Experimental Model Of Cirrhosis Research Articles

Role of Nitric Oxide in the Altered Calcium Homeostasis of Platelets from Rats with Biliary Cirrhosis.

Previously, we found that intracellular calcium (Ca2+) homeostasis is altered in platelets from an experimental model of liver cirrhosis, namely the bile-duct-ligated (BDL) rat. These alterations are compatible with the existence of a hypercoagulable state. In the present study, we analyzed the role of nitric oxide in the abnormal calcium signaling responses of an experimental cirrhosis model, the bile duct-ligated rat. Chronic treatment with L-NAME was used to inhibit NO production in a group of control and BDL animals, and the responses compared to those obtained in a control and BDL untreated group (n = 6 each). The experiments were conducted on isolated platelets loaded with fura-2, using fluorescence spectrometry. Chronic treatment with L-NAME increased thrombin-induced Ca2+ release from internal stores in both control and BDL rats. However, the effect was significantly greater in the BDL rats (p < 0.05). Thrombin-induced calcium entry from the extracellular space was also elevated but at lower doses and, similarly in both control and BDL platelets, treated with the NO synthesis inhibitor. Capacitative calcium entry was also enhanced in the control platelets but not in platelets from BDL rats treated with L-NAME. Total calcium in intracellular stores was elevated in untreated platelets from BDL rats, and L-NAME pretreatment significantly (p < 0.05) elevated these values both in controls and in BDL but significantly more in the BDL rats (p < 0.05). Our results suggest that nitric oxide plays a role in the abnormal calcium signaling responses observed in platelets from BDL rats by interfering with the mechanism that releases calcium from the internal stores.

Microbiota transplantation in portal hypertension: promises and pitfalls.

In this commentary, we discuss new findings indicating that microbiota transplantation has favorable impact on portal hypertension (PH) in the experimental model of cirrhosis induced by bile duct ligation (BDL) (Huang et al.; Clin Sci (Lond) (2021) 135(24): 2709-2728, doi: 10.1042/CS20210602). Sinusoidal PH is an ominous outcome of advanced chronic liver disease, characterized by increased intrahepatic vascular resistance (IHVR), splanchnic hyperemia, and the development of portosystemic collaterals. In the work of Huang et al., microbiota transplantation not only alleviated splanchnic hyperdynamic circulation by improving vascular responsiveness and decreasing mesenteric angiogenesis, but also reduced blood flow in portosystemic collaterals. Surprisingly, however, microbiota transplantation had no effect on intrahepatic vasoconstriction in this experimental model. We discuss these observations in the context of recent literature showing that manipulation of the gut microbiota (either by transplantation or through the use of probiotics) may improve IHVR, which is one of the earliest abnormalities in the pathogenesis of sinusoidal PH. Further research is needed to explore the specific molecular and cellular targets associated with the correction of dysbiosis in liver disease.

1-Methyl tryptophan, an indoleamine 2,3-dioxygenase inhibitor, attenuates cardiac and hepatic dysfunction in rats with biliary cirrhosis

Kynurenine Pathway (KP) is the dominant metabolic route of tryptophan which is catalyzed by indoleamine-2,3-dioxygenase (IDO). This pathway is upregulated in liver disease where the level of KP metabolites correlates with the severity of disease. Cirrhosis is associated with cardiac dysfunction, which manifests itself during severe physiological challenges such as liver transplantation. Cardiac dysfunction in cirrhosis is linked to systemic inflammation and impaired cardiac beta-adrenergic signaling pathways. The KP pathway is involved in modulation of cardiac signaling and is upregulated by systemic inflammation. Therefore, this study aimed to evaluate the effect of IDO inhibition on development of cardiac dysfunction in an experimental model of cirrhosis. Cirrhosis was induced by bile duct ligation (BDL). Experimental groups were given either 1-methyl tryptophan (1-MT, 1, 3, 9 mg/kg), or saline. 28 days after BDL, cardiac chronotropic response to epinephrine was assessed ex vivo. HPLC was employed to measure hepatic and cardiac levels of tryptophan, kynurenine and kynurenic acid. Cirrhosis in rats was associated with impaired cardiac chronotropic responsiveness to adrenergic stimulation. 1-MT dose-dependently improved cirrhosis-induced chronotropic dysfunction as well as elevated serum levels of CRP and IL-6 in BDL rats. Hepatic and cardiac kynurenine/tryptophan ratio were elevated in cirrhotic rats and were reduced following 1-MT administration. Chronic administration of 1-MT could also reduce hepatic inflammation, fibrosis and ductular proliferation. 1-MT attenuates cardiac dysfunction in rats with biliary cirrhosis. This protective effect is not limited to the cardiac function as liver histopathologic changes were also improved following chronic 1-MT administration.

