Extracellular Vesicle-Associated Neutrophil Elastase Activates Hepatic Stellate Cells and Promotes Liver Fibrogenesis via ERK1/2 Pathway.

Liver fibrosis associated with increased mortality is caused by activation of hepatic stellate cells and excessive production and accumulation of extracellular matrix in response to fibrotic insults. It has been shown that in addition to liver inflammation, systemic inflammation also contributes to liver fibrogenesis. A deeper understanding of mechanisms that control liver fibrotic response to intra- and extra-hepatic inflammation is essential to develop novel clinical strategies against this disease. Extracellular vesicles (EV) have been recognized as immune mediators that facilitate activation of hepatic stellate cells. In inflammatory diseases, activated neutrophils release neutrophil elastase (NE) bound to EV, which has been identified as a significant contributor to inflammation by promoting immune cell activation. Here, we aimed to explore the role of inflammation derived plasma EV-associated NE in liver fibrogenesis and its potential mechanisms. We show EV-associated NE induces activation, proliferation and migration of hepatic stellate cells by promoting activation of the ERK1/2 signaling pathway. This effect did not occur through EV without surface NE, and Sivelestat, a NE inhibitor, inhibited activation of the ERK1/2 signaling pathway mediated by EV-associated NE. Moreover, we found plasma EV-associated NE increases deposition of collagen1 and α-smooth muscle actin in the liver of a mouse model of liver fibrosis (Mdr2-/-). Notably, this effect does not occur in control mice without preexisting liver disease. These data suggest that EV-associated NE is a pro-fibrogenic factor for hepatic stellate cell activation via the ERK1/2 signaling pathway in pre-existing liver injuries. Inhibition of the plasma EV-associated NE in inflammatory conditions may be a therapeutic target for liver fibrosis in patients with inflammatory diseases.

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.

Culture of Mouse Liver Ductal Organoids.

3D organoid culture has become a powerful tool and model for various human diseases, including liver diseases, such as non-alcoholic steatohepatitis (NASH). Hepatic organoids have significant advantages over traditional primary cell cultures. The hepatic progenitor cells can be induced to form hepatic organoids. The established organoids can be passaged or cryopreserved for future use. The established hepatic organoids can be manipulated to study the disease progression of NASH-related fibrosis. Here, we describe a protocol to establish mouse liver ductal organoids.

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.

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.