Pentoxifylline prevents pig serum‐induced rat liver fibrosis by inhibiting interleukin‐6 production

Background/Aim: Pig serum‐induced rat liver fibrosis is a model of liver fibrosis in the absence of obvious hepatocyte injury. Penoxifylline (PTX), a xanthine derivative, which is a well‐known suppressor of tumor necrosis factor‐α (TNF‐α) production from inflammatory cells, has also been shown to inhibit the growth of hepatic stellate cells and to inhibit collagen synthesis in these cells in vitro. We investigated the effect of PTX on pig serum‐induced liver fibrosis in vivo, and assessed the mechanisms of prevention of fibrogenesis by this drug. Methods: Male Wistar rats were given intraperitoneal injections of 0.5 ml normal pig serum twice a week for 10 weeks with or without concomitant oral administration of PTX (20 mg/kg). Results: Rats that received pig serum showed significant liver fibrosis, and their serum interleukin‐6 (IL‐6) and hyaluronic acid levels were significantly increased. The serum levels of IL‐6 were well correlated with the serum levels of hyaluronic acid, and increased as the liver fibrosis progressed. Penoxifylline prevented the development of fibrosis in this animal model and reduced the serum levels of IL‐6 in a dose‐dependent manner. In vitro, by the addition of PTX to the culture medium of the rat hepatic stellate cells (HSCs), the proliferation of the HSCs was significantly inhibited and IL‐6 in the culture supernatant was also reduced significantly. Exogenous addition of IL‐6 partially restored the proliferation. Conclusion: Penoxifylline prevents pig serum‐induced rat liver fibrosis by inhibiting the proliferation of HSCs and by inhibiting the production of IL‐6 from HSCs.     

Effect of human umbilical cord blood‐derived mesenchymal stem cells in a cirrhotic rat model

Background/Aim: Cirrhosis is a long‐term consequence of chronic hepatic injury and no effective therapy is currently available for this disease. Recent reports have shown that the mesenchymal stem cells (MSCs) have the capacity to differentiate into hepatocytes, and umbilical cord blood is a rich source of MSCs. Hence, we investigated the effect of infusing of human umbilical cord blood‐derived MSCs (HMSCs) in carbon tetrachloride (CCl₄)‐induced cirrhosis in a rat model. Methods: The effect of HMSCs on cirrhosis was evaluated using haematoxylin and eosin and Masson's trichrome staining. To evaluate cirrhosis‐related factors, we measured protein and mRNA expression of transforming growth factor β₁ (TGF‐β₁), collagen type I and α‐smooth muscle actin (α‐SMA). Results: Histological findings showed that liver fibrosis in rats was alleviated by HMSCs infusion. Interestingly, CM‐DiI‐labelled HMSCs expressed the hepatocyte‐specific markers, human albumin and α‐fetoprotein. Infusion of HMSCs significantly inhibited TGF‐β₁, collagen type I and α‐SMA expressions in CCl₄‐induced cirrhotic rats. Conclusion: Our results showed that HMSCs infusion could improve liver fibrosis in rats with CCl₄‐induced cirrhosis, raising the possibility for clinical use of HMSCs in the treatment of cirrhosis.     

Cytokine blockade inhibits hepatic tissue inhibitor of metalloproteinase‐1 expression and up‐regulates matrix metalloproteinase‐9 in toxic liver injury