Liver Sinusoidal Endothelial Cells Contribute to Hepatic Antigen-Presenting Cell Function and Th17 Expansion in Cirrhosis.

Hepatic immune function is compromised during cirrhosis. This study investigated the immune features of liver sinusoidal endothelial cells (LSECs) in two experimental models of cirrhosis. Dendritic cells, hepatic macrophages, and LSECs were isolated from carbon tetrachloride and bile duct-ligated rats. Gene expression of innate receptors, bacterial internalization, co-stimulatory molecules induction, and CD4+ T cell activation and differentiation were evaluated. Induced bacterial peritonitis and norfloxacin protocols on cirrhotic rats were also carried out. LSECs demonstrated an active immunosurveillance profile, as shown by transcriptional modulation of different scavenger and cell-adhesion genes, and their contribution to bacterial internalization. LSECs significantly increased their expression of CD40 and CD80 and stimulated CD4+ T cell activation marker CD71 in both models. The pro-inflammatory Th17 subset was expanded in CCl4-derived LSECs co-cultures. In the bile duct ligation (BDL) model, CD4+ T cell differentiation only occurred under induced bacterial peritonitis conditions. Differentiated pro-inflammatory Th cells by LSECs in both experimental models were significantly reduced with norfloxacin treatment, whereas Foxp3 tolerogenic Th CD4+ cells were expanded. Conclusion: LSECs’ participation in the innate-adaptive immune progression, their ability to stimulate pro-inflammatory CD4+ T cells expansion during liver damage, and their target role in norfloxacin-induced immunomodulation granted a specific competence to this cell population in cirrhosis.

The protective effects of curcumin and curmumin nanomicelle against cirrhotic cardiomyopathy in bile duct-ligated rats

Objective(s): Cirrhotic cardiomyopathy refers to cardiac muscle dysfunction caused by liver cirrhosis. Seemingly, free radicals and inflammatory factors play a critical role in the pathophysiology of cardiomyopathy. Curcumin has the anti-inflammatory, antioxidant, and anticancer properties . However, the therapeutic indications of this compound are limited due to its low absorption, rapid metabolism, and low bioavailability. Curcumin nanomicelle is a form of nanoparticle developed to overcome the poor kinetic profile of curcumin and enhance its bioavailability and therapeutic effects. The present study aimed to develop an experimental model of cirrhosis induced by biliary duct ligation in rats. Materials and Methods: The animals were kept until 28 days after the bile duct ligation and received curcumin or curcumin nanomicelle via oral gavage at various doses during days 7-28. After the intervention, the effects of curcumin and curcumin nanomicelle on cardiovascular function, some inflammatory and antioxidant biomarkers, and histopathological changes were assessed. Results: According to the findings, cardiac electrophysiology function and contractile force improved only in the curcumin nanomicelle groups. In addition, curcumin nanomicelle significantly reduced inflammatory factors and increased antioxidant enzymes. In the histopathological studies, cardiac tissue damage and destruction were observed to decrease in the curcumin nanomicelle groups. Conclusion: Therefore, it was concluded that curcumin nanomicelle plays a protective role in cirrhotic cardiomyopathy by reducing inflammatory and oxidative factors and improving the cardiac function. Furthermore, curcumin nanomicelle exhibited more significant therapeutic effects compared to the curcumin treatment groups.

Mitochondria-targeted antioxidant mitoquinone attenuates liver inflammation and fibrosis in cirrhotic rats.

