Reduced Liver Fibrosis Research Articles

From the Editor’s desk…: April 2019

From the Editor’s desk…: April 2019

Efficacy of diacerein in reducing liver steatosis and fibrosis in patients with type 2 diabetes and non-alcoholic fatty liver disease: A randomized, placebo-controlled trial.

The aim was to assess, in a randomized, double-blinded, placebo-controlled trial, the efficacy of diacerein, an anti-inflammatory drug, in improving liver fibrosis and steatosis in patients with type 2 diabetes and non-alcoholic fatty liver disease (NAFLD). Sixty-nine diabetic patients with NAFLD were randomized to 24-month treatment with placebo (35 patients) or diacerein 100 mg/day (34 patients). Liver stiffness and steatosis were assessed by transient elastography (Fibroscan®) at baseline, and 12 and 24 months of follow-up. The primary outcome was the difference in mean liver stiffness and steatosis changes during treatment. Adjusted differences in mean changes on intention-to-treat analyses were estimated by generalized repeated-measures mixed-effects regressions. Diacerein significantly reduced liver stiffness in contrast to placebo by 1.6 kPa (95% CI: -2.6 to -0.5 kPa; p = 0.003), whereas no significant difference in mean changes in liver steatosis was observed. The reduction in liver stiffness was already evident at the 12-month examination, and accentuated at the 24-month examination. Eight patients reduced liver fibrosis stage during treatment, seven of whom were in the diacerein group (p = 0.020). In conclusion, a 2-year treatment with diacerein significantly reduced liver fibrosis in diabetic patients with NAFLD.

Vitamin A-coupled liposomal Rho-kinase inhibitor ameliorates liver fibrosis without systemic adverse effects.

Rho-kinase (ROCK) inhibitor could ameliorate liver fibrosis by suppressing hepatic stellate cell (HSC) activation. However, because systemic administration of ROCK inhibitor causes serious adverse effects, we developed a drug delivery system selectively delivering ROCK inhibitor to HSCs. Here, we examined whether our developed vitamin A (VA)-coupled liposomal ROCK inhibitor reduced liver fibrosis in rats without causing systemic adverse effects. LX-2 HSCs were analyzed for morphological changes and the expression of profibrotic proteins. The inhibitory effects of VA-coupled liposomal ROCK inhibitor on liver fibrosis were confirmed in a rat model of liver fibrosis induced by i.p. injection of carbon tetrachloride. The degree of liver fibrosis, biochemical changes, and survival rates were also investigated. Vitamin A-coupled liposomal ROCK inhibitor had an effect at approximately 1/100 the amount of the free ROCK inhibitor for inhibiting the activation of LX-2 cells and caused significant decreases in the expression levels of α-smooth muscle actin (SMA) and transforming growth factor (TGF)-β1. The degree of liver fibrosis was suppressed by treatment with VA-coupled liposomal ROCK inhibitor, and the expression of α-SMA and TGF-β1 in liver tissues was also significantly suppressed. In addition, serum levels of alanine aminotransferase and hyaluronic acid were significantly reduced, and there was no decline in kidney function, which has been noted as a systemic adverse effect of ROCK inhibitor. Furthermore, VA-coupled liposomal ROCK inhibitor improved survival rates in rats with liver fibrosis. Vitamin A-coupled liposomal ROCK inhibitor efficiently suppressed liver fibrosis without causing systemic adverse effects.

