses were used to assess fibrotic liver injury severity. Total RNAseq and pathway analyses were used to identify the potential signaling pathways modulated by BBR in the liver. The expression levels of key genes involved in regulating hepatic fibrosis, bile duct proliferation, inflammation, and bile acid metabolism were validated by qRT-PCR or Western blot analysis. The bile acid composition and levels in the serum, liver, small intestine, and feces and tissue distribution of BBR were measured by LC-MS/MS. Intestinal inflammation and injury were assessed by gene expression profiling and histological analysis. The impact on the gut microbiome was assessed using 16S rRNA gene sequencing. RESULTS: BBR treatment significantly ameliorated cholestatic liver injury, evidenced by decreased serum levels of AST, ALT, and ALP, and reduced bile duct proliferation and hepatic fibrosis, as shown by H&E, Picro-Sirius Red, and CK19 IHC staining. RNAseq and qRT-PCR analyses indicated a substantial inhibition of fibrotic and inflammatory gene expression. BBR also mitigated ER stress by downregulating Chop, Atf4 and Xbp-1 expression. In addition, BBR modulated bile acid metabolism by altering key gene expressions in the liver and small intestine, resulting in restored bile acid homeostasis characterized by reduced total bile acids in serum, liver, and small intestine and increased fecal excretion. Furthermore, BBR significantly improved intestinal barrier function and reduced bacterial translocation by modulating the gut microbiota. CONCLUSION: BBR effectively attenuates cholestatic liver injury, suggesting its potential as a therapeutic agent for PSC and other cholestatic liver diseases.

RNA binding protein HuR protects against NAFLD by suppressing long noncoding RNA H19 expression.

BACKGROUND: NAFLD has become the most common chronic liver disease worldwide. Human antigen R (HuR), an RNA-binding protein, is an important post-transcriptional regulator. HuR has been reported as a key player in regulating lipid homeostasis in the liver and adipose tissues by using tissue-specific HuR knockout mice. However, the underlying mechanism by which hepatocyte-specific HuR regulates hepatic lipid metabolism under metabolic stress remains unclear and is the focus of this study. METHODS: Hepatocyte-specific HuR deficient mice (HuRhKO) and age-/gender-matched control mice, as well as long-noncoding RNA H19 knockout mice (H19-/-), were fed a Western Diet plus sugar water (WDSW). Hepatic lipid accumulation, inflammation and fibrosis were examined by histology, RNA transcriptome analysis, qRT-PCR, and Western blot analysis. Bile acid composition was measured using LC-MS/MS. RESULTS: Hepatocyte-specific deletion of HuR not only significantly increased hepatic lipid accumulation by modulating fatty acid synthesis and metabolism but also markedly induced inflammation by increasing immune cell infiltration and neutrophil activation under metabolic stress. In addition, hepatic deficiency of HuR disrupted bile acid homeostasis and enhanced liver fibrosis. Mechanistically, HuR is a repressor of H19 expression. Analysis of a recently published dataset (GSE143358) identified H19 as the top-upregulated gene in liver-specific HuR knockout mice. Similarly, hepatocyte-specific deficiency of HuR dramatically induced the expression of H19 and sphingosine-1 phosphate receptor 2 (S1PR2), but reduced the expression of sphingosine kinase 2 (SphK2). WDSW-induced hepatic lipid accumulation was alleviated in H19-/- mice. Furthermore, the downregulation of H19 alleviated WDSW-induced NAFLD in HuRhKO mice. CONCLUSIONS: HuR not only functions as an RNA binding protein to modulate post-transcriptional gene expression but also regulates H19 promoter activity. Hepatic HuR is an important regulator of hepatic lipid metabolism via modulating H19 expression.

Reduced alcohol preference and intake after fecal transplant in patients with alcohol use disorder is transmissible to germ-free mice.