In liver cirrhosis, oxidative stress plays a major role in promoting liver inflammation and fibrosis. Mitochondria dysregulation is responsible for excessive reactive oxygen species production. Therefore, in an experimental model of cirrhosis, we investigated the effect of mitochondria-targeted antioxidant mitoquinone. Liver cirrhosis was induced in Spraque-Dawley rats by common bile duct ligation (CBDL). Mitoquinone (10 mg·kg-1·day-1, oral gavage) or vehicle was administered from 3rd to 28th day after CBDL, when animals were euthanized; liver oxidative stress, inflammation, fibrosis, mitophagy were evaluated; and in vivo and ex vivo hemodynamic studies were performed. In cirrhotic rats, mitoquinone prevented liver inflammation, hepatocyte necrosis, and fibrosis at histological examination; decreased circulating TNF-α, gene expression of transforming growth factor-β1, collagen type 1a1, TNF-α, IL-6, IL-1β, tissue inhibitor of metalloproteinase-1, matrix metalloproteinase (MMP)-2, and MMP-13; and reduced hepatic oxidative stress, as shown by reduced oxidative carbonylation of the proteins, by modulating antioxidants catalase, Mn superoxide dismutase, and Cu/Zn superoxide dismutase. Furthermore, mitoquinone attenuated apoptosis by reducing hepatic protein expression of cleaved caspase-3. A selective removal of dysfunctional mitochondria was improved by mitoquinone, as shown by the increase in Parkin translocation to mitochondria. Treatment with mitoquinone normalized the weight of the spleen; however, it increased portal blood flow and reduced splenic artery intrahepatic resistance, suggesting an effect on resistance index. Mitochondria-targeted antioxidant mitoquinone improves liver inflammation and fibrosis in cirrhotic rats by reducing hepatic oxidative stress, preventing apoptosis, and promoting removal of dysfunctional mitochondria. Therefore, it may represent a promising strategy for the prevention and treatment of liver cirrhosis.

Impairment of natriuresis and diuresis induced by intrarenal adrenoceptor mechanisms in an experimental model of cirrhosis in rats

The contribution of intrarenal alpha-2 adrenergic receptors in mediating the enhanced renal excretory responses evoked by the alpha-2-agonist xylazine was examined in a model of cirrhosis in rats. In sham-operated rats, xylazine (0.2 mg/kg, i.v.) increased diuresis and natriuresis (urine flow, control: 78 ± 12.1, 10 min: 155 ± 17, 20 min: 194 ± 19, 30 min: 146 ± 16, 40 min: 114 ± 13, 50 min: 95 ± 10.5 μl/min/g; urinary sodium excretion, control: 6.75 ± 2.08, 10 min: 7.12 ± 2.1, 20 min: 13.4 ± 4.6, 30 min: 14.6 ± 4.02, 40 min: 12.05 ± 2.35, 50 min: 12.7 ± 2.45 μeq/min/g), which was accompanied by a significant reduction in renal sympathetic nerve activity (RSNA) (control: 100, 10 min: 39.5 ± 5.8, 20 min: 53 ± 8.8, 30 min: 72 ± 7.0, 40 min: 83 ± 5.0, 50 min: 94 ± 6.1 AU). Xylazine (0.2 mg/kg) in cirrhotic animals, despite resulting in a significant reduction in RSNA (control: 100, 10 min: 73 ± 4.3*, 20 min: 70 ± 5.0*, 30 min: 76 ± 7.0*, 40 min: 85 ± 5.5*, 50 min: 92 ± 4.8* AU), was unable to increase natriuresis. A higher dose (20 mg/kg) of xylazine was not capable of increasing natriuresis and diuresis, even in the presence of a robust reduction in RSNA. Renal denervation did not alter the onset and time course of cirrhosis. The results indicated that during the development of cirrhosis, there is an adaptive process that disables the intrarenal alpha-2 adrenoceptor mechanisms that selectively promote water and urinary sodium excretion via a sympathetic renal nerve-independent mechanism. Thus, in cirrhotic rats, the diuresis/natriuresis induced by xylazine is independent on RSNA. Intrarenal and/or hormonal changes are probably involved in the impairment of xylazine-induced diuresis/natriuresis in cirrhosis.

