Hydroxyurea attenuates hepatic stellate cell proliferation in vitro and liver fibrogenesis in vivo.

Liver fibrosis results from excessive proliferation of, and collagen production by hepatic stellate cells (HSCs) that is caused by chronic liver injury. No drugs are available to cure liver fibrosis. Hydroxyurea is an anti-proliferative drug that is used in benign and malignant disorders. Here, we studied the effect of hydroxyurea on primary HSCs and its anti-fibrotic effect in the CCl4 mouse model of liver fibrosis. Primary rat HSCs were cultured in the absence or presence of hydroxyurea (0.1-1.0 mmol/L). CCl4 or vehicle was administered to C57BL/6/J mice for 4 weeks, with or without hydroxyurea (100 mg/kg/day) co-treatment. We used real-time cell proliferation analysis, Oil Red O (lipid droplet) staining, immunohistochemistry, Acridine Orange staining (apoptosis), Sytox green staining (necrosis), RT-qPCR, ELISA, and Western Blotting for analysis. Hydroxyurea dose-dependently suppressed lipid droplet-loss and mRNA levels of Col1α1 and Acta2 in transdifferentiating HSCs. In fully-activated HSCs, hydroxyurea dose-dependently attenuated PCNA protein levels and BrdU incorporation, but did not reverse Col1α1 and Acta2 mRNA expression. Hydroxyurea did not induce apoptosis or necrosis in HSCs or hepatocytes. Hydroxyurea suppressed accumulation of desmin-positive HSCs and hepatic collagen deposition after CCl4 treatment. CCl4 -induced regenerative hepatocyte proliferation, Col1α1 and Acta2 mRNA expression and α-SMA protein levels were not affected. This study demonstrates that hydroxyurea inhibits HSC proliferation in vitro and attenuates early development of liver fibrosis in vivo, while preserving hepatocyte regeneration after toxic insults by CCl4. Thus, hydroxyurea may have therapeutic value against liver fibrosis.

Extracellular vesicles originating from steatotic hepatocytes promote hepatic stellate cell senescence via AKT/mTOR signaling.

The prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) is increasing rapidly due to the obesity epidemic. In the inflammatory stages of MASLD (MASH), activation of hepatic stellate cells (HSCs) leads to initiation and progression of liver fibrosis. Extracellular vesicles (EVs) are released from all cell types and play an important role in intercellular communication. However, the role of EVs released from hepatocytes in the context of MASLD is largely unknown. Therefore, the present study aimed to investigate the role of EVs derived from both normal and steatotic (free fatty acid-treated) hepatocytes on the phenotype of HSCs via the senescence pathway. Primary rat hepatocytes were treated with free fatty acids (FFAs: oleic acid and palmitic acid). EVs were collected by ultracentrifugation. EVs markers and HSCs activation and senescence markers were assessed by Western blot analysis, qPCR and cytochemistry. Reactive oxygen species (ROS) production was assessed by fluorescence assay. RNA profiles of EVs were evaluated by sequencing. We found that EVs from hepatocytes treated with FFAs (FFA-EVs) inhibit collagen type 1 and α-smooth muscle actin expression, increase the production of ROS and the expression of senescence markers (IL-6, IL-1ß, p21 and senescence-associated ß-galactosidase activity) in early activating HSCs via the AKT-mTOR pathway. Sequencing showed differentially enriched RNA species between the EVs groups. In conclusion, EVs from FFA-treated hepatocytes inhibit HSC activation by inducing senescence via the AKT-mTOR signaling pathway. Determining the components in EVs from steatotic hepatocytes that induce HSC senescence may lead to the identification of novel targets for intervention in the treatment of MASLD in the future.

Circulating Trimethylamine-N-Oxide Is Elevated in Liver Transplant Recipients.

Liver transplant recipients (LTRs) have lower long-term survival rates compared with the general population. This underscores the necessity for developing biomarkers to assess post-transplantation mortality. Here we compared plasma trimethylamine-N-oxide (TMAO) levels with those in the general population, investigated its determinants, and interrogated its association with all-cause mortality in stable LTRs. Plasma TMAO was measured in 367 stable LTRs from the TransplantLines cohort (NCT03272841) and in 4837 participants from the population-based PREVEND cohort. TMAO levels were 35% higher in LTRs compared with PREVEND participants (4.3 vs. 3.2 µmol/L, p < 0.001). Specifically, TMAO was elevated in LTRs with metabolic dysfunction-associated steatotic liver disease, alcohol-associated liver disease, and polycystic liver disease as underlying etiology (p < 0.001 for each). Among LTRs, TMAO levels were independently associated with eGFR (std. ß = -0.43, p < 0.001) and iron supplementation (std. ß = 0.13, p = 0.008), and were associated with mortality (29 deaths during 8.6 years follow-up; log-rank test p = 0.017; hazard ratio of highest vs. lowest tertile 4.14, p = 0.007). In conclusion, plasma TMAO is likely elevated in stable LTRs, with impaired eGFR and iron supplementation as potential contributory factors. Our preliminary findings raise the possibility that plasma TMAO could contribute to increased mortality risk in such patients, but this need to be validated through a series of rigorous and methodical studies.

