Soluble Guanylyl Cyclase Activator BI 685509 Reduces Portal Hypertension and Portosystemic Shunting in a Rat Thioacetamide-Induced Cirrhosis Model.

Portal hypertension (PT) commonly occurs in cirrhosis. Nitric oxide (NO) imbalance contributes to PT via reduced soluble guanylyl cyclase (sGC) activation and cGMP production, resulting in vasoconstriction, endothelial cell dysfunction, and fibrosis. We assessed the effects of BI 685509, an NO-independent sGC activator, on fibrosis and extrahepatic complications in a thioacetamide (TAA)-induced cirrhosis and PT model. Male Sprague-Dawley rats received TAA twice-weekly for 15 weeks (300-150 mg/kg i.p.). BI 685509 was administered daily for the last 12 weeks (0.3, 1, and 3 mg/kg p.o.; n = 8-11 per group) or the final week only (Acute, 3 mg/kg p.o.; n = 6). Rats were anesthetized to measure portal venous pressure. Pharmacokinetics and hepatic cGMP (target engagement) were measured by mass spectrometry. Hepatic Sirius Red morphometry (SRM) and alpha-smooth muscle actin (αSMA) were measured by immunohistochemistry; portosystemic shunting was measured using colored microspheres. BI 685509 dose-dependently increased hepatic cGMP at 1 and 3 mg/kg (3.92 ± 0.34 and 5.14 ± 0.44 versus 2.50 ± 0.19 nM in TAA alone; P < 0.05). TAA increased hepatic SRM, αSMA, PT, and portosystemic shunting. Compared with TAA, 3 mg/kg BI 685509 reduced SRM by 38%, αSMA area by 55%, portal venous pressure by 26%, and portosystemic shunting by 10% (P < 0.05). Acute BI 685509 reduced SRM and PT by 45% and 21%, respectively (P < 0.05). BI 685509 improved hepatic and extrahepatic cirrhosis pathophysiology in TAA-induced cirrhosis. These data support the clinical investigation of BI 685509 for PT in patients with cirrhosis. SIGNIFICANCE STATEMENT: BI 685509 is an NO-independent sGC activator that was tested in a preclinical rat model of TAA-induced nodular, liver fibrosis, portal hypertension, and portal systemic shunting. BI 685509 reduced liver fibrosis, portal hypertension, and portal-systemic shunting in a dose-dependent manner, supporting its clinical assessment to treat portal hypertension in patients with cirrhosis.

Metformin slows liver cyst formation and fibrosis in experimental model of polycystic liver disease.

Polycystic liver disease (PLD) is a hereditary liver disease in which the number of cysts increases over time, causing various abdominal symptoms and poor quality of life. Although effective treatment for PLD has not been established, we recently reported that long-term exercise ameliorated liver cyst formation and fibrosis with the activation of AMP-activated protein kinase (AMPK) in polycystic kidney (PCK) rats, a PLD model. Therefore, the aim of this study was to investigate whether metformin, an indirect AMPK activator, was effective in PCK rats. PCK rats were randomly divided into a control (Con) group and a metformin-treated (Met) group. The Met group was treated orally with metformin in drinking water. After 12 wk, liver function, histology, and signaling cascades of PLD were examined in the groups. Metformin did not affect the body weight or liver weight, but it reduced liver cyst formation, cholangiocyte proliferation, and fibrosis around the cyst. Metformin increased the phosphorylation of AMPK and tuberous sclerosis complex 2 and decreased the phosphorylation of mammalian target of rapamycin, S6, and extracellular signal-regulated kinase and the expression of cystic fibrosis transmembrane conductance regulator, aquaporin I, transforming growth factor-ß, and type 1 collagen without changes in apoptosis or collagen degradation factors in the liver. Metformin slows the development of cyst formation and fibrosis with the activation of AMPK and inhibition of signaling cascades responsible for cellular proliferation and fibrosis in the liver of PCK rats.NEW & NOTEWORTHY This study indicates that metformin, an indirect AMPK activator slows liver cyst formation and fibrosis in PLD rat model. Metformin attenuates excessive cell proliferation in the liver with the inactivation of mTOR and ERK pathways. Metformin also reduces the expression of proteins responsible for cystic fluid secretion and liver fibrosis. Metformin and AMPK activators may be potent drugs for polycystic liver disease.

Inhibition of the transient receptor potential vanilloid 3 channel attenuates carbon tetrachloride-induced hepatic fibrosis.