P: 82 Genetically Engineered E. coli Nissle Attenuates Hyperammonemia and Improves Memory in an Experimental Model of Cirrhosis and Hepatic Encephalopathy

BACKGROUND: Hyperammonemia associated with chronic liver disease (CLD) is implicated in the pathogenesis of hepatic encephalopathy (HE). The gut is a major source of ammonia production that contributes to systemic hyperammonemia in CLD and HE and remains the primary therapeutic target for lowering circulating ammonia. As a therapeutic strategy, Escherichia coli Nissle 1917 bacterium (EcN), a well characterized probiotic, was genetically modified to consume and convert ammonia to arginine (SYNARG), and its administration to thioacetamide-treated mice resulted in a significant reduction of ammonia levels.1 SYNARG was further modified to synthesize butyrate (SYNARG+BUT), a short-chain fatty acid with anti-inflammatory/anti-oxidant properties, and both strains were tested in an experimental model of cirrhosis and HE, the bile duct ligation (BDL). METHODS: One week post surgery, BDL rats were gavaged with SYNARG, SYNARG+BUT (3 × 10 11 CFU/day, BID) or vehicle until they were sacrificed at 3- or 5-weeks along with respective sham controls. Plasma ammonia and live markers were measured at 3 and 5 weeks. Recognition-memory, motor-coordination, muscle-strength, locomotion and anxiety were assessed in the 5-week BDL groups. RESULTS: BDL significantly increased ammonia over time, with levels of 109.1 ± 9.2 µM (Shams 56.7 ± 3.5 µM, P &lt; 0.001) and 150.2 ± 25.6 µM (Shams 58.3 ± 3.0µM, P &lt; 0.001) at 3- and 5-weeks, respectively. In addition, plasma liver markers alanine-transaminase, aspartate-transaminase, bilirubin, and gamma-glutamyl transferase were significantly increased in BDL rats at both timepoints while albumin was significantly lowered. As compared to BDL-Veh rats, hyperammonemia was attenuated by SYNARG (103.9 ± 12.3 µM) and SYNARG+BUT (110.8 ± 8.5 µM) at 5, but not 3 weeks post-surgery, while liver fibrosis (hydroxyproline content) was attenuated at 3, but not 5 weeks post-surgery. None of the circulating liver markers were changed by the treatments at any timepoint. Motor-coordination, muscle-strength, locomotion and anxiety were affected in all BDL groups without protective effect of treatments. Short-term memory (STM) was impaired in BDL-Veh (P &lt; 0.001) and BDL-SYNARG (P &lt; 0.05) versus Shams, while STM was resolved in BDL-SYNARG+BUT (P &lt; 0.05 vs BDL-Veh). Long-term memory (LTM) was impaired in BDL-Veh vs Shams (P &lt; 0.05), but BDL-SYNARG and BDL-SYNARG+BUT were protected. CONCLUSIONS: EcN, engineered to consume ammonia in the gut and synthesize butyrate, is an effective approach to lower plasma ammonia in a model of cirrhosis and HE. Moreover, the attenuation of hyperammonemia in cirrhotic rats is associated with a protective effect on memory in this model. The therapeutic potential of these engineered EcN strains should be further evaluated in patients with CLD and HE.

FRI-230 - Regulatory T cells modulate the concentration of short chain fatty acids and the Th function in response to induced bacterial traslocation episodes, and improve the integrity of the gut barrier in an experimental cirrhosis model

FRI-230 - Regulatory T cells modulate the concentration of short chain fatty acids and the Th function in response to induced bacterial traslocation episodes, and improve the integrity of the gut barrier in an experimental cirrhosis model

Role of Tissue Inhibitors of Matrix Metalloproteases-1 (TIMP-1) in Evolution of CCl4-Induced Liver Cirrhosis in Mice.

Background Cirrhosis is a worldwide end stage liver disease. It is characterized by diffuse fibrosis and conversion of normal liver lobules into structurally abnormal nodules. Aim of the work This study was conducted to investigate the role of tissue inhibitors of matrix metalloproteases-1 (TIMP-1) in during the evolution of cirrhosis, and in fibers resolution after cessation of the insult. Materials and methods 54 adult male Balb/c mice; about 2 months old were randomly divided into control and treated groups;Control:24 animals.In the treated groups; 30 animals were subcutaneously injected with CCl4 (20% # diluted in sunflower oil) in a dose of 1 ml per Kg twice weekly.6 animals of them were sacrificed72 hours after the last dose at the 4th week, 8th week, 12th week, and 16th week.6 animals were kept for two weeks without injection after the 16 weeks of CCl4 treatment. Results Our results indicated that TIMP-1 is involved in the process of fibrogenesis and fibers resolution in an experimental model of cirrhosis in mice. Conclusion TIMP-1 is involved in the process of fibrogenesis in mice which can be applied in new strategies for the treatment of liver cirrhosis.