Effects of Atorvastatin Alongside Conventional Medical Treatment on Liver Fibrosis and Dysfunction in Patients with Chronic Hepatitis B: A Double-Blinded Clinical Trial

Background: Liver fibrosis and related hepatic dysfunction are among major medical issues in patients with chronic hepatitis B (CHB). It finally may lead to cirrhosis, hepatocellular carcinoma (HCC), and liver-related death. Objectives: This study aimed to investigate the effects of adding atorvastatin to the standard antiviral therapy on the hepatic fibrosis and progression of cirrhosis in CHB patients. Methods: In this double-blinded clinical trial, 77 CHB patients referring to the gastroenterology and hepatology clinics in Qom, Iran, were selected by simple random sampling from 2016 to 2017. The participants were randomly divided into an intervention group that received tenofovir (300 mg) with atorvastatin (20 mg) daily and a control group that received tenofovir (300 mg) and placebo of atorvastatin daily. Results: According to the findings, changes in the aspartate aminotransferase (AST) level were not significant in the control (P = 0.771) and intervention (P = 0.266) groups. Changes in the alanine aminotransferase (ALT) level were non-significant in the control group (P = 0.893) but significant in the intervention group (P = 0.018). Changes in the liver fibrosis were significant in the intervention group (P = 0.001) and between the two groups (P < 0.001). Conclusions: According to the study results, adding atorvastatin to the standard antiviral regimen improves the liver function and reduces liver fibrosis in CHB patients. Therefore, it is suggested that atorvastatin be used as complementary therapy.

HOMA-IR: An independent predictor of advanced liver fibrosis in nondiabetic non-alcoholic fatty liver disease.

Although non-alcoholic fatty liver disease (NAFLD) is common in the general population, identifying patients with advanced fibrosis remains a challenge. We investigated whether the homeostasis model assessment parameter of insulin resistance (HOMA-IR), an index of IR and one of the most important metabolic factors, is an independent predictive factor for advanced fibrosis in nondiabetic patients with NAFLD. This was a retrospective, cross-sectional multicenter study. We included 361 patients with biopsy-proven NAFLD who had not been diagnosed with type 2 diabetes mellitus: 175 (48%) were women and 48 (13%) had advanced fibrosis. We used simple random sampling; the sampling ratio of the estimation and validation groups was 7:3. A logistic model was constructed for both the estimation and validation groups. The explanatory variables were age ≥49years, sex (women), body mass index ≥26.7kg/m2 , the presence of hypertension, presence of dyslipidemia, fasting plasma glucose level≥98mg/dL, fasting immune reactive insulin level≥12.0μU/mL, and HOMA-IR≥2.90. The median HOMA-IR of the patients was 2.88 (interquartile range: 2.1-4.8). In the estimation group, univariate and multivariate analyses showed that age, dyslipidemia, and HOMA-IR were independent predictors of advanced fibrosis. In the validation group, only age and HOMA-IR were found to be independent predictors of advanced fibrosis. Homeostasis model assessment parameter of insulin resistance was an independent predictor of advanced liver fibrosis in nondiabetic patients with NAFLD. Given that most patients with NAFLD are nondiabetic, it is important to set goals with respect to improving IR to subsequently reduce liver fibrosis.

Spirulina Liquid Extract Protects against Fibrosis Related to Non-Alcoholic Steatohepatitis and Increases Ursodeoxycholic Acid.

Non-alcoholic steatohepatitis (NASH) is characterized by an excess of lipids and oxidative stress in the liver. Spirulina was reported to possess hypolipemic and antioxidative effects and might counteract NASH development. C57Bl/6J mice were fed a western diet (WD) during 25 weeks with or without spirulina liquid extract (SLE) at 2 different doses (WDS1 and WDS2 groups) in drinking water. Liver histology, inflammation, and oxidative stress were assessed as well as glucose tolerance status, lipid metabolism, and gallbladder bile acid profile. WDS2 gained significantly less weight than WD. Liver weight-to-body weight ratio and plasma alanine aminotransferase were significantly lower in WDS2 mice. A reduced liver fibrosis and NFκBp65 protein expression were measured in the supplemented group as a lower accumulation of superoxide anion, nitric oxide, and thiobarbituric reactive substances. WDS2 mice showed also a preserved glucose tolerance, a strong decrease of plasma cholesterol, and a significant increase of gallbladder ursodeoxycholic acid and β-muricholic acid. Our findings demonstrate a protective effect of SLE against WD induced NASH that is related to less inflammation and oxidative stress, a preserved glucose tolerance, and less hepatotoxic bile acid profile.