Transient receptor potential vanilloid 3 (TRPV3) is a member of the TRP superfamily. Previous studies have demonstrated that TRPV3 is associated with myocardial fibrosis. However, the role of TRPV3 in hepatic fibrosis and its underlying mechanisms are still unclear. This study aimed to elucidate the underlying effects of TRPV3 on hepatic fibrosis at multiple biological levels. First, immunohistochemical staining was performed to examine TRPV3 expression in human hepatic cirrhosis tissues. Then, we established a CCl4-induced hepatic fibrosis mouse model. The TRPV3 selective agonist drofenine and its inhibitor, forsythoside B, were intraperitoneally injected to investigate the relationship between TRPV3 and liver fibrosis progression. Finally, in vitro studies were performed using hepatic stellate cells (HSCs) to discover the potential molecular biological mechanisms. Immunohistochemistry revealed TRPV3 overexpression in liver cirrhosis. In the liver fibrosis groups, TRPV3 inhibitor treatment significantly reduced liver fibrosis, while TRPV3 agonist exacerbated its progression. In HSCs, knocking down TRPV3 with siRNA impaired DNA synthesis and cell proliferation and increased cell apoptosis. Furthermore, we found that knockdown of TRPV3 could reduce the lectin like oxidized lowdensity lipoprotein receptor-1 (LOX-1) protein levels. Our research suggests that lower expression or functional levels of TRPV3 can ameliorate the inflammatory response and fibrotic tissue proliferation.

Glycyrrhizin Attenuates Portal Hypertension and Collateral Shunting via Inhibition of Extrahepatic Angiogenesis in Cirrhotic Rats.

Portal hypertension develops along with liver cirrhosis then induces the formation of portal-systemic collaterals and lethal complications. Extrahepatic angiogenesis plays an important role. Glycyrrhizin has been found to exhibit anti-angiogenic features, which leads to its extensive use. However, the relevant effects of glycyrrhizin on liver cirrhosis and portal hypertension have not been evaluated. This study thus aimed to investigate the impact of glycyrrhizin on portal hypertension-related derangements in cirrhotic rats. Male Sprague-Dawley rats received bile duct ligation (BDL) to induce cirrhosis or sham operation as control. The rats were subdivided to receive glycyrrhizin (150 mg/kg/day, oral gavage) or vehicle beginning on the 15th day post operation, when BDL-induced liver fibrosis developed. The effects of glycyrrhizin were determined on the 28th day, the typical timing of BDL-induced cirrhosis. Glycyrrhizin significantly reduced portal pressure (p = 0.004). The splanchnic inflow as measured by superior mesenteric arterial flow decreased by 22% (p = 0.029). The portal-systemic collateral shunting degree reduced by 30% (p = 0.024). The mesenteric angiogenesis and phospho-VEGFR2 protein expression were also downregulated (p = 0.038 and 0.031, respectively). Glycyrrhizin did not significantly influence the liver biochemistry data. Although glycyrrhizin tended to reverse liver fibrosis, statistical significance was not reached (p = 0.069). Consistently, hepatic inflow from portal side, hepatic vascular resistance, and liver fibrosis-related protein expressions were not affected. Glycyrrhizin treatment at the stage of hepatic fibrosis still effectively attenuated portal hypertension and portosystemic collateral shunting. These beneficial effects were attributed to, at least in part, the suppression of mesenteric angiogenesis by VEGF signaling pathway downregulation.

Anti-Fibrotic and Anti-Angiogenic Activities of Osbeckia octandra Leaf Extracts in Thioacetamide-Induced Experimental Liver Cirrhosis.

Chronic liver inflammation has become a major global health concern. In the absence of clinical surrogate markers to diagnose inflammatory liver disease, the intervention with effective drugs in modern medicine tends to be late. In Sri Lanka, traditional medical practitioners prescribe herbal preparations from Osbeckia octandra for the prevention and treatment of liver disorders. To test the efficacy of such treatments, we have administered thioacetamide (TAA) to male Wistar rats to induce chronic liver damage (disease control; DC) and examined how various leaf extracts: crude leaf suspension (CLS), boiled leaf extract (BLE), sonicated leaf extract (SLE), methanol leaf extract (MLE) and hexane leaf extract (HLE) of O. octandra ameliorate TAA-induced liver disease. The CLS, BLE and SLE treatments in cirrhotic rats significantly attenuated disease-related changes, such as liver weight and hepato-enzymes. The mRNA levels of Tnf-α were significantly decreased by 3.6, 10 and 3.9 times in CLS, BLE and SLE compared to DC. The same treatments resulted in significantly lower (19.5, 4.2 and 2.4 times) α-Sma levels compared to DC. In addition, Tgf-ß1 and Vegf-R2 mRNA expressions were significantly lower with the treatments. Moreover, BLE expressed a strong anti-angiogenic effect. We conclude that CLS, BLE and SLE from O. octandra have potent hepatic anti-fibrotic effects in TAA-induced liver cirrhosis.