Hepatic stellate cells induce an inflammatory phenotype in Kupffer cells via the release of extracellular vesicles.

Liver fibrosis is the response of the liver to chronic liver inflammation. The communication between the resident liver macrophages (Kupffer cells [KCs]) and hepatic stellate cells (HSCs) has been mainly viewed as one-directional: from KCs to HSCs with KCs promoting fibrogenesis. However, recent studies indicated that HSCs may function as a hub of intercellular communications. Therefore, the aim of the present study was to investigate the role of HSCs on the inflammatory phenotype of KCs. Primary rat HSCs and KCs were isolated from male Wistar rats. HSCs-derived conditioned medium (CM) was harvested from different time intervals (Day 0-2: CM-D2 and Day 5-7: CM-D7) during the activation of HSCs. Extracellular vesicles (EVs) were isolated from CM by ultracentrifugation and evaluated by nanoparticle tracking analysis and western blot analysis. M1 and M2 markers of inflammation were measured by quantitative PCR and macrophage function by assessing phagocytic capacity. CM-D2 significantly induced the inflammatory phenotype in KCs, but not CM-D7. Neither CM-D2 nor CM-D7 affected the phagocytosis of KCs. Importantly, the proinflammatory effect of HSCs-derived CM is mediated via EVs released from HSCs since EVs isolated from CM mimicked the effect of CM, whereas EV-depleted CM lost its ability to induce a proinflammatory phenotype in KCs. In addition, when the activation of HSCs was inhibited, HSCs produced less EVs. Furthermore, the proinflammatory effects of CM and EVs are related to activating Toll-like receptor 4 (TLR4) in KCs. In conclusion, HSCs at an early stage of activation induce a proinflammatory phenotype in KCs via the release of EVs. This effect is absent in CM derived from HSCs at a later stage of activation and is dependent on the activation of TLR4 signaling pathway.

Arginase 1 expression is increased during hepatic stellate cell activation and facilitates collagen synthesis.

Activation of hepatic stellate cells (HSC) is a key event in the initiation of liver fibrosis. Activated HSCs proliferate and secrete excessive amounts of extracellular matrix (ECM), disturbing liver architecture and function, leading to fibrosis and eventually cirrhosis. Collagen is the most abundant constituent of ECM and proline is the most abundant amino acid of collagen. Arginine is the precursor in the biosynthetic pathway of proline. Arginine is the exclusive substrate of both nitric oxide synthase (NOS) and arginase. NOS is an M1 (proinflammatory) marker of macrophage polarization whereas arginase-1 (Arg1) is an M2 (profibrogenic) marker of macrophage polarization. Differential expression of NOS and Arg1 has not been studied in HSCs yet. To identify the expression profile of arginine catabolic enzymes during HSC activation and to investigate their role in HSC activation, primary rat HSCs were cultured-activated for 7 days and expression of iNOS and Arg1 were investigated. Nor-NOHA was used as a specific and reversible arginase inhibitor. During HSC activation, iNOS expression decreased whereas Arg1 expression increased. Inhibition of Arg1 in activated HSCs efficiently inhibited collagen production but not cell proliferation. HSC activation is accompanied by a switch of arginine catabolism from iNOS to Arg1. Inhibition of Arg1 decreases collagen synthesis. Therefore, we conclude that Arg1 can be a therapeutic target for the inhibition of liver fibrogenesis.

Scopoletin and umbelliferone protect hepatocytes against palmitate- and bile acid-induced cell death by reducing endoplasmic reticulum stress and oxidative stress.