Platelet-derived growth factor in patients with liver fibrosis

Objective To asses the changes in platelet-derived growth factor (PDGF) in liver fibrosis. Data sources The data were sourced from the PubMed database from the start of the database to 2017 and from all materials available in the Internet. Study selection The initial search presented 423 articles of which 39 met the inclusion criteria. The articles studied the relation between PDGF and liver fibrosis. Data extraction If the studies did not fulfill the inclusion criteria, they were excluded. Study quality assessment included when ethical approval was obtained, eligibility criteria defined, sufficient information, convenient controls, and known assessment measures. Data synthesis Comparisons were made through structured review with the results tabulated. Findings In total, 39 potentially relevant publications were included. The studies indicate an association between PDGF and liver fibrosis as PDGF increases in patients with hepatic fibrosis and correlates well with the grade of fibrosis. The majority of studies were performed on the effect of PDGF antagonists on liver fibrosis and they found that interference of PDGF pathway leads to reduction of liver fibrosis. Conclusion We found that PDGF levels increase in patients with liver fibrosis and its level correlates well with the stage of fibrosis; thus, PDGF can be used as a potential noninvasive marker for liver fibrosis either alone or in association with other fibrosis markers. Further, antagonizing the pathway of PDGF may offer a promising strategy for the treatment of liver fibrosis.

Hepatocyte-specific suppression of microRNA-221-3p mitigates liver fibrosis

Fibrosis, a cardinal feature of a dysfunctional liver, significantly contributes to the ever-increasing mortality due to end-stage chronic liver diseases. The crosstalk between hepatocytes and hepatic stellate cells (HSCs) plays a key role in the progression of fibrosis. Although ample efforts have been devoted to elucidate the functions of HSCs during liver fibrosis, the regulatory functions of hepatocytes remain elusive. Using an unbiased functional microRNA (miRNA) screening, we investigated the ability of hepatocytes to regulate fibrosis by fine-tuning gene expression via miRNA modulation. The in vivo functional analyses were performed by inhibiting miRNA in hepatocytes using adeno-associated virus in carbon-tetrachloride- and 3,5-di-diethoxycarbonyl-1,4-dihydrocollidine-induced liver fibrosis. Blocking miRNA-221-3p function in hepatocytes during chronic liver injury facilitated recovery of the liver and faster resolution of the deposited extracellular matrix. Furthermore, we demonstrate that reduced secretion of C-C motif chemokine ligand 2, as a result of post-transcriptional regulation of GNAI2 (G protein alpha inhibiting activity polypeptide 2) by miRNA-221-3p, mitigates liver fibrosis. Collectively, miRNA modulation in hepatocytes, an easy-to-target cell type in the liver, may serve as a potential therapeutic approach for liver fibrosis. Liver fibrosis majorly contributes to mortality resulting from various liver diseases. We discovered a small RNA known as miRNA-221-3p, whose downregulation in hepatocytes results in reduced liver fibrosis. Thus, inhibition of miRNA-221-3p may serve as one of the therapeutic approaches for treatment of liver fibrosis.

Mesenchymal Stem Cells and Induced Bone Marrow-Derived Macrophages Synergistically Improve Liver Fibrosis in Mice.