Yiguanjian decoction inhibits macrophage M1 polarization and attenuates hepatic fibrosis induced by CCl4/2-AAF.

CONTEXT: Our previous studies indicated that Yiguanjian decoction (YGJ) has an anti-hepatic-fibrosis effect and could regulate macrophage status. OBJECTIVE: To elucidate the mechanism of YGJ in regulating macrophages. MATERIALS AND METHODS: Liver cirrhosis was induced by CCl4 for 12 weeks combined with 2-acetylaminofluorene (2-AAF) for the last 4 weeks in male Wistar rats. YGJ (3.56 mg/kg) orally administered in the last 4 weeks, and SORA (1 mg/kg) as control. In vitro, RAW264.7 cells were treated with lipopolysaccharides (LPSs) to induce macrophage polarization to the M1 phenotype, and they were co-cultured with WB-F344 cells and allocated to M group, YGJ group (2 µg/mL) and WIF-1 group (1 µg/mL) with untreated cells as control. The differentiation direction of WB-F344 cell line was observed in the presence or absence of YGJ. Pathology, fibrosis-related cytokines, macrophage polarization-related components, and Wnt signalling pathway components were detected. RESULTS: In vivo, the expression levels of α-SMA, Col (1), OV6, SOX9, EpCAM and M1 macrophage-related components (STAT1, IRF3, IRF5, IRF8, SOCS3) significantly decreased in the YGJ group compared with those in the 2-AAF/CCl4 group (p < 0.01 or 0.05). In vitro, the expression levels of M1 macrophage-related components, including STAT1, NF-κB, IRF3, IRF5, and SOCS3, in RAW264.7 cells decreased significantly in the YGJ group compared with those in the M group (p < 0.05 or p < 0.01). The expression levels of Wnt3A, FZD5, LRP-5/-6, and ß-catenin significantly increased in the YGJ group compared with those in the M group (p < 0.05 or p < 0.01). In addition, the expression levels of Wnt-4/-5A/-5B, and FZD2 significantly decreased in the YGJ group compared with those in the M group (p < 0.05 or p < 0.01). CONCLUSION: This study suggests that the anti-cirrhosis effect of YGJ is associated with its ability to inhibit macrophage M1-polarization, which provides a scientific basis for the clinical application of YGJ.

Gant61 ameliorates CCl4-induced liver fibrosis by inhibition of Hedgehog signaling activity.

As an intercellular signaling molecule, Hedgehog (Hh) plays a critical role in liver fibrosis/regeneration. Transcription effectors Gli1 and Gli2 are key components of the Hh signaling pathway. However, whether inhibition of Gli1/2 activity can affect liver fibrogenesis is largely unknown. In the present study, we investigated the effect of Gant61 (a Gli1/2 transcription factor inhibitor) on liver fibrosis and its possible mechanism. Wild-type and Shh-EGFP-Cre male mice were exposed to CCl4, and then treated with or without Gant61 for four weeks. The level of liver injury/fibrosis and expression levels of mRNA and protein related to the Hh ligand/pathway were assessed. In our study, CCl4 treatment induced liver injury/fibrosis and promoted activation of hepatic stellate cells (HSCs). In addition, CCl4 induced the expression of Shh ligands in and around the fibrotic lesion, accompanied by induction of mRNA and protein expression of Hh components (Smo, Gli1 and Gli2). However, administration of Gant61 decreased liver fibrosis by reduction in HSC number, down-regulation of mRNA and protein expression of Hh components (Smo, Gli1 and Gli2), and cell-cycle arrest of HSCs. Our data highlight the importance of the Shh pathway for the development of liver fibrosis, and also suggest Glis as potential therapeutic targets for the treatment of liver fibrosis.

SB431542-Loaded Liposomes Alleviate Liver Fibrosis by Suppressing TGF-ß Signaling.

Liver fibrosis is a common outcome of most chronic liver diseases, but there is no clinically approved drug for its treatment. Previous studies have reported the potential of SB431542 as an inhibitor of TGF-ß signaling in the treatment of liver fibrosis, but it shows poor water solubility and low bioavailability. Here, we improve these characteristics of SB431542 by loading it into liposomes (SB-Lips) with two FDA-approved excipients: soya phosphatidyl S100 and Solutol HS15. In vitro, SB-Lips had stronger inhibitory effects on the proliferation and activation of hepatic stellate cells LX-2 than free SB. After an intravenous injection in a CCl4-induced liver fibrosis mouse model, SB-Lips accumulated preferentially in the liver, its area under the concentration-time curve was significantly higher than that of free SB431542, and it alleviated hepatic fibrosis significantly more than free drug, which was associated with greater inhibition of TGF-ß signaling. Furthermore, SB-Lips did not cause significant injury to other organs. These results suggest that our liposomal system is safe and effective for delivering SB431542 to fibrotic liver.