Skeletal muscle myopenia in mice model of bile duct ligation and carbon tetrachloride-induced liver cirrhosis.

Skeletal muscle myopathy is universal in cirrhotic patients, however, little is known about the main mechanisms involved. The study aims to investigate skeletal muscle morphological, histological, and functional modifications in experimental models of cirrhosis and the principal molecular pathways responsible for skeletal muscle myopathy. Cirrhosis was induced by bile duct ligation (BDL) and carbon tetrachloride (CCl4) administration in mice. Control animals (CTR) underwent bile duct exposure or vehicle administration only. At sacrifice, peripheral muscles were dissected and weighed. Contractile properties of extensor digitorum longus (EDL) were studied in vitro. Muscle samples were used for histological and molecular analysis. Quadriceps muscle histology revealed a significant reduction in cross‐sectional area of muscle and muscle fibers in cirrhotic mice with respect to CTR. Kinetic properties of EDL in both BDL and CCl4 were reduced with respect to CTR; BDL mice also showed a reduction in muscle force and a decrease in the resistance to fatigue. Increase in myostatin expression associated with a decrease in AKT‐mTOR expressions was observed in BDL mice, together with an increase in LC3 protein levels. Upregulation of the proinflammatory citochines TNF‐a and IL6 and an increased expression of NF‐kB and MuRF‐1 were observed in CCl4 mice. In conclusion, skeletal muscle myopenia was present in experimental models of BDL and CCl4‐induced cirrhosis. Moreover, reduction in protein synthesis and activation of protein degradation were the main mechanisms responsible for myopenia in BDL mice, while activation of ubiquitin‐pathway through inflammatory cytokines seems to be the main potential mechanism involved in CCl4 mice.

ALTERATION OF TASTE BUDS IN EXPERIMENTAL CIRRHOSIS. Is there correlation with human hypogeusia?

The inherent complications of cirrhosis include protein-calorie malnutrition and micronutrient deficiencies.Changes in taste are detrimental to the nutritional status, and the mechanism to explain these changes is not well documented in the cirrhotic patients. To evaluate the taste buds of cirrhotic rats. Fourteen male Wistar rats were evaluated. After 16 weeks, the liver was removed to histologically diagnose cirrhosis, and blood was collected to perform liver integrity tests. The tongue was removed for histological examination and immunohistochemistry using antibodies against protein gene product PGP 9.5 and the sweet taste receptors T1R2 and T1R3. Morphological changes were determined by scanning electron microscopy. Serum zinc levels were measured. The cirrhotic animals, but not the control animals, exhibited zinc deficiency. In both groups, there was positive immunoreactivity for type II and III cells and T1R2 receptors. The cirrhotic animals had no immunoreactivity for T1R3 receptors. Scanning electron microscopy analysis of the cirrhotic group revealed a uniform tapering of the gustatory papillae. In conclusion the experimental cirrhosis model mimicked the biochemical and histological parameters of human cirrhosis, therefore enabling a study of the gustatory papillae and taste buds.