Abstract: Background: Tissue inhibitor of metalloproteinases (TIMP)‐1, the most important endogenous inhibitor of matrix metalloproteinases, plays a pivotal role in the pathogenesis of liver fibrosis and may represent an effective therapeutic target in the design of antifibrotic strategies for chronic liver diseases. Methods: Intraperitoneal application of a single dose of either tumor necrosis factor (TNF)‐α or interleukin (IL)‐1β in mice led to an enhanced expression of hepatic TIMP‐1 after 4–16 h. Male Sprague–Dawley rats were treated with carbon tetrachloride (CCl₄) in the presence and absence of specific TNF‐α and IL‐1β inhibitors. Results: Real‐time PCR revealed a significant increase of TIMP‐1 mRNA in total rat liver 24 h after CCl₄ injection. Repetitive injection of both, etanercept and anakinra, before and after CCl₄ injection effectively inactivated TNF‐α and IL‐1β. Anticytokine pretreatment reduced the increase of TIMP‐1 expression after a single CCl₄ injection by 50% and 75%, respectively. In contrast to CCl₄‐treated rats with and without TNF‐α blockade, IL‐1β inactivation caused a sevenfold increase in matrix metalloproteinases‐9 mRNA levels. Conclusions: In conclusion, TIMP‐1 expression is up‐regulated in the early phase of toxic liver injury by proinflammatory cytokines such as TNF‐α and IL‐1β in rodents. Pharmacological inactivation of these cytokines significantly reduces TIMP‐1 gene expression. Our data provide a potential new antifibrotic approach.     

RNA interference targeting the platelet‐derived growth factor receptor β subunit ameliorates experimental hepatic fibrosis in rats

Background/Aims: Platelet‐derived growth factor (PDGF) is the strongest stimulator of the proliferation of hepatic stellate cells (HSCs). PDGF receptor β subunit (PDGFR‐β) is acquired on HSCs proliferation induced by PDGF. In this study, we aim to investigate the effect of PDGFR‐β small interference RNA (siRNA) on experimental hepatic fibrosis. Methods: We constructed a PDGFR‐β siRNA expression plasmid and investigated its effect on the activation of HSCs. Bromodeoxyuridine incorporation was performed to investigate the effect of PDGFR‐β siRNA on HSCs proliferation. A hydrodynamics‐based transfection method was used to deliver PDGFR‐β siRNA to rats with hepatic fibrosis. The distribution of transgenes in the liver was observed by immunofluorescence. The antifibrogenic effect of PDGFR‐β siRNA was investigated pathologically. Results: Platelet‐derived growth factor receptor‐β subunit siRNA could significantly downregulate PDGFR‐β expression, suppress HSCs activation, block the mitogen‐activated protein kinase signalling pathway and inhibit HSCs proliferation in vitro. PDGFR‐β siRNA expression plasmid could be delivered into activated HSCs by the hydrodynamics‐based transfection method, and remarkably improve the liver function of the rat model induced by dimethylnitrosamine and bile duct ligation. Furthermore, the progression of fibrosis in the liver was significantly suppressed by PDGFR‐β siRNA in both animal models. Conclusions: Platelet‐derived growth factor receptor‐β subunit siRNA may be presented as an effective antifibrogenic gene therapeutic method for hepatic fibrosis.     

Overexpression of NK2 promotes liver fibrosis in carbon tetrachloride‐induced chronic liver injury

Background/Aims: Hepatocyte growth factor (HGF) inhibits liver fibrosis induced by carbon tetrachloride (CCl₄) in animal models. NK2 is a natural splice variant of HGF, but its in vivo function remains to be elucidated. We investigated the in vivo effects of NK2 on CCl₄‐induced liver fibrosis. Methods: NK2 transgenic mice and wild‐type (WT) mice were injected intraperitoneally with CCl₄ twice a week. The extent of hepatic fibrosis was evaluated by Azan–Mallory staining. Expression levels of mRNAs of transforming growth factor‐β1 (TGF‐β1) and matrix metalloproteinase‐13 (MMP‐13) were examined by real‐time polymerase chain reaction. The protein levels of α‐smooth muscle actin (α‐SMA), c‐Met and its phosphorylation were determined by Western blot analysis. Results: Liver fibrosis was significantly more severe in NK2 transgenic mice than in WT mice. CCl₄ administration increased the expression levels of TGF‐β1 mRNA and α‐SMA protein, and decreased the expression of MMP‐13 mRNA in livers of NK2 transgenic mice compared with those of WT mice. c‐Met protein expression in the liver was compatible with the degree of fibrosis. As for c‐Met activation, no difference was found between NK2 and WT livers. Conclusion: Overexpression of NK2 acts as an antagonist of HGF and promotes liver fibrosis in CCl₄‐induced chronic liver injury.     