Protective Effects of Spermidine Against Cirrhotic Cardiomyopathy in Bile Duct-Ligated Rats.

Cirrhotic cardiomyopathy is a critical factor that causes morbidity and mortality in crucial conditions such as liver transplantation. In animal model, the common pathophysiologic mechanisms of cirrhotic cardiomyopathy are similar to those associated with bile duct ligation (BDL). Overproduction of inflammatory and oxidant markers plays a crucial role in cirrhotic cardiomyopathy. Spermidine, a multifunctional polyamine, is known for its antioxidant and anti-inflammatory effects. In this study, we investigated the effects of spermidine on development of cirrhotic cardiomyopathy in BDL rats. Rats were randomly housed in 6 groups. Except the normal and sham groups, BDL was performed for all the control and spermidine groups. Seven days after operation, 3 different doses of spermidine (5, 10 and 50 mg/kg) were administrated until day 28, in spermidine groups. At the end of the fourth week, the electrocardiography (ECG) and papillary muscle isolation were performed. The serum level of tumor necrosis factor-a (TNF-α), interleukin-1ß (IL-1ß), and IL-10 and cardiac level of superoxide dismutase, glutathione (GSH). and malondialdehyde (MDA) were assessed. Furthermore, the nuclear factor-κB (NF-κB) expression was assessed by western blot. Cardiac histopathological changes were monitored. The serum levels of magnesium (Mg) and potassium (K) were investigated. Control group, exhibited exaggerated signs of cirrhotic cardiomyopathy in comparison with the sham group. Co-administration of spermidine at the dose of 10 mg/kg in BDL rats significantly improved the cardiac condition, reduced the inflammatory mediators, and increased antioxidant enzymes. In addition, the histopathologic findings were in accordance with the other results of the study. Besides, there was no significant alteration in serum levels of Mg and K. This study demonstrates that spermidine at the dose of 10 mg/kg significantly improved the cirrhotic cardiomyopathy in BDL model in rats.

Inhibition of monoacylglycerol lipase, an anti-inflammatory and antifibrogenic strategy in the liver.

OBJECTIVE: Sustained inflammation originating from macrophages is a driving force of fibrosis progression and resolution. Monoacylglycerol lipase (MAGL) is the rate-limiting enzyme in the degradation of monoacylglycerols. It is a proinflammatory enzyme that metabolises 2-arachidonoylglycerol, an endocannabinoid receptor ligand, into arachidonic acid. Here, we investigated the impact of MAGL on inflammation and fibrosis during chronic liver injury. DESIGN: C57BL/6J mice and mice with global invalidation of MAGL (MAGL -/- ), or myeloid-specific deletion of either MAGL (MAGLMye-/-), ATG5 (ATGMye-/-) or CB2 (CB2Mye-/-), were used. Fibrosis was induced by repeated carbon tetrachloride (CCl4) injections or bile duct ligation (BDL). Studies were performed on peritoneal or bone marrow-derived macrophages and Kupffer cells. RESULTS: MAGL -/- or MAGLMye-/- mice exposed to CCl4 or subjected to BDL were more resistant to inflammation and fibrosis than wild-type counterparts. Therapeutic intervention with MJN110, an MAGL inhibitor, reduced hepatic macrophage number and inflammatory gene expression and slowed down fibrosis progression. MAGL inhibitors also accelerated fibrosis regression and increased Ly-6Clow macrophage number. Antifibrogenic effects exclusively relied on MAGL inhibition in macrophages, since MJN110 treatment of MAGLMye-/- BDL mice did not further decrease liver fibrosis. Cultured macrophages exposed to MJN110 or from MAGLMye-/- mice displayed reduced cytokine secretion. These effects were independent of the cannabinoid receptor 2, as they were preserved in CB2Mye-/- mice. They relied on macrophage autophagy, since anti-inflammatory and antifibrogenic effects of MJN110 were lost in ATG5Mye-/- BDL mice, and were associated with increased autophagic flux and autophagosome biosynthesis in macrophages when MAGL was pharmacologically or genetically inhibited. CONCLUSION: MAGL is an immunometabolic target in the liver. MAGL inhibitors may show promising antifibrogenic effects during chronic liver injury.