Reply

We thank the editors for giving us an opportunity to comment on Drs Kalman and Goldberg’s excellent summary of our paper on the use of obeticholic acid (OCA) in an experimental model of cirrhosis. Their commentary raises several issues regarding the relevance of the inflamed gut as a target for the management of portal hypertension and related complications in cirrhosis. Our main finding was that OCA reduces gut bacterial translocation (GBT) by mitigating intestinal inflammation in a model of noncholestatic cirrhosis. Intestinal inflammation was measured as increased activated immune system cells and inflammation-related gene expression in the gut wall of rats with cirrhosis, especially in those with GBT. In this last setting, inflammation products damage the intestinal barrier and are major contributors to increased intestinal permeability and GBT. In turn, gut dysbiosis seems to be the driver of intestinal inflammation in cirrhosis, because the latter is ameliorated after lowering the intestinal bacterial burden with antibiotics (Hepatology 2012;56:1861–1869). FXR stimulation by OCA normalized increased nuclear factor-κB activation, the expression of inflammation-related cytokine genes and innate defense mechanisms in the bowel wall of cirrhotic rats, as the main mechanism leading to improved intestinal barrier function and reduced GBT. Bacterial infections such as spontaneous peritonitis are an increasing cause of mortality in cirrhosis, and their prophylaxis is circumscribed to selective bowel decontamination with quinolones, a risk factor for infection with MDR strains (Hepatology 2012;55:1551–1561). Targeting gut inflammation to halt GBT is a novel alternative to antibiotics as primary or secondary prophylaxis for bacterial infection that warrants further clinical investigation. As indicated by Kalman and Goldberg, a remarkable finding of our study was the improvement of hepatic fibrogenesis, liver function, and ascites in cirrhotic rats on OCA. Although we did not address the mechanisms involved, it is tempting to speculate that the beneficial effects of OCA at the hepatic level could result from reduced stimulation of liver immune cells owing to lowered endotoxinemia and passage of bacterial products from the gut, and direct inhibition of activated hepatic stellate cells. Additionally, OCA can lower portal pressure by improving the vascular tone of the cirrhotic liver through (1) increased intrahepatic nitric oxide bioavailability (Hepatology 2014;59:2286–2298) and (2) decreased hepatic macrophage activation due to reduced passage of bacterial products from the gut to the portal circulation. Our preclinical study in a noncholestatic model of cirrhosis opens the window to managing portal hypertension and related complications with drugs mechanistically targeting the gut. The Role of Obeticholic Acid in Gut Bacterial Translocation and InflammationGastroenterologyVol. 151Issue 4PreviewUbeda M, Lario M, and Munoz L, et al. Obetocholic acid reduces bacterial translocation and inhibits intestinal inflammation in cirrhotic rats. J Hepatol 2016;64:1049–1057. Full-Text PDF

Splenectomy enhances the therapeutic effect of adipose tissue-derived mesenchymal stem cell infusion on cirrhosis rats.

Clinical studies suggest that splenectomy improves liver function in cirrhotic patients, but the influence of splenectomy on stem cell transplantation is poorly understood. This study investigated the effect ofsplenectomy on stem cell infusion and elucidated its mechanism. Rat adipose tissue-derived mesenchymal stem cells were infused into cirrhosis rats with or without splenectomy, followed by the assessment of the invivo distribution of stem cells and pathological changes. Stromal cell-derived factor-1 and hepatocyte growth factor expression were also investigated in splenectomized cirrhosis patients and rats. Splenectomy, prior to cell infusion, improved liver function and suppressed fibrosis progression more efficiently than cell infusion alone in the experimental cirrhosis model. Stromal cell-derived factor-1 and hepatocyte growth factor levels after splenectomy were increased in patients and rats. These upregulated cytokines significantly facilitated stem cell motility, migration and proliferation invitro. C-X-C chemokine receptor type 4 neutralization weakened the promotion of cell migration by these cytokines. The infused cells integrated into liver fibrosis septa and participated in regeneration more efficiently in splenectomized rats. Direct coculture with stem cells led to inhibition of hepatic stellate cell proliferation. In addition, hepatocyte growth factor induced hepatic stellate cell apoptosis via the c-jun N-terminal kinase-p53 pathway. Splenectomy prior to cell infusion enhanced the therapeutic effect of stem cells on cirrhosis, which involved upregulation of stromal cell-derived factor-1 and hepatocyte growth factor after splenectomy.

Diethylnitrosamine-induced cirrhosis in Wistar rats: an experimental feasibility study.

The experimental models of the development of cirrhosis in rats require a long time. Many studies in animals have demonstrated similarities in histological pattern with human cirrhosis. Just like the relation between cirrhosis and increased lipid peroxidation (LPO), which contributes to the worsening of the disease. However, few studies have focused on the reduction of time to establish cirrhosis and evaluated the expression of heat-shock protein 70 (HSP70) in cirrhotic livers of rodents. The present study proposes the adaptation of an experimental cirrhosis model using diethylnitrosamine (DEN). Twenty-six male Wistar rats, weighing ±270 g, divided into two groups: (i) CO-control and (ii) DEN-diethylnitrosamine. The DEN group received 50 mg/kg of DEN twice a week intraperitoneally for 7 weeks. The model developed cirrhosis in 7 weeks. The liver function tests showed that the animals with DEN-induced cirrhosis had increased levels when compared to control. The histological examination showed changes in the liver architecture, with severe ductal proliferation, signs of chronic damage, cholestasis, lymphocytic infiltrate, steatosis, and extensive parenchymal loss. We also found nodular formations with homogeneous pattern, increased LPO, increased expression of iNOS, TGF beta, α-SMA, and NQO1. However, the HSP70 expression was reduced in cirrhotic animals. This study showed signs of cirrhosis in liver based on biochemical, histological, and molecular analysis. The reduced expression of HSP70 appears to be associated with increased oxidative stress, contributing to the worsening of the disease.