Alcohol use disorder is a major cause of morbidity, which requires newer treatment approaches. We previously showed in a randomized clinical trial that alcohol craving and consumption reduces after fecal transplantation. Here, to determine if this could be transmitted through microbial transfer, germ-free male C57BL/6 mice received stool or sterile supernatants collected from the trial participants pre-/post-fecal transplant. We found that mice colonized with post-fecal transplant stool but not supernatants reduced ethanol acceptance, intake and preference versus pre-fecal transplant colonized mice. Microbial taxa that were higher in post-fecal transplant humans were also associated with lower murine alcohol intake and preference. A majority of the differentially expressed genes (immune response, inflammation, oxidative stress response, and epithelial cell proliferation) occurred in the intestine rather than the liver and prefrontal cortex. These findings suggest a potential for therapeutically targeting gut microbiota and the microbial-intestinal interface to alter gut-liver-brain axis and reduce alcohol consumption in humans.

Bile Acid Receptors and the Gut-Liver Axis in Nonalcoholic Fatty Liver Disease.

The prevalence of nonalcoholic fatty liver disease (NAFLD) has been significantly increased due to the global epidemic of obesity. The disease progression from simple steatosis (NAFL) to nonalcoholic steatohepatitis (NASH) is closely linked to inflammation, insulin resistance, and dysbiosis. Although extensive efforts have been aimed at elucidating the pathological mechanisms of NAFLD disease progression, current understanding remains incomplete, and no effective therapy is available. Bile acids (BAs) are not only important physiological detergents for the absorption of lipid-soluble nutrients in the intestine but also metabolic regulators. During the last two decades, BAs have been identified as important signaling molecules involved in lipid, glucose, and energy metabolism. Dysregulation of BA homeostasis has been associated with NAFLD disease severity. Identification of nuclear receptors and G-protein-coupled receptors activated by different BAs not only significantly expanded the current understanding of NAFLD/NASH disease progression but also provided the opportunity to develop potential therapeutics for NAFLD/NASH. In this review, we will summarize the recent studies with a focus on BA-mediated signaling pathways in NAFLD/NASH. Furthermore, the therapeutic implications of targeting BA-mediated signaling pathways for NAFLD will also be discussed.

Hepatic Branch Vagotomy Modulates the Gut-Liver-Brain Axis in Murine Cirrhosis.

BACKGROUND: Cirrhosis and hepatic encephalopathy (HE) are linked with an altered gut-liver-brain axis, however, the relative contribution of hepatic vagal innervation is unclear. We aimed to determine the impact of hepatic vagotomy on the gut microbiome, brain, and liver in murine cirrhosis. METHODS: 10-15-week-old male C57BL/6 mice with and without hepatic vagotomy underwent carbon tetrachloride (CCl4) gavage for 8 weeks. Frontal cortex [inflammation, glial/microglial activation, BDNF (brain-derived neurotrophic factor)], liver [histology including inflammation and steatosis, fatty acid synthesis (sterol-responsive binding protein-1) SREBP-1, insulin-induced gene-2 (Insig2) and BDNF], and colonic mucosal microbiota (16srRNA microbial sequencing) were evaluated on sacrifice. Conventional mice with and without cirrhosis were compared to vagotomized counterparts. RESULTS: Conventional control vs. cirrhosis: Cirrhosis resulted in dysbiosis, hepatic/neuro-inflammation with glial/microglial activation, and low brain BDNF vs. controls. Conventional control vs. vagotomy controls: Vagotomized control mice had a lower colonic dysbiosis than conventional mice but the rest of the hepatic/brain parameters were similar. Conventional cirrhosis vs. vagotomized cirrhosis: After vagotomy + cirrhosis, we found lower dysbiosis but continuing neuroinflammation in the absence of glial/microglial activation vs. conventional cirrhosis. Vagotomy + Cirrhosis groups showed higher hepatic steatosis due to higher SREBP1 and low Insig2 protein and altered activation of key genes involved in hepatic lipid metabolism and inflammation. BDNF levels in the brain were higher but low in the liver in vagotomy + cirrhosis, likely a protective mechanism. CONCLUSIONS: Hepatic vagal innervation affects the gut microbial composition, hepatic inflammation and steatosis, and cortical inflammation and BDNF expression and could be a critical modulator of the gut-liver-brain axis with consequences for HE development.

Berberine Prevents Disease Progression of Nonalcoholic Steatohepatitis through Modulating Multiple Pathways.