γ-Mangostin alleviates liver fibrosis through Sirtuin 3-superoxide-high mobility group box 1 signaling axis.

The activation of hepatic stellate cells (HSCs) plays a critical role in liver fibrosis. In the current study, γ-mangostin (γ-man), one of the major xanthones from mangosteen (Garcinia mangostana), was found to alleviate fibrogenesis in human immortalized HSCs (LX-2 cells) and in liver from chronic carbon tetrachloride (CCl4) injured mice. γ-Man suppressed the expression levels of collagen I and α-smooth muscle actin (α-SMA) in LX-2 cells in both dose and time dependent manners. Furthermore, γ-man inhibited NAD(P)H oxidase activity through induction of sirtuin 3 (SIRT3), resulting in reduced intracellular oxidative stress in LX-2 cells. Moreover, γ-man stimulated the expression of histone deacetylase 1, which in turn decreased the acetylation and cytoplasmic shuttling of high mobility group box 1 (HMGB1), to impair autocrine HMGB1-induced HSC activation. In CCl4-injured mice, γ-man enhanced the expression of SIRT3 and decreased the expression of HMGB1, resulting in decreased accumulation of collagen I and α-SMA in liver. Consequently, γ-man might be a potent candidate to treat oxidative stress induced liver fibrosis.

Treatment with Melatonin Improves Cognitive Behavior and Motor Skills in a Rat Model of Liver Fibrosis.

INTRODUCTION AND AIM: Patients with liver cirrhosis (LC) and minimal hepatic encephalopaty have a higher accident rate. LC impairs the normal sleep-awake cycle and produces disturbances in behavior, cognition and motor skills. Abnormal melatonin (MT) levels have also been identified in LC. Administration of MT may regulate circadian rhythms and prevent the oxidative damage. We studied the effects of MT on spatial memory acquisition (SMA) and motor skills in a liver fibrosis model (LF)s. MATERIALS AND METHODS: Forty-five rats, divided into 4 groups. [G1: LF; G2: LF + MT; G3: MT; G4: Healthy control (HC)]. LF was induced by carbon tetrachloride intraperitoneal injection (0.2 mL/kg) for 5 months. MT was administered during 5 weeks (0.4 mg/kg/day). SMA was evaluated by using the Morris Water Maze protocol where the escape latency (EL) and mean speed were measured. Data were registered by SMART®. RESULTS: The EL measurement analyzed by two way ANOVA: cirrhosis presented a higher EL than controls or those treated with MT suggesting impaired memory acquisition which is rescued by MT treatment. The mean speed analysis revealed that LF presented higher speed than LF+MT or HC, suggesting that LF affects motor skills, which are improved by MT. To discard whether EL is affected by altered motor skills in LF treated with MT, we compared the average EL and speed between days 2 and 6 of the training protocol. Speed was not improved during the trials unlike EL, suggesting that memory acquisition is independent of motor skills. CONCLUSION: These findings suggest that MT improves cognition and motor skills in the LF model.

[Study of compound glutathione inosine injection effect on expression of regucalcin in liver tissue of rats with immune hepatic fibrosis].

Objective: To investigate the change in expression of anti-senescence marker protein calmodulin (RGN) in liver tissues of rats with immune hepatic fibrosis, and to observe the effect of compound glutathione inosine injection (CGII) on it. Methods: Rat liver fibrosis model was induced by intraperitoneal injection of porcine serum, and CGII intervention was administered at the appropriate time. Rat liver tissues were stained with HE and Masson. RGN and protein expression at mRNA in liver tissues was detected by fluorescence quantitative PCR and immunohistochemistry. One-way Anova was used for measurement data. LDS test was used for two-way comparison, and pathological semi-quantitative results were analyzed by rank-sum test. Results: The relative expression of RGN mRNA and protein in liver tissue of fibrotic rats was 82.23 ± 15.21 and 12.52 ± 3.23, respectively, which were significantly lower than that of normal rats 176.39 ± 11.35 and 59.23 ± 9.13 (P < 0.01). The degree of liver fibrosis in fibrotic rats after CGII intervention was significantly lower than fibrotic rats. The relative expression of RGN mRNA and protein in the intervention group was 168.78 ± 21.31 and 46.42 ± 4.71, respectively, which were significantly higher than fibrosis and spontaneous recovery group. The difference was statistically significant (P < 0.01). The relative expression of RGN mRNA and protein in the spontaneous recovery group was 86.23 ± 17.16 and 14.34 ± 5.16, which was higher than model group. The difference was not statistically significant (P > 0.05). Conclusion: The expression of RGN in liver tissue of rats with hepatic fibrosis induced by porcine serum is decreased, while the expression of RGN increases with the decrease of fibrosis after CGII intervention, suggesting that the protein may play an important role in the development of liver fibrosis.