BACKGROUND: The number of patients with non-alcoholic fatty liver disease (NAFLD) is rapidly increasing due to the growing epidemic of obesity. Non-alcoholic steatohepatitis (NASH), the inflammatory stage of NAFLD, is characterized by lipid accumulation in hepatocytes, chronic inflammation and hepatocyte cell death. Scopoletin and umbelliferone are coumarin-like molecules and have antioxidant, anti-cancer and anti-inflammatory effects. Cytoprotective effects of these compounds have not been described in hepatocytes and the mechanisms of the beneficial effects of scopoletin and umbelliferone are unknown. AIM: To investigate whether scopoletin and/or umbelliferone protect hepatocytes against palmitate-induced cell death. For comparison, we also tested the cytoprotective effect of scopoletin and umbelliferone against bile acid-induced cell death. METHODS: Primary rat hepatocytes were exposed to palmitate (1 mmol/L) or the hydrophobic bile acid glycochenodeoxycholic acid (GCDCA; 50 µmol/L). Apoptosis was assessed by caspase-3 activity assay, necrosis by Sytox green assay, mRNA levels by qPCR, protein levels by Western blot and production of reactive oxygen species (ROS) by fluorescence assay. RESULTS: Both scopoletin and umbelliferone protected against palmitate and GCDCA-induced cell death. Both palmitate and GCDCA induced the expression of ER stress markers. Scopoletin and umbelliferone decreased palmitate- and GCDCA-induced expression of ER stress markers, phosphorylation of the cell death signaling intermediate JNK as well as ROS production. CONCLUSION: Scopoletin and umbelliferone protect against palmitate and bile acid-induced cell death of hepatocytes by inhibition of ER stress and ROS generation and decreasing phosphorylation of JNK. Scopoletin and umbelliferone may hold promise as a therapeutic modality for the treatment of NAFLD.

Elevated cAMP Protects against Diclofenac-Induced Toxicity in Primary Rat Hepatocytes: A Protective Effect Mediated by the Exchange Protein Directly Activated by cAMP/cAMP-Regulated Guanine Nucleotide Exchange Factors.

Chronic consumption of the nonsteroidal anti-inflammatory drug diclofenac may induce drug-induced liver injury (DILI). The mechanism of diclofenac-induced liver injury is partially elucidated and involves mitochondrial damage. Elevated cAMP protects hepatocytes against bile acid-induced injury. However, it is unknown whether cAMP protects against DILI and, if so, which downstream targets of cAMP are implicated in the protective mechanism, including the classic protein kinase A (PKA) pathway or alternative pathways like the exchange protein directly activated by cAMP (EPAC). The aim of this study was to investigate whether cAMP and/or its downstream targets protect against diclofenac-induced injury in hepatocytes. Rat hepatocytes were exposed to 400 µmol/l diclofenac. Apoptosis and necrosis were measured by caspase-3 activity assay and Sytox green staining, respectively. Mitochondrial membrane potential (MMP) was measured by JC-10 staining. mRNA and protein expression were assessed by quantitative polymerase chain reaction (qPCR) and Western blot, respectively. The cAMP-elevating agent 7ß-acetoxy-8,13-epoxy-1α,6ß,9α-trihydroxylabd-14-en-11-one (forskolin), the pan-phosphodiesterase inhibitor IBMX, and EPAC inhibitors 5,7-dibromo-6-fluoro-3,4-dihydro-2-methyl-1(2H)-quinoline carboxaldehyde (CE3F4) and ESI-O5 were used to assess the role of cAMP and its effectors, PKA or EPAC. Diclofenac exposure induced apoptotic cell death and loss of MMP in hepatocytes. Both forskolin and IBMX prevented diclofenac-induced apoptosis. EPAC inhibition but not PKA inhibition abolished the protective effect of forskolin and IBMX. Forskolin and IBMX preserved the MMP, whereas both EPAC inhibitors diminished this effect. Both EPAC1 and EPAC2 were expressed in hepatocytes and localized in mitochondria. cAMP elevation protects hepatocytes against diclofenac-induced cell death, a process primarily involving EPACs. The cAMP/EPAC pathway may be a novel target for treatment of DILI. SIGNIFICANCE STATEMENT: This study shows two main highlights. First, elevated cAMP levels protect against diclofenac-induced apoptosis in primary hepatocytes via maintenance of mitochondrial integrity. In addition, this study proposes the existence of mitochondrial cAMP-EPAC microdomains in rat hepatocytes, opening new avenues for targeted therapy in drug-induced liver injury (DILI). Both EPAC1 and EPAC2, but not protein kinase A, are responsible for this protective effect. Our findings present cAMP-EPAC as a potential target for the treatment of DILI and liver injury involving mitochondrial dysfunction.