We describe a novel therapeutic approach for cirrhosis using mesenchymal stem cells (MSCs) and colony‐stimulating factor‐1‐induced bone marrow‐derived macrophages (id‐BMMs) and analyze the mechanisms underlying fibrosis improvement and regeneration. Mouse MSCs and id‐BMMs were cultured from mouse bone marrow and their interactions analyzed in vitro. MSCs, id‐BMMs, and a combination therapy using MSCs and id‐BMMs were administered to mice with CCl4‐induced cirrhosis. Fibrosis regression, liver regeneration, and liver‐migrating host cells were evaluated. Administered cell behavior was also tracked by intravital imaging. In coculture, MSCs induced switching of id‐BMMs toward the M2 phenotype with high phagocytic activity. In vivo, the combination therapy reduced liver fibrosis (associated with increased matrix metalloproteinases expression), increased hepatocyte proliferation (associated with increased hepatocyte growth factor, vascular endothelial growth factor, and oncostatin M in the liver), and reduced blood levels of liver enzymes, more effectively than MSCs or id‐BMMs monotherapy. Intravital imaging showed that after combination cell administration, a large number of id‐BMMs, which phagocytosed hepatocyte debris and were retained in the liver for more than 7 days, along with a few MSCs, the majority of which were trapped in the lung, migrated to the fibrotic area in the liver. Host macrophages and neutrophils infiltrated after combination therapy and contributed to liver fibrosis regression and promoted regeneration along with administered cells. Indirect effector MSCs and direct effector id‐BMMs synergistically improved cirrhosis along with host cells in mice. These studies pave the way for new treatments for cirrhosis. stem cells translational medicine 2019;8:271&284

Dabigatran Reduces Liver Fibrosis in Thioacetamide-Injured Rats.

Liver fibrosis can progress to cirrhosis, hepatocellular carcinoma, or liver failure. Unfortunately, the antifibrotic agents are limited. Thrombin activates hepatic stellate cells (HSCs). Therefore, we investigated the effects of a direct thrombin inhibitor, dabigatran, on liver fibrosis. Adult male Sprague-Dawley rats were injected intraperitoneally with thioacetamide (TAA, 200mg/kg twice per week) for 8 or 12weeks to induce liver fibrosis. The injured rats were assigned an oral gavage of dabigatran etexilate (30mg/kg/day) or vehicle in the last 4weeks of TAA administration. Rats receiving an injection of normal saline and subsequent oral gavage of dabigatran etexilate or vehicle served as controls. In the 8-week TAA-injured rats, dabigatran ameliorated fibrosis, fibrin deposition, and phosphorylated ERK1/2 in liver, without altering the transcript expression of thrombin receptor protease-activated receptor-1. In vitro, dabigatran inhibited thrombin-induced HSC activation. Furthermore, dabigatran reduced intrahepatic angiogenesis and portal hypertension in TAA-injured rats. Similarly, in the 12-week TAA-injured rats, a 4-week treatment with dabigatran reduced liver fibrosis and portal hypertension. By inhibiting thrombin action, dabigatran reduced liver fibrosis and intrahepatic angiogenesis. Dabigatran may be a promising therapeutic agent for treatment of liver fibrosis.

Enhancer of Zeste Homologue 2 Inhibition Attenuates TGF-β Dependent Hepatic Stellate Cell Activation and Liver Fibrosis.

Background & AimsTransdifferentiation of hepatic stellate cells (HSCs) into myofibroblasts is a key event in the pathogenesis of liver fibrosis. Transforming growth factor β (TGF-β) and platelet-derived growth factor (PDGF) are canonical HSC activators after liver injury. The aim of this study was to analyze the epigenetic modulators that differentially control TGF-β and PDGF signaling pathways.MethodsWe performed a transcriptomic comparison of HSCs treated with TGF-β or PDGF-BB using RNA sequencing. Among the targets that distinguish these 2 pathways, we focused on the histone methyltransferase class of epigenetic modulators.ResultsEnhancer of zeste homolog 2 (EZH2) was expressed differentially, showing significant up-regulation in HSCs activated with TGF-β but not with PDGF-BB. Indeed, EZH2 inhibition using either a pharmacologic (GSK-503) or a genetic (small interfering RNA) approach caused a significant attenuation of TGF-β–induced fibronectin, collagen 1α1, and α-smooth muscle actin, both at messenger RNA and protein levels. Conversely, adenoviral overexpression of EZH2 in HSCs resulted in a significant stimulation of fibronectin protein and messenger RNA levels in TGF-β–treated cells. Finally, we conducted in vivo experiments with mice chronically treated with carbon tetrachloride or bile duct ligation. Administration of GSK-503 to mice receiving either carbon tetrachloride or bile duct ligation led to attenuated fibrosis as assessed by Trichrome and Sirius red stains, hydroxyproline, and α-smooth muscle actin/collagen protein assays.ConclusionsTGF-β and PDGF share redundant and distinct transcriptomic targets, with the former predominating in HSC activation. The EZH2 histone methyltransferase is preferentially involved in the TGF-β as opposed to the PDGF signaling pathway. Inhibition of EZH2 attenuates fibrogenic gene transcription in TGF-β–treated HSCs and reduces liver fibrosis in vivo. The data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE119606 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE119606)