Epigenetic Effects of an Adenosine Derivative in a Wistar Rat Model of Liver Cirrhosis.

The pathological characteristic of cirrhosis is scarring which results in a structurally distorted and dysfunctional liver. Previously, we demonstrated that Col1a1 and Pparg genes are deregulated in CCl4 -induced cirrhosis but their normal expression levels are recovered upon treatment with IFC-305, an adenosine derivative. We observed that adenosine was able to modulate S-adenosylmethionine-dependent trans-methylation reactions, and recently, we found that IFC-305 modulates HDAC3 expression. Here, we investigated whether epigenetic mechanisms, involving DNA methylation processes and histone acetylation, could explain the re-establishment of gene expression mediated by IFC-305 in cirrhosis. Therefore, Wistar rats were CCl4 treated and a sub-group received IFC-305 to reverse fibrosis. Global changes in DNA methylation, 5-hydroxymethylation, and histone H4 acetylation were observed after treatment with IFC-305. In particular, during cirrhosis, the Pparg gene promoter is depleted of histone H4 acetylation, whereas IFC-305 administration restores normal histone acetylation levels which correlates with an increase of Pparg transcript and protein levels. In contrast, the promoter of Col1a1 gene is hypomethylated during cirrhosis but gains DNA methylation upon treatment with IFC-305 which correlates with a reduction of Col1a1 transcript and protein levels. Our results suggest a model in which cirrhosis results in a general loss of permissive chromatin histone marks which triggers the repression of the Pparg gene and the upregulation of the Col1a1 gene. Treatment with IFC-305 restores epigenetic modifications globally and specifically at the promoters of Pparg and Col1a1 genes. These results reveal one of the mechanisms of action of IFC-305 and suggest a possible therapeutic function in cirrhosis. J. Cell. Biochem. 119: 401-413, 2018. © 2017 Wiley Periodicals, Inc.

PBI-4050 Reduces Stellate Cell Activation and Liver Fibrosis through Modulation of Intracellular ATP Levels and the Liver Kinase B1/AMP-Activated Protein Kinase/Mammalian Target of Rapamycin Pathway.

Hepatic fibrosis is a major cause of morbidity and mortality for which there is currently no effective therapy. We previously showed that 2-(3-pentylphenyl)acetic acid (PBI-4050) is a dual G protein-coupled receptor GPR40 agonist/GPR84 antagonist that exerts antifibrotic, anti-inflammatory, and antiproliferative action. We evaluated PBI-4050 for the treatment of liver fibrosis in vivo and elucidated its mechanism of action on human hepatic stellate cells (HSCs). The antifibrotic effect of PBI-4050 was evaluated in carbon tetrachloride (CCl4)- and bile duct ligation-induced liver fibrosis rodent models. Treatment with PBI-4050 suppressed CCl4-induced serum aspartate aminotransferase levels, inflammatory marker nitric oxide synthase, epithelial to mesenchymal transition transcription factor Snail, and multiple profibrotic factors. PBI-4050 also decreased GPR84 mRNA expression in CCl4-induced injury, while restoring peroxisome proliferator-activated receptor γ (PPARγ) to the control level. Collagen deposition and α-smooth muscle actin (α-SMA) protein levels were also attenuated by PBI-4050 treatment in the bile duct ligation rat model. Transforming growth factor-ß-activated primary HSCs were used to examine the effect of PBI-4050 and its mechanism of action in vitro. PBI-4050 inhibited HSC proliferation by arresting cells in the G0/G1 cycle phase. Subsequent analysis demonstrated that PBI-4050 signals through a reduction of intracellular ATP levels, activation of liver kinase B1 (LKB1) and AMP-activated protein kinase (AMPK), and blockade of mammalian target of rapamycin (mTOR), resulting in reduced protein and mRNA levels of α-SMA and connective tissue growth factor and restored PPARγ mRNA expression. Our findings suggest that PBI-4050 may exert antifibrotic activity in the liver through a novel mechanism of action involving modulation of intracellular ATP levels and the LKB1/AMPK/mTOR pathway in stellate cells, and PBI-4050 may be a promising agent for treating liver fibrosis.

Norfloxacin is more effective than Rifaximin in avoiding bacterial translocation in an animal model of cirrhosis.