The disease progression of nonalcoholic fatty liver disease (NAFLD) from simple steatosis (NAFL) to nonalcoholic steatohepatitis (NASH) is driven by multiple factors. Berberine (BBR) is an ancient Chinese medicine and has various beneficial effects on metabolic diseases, including NAFLD/NASH. However, the underlying mechanisms remain incompletely understood due to the limitation of the NASH animal models used. Methods: A high-fat and high-fructose diet-induced mouse model of NAFLD, the best available preclinical NASH mouse model, was used. RNAseq, histological, and metabolic pathway analyses were used to identify the potential signaling pathways modulated by BBR. LC-MS was used to measure bile acid levels in the serum and liver. The real-time RT-PCR and Western blot analysis were used to validate the RNAseq data. Results: BBR not only significantly reduced hepatic lipid accumulation by modulating fatty acid synthesis and metabolism but also restored the bile acid homeostasis by targeting multiple pathways. In addition, BBR markedly inhibited inflammation by reducing immune cell infiltration and inhibition of neutrophil activation and inflammatory gene expression. Furthermore, BBR was able to inhibit hepatic fibrosis by modulating the expression of multiple genes involved in hepatic stellate cell activation and cholangiocyte proliferation. Consistent with our previous findings, BBR's beneficial effects are linked with the downregulation of microRNA34a and long noncoding RNA H19, which are two important players in promoting NASH progression and liver fibrosis. Conclusion: BBR is a promising therapeutic agent for NASH by targeting multiple pathways. These results provide a strong foundation for a future clinical investigation.

Berberine inhibits free fatty acid and LPS-induced inflammation via modulating ER stress response in macrophages and hepatocytes.

Inflammation plays an essential role in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Berberine (BBR), an isoquinoline alkaloid isolated from Chinese medicinal herbs, has been widely used to treat various diseases, including liver diseases for hundreds of years. The previous studies have shown that BBR inhibits high fat-diet-induced steatosis and inflammation in rodent models of NAFLD. However, the underlying molecular mechanisms remain unclear. This study is aimed to identify the potential mechanisms by which BBR inhibits free fatty acid (FFA) and LPS-induced inflammatory response in mouse macrophages and hepatocytes. Mouse RAW264.7 macrophages and primary mouse hepatocytes were treated with palmitic acid (PA) or LPS or both with or without BBR (0-10 µM) for different periods (0-24 h). The mRNA and protein levels of proinflammatory cytokines (TNF-α, IL-6, IL-1ß, MCP-1) and ER stress genes (CHOP, ATF4, XBP-1) were detected by real-time RT-PCR, Western blot and ELISA, respectively. The results indicated that BBR significantly inhibited PA and LPS-induced activation of ER stress and expression of proinflammatory cytokines in macrophages and hepatocytes. PA/LPS-mediated activation of ERK1/2 was inhibited by BBR in a dose-dependent manner. In summary, BBR inhibits PA/LPS-induced inflammatory responses through modulating ER stress-mediated ERK1/2 activation in macrophages and hepatocytes.

Cholangiocyte-Derived Exosomal lncRNA H19 Promotes Macrophage Activation and Hepatic Inflammation under Cholestatic Conditions.

Activation of hepatic macrophages represents the critical driving force to promote cholestatic liver injury. Exosomes, as important small extracellular vesicles released by almost all types of cells, contribute to intercellular communication. We previously reported that cholangiocyte-derived exosomal long noncoding RNA (lncRNA) H19 plays a vital role in disrupting bile acid homeostasis in hepatocytes and promoting the activation of hepatic stellate cells (HSCs). Exosomal H19 derived from cholangiocytes was rapidly taken up by Kupffer cells. However, the mechanistic links between exosomal lncRNA H19 and macrophage-driven inflammation in cholestasis remain unclear. Here, we reported that the hepatic H19 level was closely correlated with macrophage activation and hepatic fibrosis in both Mdr2-/- and bile duct ligation (BDL) cholestatic mouse models, as well as in human primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC) patients. Exosomal H19 significantly induced the expression and secretion of chemokine (C-C motif) ligand 2 (CCL-2) and interleukin 6 (IL-6) in Kupffer cells. H19-enriched exosomes enhanced the activation M1 polarization of Kupffer cells and promoted the recruitment and differentiation of bone marrow-derived macrophages, which were inhibited by a CCL-2 pharmacological inhibitor. In conclusion, Cholangiocyte-derived exosomal H19 played a critical role in macrophage activation, differentiation, and chemotaxis through CCL-2/CCR-2 signaling pathways, which represent a therapeutic target for cholestatic liver diseases.