Vitamin A can ameliorate fibrosis of liver in an established rat model of biliary atresia and Kasai portoenterostomy

BackgroundAntifibrosis therapy may prevent progressive liver fibrosis after successful Kasai portoenterostomy (KPE) in biliary atresia (BA) patients. The aim of this study is to evaluate the efficacy of antifibrosis therapy in a rat model of BA and KPE. MethodsBA model was created on three-week-old Sprague–Dawley rats by intrabiliary alcohol injection as previously described, and KPE was performed at postoperative week (POW) 5 by cystoenterostomy. Liver biopsies were performed at the time of BA creation, during KPE, POW 9, and at sacrifice (POW 17). Prednisolone (0.1 mg/100 g/day, group 1, n = 20), Vitamin A (0.5 mg/100 g/day, group 2, n = 20), and ursodeoxycholic acid (UDCA, 1.5 mg/100 g/day, group 3, n = 20) were respectively given to three groups after KPE and continued daily until sacrifice. Histological evaluation of fibrosis and immunohistochemistry stains for 8 fibrosis markers were compared to the control group (without medication, n = 10). ResultsAmong the four markers, namely ɑ-smooth muscle actin (ɑ-SMA), glial fibrillary acidic protein (GFAP), tumor growth factor β1 (TGFβ1) receptors 1 and 2, which showed persistently high expression after successful KPE in the examined 8 markers, only the expression of ɑ-SMA was significantly reduced in all treatment groups at POW17. However, the fibrosis grade at POW 17 was only significantly reduced in group 2 in comparison with the control group (Vitamin A vs. control group, Ishak score 3 vs. 1.8, p < 0.05). ConclusionIn our rat model of BA with KPE, Vitamin A was effective in reducing liver fibrosis, and the mechanisms deserve further study. Level of evidenceBasic science.

Publisher Correction: Reduction of liver fibrosis by rationally designed macromolecular telmisartan prodrugs

In the version of this Article originally published, the author Peter Blume-Jensen was not denoted as a corresponding author; this has now been amended and the author's email address has been added. The 'Correspondence and requests for materials' statement was similarly affected and has now been updated with the author's initials 'P.B-J.'

Reduction of liver fibrosis by rationally designed macromolecular telmisartan prodrugs.

At present there are no drugs for the treatment of chronic liver fibrosis that have been approved by the Food and Drug administration of the United States. Telmisartan, a small-molecule antihypertensive drug, displays antifibrotic activity, but its clinical use is limited because it causes systemic hypotension. Here, we report the scalable and convergent synthesis of macromolecular telmisartan prodrugs optimized for preferential release in diseased liver tissue. We optimized the release of active telmisartan in fibrotic liver to be depot-like (that is, a constant therapeutic concentration) through the molecular design of telmisartan brush-arm star polymers, and show that these lead to improved efficacy and to the avoidance of dose-limiting hypotension in both metabolically and chemically induced mouse models of hepatic fibrosis, as determined by histopathology, enzyme levels in the liver, intact-tissue protein markers, hepatocyte necrosis protection, and gene-expression analyses. In rats and dogs, the prodrugs are retained long-term in liver tissue and have a well-tolerated safety profile. Our findings support the further development of telmisartan prodrugs that enable infrequent dosing in the treatment of liver fibrosis.

A novel dual PPAR-γ agonist/sEH inhibitor treats diabetic complications in a rat model of type 2 diabetes.