Effects of 1‐O‐hexyl‐2,3,5‐trimethylhydroquinone on carbon tetrachloride‐induced hepatic cirrhosis in rats

Aim: The present study was undertaken to evaluate the effects of 1‐O‐hexyl‐2,3,5‐trimethylhydroquinone (HTHQ), a synthesized vitamin E derivative, on carbon tetrachloride (CCl₄)‐induced cirrhosis. Methods: Rats were treated with hypodermic injections of CCl₄ twice a week to induce the hepatic cirrhosis, and given drinking water containing HTHQ or solvent. Primary cultures of rat hepatocytes were performed to evaluate the effects of HTHQ on the expression of inducible nitric oxide synthase (iNOS). Results: Masson's staining of rat livers showed fibrosis around pseudo‐lobules in the CCl₄ group, the lesions being reduced in the CCl₄ HTHQ group. Increases in liver tissue hydroxyproline and α₁(I) collagen, α‐smooth muscle actin and iNOS induced by CCl₄, were also markedly diminished by HTHQ. Furthermore, both HTHQ and vitamin E attenuated interleukin‐1β‐induced iNOS protein expression in cultured hepatocytes, the potency of HTHQ being 10‐times higher than that of vitamin E. Conclusion: HTHQ may inhibit development of hepatic cirrhosis in rats, more potently than vitamin E, by inhibiting the iNOS expression in hepatocytes. Because vitamin E has a radical scavenging action, roles of NO and peroxynitrite will be discussed in the effects of HTHQ on the fibrosis.     

Evaluation of Fibrosis Markers: Apelin and Transforming Growth Factor‐β1 in Autosomal Dominant Polycystic Kidney Disease Patients

Renal interstitial fibrosis is an important pathological feature of autosomal dominant polycystic kidney disease (ADPKD), which progressively develops to end‐stage renal disease (ESRD). It has been shown that apelin and transforming growth factor‐β1 (TGF‐β1) play important roles in the renal fibrosis process. The aim of the present study is to evaluate the relationship of these fibrosis markers and ADPKD. Forty‐five patients with ADPKD and 28 healthy controls were studied cross‐sectionally. Estimated glomerular filtration rate (eGFR), apelin, TGF‐β1 were measured in all participants, using conventional methods. Apelin levels were lower (1.2 ± 0.9 ng/mL vs. 2.5 ± 1.3 ng/mL, P < 0.001), while TGF‐β1 levels were higher in the patient group according to healthy controls (466.5 ± 200.5 ng/L vs. 367.1 ± 163.45 ng/L, P = 0.031), respectively. Apelin was negatively correlated with TGF‐β1 and highly sensitive C‐reactive protein (hs‐CRP); and positively correlated with eGFR. In all subjects, eGFR was independently predicted by TGF‐β1 and apelin. Apelin and TGF‐β1 may be used as biomarkers of renal fibrosis that is an important pathological feature of ADPKD, which progressively develops to ESRD in ADPKD patients.     

Endogenous α‐calcitonin gene‐related peptide mitigates liver fibrosis in chronic hepatitis induced by repeated administration of concanavalin A

Background: α‐Calcitonin gene‐related peptide (αCGRP) is a 37‐amino acid pleiotropic peptide that we previously showed to exert a hepatoprotective effect during concanavalin A (Con A)‐induced acute hepatitis. In the present study, we used αCGRP^?/? mice to further investigate the antifibrogenic and hepatoprotective effects of endogenous αCGRP in Con A‐induced chronic hepatitis. Methods: Chronic hepatitis was induced in αCGRP^?/? and wild‐type mice by repeated administration of Con A. Serum transaminases were measured to assess hepatic injury. The severity of fibrosis and the activation of hepatic stellate cells (HSCs) were analysed by Masson trichrome staining and immunohistochemical staining of α‐smooth muscle actin (α‐SMA) respectively. Altered expression of fibrosis‐ and inflammation‐related genes was evaluated using a quantitative real‐time polymerase chain reaction. Activation and proliferation of HSCs were analysed using both primary cultured HSCs from the mice and the LI90 HSC cell line. Results: αCGRP^?/? mice showed more severe liver fibrosis than wild‐type mice in a Con A‐induced chronic hepatitis model. In histological and gene expression analyses, αCGRP^?/? mice showed greater inflammatory and fibrotic changes, greater HSC activation and a higher incidence of apoptosis among nonparenchymal cells than wild‐type mice. Conclusions: Endogenous αCGRP mitigates liver fibrosis in chronic hepatitis induced by repeated administration of Con A. αCGRP could be a useful therapeutic target for the treatment of chronic hepatitis.     