Overexpression of angiopoietin‐2 in rats and patients with liver fibrosis. Therapeutic consequences of its inhibition

Studies in experimental models of cirrhosis showed that anti-angiogenic treatments may be effective for the treatment of liver fibrosis. In this context, angiopoietins are potential therapeutic targets as they are involved in the maintenance and stabilization of newly formed blood vessels. In addition, angiopoietin-2 is expressed in fibrotic livers and its inhibition in tumours results in vessel stability. Therefore, our study was aimed to assess the therapeutic utility of inhibiting angiopoietin-2. Circulating levels of angiopoietin-1 and angiopoietin-2 were quantified by ELISA in CCl4 -treated rats and in patients with cirrhosis. In vivo blockade of angiopoietin-2 in rats with liver fibrosis was performed with a chemically programmed antibody, CVX-060. High levels of angiopoietin-2 were found in the systemic and suprahepatic circulation of cirrhotic patients and the ratio angiopoietin-1/angiopoietin-2 inversely correlated with prognostic models for alcoholic liver disease. Chronic treatment of CCl4 -treated rats with CVX-060 was associated with a significant decrease in inflammatory infiltrate, normalization of the hepatic microvasculature and reduction in VCAM-1 vascular expression. The anti-angiopoietin-2 treatment was also associated with less liver fibrosis and with lower levels of circulating transaminases. CVX-060 treatment was not associated with either vascular pruning in healthy tissue or compensatory overexpression of VEGF. Inhibition of angiopoietin-2 is an effective and safe treatment for liver fibrosis in CCl4 -treated rats, acting mainly through the induction of vessel normalization and the attenuation of hepatic inflammatory infiltrate. Therefore, inhibition of angiopoietin-2 offers a therapeutic alternative for liver fibrosis.

Effect of endotoxin on heart rate dynamics in rats with cirrhosis

Reduced heart rate variability (HRV) is a hallmark of systemic inflammation which carries negative prognostic information in sepsis. Decreased HRV is associated with partial uncoupling of cardiac pacemaker from cholinergic neural control during systemic inflammation. Sepsis is a common complication in liver cirrhosis with high mortality. The present study was aimed to explore the hypothesis that endotoxin uncouples cardiac pacemaker from autonomic neural control and reduces HRV in an experimental model of cirrhosis. Cirrhosis was induced by surgical ligation of the bile duct in rats. Cirrhotic rats were given intraperitoneal injection of either saline or lipopolysaccharide (endotoxin, 1mg/kg). Changes in HRV indices were studied in conscious rats using implanted telemetric probes. The atria were isolated and chronotropic responsiveness to cholinergic stimulation was assessed in vitro. Endotoxin injection induced a significant tachycardia and decreased short-term and long-term HRV indices in control rats. However, endotoxin was unable to increase heart rate in cirrhotic animals. In contrast with control rats, endotoxin induced biphasic changes in short-term HRV in cirrhotic rats. Acute endotoxin challenge reduced long-term HRV with 60-min delay in comparison with control animals. Endotoxin injection was associated with a significant hypo-responsiveness to cholinergic stimulation in control rats in vitro. Endotoxin did not change atrial chronotropic responsiveness to cholinergic stimulation in cirrhotic rats. Our data shows that cirrhosis is associated with development of tolerance to cardiac chronotropic effect of endotoxin in rats.

