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.     

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.     

All‐trans‐retinoic acid ameliorates carbon tetrachloride‐induced liver fibrosis in mice through modulating cytokine production

Background/Aims: Liver fibrosis with any aetiology, induced by the transdifferentiation and proliferation of hepatic stellate cells (HSCs) to produce collagen, is characterized by progressive worsening in liver function, leading to a high incidence of death. We have recently reported that all‐trans‐retinoic acid (ATRA) suppresses the transdifferentiation and proliferation of lung fibroblasts and prevents radiation‐ or bleomycin‐induced lung fibrosis. Methods: We examined the impact of ATRA on carbon tetrachloride (CCl₄)‐induced liver fibrosis. We performed histological examinations and quantitative measurements of transforming growth factor (TGF)‐β1 and interleukin (IL)‐6 in CCl₄‐treated mouse liver tissues with or without the administration of ATRA, and investigated the effect of ATRA on the production of the cytokines in quiescent and activated HSCs. Results: CCl₄‐induced liver fibrosis was attenuated in histology by intraperitoneal administration of ATRA, and the overall survival rate at 12 weeks was 26.5% without ATRA (n=25), whereas it was 75.0% (n=24) in the treatment group (P=0.0187). In vitro studies disclosed that the administration of ATRA reduced (i) the production of TGF‐β1, IL‐6 and collagen from HSCs, (ii) TGF‐β‐dependent transdifferentiation of the cells and IL‐6‐dependent cell proliferation and (iii) the activities of nuclear factor‐κB p65 and p38mitogen‐activated protein kinase, which stimulate the production of TGF‐β1 and IL‐6, which could be the mechanism underlying the preventive effect of ATRA on liver fibrosis. Conclusions: Our findings indicate that ATRA ameliorates liver fibrosis. As the oral administration of the drug results in good compliance, ATRA could be a novel approach in the treatment of liver fibrosis.     

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.     

Loss of clock gene mPer2 promotes liver fibrosis induced by carbon tetrachloride

Aim: The clock gene mPer2 controls circadian periods and plays a critical role in clock resetting and responses to drugs of abuse. Mice deficient in mPer2 exhibit a marked susceptibility to acute liver injury. Clinical observations have demonstrated the existence of a relationship between circadian rhythm and liver cirrhosis. Here, we sought direct evidence for clock function to liver fibrosis using mPer2‐deficient mice. Methods: Hepatic fibrosis was induced in wild‐type (WT) and mPer2^?/? mice by repetitive intraperitoneal carbon tetrachloride (CCl₄) injection. Masson trichrome staining and analysis of α‐smooth muscle actin (α‐SMA) immunohistochemistry were performed to show the collagen accumulation and the hepatic stellate cell (HSC) activation, respectively. The mRNA levels of fibrosis‐related genes were monitored by quantitative real‐time polymerase chain reaction. The protein level of TIMP‐1 was determined by immunohistochemistry. Transferase deoxytidyl uridine end labeling, α‐SMA double staining and 4′,6′‐diamidino‐2‐phenylindole dihydrochloride staining were performed to show HSC apoptosis in vivo and in vitro, respectively. Results: CCl₄ caused much more severe liver fibrosis and activated more HSC in mPer2 null mice as compared to WT animals. Meanwhile, mPer2 null mice exhibited less efficiency in fibrosis resolution. Apoptotic HSC were significantly fewer in mPer2 null mice compared with WT mice after CCl₄; transfected Per2 cDNA into cultured HSC resulted in more HSC apoptosis with upregulation of TRAIL‐R2/DR5 expression. Conclusion: Loss of clock gene mPer2 predisposes liver fibrosis by increasing HSC activation and inhibiting HSC apoptosis.     