BACKGROUND & AIMS: Norfloxacin administration is useful in preventing bacterial infections in cirrhosis but associated to the generation of resistant species. Rifaximin is known to reach high concentrations in the intestinal lumen without generating relevant resistance in the intestinal flora. Our aim was to compare the effect of Norfloxacin and Rifaximin on intestinal flora composition, bacterial translocation and survival in cirrhotic rats. METHODS: Cirrhosis was induced in rats by oral administration of CCl4 . Animals were divided into three groups: only CCl4 (group I, n = 10); CCl4 + Norfloxacin (group II, n = 17) and CCl4 + Rifaximin (group III, n = 14). Gut bacterial composition, bacterial translocation and cytokine levels were measured. RESULTS: Forty-one rats were finally included. The incidence of viable and non-viable bacterial translocation was significantly reduced in animals receiving Norfloxacin; Rifaximin also decreased the incidence of viable and non-viable bacterial translocation, but did not reach statistical significance. Serum TNF-α levels were significantly lower in antibiotic groups. Norfloxacin modified intestinal microbiota, depleting significantly more pathobionts than Rifaximin. CONCLUSION: Norfloxacin is more effective than Rifaximin in preventing bacterial translocation in rats with cirrhosis probably because of its capacity to reduce pathobionts from intestinal microbiota.

BRD4 is a novel therapeutic target for liver fibrosis.

Liver fibrosis is characterized by the persistent deposition of extracellular matrix components by hepatic stellate cell (HSC)-derived myofibroblasts. It is the histological manifestation of progressive, but reversible wound-healing processes. An unabated fibrotic response results in chronic liver disease and cirrhosis, a pathological precursor of hepatocellular carcinoma. We report here that JQ1, a small molecule inhibitor of bromodomain-containing protein 4 (BRD4), a member of bromodomain and extraterminal (BET) proteins, abrogate cytokine-induced activation of HSCs. Cistromic analyses reveal that BRD4 is highly enriched at enhancers associated with genes involved in multiple profibrotic pathways, where BRD4 is colocalized with profibrotic transcription factors. Furthermore, we show that JQ1 is not only protective, but can reverse the fibrotic response in carbon tetrachloride-induced fibrosis in mouse models. Our results implicate that BRD4 can act as a global genomic regulator to direct the fibrotic response through its coordinated regulation of myofibroblast transcription. This suggests BRD4 as a potential therapeutic target for patients with fibrotic complications.

[Effects of blueberry on hepatic fibrosis and expression of nuclear transcription factor-кB in rats].

Objective: To observe the effects of blueberry and nuclear expression of transcription factor-кb (NF-кb) p65 in an experimental rat model of liver fibrosis. Methods: Forty-five Sprague-Dawley rats were randomly divided into isotonic saline control group (A); model group (B); blueberry juice prevention group (C, 15 g/kg); dan-shao-hua-xian capsule prevention group (D, 1 g/kg); and blueberry juice + dan-shao-hua-xian capsule prevention group (E). Rat liver fibrosis model was established by covalent compound carbon tetrachloride (CCl(4)). Each prevention group was given the corresponding dose of blueberry juice or (and) dan-shao-hua-xian capsule, and the rats were sacrificed after 8 weeks. The degree of liver fibrosis was evaluated by hematoxylin and eosin stain. A liver tissue of NF-κBp65 was detected by immunohistochemical method. The NF-κBp65 protein expression of liver tissue and transforming growth factor (TGF) ß1 was detected by Western blot. Data of multiple groups were compared by one-way analysis of variance, and rank sum test. Results: Immunohistochemistry detected that TGFß1 protein was mainly expressed in mesenchymal origin of hepatic stellate cells. The expression level of group A (3.75 ± 1.67) was low, while those of group B (9.00 ± 2.07), C (7.33 ± 1.00), D (6.00 ± 1.51), and E (3.5 ± 1.41) were high. However, the expression level of TGF-ß1 protein in hepatic tissues of group B was significantly higher than that of group C, D and E [group E: 3.5 ± 1.41, F = 18.350, P < 0.05]. In addition, group D was higher than group E (P < 0.05). The expression of NF- kappa Bp65 protein was very complex, and the expression patterns in different groups were different (Statistical calculation of experimental data were based on expression of liver cells). Compared with group B (4.37 ± 2.13), the relative expression levels of NF-κBp65 protein in-group A (0.46 ± 0.25), group C (2.76 ± 1.01), group D (2.13 ± 1.51), group E (1.88 ± 0.99) were significantly decreased (F = 27.490, P < 0.05), and the expression trend was consistent with TGFß1 protein. Western blot detected NF-κBp65 protein in liver tissues of rats. Compared with group A, levels in groups B, C, D and E were significantly increased (F = 96.983, P < 0.05), and groups C, D and E were significantly lower. The E group was significantly lower than the C group (F = 96.983, P < 0.05), and the degree of hepatic fibrosis was lower in each prevention group than in the B group (T = 24.1, P < 0.05). Conclusion: Blueberries have preventive effect on CCl4-induced hepatic fibrosis in rats, and its preventive mechanism may inhibit the expression and activation of NF-κBp65 in hepatocytes, thereby reducing TGFß1- mediated production or activation.