Elevated concentrations of 15‐deoxy‐Δ12,14‐prostaglandin J2 in chronic liver disease propose therapeutic trials with peroxisome proliferator activated receptor γ‐inducing drugs

Background/Aims: Current knowledge confers a crucial role to connective tissue growth factor (CTGF/CCN2) in hepatic fibrogenesis. Hepatocytes are likely to be the major cellular source of CTGF in the liver in which CTGF is sensitively upregulated by TGF‐β. Recently, we demonstrated that the methylxanthine derivate caffeine leads to an upregulation of peroxisome proliferator activated receptor γ (PPARγ) expression in hepatocytes, thus sensitizing these cells to the well‐known inhibitory effect of 15‐deoxy‐Δ^12,14‐prostaglandin J₂ (15‐d‐PGJ₂) on CTGF expression. However, upregulation of the receptor alone is not sufficient per se; its physiological ligand 15‐d‐PGJ₂ is required to exert an inhibitory effect on transforming growth factor‐β (TGF‐β) target genes such as CTGF. Methods: This study compared serum concentrations of 15‐d‐PGJ₂ in Caucasian patients with fibrotic liver diseases (n=289), Caucasian controls (n=136) and Caucasian non‐liver disease (NLD) sick (n=307), as well as of Chinese patients with hepatocellular carcinoma (HCC) (n=43) and Chinese healthy controls (n=63) in order to characterize their suitability for therapeutic approaches with PPARγ‐inducing (i.e. CTGF inhibitory) drugs such as caffeine. Results: The presented data showed that Caucasian patients with ongoing hepatic fibrogenesis (mean 6.2±5.9 μg/L) displayed strikingly higher serum concentrations of 15‐d‐PGJ₂ than healthy probands (mean 2.3±1.0) and Caucasian patients with NLD (mean 2.7±1.4 μg/L). Similar results were found in Chinese patients with fully developed HCC (mean 1.3±0.7 μg/L) compared with Chinese healthy controls (mean 0.4±0.2 μg/L). Conclusions: In conclusion, our data thus proposed an increased suitability of these patient groups for therapeutic approaches with drugs inducing PPARγ expression, such as methylxanthine derivates.     

CD14 expression on Kupffer cells during the course of carbon tetrachloride‐mediated liver injury

OBJECTIVE: To investigate the expression of CD14 on Kupffer cells during the course of carbon tetrachloride (CCl₄)‐mediated liver injury and its role in the activation of Kupffer cells. METHODS: Rats were administered CCl₄ twice weekly for up to 8 weeks. Kupffer cells were isolated from normal and CCl₄‐treated rats by the combined ‘collagenase‐pronase’ perfusion method, discontinuous density gradient centrifugation. On the day after isolation, the cells were incubated with RPMI‐1640 containing varying doses of lipopolysaccharide (LPS) for 6 h. Supernatants were then collected for measuring the concentration of tumor necrosis factor‐alpha (TNF‐α) by enzyme‐linked immunosorbent assay (ELISA). The expression of CD14 mRNA on Kupffer cells were determined by RT‐PCR. The plasma concentrations of endotoxin were determined by chromogenic substrate Limulus amebocyte lysate assay. RESULTS: Basic TNF‐α production of Kupffer cells isolated from CCl₄‐treated rats at 4 and 6 weeks was significantly higher than that of normal (P < 0.05). Following LPS stimulation the production of TNF‐α was markedly increased in Kupffer cells from the 2‐, 4‐ and 6‐week treatment groups (P < 0.05). Moreover, LPS‐induced TNF‐α production was dose‐dependent. CD14 mRNA expression on Kupffer cells isolated from CCl₄‐treated rats was elevated following 2 weeks of CCl₄ administration and the maximum elevation occurred at 6 weeks. Gene expression was decreased in Kupffer cells after 8 weeks of CCl₄ treatment. CCl₄ administration elicited extensive changes in liver morphology, including steatosis, inflammation and necrosis. The plasma concentrations of endotoxin of CCl₄ ‐ treated rats were increased during the time of liver injury. CONCLUSION: Up‐regulation of CD14 expression in Kupffer cells during CCl₄‐mediated chronic liver injury indicates cell activation and that they are more sensitive to LPS stimulation. Kupffer cells are critical effector cells in the early stage of liver injury.     