The metabolic syndrome is a cluster of risk correlates that can progress to type 2 diabetes. The present study aims to evaluate a novel molecule with a dual action against the metabolic syndrome and type 2 diabetes. We developed and tested a novel dual modulator, RB394, which acts as a soluble epoxide hydrolase (sEH) inhibitor and a peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist in rat models of the metabolic syndrome-the obese spontaneously hypertensive (SHROB) rat and the obese diabetic Zucker fatty/spontaneously hypertensive heart failure F1 hybrid (ZSF1) rat. In SHROB rats we studied the ability of RB394 to prevent metabolic syndrome phenotypes, while in ZSF1 obese diabetic rats we compared RB394 with the ACE inhibitor enalapril in the treatment of type 2 diabetes and associated comorbid conditions. RB394 (10mg/kg daily) and enalapril (10mg/kg daily) were administered orally for 8weeks. RB394 blunted the development of hypertension, insulin resistance, hyperlipidaemia and kidney injury in SHROB rats and reduced fasting blood glucose and HbA1c, improved glucose tolerance, reduced blood pressure and improved lipid profiles in obese ZSF1 rats. A reduction in liver fibrosis and hepatosteatosis was evident in RB394-treated obese ZSF1 rats. Unlike RB394, enalapril did not demonstrate any positive effects in relation to diabetes, hyperlipidaemia or liver dysfunction in obese ZSF1 rats. RB394 ameliorated diabetic nephropathy by reducing renal interstitial fibrosis and renal tubular and glomerular injury in obese diabetic ZSF1 rats. Intriguingly, enalapril demonstrated a weaker action against diabetic nephropathy in obese ZSF1 rats. These findings demonstrate that a novel sHE inhibitor/PPAR-γ agonist molecule targets multiple risk factors of the metabolic syndrome and is a glucose-lowering agent with a strong ability to treat diabetic complications.

Yu Gan Long reduces rat liver fibrosis by blocking TGF-β1/Smad pathway and modulating the immunity

Yu Gan Long (YGL) is a Chinese traditional herbal medicine that has been used in the treatment of liver fibrosis for many years in clinical practice. However, its anti-hepatofibrotic mechanism has not been studied yet. In this study, the effect and mechanism of YGL in reducing liver fibrosis was demonstrated in vivo. Our results showed that liver fibrosis biomarkers collagen IV (Col IV), type III precollagen (PCIII), hyaluronuc acid (HA) and laminin (LN), were increased after CCl4 treatment and decreased by YGL. Among the liver fibrosis indicators, α-smooth muscle actin (α-SMA) was decreased by YGL in the CCl4-treated rats, while MMP2 and MMP9 was upregulated followed by TIMP1 downregulation. Proteins involved in liver fibrosis such as p-Smad2, p-Smad3 and Smad4 were down-regulated, while Smad7 protein was up-regulated by YGL after CCl4-induced liver damage. YGL also suppressed the increase of TGF-β1, TNF-α, IL-1β, IL-6, IL-4 and IL-17 A induced by CCl4 treatment, while promoted IFN-γ expression. Finally, the transcription factors ROR-γt and GATA3 were decreased, while T-bet was increased after YGL treatment. These results suggested that YGL attenuated CCl4-induced hepatic fibrosis by accelerating the extracellular matrix degradation, blocking the TGF-β1/Smad signaling pathway and modulating the balance among IL-4, IL-17 A and IFN-γ, demonstrating YGL protective effect and its potential mechanisms in treating liver fibrosis.

Salvia Miltiorrhiza Ameliorates Liver Fibrosis by Activating Hepatic Natural Killer Cells in Vivo and in Vitro.