Inhibition of epoxyeicosatrienoic acid production in rats with cirrhosis has beneficial effects on portal hypertension by reducing splanchnic vasodilation.

UNLABELLED: In cirrhosis, 11,12-epoxyeicosatrienoic acid (EET) induces mesenteric arterial vasodilation, which contributes to the onset of portal hypertension. We evaluated the hemodynamic effects of in vivo inhibition of EET production in experimental cirrhosis. Sixteen control rats and 16 rats with carbon tetrachloride-induced cirrhosis were studied. Eight controls and eight rats with cirrhosis were treated with the specific epoxygenase inhibitor N-(methylsulfonyl)-2-(2-propynyloxy)-benzenehexanamide (MS-PPOH; 20 mg/kg/day) for 3 consecutive days. Portal blood flow and renal and splenic resistive indexes were calculated through echographic measurements, while portal and systemic pressures were measured through polyethylene-50 catheters. Small resistance mesenteric arteries were connected to a pressure servo controller in a video-monitored perfusion system, and concentration-response curves to phenylephrine and acetylcholine were evaluated. EET levels were measured in tissue homogenates of rat liver, kidney, and aorta, using an enzyme-linked immunosorbent assay. Urinary Na(+) excretion function was also evaluated. In rats with cirrhosis, treatment with MS-PPOH significantly reduced portal blood flow and portal pressure compared to vehicle (13.6 ± 5.7 versus 25.3 ± 7.1 mL/min/100 g body weight, P < 0.05; 9.6 ± 1.1 versus 12.2 ± 2.3 mm Hg, P < 0.05; respectively) without effects on systemic pressure. An increased response to acetylcholine of mesenteric arteries from rats with cirrhosis (50% effect concentration -7.083 ± 0.197 versus -6.517 ± 0.73 in control rats, P < 0.05) was reversed after inhibition of EET production (-6.388 ± 0.263, P < 0.05). In liver, kidney, and aorta from animals with cirrhosis, treatment with MS-PPOH reversed the increase in EET levels. In both controls and rats with cirrhosis, MS-PPOH increased urinary Na(+) excretion. CONCLUSION: In rats with cirrhosis, in vivo inhibition of EET production normalizes the response of mesenteric arteries to vasodilators, with beneficial effects on portal hypertension. (Hepatology 2016;64:923-930).

Mitochondria-targeted antioxidant mitoquinone deactivates human and rat hepatic stellate cells and reduces portal hypertension in cirrhotic rats.

BACKGROUND & AIMS: In cirrhosis, activated hepatic stellate cells (HSC) play a major role in increasing intrahepatic vascular resistance and developing portal hypertension. We have shown that cirrhotic livers have increased reactive oxygen species (ROS), and that antioxidant therapy decreases portal pressure. Considering that mitochondria produce many of these ROS, our aim was to assess the effects of the oral mitochondria-targeted antioxidant mitoquinone on hepatic oxidative stress, HSC phenotype, liver fibrosis and portal hypertension. METHODS: Ex vivo: Hepatic stellate cells phenotype was analysed in human precision-cut liver slices in response to mitoquinone or vehicle. In vitro: Mitochondrial oxidative stress was analysed in different cell type of livers from control and cirrhotic rats. HSC phenotype, proliferation and viability were assessed in LX2, and in primary human and rat HSC treated with mitoquinone or vehicle. In vivo: CCl4 - and thioacetamide-cirrhotic rats were treated with mitoquinone (5 mg/kg/day) or the vehicle compound, DecylTPP, for 2 weeks, followed by measurement of oxidative stress, systemic and hepatic haemodynamic, liver fibrosis, HSC phenotype and liver inflammation. RESULTS: Mitoquinone deactivated human and rat HSC, decreased their proliferation but with no effects on viability. In CCl4 -cirrhotic rats, mitoquinone decreased hepatic oxidative stress, improved HSC phenotype, reduced intrahepatic vascular resistance and diminished liver fibrosis. These effects were associated with a significant reduction in portal pressure without changes in arterial pressure. These results were further confirmed in the thioacetamide-cirrhotic model. CONCLUSION: We propose mitochondria-targeted antioxidants as a novel treatment approach against portal hypertension and cirrhosis.