The −509C/T polymorphism of transforming growth factor‐β1 is associated with increased risk for development of chronic idiopathic neutropenia

Objective: Impaired granulopoiesis in chronic idiopathic neutropenia (CIN) has been associated with an inflammatory bone marrow (BM) microenvironment consisting of pro‐inflammatory and pro‐apoptotic mediators, such as tumor necrosis factor (TNF)‐α, transforming growth factor (TGF)‐β1, and Fas‐Ligand (Fas‐L). In this study, we evaluated the frequency of TNF‐α, TGF‐β1 and Fas‐L gene polymorphisms in CIN patients and explored their role in excessive cytokine production and their association with CIN development. Methods: The TNF‐α?308G/A, TGF‐β1 ?509C/T, +869T/C, +915G/C, and Fas‐L ?844T/C polymorphisms were studied in 57 CIN patients, and 100 healthy controls from Crete, a well‐defined area with genetically homogeneous population, using a polymerase chain reaction‐based restriction fragment length polymorphism assay. Results: The mutant genotype C/T or T/T of TGF‐β1 ?509C/T polymorphism was more common in CIN patients than in controls (P = 0.033). Compared to wild‐type genotype, the TT genotype was associated with increased risk for CIN development (OR: 5.7; 95% CI: 1.18–27.26; P = 0.033). Compared to controls, patients with CT and TT genotypes displayed increased TGF‐β1 levels in serum (P < 0.0001 and P = 0.0002, respectively) and BM (P < 0.0001 and P = 0.0002, respectively). No significant difference was found between patients and controls in the frequency of TNF‐α?308G/A, TGF‐β1 +869T/C and +915G/C and Fas‐L ‐844T/C polymorphisms. Conclusions: The TGF‐β1 ?509C/T polymorphism is associated with increased risk for CIN and contributes to the pathophysiology of the disorder by inducing TGF‐β1 overproduction. This is the first study providing evidence that genetic factors may predispose to CIN and may have a role in the pathophysiology of the disorder.     

Inhibition of hepatic tumour necrosis factor‐α attenuates the anandamide‐induced vasoconstrictive response in cirrhotic rat livers

Background: Increased anandamide, an endocannabinoid that interacts with both cannabinoid CB₁ and CB₂ receptors, can induce hepatic vasoconstrictive responses that contribute to the increased intrahepatic resistance (IHR) in cirrhotic rats. Chronic endotoxaemia and the subsequent release of tumour necrosis factor‐α (TNF‐α) are suggested to result in increased anandamide in cirrhotic livers. Thalidomide, which inhibited TNF‐α effectively, has been used clinically in states of chronic TNF‐α elevation with encouraging results. Aims: This study explores the possible effects of thalidomide on hepatic endocannabinoids and microcirculation of cirrhotic rats. Methods: Portal venous pressure (PVP), superior mesenteric arterial blood flow (SMA BF), hepatic TNF‐α, interleukin (IL‐6), protein expression of CB₁ and CB₂ receptor and thromboxane synthase (TXS) were measured in bile duct‐ligated (BDL) rats receiving 1‐month of vehicle (BDL‐V) or thalidomide (BDL‐thalido). The degree of hepatic fibrosis was also assessed. In the liver perfusion system, IHR and concentration–response curves of the portal perfusion pressure to anandamide were evaluated. Results: In BDL‐thalido rats, PVP, IHR and hepatic levels of TNF‐α and IL‐6, protein expression of CB₁ receptors, TXS and hepatic fibrosis were lower than in BDL‐V rats. In BDL‐thalido rat livers, the attenuation of the vasoconstrictive response to anandamide was associated with an upregulation of the CB₂ receptor and a downregulation of the CB₁ receptor. Nevertheless, SMA BF was not different between BDL‐thalido and BDL‐V rats. Conclusions: Thalidomide decreased the PVP and IHR through the attenuation of anandamide‐induced constrictive response, decreasing the production of TNF‐α, IL‐6 and TXA₂ in the liver and the suppression of hepatic fibrogenesis of rats with biliary cirrhosis of this study.     