Natural killer (NK) cells are known for their ability to kill activated hepatic stellate cells (HSCs), which has been confirmed both in patients and animal models. But the killing function is depressed in period of advanced liver injury. Salvia Miltiorrhiza (SM), a Chinese herbal medicine for invigorating blood circulation and eliminating stasis, is widely used to treat liver fibrosis in clinic. Nevertheless, the immunological mechanism remains unclearly. Here, we put forward the hypothesis that the anti-fibrotic effect of SM is concerned with boosting the activation of hepatic NK cells. Liver fibrosis was induced with carbon tetrachloride (CCl4) and effects of SM on NK cells and HSC (JS-1 cell line, HSC) were investigated in vivo and in vitro. Hepatic NK cells were isolated from C57BL/6 mice, and pre-incubated with SM before they were co-cultured with HSCs. We found that SM increased frequency of NK cells, enhanced activities of NKG2D and Nkp46 on NK cells and inhibited activation of HSCs in vivo and in vitro. SM could promote the activities of NK cells by increasing the expressions of NKG2D and IFN-γ before or after co-cultured with HSCs in vitro. Besides, SM could partially antagonize ASGM-1-induced NK cell depletion and enhance the cell activities to inhibit HSCs activation in vitro. Therefore, our work provided a new insight into the anti-fibrotic mechanism that SM could enhance the activities of NK cell to reduce liver fibrosis in vivo and in vitro.

Abstract 4004: The H2 receptor antagonist nizatidine inhibits carcinogenesis in two rodent models of hepatocellular carcinoma

Abstract Introduction: The purpose of this study was to examine whether nizatidine could reduce liver fibrosis and subsequent tumor burden in two rodent models of hepatocellular carcinoma (HCC). Methods: An in silico screen identified nizatidine as a compound that can reverse a previously identified gene signature associated with disease progression and HCC development in human cirrhosis patients. We tested the ability of nizatidine to inhibit HCC development in two rodent models. In the first study, male Wistar rats received weekly intraperitoneal injections of 50 mg/kg diethylnitrosamine (DEN). This model has previously been shown to reliably recapitulate the histologic and molecular features of human HCC development including fibrosis after 8 weeks, cirrhosis after 12 weeks, and HCC by 18 weeks. DEN-injured rats were randomized to receive oral gavage of nizatidine (n=7) or vehicle control (n=9) after 8 weeks. In the second study, male C57BL/6J mice received a single intraperitoneal injection of 25 mg/kg DEN at day 15 followed by initiation of a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) at 6 weeks of age. DEN+CDAHFD-injured mice were randomized to receive oral gavage of nizatidine (n=7) or vehicle control (n=9) at 12 weeks of age and were sacrificed at 30 weeks of age after development of HCC in the setting of nonalcoholic steatohepatitis (NASH). Results: As expected, repeated injections of DEN in rats resulted in progressive fibrosis followed by HCC formation. Treatment with nizatidine resulted in a 35% reduction of tumor nodules relative to vehicle controls (p&amp;lt;0.18). Liver sections were stained by picrosirius red to assess fibrosis and nizatidine reduced collagen deposition in DEN-injured rats (collagen proportional area = 5±0.03 vs. 9.2±0.04; p&amp;lt;0.05). This histologic observation was further confirmed by gene expression analysis with reduction in several profibrotic markers, such as alpha-smooth muscle actin and collagen type I, after treatment with nizatidine. All mice receiving DEN+CDAHFD developed HCC. Treatment with nizatidine resulted in a 60% reduction in tumor nodules relative to vehicle controls (p&amp;lt;0.0001). Nizatidine treatment also resulted in a significant reduction in liver to body weight (p&amp;lt;0.01). Nizatidine treatment reduced collagen proportional area (11±0.05 vs. 15±.01; p&amp;lt;0.05) and expression of profibrotic markers. Nizatidine treatment also reduced the expression of several proinflammatory markers including CD68, interferon gamma, and interleukin-6. Conclusion: Our data suggest that the H2 receptor antagonist nizatidine reduces fibrosis and subsequent HCC development. This could be beneficial in patient management given the low cost and ready availability of this agent. Citation Format: Shen Li, Sarani Ghoshal, Gunisha Arora, Derek J. Erstad, Mozhdeh Sojoodi, Michael Lanuti, Yujin Hoshida, Thomas Baumert, Kenneth K. Tanabe, Bryan C. Fuchs. The H2 receptor antagonist nizatidine inhibits carcinogenesis in two rodent models of hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4004.