Anti-TNFα treatment in Crohn's disease: Impact on hepatic steatosis, gut-derived hormones and metabolic status.

BACKGROUND AND AIMS: An association between Crohn's disease (CD) and hepatic steatosis has been reported. However, the underlying mechanisms of steatosis progression in CD are not clear. Among the most effective CD treatments are agents that inhibit Tumor-Necrosis-Factor (TNF) activity, yet it is unclear why anti-TNFα agents would affect steatosis in CD. Recent studies suggest that microbiome can affect both, CD and steatosis pathogenesis. Therefore, we here analysed a potential relationship between anti-TNF treatment and hepatic steatosis in CD, focusing on the gut-liver axis. METHODS: This cross-sectional study evaluated patients with established CD, with and without anti-TNFα treatment, analysing serum markers of liver injury, measurement of transient elastography, controlled attenuation parameter (CAP) and MRI for fat detection. Changes in lipid and metabolic profiles were assessed by serum and stool lipidomics and metabolimics. Additionally, we analysed gut microbiota composition and mediators of bile acid (BA) signalling via stool and serum analysis. RESULTS: Patients on anti-TNFα treatment had less hepatic steatosis as assessed by CAP and MRI. Serum FGF19 levels were significantly higher in patients on anti-TNFα therapy and associate with reduced steatosis and increased bowel motility. Neutral lipids including triglycerides were reduced in the serum of patients on anti-TNF treatment. Bacteria involved in BA metabolism and FGF19 regulation, including Firmicutes, showed group-specific alterations with low levels in patients without anti-TNFα treatment. Low abundance of Firmicutes was associated with higher triglyceride levels. CONCLUSIONS: Anti-TNFα treatment is associated with reduced steatosis, lower triglyceride levels, alterations in FXR-signalling (eg FGF19) and microbiota composition in CD.

Hesperetin protects against palmitate-induced cellular toxicity via induction of GRP78 in hepatocytes.

Lipotoxicity plays a critical role in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Hesperetin, a flavonoid derivative, has anti-oxidant, anti-inflammatory and cytoprotective properties. In the present study, we aim to examine whether hesperetin protects against palmitate-induced lipotoxic cell death and to investigate the underlying mechanisms in hepatocytes. Primary rat hepatocytes and HepG2 cells were pretreated with hesperetin for 30 min and then exposed to palmitate (1.0 mmol/L in primary rat hepatocytes; 0.5 mmol/L in HepG2 cells) in the presence or absence of hesperetin. Necrotic cell death was measured via Sytox green nuclei staining and quantified by LDH release assay. Apoptotic cell death was determined by caspase 3/7 activity and the protein level of cleaved-PARP. The unfolded protein response (UPR) was assessed by measuring the expression of GRP78, sXBP1, ATF4 and CHOP. Results show that hesperetin (50 µmol/L and 100 µmol/L) protected against palmitate-induced cell death and inhibited palmitate-induced endoplasmic reticulum (ER) stress in both primary rat hepatocytes and HepG2 cells. Hesperetin (100 µmol/L) significantly activated sXBP1/GRP78 signaling, whereas a high concentration of hesperetin (200 µmol/L) activated p-eIF2α and caused hepatic cell death. Importantly, GRP78 knockdown via siRNA abolished the protective effects of hesperetin in HepG2 cells. In conclusion, hesperetin protected against palmitate-induced hepatic cell death via activation of the sXBP1/GRP78 signaling pathway, thus inhibiting palmitate-induced ER stress. Moreover, high concentrations of hesperetin induce ER stress and subsequently cause cell death in hepatocytes.

Oxidative stress is associated with suspected non-alcoholic fatty liver disease and all-cause mortality in the general population.