Attenuated liver fibrosis after bile duct ligation and defective hepatic stellate cell activation in neural cell adhesion molecule knockout mice

Aim: Neural cell adhesion molecule (N‐CAM) is expressed by activated hepatic stellate cells (HSC), portal fibroblasts, cholangiocytes and hepatic progenitor cells during liver injury. Its functional role in liver disease and fibrogenesis is unknown. The aim of this study was to investigate the role of N‐CAM in liver fibrogenesis. Methods: To induce fibrosis, N‐CAM knockout mice and wild‐type controls were subjected to bile duct ligation (BDL) or repeated carbon tetrachloride (CCl₄) injections. Fibrosis was quantified by hydroxyproline, immunhistochemistry staining and image analysis. Protein levels were determined with immunoblotting. HSCs were isolated by ultracentrifugation in a Larcoll gradient and thereafter in vitro stimulated with recombinant transforming growth factor (TGF)‐β1. Results: Two weeks after BDL, wild‐type mice had developed pronounced liver fibrosis while N‐CAM?/? mice had less such alterations. N‐CAM?/? mice had less deposition of collagen and fibronectin seen in immunhistochemistry. The protein levels of fibronectin were higher in the liver from the wild type, while laminin were unaltered. CCl₄‐treated N‐CAM?/? and wild‐type mice showed no significant difference in the extent of liver fibrosis or the expression levels of the above‐mentioned genes. HSC isolated from N‐CAM?/? mice showed declined levels of smooth muscle actin and desmin after stimulation in vitro with TGF‐β1. Conclusions: Loss of N‐CAM results in decreased hepatic collagen and fibronectin deposition in mice subjected to BDL, but not in animals exposed to repeated CCl₄ injections. HSC isolated from N‐CAM null mice show impaired activation in vitro. This indicates a role of N‐CAM in cholestatic liver disease and HSC activation.     

The primary mitogen (TCPOBOP)‐induced hepatocyte proliferation is resistant to transforming growth factor‐ β‐1 inhibition

Background: Transforming growth factor (TGF)‐β‐1 is a very efficient inhibitor of hepatocyte proliferation in various in vivo and in vitro experimental systems. However, there are no data on whether it can influence the mitogenic response induced by primary hepatocyte mitogens. Aims: In this study, we compared the proliferative response in the liver between wild‐type and transgenic mice, overexpressing active TGF‐β‐1 in their liver following the treatment by a primary hepatocyte mitogen TCPOBOP (1,4‐bis[2‐(3,5‐dichloropyridyloxy)]benzene). Methods: The proliferative response was characterized by the immunohistochemical examination of pulse and cumulative bromodeoxyuridine labelling and by quantitative real‐time polymerase chain reaction analysis of cell cycle‐related genes. Results: Neither of the applied techniques revealed significant differences between the two groups of mice; furthermore, we observed the upregulation of TGF‐β‐1 expression following the mitogenic treatment. Conclusions: TGF‐β‐1 does not inhibit the primary mitogen‐induced proliferative response of the hepatocytes. This observation may provide an explanation for the divergent consequences of hepatic proliferations induced by partial hepatectomy or primary mitogenic treatment.