Antifibrotic and fibrolytic properties of celecoxib in liver damage induced by carbon tetrachloride in the rat

Background: Transforming growth factor‐β (TGF‐β) plays a pivotal role in liver fibrosis, because it activates hepatic stellate cells, stimulating extracellular matrix deposition. Cyclooxygenase‐2 (COX‐2) has been associated with TGF‐β because its inhibition decreases TGF‐β expression and collagen production in some cultured cell types. Aim: The aim of this work was to evaluate the ability of celecoxib (a selective COX‐2 inhibitor) to prevent and to reverse the liver fibrosis induced by CCl₄. Methods: We established experimental groups of rats including vehicle and drug controls, damage induced by chronic CCl₄ administration and CCl₄ plus pharmacological treatment in both prevention and reversion models. We determined: alanine aminotransferase, alkaline phosphatase, γ‐glutamyl transpeptidase, COX and metalloproteinase‐2 and ‐9 activities, lipid peroxidation, glutathione levels, glycogen and collagen content and TGF‐β expression. Results: Celecoxib prevented and aided to the recovery of livers with necrotic and cholestatic damage. Celecoxib exhibited anti‐oxidant properties by restoring the redox equilibrium (lipid peroxidation and glutathione levels). Glycogen was decreased by CCl₄, while celecoxib partially prevented and reversed this effect. Celecoxib inhibited COX‐2 activity, decreased TGF‐β expression, induced metalloproteinase‐2 activity and, consequently, prevented and reversed collagen accumulation. Conclusion: Our findings indicate that celecoxib exerts strong antifibrogenic and fibrolytic effects in the CCl₄ model of cirrhosis.     

(Z)2-(5-(4-methoxybenzylidene)-2, 4-dioxothiazolidin-3-yl) acetic acid protects rats from CCl(4) -induced liver injury

Background and Aim: (Z)2‐(5‐(4‐methoxybenzylidene)‐2, 4‐dioxothiazolidin‐3‐yl) acetic acid (MDA) is an aldose reductase (AR) inhibitor. Recent studies suggest that AR contributes to the pathogenesis of inflammation by affecting the nuclear factor κB (NF‐κB)‐dependent expression of cytokines and chemokines and therefore could be a novel therapeutic target for inflammatory pathology. The current study evaluated the in vivo role of MDA in protecting the liver against injury and fibrogenesis caused by CCl₄ in rats, and the underlying mechanisms. Methods: A single injection of CCl₄ induced acute hepatitis, and repeated injections were used to induce hepatic fibrosis in rats. Therapeutic efficacy was assessed by comparison of the severity of hepatic injury and fibrosis in MDA ‐ treated rats versus untreated controls. Results: MDA significantly protected the liver from injury by reducing the activity of serum alanine aminotransferase, and improving the histological architecture of the liver. MDA modulated NF‐κB‐dependent activation of inflammatory cytokines by reducing hepatic mRNA levels of tumor necrosis factor‐α, interleukin‐1β, inducible nitric oxide (NO) synthase and transforming growth factor‐β. In addition, MDA attenuated oxidative stress by increasing the content of hepatic glutathione. These favorable changes were associated with suppressed hepatic NF‐κB activation by MDA. MDA treatment improved liver fibrosis in rats that received repeated CCl₄ injections. In vitro, MDA attenuated phosphorylation of IκB and activation of NF‐κB, and thus prevented biosynthesis of NO in lipopolysaccharide‐activated RAW264.7 cells. Conclusions: The present study suggests that AR is a novel therapeutic anti‐inflammatory target for the treatment of hepatitis and liver fibrosis.     

Effects of salviainolic acid A (SA‐A) on liver injury: SA‐A action on hepatic peroxidation

Abstract: Background/Aims: This study aimed to investigate the actions of salviainolic acid A (SA‐A), an antiperoxidative component of Salvia miltiorrhiza (Sm), on rat liver injury and fibrosis. Methods: Acute and chronic rat liver injury models were established using carbon tetrachloride (CCl₄). After 48 h (acute) or during 6 weeks of CCl₄ injection, rats were further divided and treated with biphenyl dimethyl‐dicarboxylate (BDD) or colchicine, as a control antifibrotic treatment, with Sm, a herbal compound, or SA‐A, a water‐soluble extract of Sm. Liver function was investigated by assessing alanine transaminase (ALT) and aspartate transaminase (AST) activities, histological analysis, hydroxyproline (Hyp) and malondiadehyde (MDA) content. In vitro, isolated cultured hepatocytes were injured with CCl₄ gas for 24 h, followed by treatment with either vitamin E or various concentrations of SA‐A. The extent of hepatocyte injury was monitored by analyzing various lipid peroxidative parameters such as superoxide dismutase (SOD), glutathione (GSH), catalase (CAT), lactase dehydrogenase (LDH), and glutathione peroxidase (GSH‐PX) levels in hepatocyte supernatants. Results: SA‐A significantly decreased abnormal serum ALT activity both in acutely and chronically injured rat livers, decreased abnormal serum AST activity, Hyp and MDA content and attenuated hepatic collagen deposition. After CCl₄ incubation and injury, the activities of AST, ALT CAT, GSH‐PX and LDH and MDA content in hepatocyte supernatants increased significantly, but GSH levels decreased significantly. SA‐A markedly improved these pathological changes in a dose‐dependent manner. 10^?4 mol/l SA‐A had stronger inhibitory action than vitamin E. Conclusions: Our studies suggest that SA‐A has antiperoxidative effects on injured hepatocytes in liver injury and fibrosis induced by CCl₄.     