BACKGROUND & AIMS: Non-alcoholic fatty liver disease (NAFLD) is characterized by excessive lipid accumulation, inflammation and an imbalanced redox homeostasis. We hypothesized that systemic free thiol levels, as a proxy of systemic oxidative stress, are associated with NAFLD. METHODS: Protein-adjusted serum free thiol concentrations were determined in participants from the Prevention of Renal and Vascular End-Stage Disease (PREVEND) cohort study (n = 5562). Suspected NAFLD was defined by the Fatty Liver Index (FLI ≥ 60) and Hepatic Steatosis Index (HSI > 36). RESULTS: Protein-adjusted serum free thiols were significantly reduced in subjects with FLI ≥ 60 (n = 1651). In multivariable logistic regression analyses, protein-adjusted serum free thiols were associated with NAFLD (FLI ≥ 60) (OR per doubling of concentration: 0.78 [95% CI 0.64-0.96], P = .016) even when adjusted for potential confounding factors, including systolic blood pressure, diabetes, current smoking, use of alcohol and total cholesterol (OR 0.80 [95% CI 0.65-0.99], P = .04). This association lost its significance (OR 0.94 [95% CI 0.73-1.21], P = .65) after additional adjustment for high-sensitive C-reactive protein. Stratified analyses showed significantly differential associations of protein-adjusted serum free thiol concentrations with suspected NAFLD for gender (P < .02), hypertension (P < .001) and hypercholesterolemia (P < .003). Longitudinally, protein-adjusted serum free thiols were significantly associated with the risk of all-cause mortality in subjects with NAFLD (FLI ≥ 60) (HR 0.27 [95% CI 0.17-0.45], P < .001). CONCLUSION: Protein-adjusted serum free thiol levels are reduced and significantly associated with all-cause mortality in subjects with suspected NAFLD. Quantification of free thiols may be a promising, minimally invasive strategy to improve detection of NAFLD and associated risk of all-cause mortality in the general population.

Hepatocyte KLF6 expression affects FXR signalling and the clinical course of primary sclerosing cholangitis.

BACKGROUND & AIMS: Primary sclerosing cholangitis (PSC) is characterized by chronic cholestasis and inflammation, which promotes cirrhosis and an increased risk of cholangiocellular carcinoma (CCA). The transcription factor Krueppel-like-factor-6 (KLF6) is a mediator of liver regeneration, steatosis, and hepatocellular carcinoma (HCC), but no data are yet available on its potential role in cholestasis. Here, we aimed to identify the impact of hepatic KLF6 expression on cholestatic liver injury and PSC and identify potential effects on farnesoid-X-receptor (FXR) signalling. METHODS: Hepatocellular KLF6 expression was quantified by immunohistochemistry (IHC) in liver biopsies of PSC patients and correlated with serum parameters and clinical outcome. Liver injury was analysed in hepatocyte-specific Klf6-knockout mice following bile duct ligation (BDL). Chromatin-immunoprecipitation-assays (ChIP) and KLF6-overexpressing HepG2 cells were used to analyse the interaction of KLF6 and FXR target genes such as NR0B2. RESULTS: Based on IHC, PSC patients could be subdivided into two groups showing either low (<80%) or high (>80%) hepatocellular KLF6 expression. In patients with high KLF6 expression, we observed a superior survival in Kaplan-Meier analysis. Klf6-knockout mice showed reduced hepatic necrosis following BDL when compared to controls. KLF6 suppressed NR0B2 expression in HepG2 cells mediated through binding of KLF6 to the NR0B2 promoter region. CONCLUSION: Here, we show an association between KLF6 expression and the clinical course and overall survival in PSC patients. Mechanistically, we identified a direct interaction of KLF6 with the FXR target gene NR0B2.

Hydrogen sulfide stimulates activation of hepatic stellate cells through increased cellular bio-energetics.

Hepatic fibrosis is caused by chronic inflammation and characterized as the excessive accumulation of extracellular matrix (ECM) by activated hepatic stellate cells (HSCs). Gasotransmitters like NO and CO are known to modulate inflammation and fibrosis, however, little is known about the role of the gasotransmitter hydrogen sulfide (H2S) in liver fibrogenesis and stellate cell activation. Endogenous H2S is produced by the enzymes cystathionine ß-synthase (CBS), cystathionine γ-lyase (CTH) and 3-mercaptopyruvate sulfur transferase (MPST) [1]. The aim of this study was to elucidate the role of endogenously produced and/or exogenously administered H2S on rat hepatic stellate cell activation and fibrogenesis. Primary rat HSCs were culture-activated for 7 days and treated with different H2S releasing donors (slow releasing donor GYY4137, fast releasing donor NaHS) or inhibitors of the H2S producing enzymes CTH and CBS (DL-PAG, AOAA). The main message of our study is that mRNA and protein expression level of H2S synthesizing enzymes are low in HSCs compared to hepatocytes and Kupffer cells. However, H2S promotes hepatic stellate cell activation. This conclusion is based on the fact that production of H2S and mRNA and protein expression of its producing enzyme CTH are increased during hepatic stellate cell activation. Furthermore, exogenous H2S increased HSC proliferation while inhibitors of endogenous H2S production reduce proliferation and fibrotic makers of HSCs. The effect of H2S on stellate cell activation correlated with increased cellular bioenergetics. Our results indicate that the H2S generation in hepatic stellate cells is a target for anti-fibrotic intervention and that systemic interventions with H2S should take into account cell-specific effects of H2S.