Sorafenib and praziquantel synergistically attenuate Schistosoma japonicum-induced liver fibrosis in mice.

Liver fibrosis is an important process that occurs in most types of chronic liver diseases and often results in the end stage of liver diseases, such as cirrhosis, portal hypertension, and hepatocellular carcinoma. Sorafenib, a multiple tyrosine kinase inhibitor, has been shown to inhibit liver fibrosis in multiple experimental fibrosis mouse and rat models. The aim of this study was to test the therapeutic effect of sorafenib on liver fibrosis induced by infection with a parasite, Schistosoma japonicum, in mice. Mice were percutaneously infected through the abdomen with Schistosoma cercariae to develop a schistosomula liver fibrosis model. Eight weeks after infection, infected mice were treated with the anti-parasitic agent praziquantel for 2days and sorafenib for 2weeks. Hepatic histopathological changes were assessed using hematoxylin and eosin (HE) and Masson's trichome staining. The hepatic expression levels of collagen I, collagen III, alpha-smooth muscle actin (α-SMA), platelet-derived growth factor (PDGF), and PDGF receptor-beta (PDGFR-β) were analyzed by immunohistochemistry and western blot. Praziquantel administration alone but not sorafenib reduced liver fibrosis, and the combination of praziquantel and sorafenib significantly attenuated liver fibrosis in S. japonicum-infected mice. Moreover, sorafenib plus praziquantel markedly decreased the hepatic deposition of collagen and expression of fibrogenic genes in these mice. In conclusion, the use of sorafenib following praziquantel treatment may represent a potential therapeutic strategy for liver fibrosis induced by S. japonicum in patients.

Evaluation of the accuracy of exchangeable copper and relative exchangeable copper (REC) in a mouse model of Wilson’s disease

Wilson's disease (WD) is caused by mutations in the ATP7B gene responsible for a toxic copper overload mainly in the liver and the central nervous system. Phenotypic heterogeneity may challenge the diagnostic confirmation. Exchangeable copper (CuEXC) has recently been proposed as a new marker of WD, and its ratio to the total serum copper (Cus), Relative Exchangeable Copper (REC = CuEXC/Cus), as a diagnostic marker. This study aimed to investigate whether this could be confirmed in Atp7b-/- mice, an engineered WD animal model. Atp7b-/- (n = 137) and wild type (WT; n = 101) mice were investigated under the same conditions at 6–8, 20, 39, or 50 weeks of age. Twenty-four Atp7b−/− mice received D-penicillamine treatment from 39 to 50 weeks of age. Serum and liver [histology and intrahepatic copper (IHCu)] data were evaluated. In the WT group, all serum and liver data were normal. Atp7b−/− livers developed a chronic injury from isolated moderate inflammation (6–8 weeks: 16/33 = 48%) to inflammatory fibrosis with cirrhosis (50 weeks: 25/25 = 100% and 16/25 = 64% respectively). Cus and CuEXC increased until week 39, whereas IHCu and REC were stable with increasing age and much higher than in WT mice (mean ± SD: 669 ± 269 vs. 13 ± 3 μg/g dry liver and 39 ± 12 vs. 11 ± 3%, respectively). A threshold value of 20% for REC provided a diagnostic sensitivity and specificity of 100%, regardless of sex, age, or the use of D-penicillamine. Eleven weeks of 100 mg/kg D-penicillamine reduced liver fibrosis (p = 0.001), IHCu (p = 0.026) and CuEXC (p = 0.175). In conclusion, this study confirms REC as a WD diagnostic marker in a mouse model of chronic liver disease caused by copper overload. Further studies are needed to assess the usefulness of CuEXC to monitor the evolution of WD, particularly during treatment.