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.     

Minocycline and N-methyl-4-isoleucine cyclosporin (NIM811) mitigate storage/reperfusion injury after rat liver transplantation through suppression of the mitochondrial permeability transition

Graft failure after liver transplantation may involve mitochondrial dysfunction. We examined whether prevention of mitochondrial injury would improve graft function. Orthotopic rat liver transplantation was performed after 18 hours' cold storage in University of Wisconsin solution and treatment with vehicle, minocycline, tetracycline, or N-methyl-4-isoleucine cyclosporin (NIM811) of explants and recipients. Serum alanine aminotransferase (ALT), necrosis, and apoptosis were assessed 6 hours after implantation. Mitochondrial polarization and cell viability were assessed by intravital microscopy. Respiration and the mitochondrial permeability transition (MPT) were assessed in isolated rat liver mitochondria. After transplantation with vehicle or tetracycline, ALT increased to 5242 U/L and 4373 U/L, respectively. Minocycline and NIM811 treatment decreased ALT to 2374 U/L and 2159 U/L, respectively (P < 0.01). Necrosis and terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) also decreased from 21.4% and 21 cells/field, respectively, after vehicle to 10.1% and 6 cells/field after minocycline and to 8.7% and 5.2 cells/field after NIM811 (P < 0.05). Additionally, minocycline decreased caspase-3 activity in graft homogenates (P < 0.05). Long-term graft survival was 27% and 33%, respectively, after vehicle and tetracycline treatment, which increased to 60% and 70% after minocycline and NIM811 (P < 0.05). In isolated mitochondria, minocycline and NIM811 but not tetracycline blocked the MPT. Minocycline blocked the MPT by decreasing mitochondrial Ca(2+) uptake, whereas NIM811 blocks by interaction with cyclophilin D. Intravital microscopy showed that minocycline and NIM811 preserved mitochondrial polarization and cell viability after transplantation (P < 0.05). Conclusion: Minocycline and NIM811 attenuated graft injury after rat liver transplantation and improved graft survival. Minocycline and/or NIM811 might be useful clinically in hepatic surgery and transplantation.     

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.     

Melatonin promotes hepatic differentiation of human dental pulp stem cells: clinical implications for the prevention of liver fibrosis

Melatonin's effect on hepatic differentiation of stem cells remains unclear. The aim of this study was to investigate the action of melatonin on hepatic differentiation as well as its related signaling pathways of human dental pulp stem cells (hDPSCs) and to examine the therapeutic effects of a combination of melatonin and hDPSC transplantation on carbon tetrachloride (CCl₄)‐induced liver fibrosis in mice. In vitro hepatic differentiation was assessed by periodic acid‐Schiff (PAS) staining and mRNA expression for hepatocyte markers. Liver fibrosis model was established by injecting 0.5 mL/kg CCl₄ followed by treatment with melatonin (5 mg/kg, twice a week) and hDPSCs. In vivo therapeutic effects were evaluated by histopathology and by means of liver function tests including measurement of alanine transaminase (ALT), aspartate transaminase (AST), and ammonia levels. Melatonin promoted hepatic differentiation based on mRNA expression of differentiation markers and PAS‐stained glycogen‐laden cells. In addition, melatonin increased bone morphogenic protein (BMP)‐2 expression and Smad1/5/8 phosphorylation, which was blocked by the BMP antagonist noggin. Furthermore, melatonin activated p38, extracellular signal‐regulated kinase (ERK), and nuclear factor‐κB (NF‐κB) in hDPSCs. Melatonin‐induced hepatic differentiation was attenuated by inhibitors of BMP, p38, ERK, and NF‐κB. Compared to treatment of CCl₄‐injured mice with either melatonin or hDPSC transplantation alone, the combination of melatonin and hDPSC significantly suppressed liver fibrosis and restored ALT, AST, and ammonia levels. For the first time, this study demonstrates that melatonin promotes hepatic differentiation of hDPSCs by modulating the BMP, p38, ERK, and NF‐κB pathway. Combined treatment of grafted hDPSCs and melatonin could be a viable approach for the treatment of liver cirrhosis.     