The role of sphingosine kinase 2 in alcoholic liver disease.

Alcoholic liver disease (ALD) is one of the most common liver diseases worldwide. However, the exact mechanisms underlying ALD remain unclear. Previous studies reported that sphingosine kinase 2 (SphK2) plays an essential role in regulating hepatic lipid metabolism. In the current study, we demonstrate that compared to wild-type (WT) mice, SphK2 deficient (SphK2-/-) mice exhibited a greater degree of liver injury and hepatic lipid accumulation after feeding with an alcohol diet for 60 days. This is accompanied by a down-regulation of steroid 7-alpha-hydroxylase (Cyp7b1) and an up-regulation of pro-inflammatory mediators (Tnfα, F4/80, Il-1ß). In vitro experiments showed that alcohol induced SphK2 expression in mouse primary hepatocytes and cultured mouse macrophages. Furthermore, alcohol feeding induced a more severe intestinal barrier dysfunction in SphK2-/- mice than WT mice. Deficiency of SphK2 impaired the growth of intestinal organoids. Finally, SphK2 expression levels were down-regulated in the livers of human patients with alcoholic cirrhosis and hepatocellular carcinoma compared to healthy controls. In summary, these findings suggest that SphK2 is a crucial regulator of hepatic lipid metabolism and that modulating the SphK2-mediated signaling pathway may represent a novel therapeutic strategy for the treatment of ALD and other metabolic liver diseases.

Effect of Increasing Age on Brain Dysfunction in Cirrhosis.

Patients with cirrhosis are growing older, which could have an impact on brain dysfunction beyond hepatic encephalopathy. Our aim was to study the effect of concomitant aging and cirrhosis on brain inflammation and degeneration using human and animal experiments. For the human study, age-matched patients with cirrhosis and controls between 65 and 85 years underwent cognitive testing, quality of life (QOL) assessment, and brain magnetic resonance (MR) spectroscopy and resting state functional MR imaging (rs-fMRI) analysis. Data were compared between groups. For the animal study, young (10-12 weeks) and old (1.5 years) C57BL/6 mice were given either CCl4 gavage to develop cirrhosis or a vehicle control and were followed for 12 weeks. Cortical messenger RNA (mRNA) expression of inflammatory mediators (interleukin [IL]-6, IL-1ß, transforming growth factor ß [TGF-ß], and monocyte chemoattractant protein 1), sirtuin-1, and gamma-aminobutyric acid (GABA)-ergic synaptic plasticity (neuroligin-2 [NLG2], discs large homolog 4 [DLG4], GABA receptor, subunit gamma 1/subunit B1 [GABRG1/B1]) were analyzed and compared between younger/older control and cirrhotic mice. The human study included 46 subjects (23/group). Patients with cirrhosis had worse QOL and cognition. On MR spectroscopy, patients with cirrhosis had worse changes related to ammonia and lower N-acetyl aspartate, whereas rs-fMRI analysis revealed that these patients demonstrated functional connectivity changes in the frontoparietal cortical region compared to controls. Results of the animal study showed that older mice required lower CCl4 to reach cirrhosis. Older mice, especially with cirrhosis, demonstrated higher cortical inflammatory mRNA expression of IL-6, IL-1ß, and TGF-ß; higher glial and microglial activation; and lower sirtuin-1 expression compared to younger mice. Older mice also had lower expression of DLG4, an excitatory synaptic organizer, and higher NLG2 and GABRG1/B1 receptor expression, indicating a predominantly inhibitory synaptic organization. Conclusion: Aging modulates brain changes in cirrhosis; this can affect QOL, cognition, and brain connectivity. Cortical inflammation, microglial activation, and altered GABA-ergic synaptic plasticity could be contributory.