Relaxin is a renal vasodilator in experimental models of cirrhosis and a potential novel therapy for hepatorenal syndrome in man

BackgroundHepatorenal syndrome is a feared complication of cirrhosis, with a high mortality rate and limited treatment options. The hallmarks of the disorder are functional renal failure, normal kidney histological findings, and profound renal vasoconstriction. The hormone relaxin mediates haemodynamic adaptations to pregnancy including increased renal blood flow and glomerular filtration rate (GFR). We hypothesised that relaxin could be used to modulate renal blood flow in cirrhosis. MethodsCirrhosis was induced in male rats by 16 weeks of carbon tetrachloride (intraperitoneally) and decompensated biliary cirrhosis by bile duct ligation. The effect of acute intravenous or extended (72 h) subcutaneous relaxin versus placebo was determined by analysis of systemic haemodynamics, renal blood flow, GFR, and organ histology. The effect of co-treatment with L-NG-nitroarginine methyl ester (L-NAME) was measured in subgroups of relaxin or placebo treated rats. Blood oxygen dependent-MRI (BOLD-MRI) was used to non-invasively detect changes to renal oxygenation. Hepatic and renal expression of RXFP1 (relaxin receptor) was determined by IHC and quantitative PCR (qPCR). A qPCR array was used to quantify changes in renal vasodilator/vasoconstrictor gene expression in response to relaxin. FindingsRXFP1 was detected in glomerular podocytes, renal pericytes, and renal, segmental, and interlobar arteries of cirrhotic rats. In carbon tetrachloride (CCl4) induced cirrhosis, acute intravenous relaxin (4 μg) induced a 50% increase in renal blood flow after 60 min (p<0·01 vs placebo, n=6). BOLD-MRI showed statistically significant (p<0·05) increased tissue oxygenation at the same timepoint in renal cortex and medulla. Extended subcutaneous relaxin increased renal blood flow by 54% in CCl4 (p<0·01 vs placebo, n=8) and 87% in bile duct ligated animals (p<0·05 vs placebo, n=3) and increased GFR by 138% in CCl 4 (p<0·01 vs placebo, n=8) and 70% in bile duct ligated animals (p<0·05 vs placebo, n=3). Mean arterial pressure was unaffected by relaxin. L-NAME (250 mg/L) orally abrogated the effect of relaxin on renal blood flow and GFR. Relative expression of vasoconstrictor genes in kidney was markedly reduced by relaxin treatment. InterpretationRelaxin increases renal blood flow in experimental models of cirrhosis. Crucially, relaxin also improves renal function and oxygenation and does not induce systemic hypotension even in advanced disease. These effects are modulated via augmentation of nitric oxide and downregulation of vasoconstrictors, pivotal in the pathogenesis of hepatorenal syndrome. Relaxin has potential as a novel therapy for hepatorenal syndrome, and further translational studies are warranted. FundingWellcome Trust through the Scottish Translational Medicine and Therapeutics Initiative.

Prevention of Hepatocellular Carcinoma: Potential Targets, Experimental Models, and Clinical Challenges

Chronic fibrotic liver diseases such as viral hepatitis eventually develop liver cirrhosis, which causes occurrence of hepatocellular carcinoma (HCC). Given the limited therapeutic efficacy in advanced HCC, prevention of HCC development could be an effective strategy for improving patient prognosis. However, there is still no established therapy to meet the goal. Studies have elucidated a wide variety of molecular mechanisms and signaling pathways involved in HCC development. Genetically-engineered or chemically-treated experimental models of cirrhosis and HCC have been developed and shown their potential value in investigating molecular therapeutic targets and diagnostic biomarkers for HCC prevention. In this review, we overview potential targets of prevention and currently available experimental models, and discuss strategies to translate the findings into clinical practice.

Prevention of Hepatocellular Carcinoma: Potential Targets, Experimental Models, and Clinical Challenges

Chronic fibrotic liver diseases such as viral hepatitis eventually develop liver cirrhosis, which causes occurrence of hepatocellular carcinoma (HCC). Given the limited therapeutic efficacy in advanced HCC, prevention of HCC development could be an effective strategy for improving patient prognosis. However, there is still no established therapy to meet the goal. Studies have elucidated a wide variety of molecular mechanisms and signaling pathways involved in HCC development. Genetically-engineered or chemically-treated experimental models of cirrhosis and HCC have been developed and shown their potential value in investigating molecular therapeutic targets and diagnostic biomarkers for HCC prevention. In this review, we overview potential targets of prevention and currently available experimental models, and discuss strategies to translate the findings into clinical practice.