Effect of the regulation of retinoid X receptor‐α gene expression on rat hepatic fibrosis

Aim: To study the effect of retinoid X receptor‐α (RXR‐α) expression on rat hepatic fibrosis. Methods: Rat hepatic fibrosis was induced by CCl₄, and the rats were randomly divided into an early‐phase hepatic fibrosis group (2 weeks) and a sustained hepatic fibrosis group (8 weeks). They were then divided into four groups (normal control, hepatic fibrosis, negative control and RXR‐α groups). A recombinant lentiviral expression vector carrying the rat RXR‐α gene was injected into the rats to induce RXR‐α expression by intraportal infusion, hepatic tissue pathological examination was performed, and hydroxyproline content was detected. Hepatic stellate cells (HSC) were cultured in vitro, an RXR‐α lentivirus vector was used to activate HSC, and 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) activation was assayed to detect HSC proliferation. Results: In vivo experiments indicated that in the sustained hepatic fibrosis group, there were significant differences in the hydroxyproline content, and expression of RXR‐α, α‐smooth muscle actin (α‐SMA) and type I collagen (P < 0.01). However, in the early‐phase hepatic fibrosis group, hydroxyproline content and the protein level of RXR‐α showed no significant difference compared with the normal control group (P > 0.05). In vitro studies revealed that expression of RXR‐α significantly inhibited expression of α‐SMA and type I collagen in activated HSC (P < 0.01), as well as HSC proliferation (P < 0.01). Conclusion: The increased RXR‐α gene expression inhibited HSC activation and proliferation and the degree of hepatic fibrosis.     

Hepatic clearance of tissue‐type plasminogen activator and plasma kallikrein in experimental liver fibrosis

Abstract: We have previously shown that tissue‐type plasminogen activator (tPA) and rat plasma kallikrein (RPK) share a common, but not unique, pathway for liver clearance. Aim: To evaluate the hepatic clearance of both proteases in experimental liver fibrosis. Methods: The hepatic clearance of these proteases was studied in porcine serum‐induced liver fibrosis using the isolated and perfused rat liver model. To better interpret the results, we also studied four other experimental groups: the turpentine oil‐induced acute‐phase response (AP group), AP group followed by GdCl₃ administration (AP/Gd group), CCl₄‐induced cirrhosis (CCl₄ group) and normal group. Results: The tPA clearance decreased significantly by both fibrotic and cirrhotic rat livers whereas the RPK clearance was not altered by the fibrotic rat liver. The hepatic clearance of tPA was reduced in the AP and AP/Gd groups; on the other hand, RPK clearance was increased in the AP group and, interestingly, this effect was neutralized by concomitant GdCl₃ administration. Conclusions: We observed that tPA and RPK clearances were affected differently by fibrosis as well as by different stimuli of the acute‐phase response, despite the fact that they share a common hepatic clearance mechanism in normal livers, and they were equally affected in cirrhosis.     