Prescription of statins in suspected non-alcoholic fatty liver disease and high cardiovascular risk, a population-based study.

BACKGROUND & AIMS: The prevalence of non-alcoholic fatty liver disease (NAFLD) is increasing, with concomitant high incidence of lipoprotein abnormalities. Cardiovascular disease (CVD) is the main cause of death in subjects with NAFLD and management of dyslipidaemia is pivotal for prevention. We aimed to determine cardiovascular risk and indication for statin therapy in subjects with NAFLD. METHODS: A cross-sectional analysis of the population-based Lifelines Cohort Study of 34 240 adult individuals. Subjects with reported use of lipid-lowering drugs were excluded. Suspected NAFLD was defined as Fatty Liver Index (FLI) ≥60 and advanced hepatic fibrosis as NAFLD fibrosis score (NFS) >0.676. Cardiovascular risk and indication for statin therapy were defined according to the European Society of Cardiology and European Atherosclerosis Society Guideline for the Management of Dyslipidaemias. RESULTS: FLI ≥ 60 was present in 7067 (20.6%) participants and coincided with increased prevalence of type 2 diabetes mellitus, metabolic syndrome, CVD and impaired renal function (all P < 0.001). 10-year predicted cardiovascular risk was significantly increased in subjects with elevated FLI and NFS (both P < 0.001). Indication for statin use was significantly increased in subjects with FLI ≥ 60 (31.0% vs 15.6%, P < 0.001) and NFS > 0.676 (73.2% vs 30.6%, P < 0.001). In multivariable analyses, FLI ≥ 60 (OR 1.26, 95%CI: 1.13-1.41, P < 0.001) and NFS > 0.676 (OR 5.03, 95%CI: 2.76-9.17, P < 0.001) were independent predictors for indication regarding statin therapy. CONCLUSIONS: Because of increased cardiovascular risk, substantial proportions of subjects with suspected NAFLD and/or fibrosis have an indication for lipid-lowering treatment and could benefit from statin therapy.

A liver-specific long noncoding RNA with a role in cell viability is elevated in human nonalcoholic steatohepatitis.

Hepatocyte apoptosis in nonalcoholic steatohepatitis (NASH) can lead to fibrosis and cirrhosis, which permanently damage the liver. Understanding the regulation of hepatocyte apoptosis is therefore important to identify therapeutic targets that may prevent the progression of NASH to fibrosis. Recently, increasing evidence has shown that long noncoding (lnc) RNAs are involved in various biological processes and that their dysregulation underlies a number of complex human diseases. By performing gene expression profiling of 4,383 lncRNAs in 82 liver samples from individuals with NASH (n = 48), simple steatosis but no NASH (n = 11), and healthy controls (n = 23), we discovered a liver-specific lncRNA (RP11-484N16.1) on chromosome 18 that showed significantly elevated expression in the liver tissue of NASH patients. This lncRNA, which we named lnc18q22.2 based on its chromosomal location, correlated with NASH grade (r = 0.51, P = 8.11 × 10-7 ), lobular inflammation (r = 0.49, P = 2.35 × 10-6 ), and nonalcoholic fatty liver disease activity score (r = 0.48, P = 4.69 × 10-6 ). The association of lnc18q22.2 to liver steatosis and steatohepatitis was replicated in 44 independent liver biopsies (r = 0.47, P = 0.0013). We provided a genetic structure of lnc18q22.2 showing an extended exon 2 in liver. Knockdown of lnc18q22.2 in four different hepatocyte cell lines resulted in severe phenotypes ranging from reduced cell growth to lethality. This observation was consistent with pathway analyses of genes coexpressed with lnc18q22.2 in human liver or affected by lnc18q22.2 knockdown. CONCLUSION: We identified an lncRNA that can play an important regulatory role in liver function and provide new insights into the regulation of hepatocyte viability in NASH. (Hepatology 2017;66:794-808).

Beneficial effects of mineralocorticoid receptor blockade in experimental non-alcoholic steatohepatitis.

BACKGROUND: Therapeutic options to treat Non-alcoholic steatohepatitis (NASH) are limited. Mineralocorticoid receptor (MR) activation could play a role in hepatic fibrogenesis and its modulation could be beneficial for NASH. AIM: To investigate whether eplerenone, a specific MR antagonist, ameliorates liver damage in experimental NASH. METHODS: C57bl6 mice were fed a choline-deficient and amino acid-defined (CDAA) diet for 22 weeks with or without eplerenone supplementation. Serum levels of aminotransferases and aldosterone were measured and hepatic steatosis, inflammation and fibrosis scored histologically. Hepatic triglyceride content (HTC) and hepatic mRNA levels of pro-inflammatory pro-fibrotic, oxidative stress-associated genes and of MR were also assessed. RESULTS: CDAA diet effectively induced fibrotic NASH, and increased the hepatic expression of pro-inflammatory, pro-fibrotic and oxidative stress-associated genes. Hepatic MR mRNA levels significantly correlated with the expression of pro-inflammatory and pro-fibrotic genes and were significantly increased in hepatic stellate cells obtained from CDAA-fed animals. Eplerenone administration was associated to a reduction in histological steatosis and attenuation of liver fibrosis development, which was associated to a significant decrease in the expression of collagen-α1, collagen type III, alpha 1 and Matrix metalloproteinase-2. CONCLUSION: The expression of MR correlates with inflammation and fibrosis development in experimental NASH. Specific MR blockade with eplerenone has hepatic anti-steatotic and anti-fibrotic effects. These data identify eplerenone as a potential novel therapy for NASH. Considering its safety and FDA-approved status, human studies are warranted.

Melatonin suppresses activation of hepatic stellate cells through RORα-mediated inhibition of 5-lipoxygenase.

Liver fibrosis is scar tissue resulting from an uncontrolled wound-healing process in response to chronic liver injury. Liver damage generates an inflammatory reaction that activates hepatic stellate cells (HSC) that transdifferentiate from quiescent cells that control retinol metabolism to proliferative and migratory myofibroblasts that produce excessive amounts of extracellular matrix proteins, in particular collagen 1a1 (COL1A1). Although liver fibrosis is reversible, no effective drug therapy is available to prevent or reverse HSC activation. Melatonin has potent hepatoprotective properties in a variety of acute and chronic liver injury models and suppresses liver fibrosis. However, it remains unclear whether melatonin acts indirectly or directly on HSC to prevent liver fibrosis. Here, we studied the effect of melatonin on culture-activated rat HSC. Melatonin dose-dependently suppressed the expression of HSC activation markers Col1a1 and alpha-smooth muscle actin (αSMA, Acta2), as well as HSC proliferation and loss of lipid droplets. The nuclear melatonin sensor retinoic acid receptor-related orphan receptor-alpha (RORα/Nr1f1) was expressed in quiescent and activated HSC, while the membranous melatonin receptors (Mtrn1a and Mtrn1b) were not. The synthetic RORα agonist SR1078 more potently suppressed Col1a1 and αSma expression, HSC proliferation, and lipid droplet loss, while the RORα antagonist SR1001 blocked the antifibrotic features of melatonin. Melatonin and SR1078 inhibited the expression of Alox5, encoding 5-lipoxygenase (5-LO). The pharmacological 5-LO inhibitor AA861 reduced Acta2 and Col1a1 expression in activated HSC. We conclude that melatonin directly suppresses HSC activation via RORα-mediated inhibition of Alox5 expression, which provides novel drug targets to treat liver fibrosis.

Sphingosine kinase-1 inhibition protects primary rat hepatocytes against bile salt-induced apoptosis.

Sphingosine kinases (SphKs) and their product sphingosine-1-phosphate (S1P) have been reported to regulate apoptosis and survival of liver cells. Cholestatic liver diseases are characterized by cytotoxic levels of bile salts inducing liver injury. It is unknown whether SphKs and/or S1P play a role in this pathogenic process. Here, we investigated the putative involvement of SphK1 and S1P in bile salt-induced cell death in hepatocytes. Primary rat hepatocytes were exposed to glycochenodeoxycholic acid (GCDCA) to induce apoptosis. GCDCA-exposed hepatocytes were co-treated with S1P, the SphK1 inhibitor Ski-II and/or specific antagonists of S1P receptors (S1PR1 and S1PR2). Apoptosis and necrosis were quantified. Ski-II significantly reduced GCDCA-induced apoptosis in hepatocytes (-70%, P<0.05) without inducing necrosis. GCDCA increased the S1P levels in hepatocytes (P<0.05). GCDCA induced [Ca(2+)] oscillations in hepatocytes and co-treatment with the [Ca(2+)] chelator BAPTA repressed GCDCA-induced apoptosis. Ski-II inhibited the GCDCA-induced intracellular [Ca(2+)] oscillations. Transcripts of all five S1P receptors were detected in hepatocytes, of which S1PR1 and S1PR2 appear most dominant. Inhibition of S1PR1, but not S1PR2, reduced GCDCA-induced apoptosis by 20%. Exogenous S1P also significantly reduced GCDCA-induced apoptosis (-50%, P<0.05), however, in contrast to the GCDCA-induced (intracellular) SphK1 pathway, this was dependent on S1PR2 and not S1PR1. Our results indicate that SphK1 plays a pivotal role in mediating bile salt-induced apoptosis in hepatocytes in part by interfering with intracellular [Ca(2+)] signaling and activation of S1PR1.