Transplantation of bone marrow cells reduces CCl4‐induced liver fibrosis in mice

Background: We investigated the reversibility of liver fibrosis induced with a CCl₄ injection and the role of stem cells in reversing the hepatic injury. Furthermore, the most effective cell fraction among bone marrow cells (BMCs) in the repair process was analysed. Methods: C57BL/6 mice were divided into four groups after 5 weeks of injection of CCl₄: control, sacrificed after 5 weeks, sacrificed at 10 weeks and sacrificed 5 weeks later after GFP‐donor BM transplantation. Liver function tests and real‐time polymerase chain reaction (PCR) of markers indicating liver fibrosis were compared between the groups. To identify the most effective BMC fraction that repairs liver injury, the mice were divided into three groups after the injection of CCl₄ for 2 days: granulocyte colony stimulating factor (G‐CSF) only, mononuclear cell (MNC) transplantation and Lin‐Sca‐1+c‐kit+haematopoietic stem cell (HSC) transplantation. Eight days after transplantation, the mice were harvested and morphometric, immunohistochemical analyses were performed to compare the expression of extracellular matrix and liver fibrosis‐related factors. Results: The liver fibrosis induced by CCl₄ was not spontaneously recovered but was persistent until 10 weeks, but the group injected with BMCs had less fibrosis and better liver function. Mobilization with G‐CSF increased the recovery of the injured liver and the best results were seen in those mice administered the MNC fraction and Lin‐Sca‐1+c‐kit+HSC fraction, with no difference between the two groups. Conclusion: BMC transplantation and stem cell mobilization with G‐CSF effectively treats liver injury in mice. These are promising techniques for autologous transplantation in humans with liver fibrosis.     

Cyclophilin A affects inflammation, virus elimination and myocardial fibrosis in coxsackievirus B3-induced myocarditis

Extracellular cyclophilin A (CyPA) and its receptor Extracellular Matrix Metalloproteinase Inducer (EMMPRIN, CD147) modulate inflammatory processes beyond metalloproteinase (MMP) activity. Recently, we have shown that CyPA and CD147 are upregulated in patients with inflammatory cardiomyopathy. Here we investigate the role of CyPA and CD147 in murine coxsackievirus B3 (CVB3)-induced myocarditis. CVB3-infected CyPA(-/-) mice (129S6/SvEv) revealed a significantly reduced T-cell and macrophage recruitment at 8 days p.i. compared to wild-type mice. In A.BY/SnJ mice, treatment with the cyclophilin-inhibitor NIM811 was associated with a reduction of inflammatory lesions and MMP-9 expression but with enhanced virus replication 8 days p.i. At 28 days p.i. the extent of lesion areas was not affected bei NIM811, whereas the collagen content was reduced. Initiation of NIM811-treatment on day 12 (after an effective virus defense) resulted in an even more pronounced reduction of myocardial fibrosis. In conclusion, in CVB3-induced myocarditis CyPA is important for macrophage and T cell recruitment and effective virus defense and may represent a pharmacological target to modulate myocardial remodeling in myocarditis.     

Inhibition of hepatic stellate cell activation following Yinchenhao decoction administration to dimethylnitrosamine-treated rats

Aim: In an effort to investigate the mechanism by which Yinchenhao decoction (YCHD) acts on liver injury, we investigated the potential antifibrogenic effects of YCHD in an experimental liver fibrosis rat model, with special focus on the mechanisms inhibiting the activation and promoting apoptosis of hepatic stellate cells (HSC). Methods: The rats were initially randomized into two groups: the control (n = 10) and dimethylnitrosamine‐treated (DMN; n = 30) groups. DMN (10 mg/kg body weight) was administered intraperitoneally to the DMN‐treated rats for three consecutive days each week. At the end of the second week, three rats from the control and six rats from the DMN‐treated groups were killed for the fibrosis development assessment. The remaining DMN rats were further randomized into two groups: the DMN–water group (n = 12) and the DMN–YCHD group (n = 12). Both groups continued to receive weekly DMN treatment for another 2 weeks in addition to daily administration of either water or YCHD, which were given intragastrically at a dose of 0.418 g/100 g body weight. Results: Hepatic hydroxyproline content decreased and had improved histopathology in the DMN–YCHD rats. Compared to the DMN group, α‐smooth muscle actin (SMA) and CD68 expression in the DMN–YCHD group was reduced significantly; however, α‐SMA‐positive HSC apoptosis was not observed by confocal microscopy; Fibrogenic proteins (tissue inhibitor matrix proteinases‐1 and 2 and matrix metalloproteinase [MMP]‐2/14) and cytokines (tumor necrosis factor‐α and transforming growth factor‐β₁) were decreased; MMP‐9 was significantly upregulated. Conclusion: Yinchenhao administration attenuates liver fibrosis at least in part by inhibiting HSC activation directly, rather than promoting cell apoptosis of activated HSC, and the suppressive activation of Kupffer cells.     

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.