Glutathione and antioxidant enzymes serve complementary roles in protecting activated hepatic stellate cells against hydrogen peroxide-induced cell death.

BACKGROUND: In chronic liver disease, hepatic stellate cells (HSCs) are activated, highly proliferative and produce excessive amounts of extracellular matrix, leading to liver fibrosis. Elevated levels of toxic reactive oxygen species (ROS) produced during chronic liver injury have been implicated in this activation process. Therefore, activated hepatic stellate cells need to harbor highly effective anti-oxidants to protect against the toxic effects of ROS. AIM: To investigate the protective mechanisms of activated HSCs against ROS-induced toxicity. METHODS: Culture-activated rat HSCs were exposed to hydrogen peroxide. Necrosis and apoptosis were determined by Sytox Green or acridine orange staining, respectively. The hydrogen peroxide detoxifying enzymes catalase and glutathione-peroxidase (GPx) were inhibited using 3-amino-1,2,4-triazole and mercaptosuccinic acid, respectively. The anti-oxidant glutathione was depleted by L-buthionine-sulfoximine and repleted with the GSH-analogue GSH-monoethylester (GSH-MEE). RESULTS: Upon activation, HSCs increase their cellular glutathione content and GPx expression, while MnSOD (both at mRNA and protein level) and catalase (at the protein level, but not at the mRNA level) decreased. Hydrogen peroxide did not induce cell death in activated HSCs. Glutathione depletion increased the sensitivity of HSCs to hydrogen peroxide, resulting in 35% and 75% necrotic cells at 0.2 and 1mmol/L hydrogen peroxide, respectively. The sensitizing effect was abolished by GSH-MEE. Inhibition of catalase or GPx significantly increased hydrogen peroxide-induced apoptosis, which was not reversed by GSH-MEE. CONCLUSION: Activated HSCs have increased ROS-detoxifying capacity compared to quiescent HSCs. Glutathione levels increase during HSC activation and protect against ROS-induced necrosis, whereas hydrogen peroxide-detoxifying enzymes protect against apoptotic cell death.

Extracellular vesicles derived from liver sinusoidal endothelial cells inhibit the activation of hepatic stellate cells and Kupffer cells in vitro.

Liver sinusoidal endothelial cells (LSECs) play a crucial role in maintaining liver microcirculation and exchange of nutrients in the liver and are thought to be involved in the pathogenesis of metabolic dysfunction-associated steatotic liver disease (MASLD). The activation of hepatic stellate cells (HSCs) and Kupffer cells (KCs) has been considered to be responsible for the onset of liver fibrosis and the aggravation of liver injury. However, the paracrine regulatory effects of LSECs in the development of MASLD, in particular the role of LSEC-derived extracellular vesicles (EVs) remains unclear. Therefore, the aim of the present study was to investigate the influence of LSEC-derived EVs on HSCs and KCs. Primary rat LSECs, HSCs and KCs were isolated from male Wistar rats. LSEC-derived EVs were isolated from conditioned medium by ultracentrifugation and analyzed by nanoparticle tracking analysis, and expression of specific markers. LSEC-derived EVs reduced the expression of activation markers in activated HSCs but did not affect quiescent HSCs. Also, LSEC-derived EVs suppressed proliferation of activated HSCs activation, as assessed by Xcelligence and BrdU assay. LSEC-derived EVs also increased the expression of inflammatory genes in HSCs that normally are lowly expression during their activation. In contrast, EVs decreased the expression of inflammatory genes in activated KCs. In summary, our results suggest that LSEC-derived EVs may attenuate the fibrogenic phenotype of activated HSCs and the inflammatory phenotype of KCs. Our results show promise for LSEC-derived EVs as therapeutic moieties to treat MASLD. In addition, these EVs might prove of diagnostic value.