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.     

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.     

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.     

Targeted disruption of Smad3 confers resistance to the development of dimethylnitrosamine‐induced hepatic fibrosis in mice

Background: Hepatic fibrosis is characterized by a progressive accumulation of fibrillar extracellular matrix (ECM) proteins including collagen, which occurs in most types of chronic liver diseases. Transforming growth factor‐β (TGF‐β)/Smad3 signalling plays a central role in tissue fibrogenesis, acting as a potent stimulus of ECM accumulation. Aim: To evaluate the potential protective role of Smad3 deficiency in the pathogenesis of liver fibrosis induced by dimethylnitrosamine (DMN) in Smad3 null mice. Methods: Chronic hepatitis‐associated fibrosis was induced in 13 Smad3 null and 13 wild‐type (WT) mice by intraperitoneal DMN administration (10 μg/g body weight/day) for three consecutive days per week for 6 weeks. The liver was excised for macroscopic examination and histological, morphometric and immunohistochemical (IHC) analyses. For IHC, α‐smooth muscle actin (α‐SMA), collagen types I–III, TGF‐β1, connective tissue growth factor (CTGF), Smad3, Smad7 and CD3 antibodies were used. Results: At macroscopic examination, the liver of DMN‐treated Smad3 WT appeared harder with a dark brown colouring and necrotic areas compared with that from null mice. Histological and morphometric evaluation revealed a significantly higher degree of hepatic fibrosis and accumulation of connective tissue in the Smad3 WT compared with null mice. IHC evaluation showed a marked increase in α‐SMA, CTGF, collagen I‐III, TGF‐β and Smad3 staining in the liver of Smad3 WT compared with that in null mice, whereas Smad7 was increased only in null mice. Conclusions: The results indicate that Smad3 loss confers resistance to the development of DMN‐induced hepatic fibrosis. The reduced fibrotic response appears to be due to a reduction of fibrogenic myofibroblast activation and ECM production and accumulation. Smad3 could be a novel target for potential treatment of fibrosis complicating chronic hepatitis.     

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.     

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.     

Bcl‐2 overexpression in hepatic stellate cell line CFSC‐2G,induces a pro‐fibrotic state

Background and Aim: Development of hepatic fibrosis is a complex process that involves oxidative stress (OS) and an altered balance between pro‐ and anti‐apoptotic molecules. Since Bcl‐2 overexpression preserves viability against OS, our objective was to address the effect of Bcl‐2 overexpression in the hepatic stellate cells (HSC) cell‐line CFSC‐2G under acetaldehyde and H₂O₂ challenge, and explore if it protects these cells against OS, induces replicative senescence and/or modify extracellular matrix (ECM) remodeling potential. Methods: To induce Bcl‐2 overexpression, HSC cell line CFSC‐2G was transfected by lipofection technique. Green fluorescent protein‐only CFSC‐2G cells were used as a control. Cell survival after H₂O₂ treatment and total protein oxidation were assessed. To determine cell cycle arrest, proliferation‐rate, DNA synthesis and senescence were assessed. Matrix metalloproteinases (MMP), tissue‐inhibitor of MMP (TIMP), transglutaminases (TG) and smooth muscle a‐actin (α‐SMA) were evaluated by western blot in response to acetaldehyde treatment as markers of ECM remodeling capacity in addition to transforming growth factor‐β (TGF‐β) mRNA. Results: Cells overexpressing Bcl‐2 survived ≈ 20% more than control cells when exposed to H₂O₂ and ≈ 35% proteins were protected from oxidation, but Bcl‐2 did not slow proliferation or induced senescence. Bcl‐2 overexpression did not change α‐SMA levels, but it increased TIMP‐1 (55%), tissue transglutaminases (tTG) (25%) and TGF‐β mRNA (49%), when exposed to acetaldehyde, while MMP‐13 content decreased (47%). Conclusions: Bcl‐2 overexpression protected HSC against oxidative stress but it did not induce replicative senescence. It increased TIMP‐1, tTG and TGF‐β mRNA levels and decreased MMP‐13 content, suggesting that Bcl‐2 overexpression may play a key role in the progression of fibrosis in chronic liver diseases.     

N-methyl-4-isoleucine cyclosporine attenuates CCl -induced liver fibrosis in rats by interacting with cyclophilin B and D

Background and Aim: N‐methyl‐4‐isoleucine cyclosporine (NIM811), a new analogue of cyclosporine A, can inhibit collagen deposition in vitro and reduce liver necrosis in a bile‐duct‐ligation animal model. However, whether NIM811 effects on CCl₄‐induced rat liver fibrosis, and the related mechanism has not been determined. Methods: A liver fibrosis model was induced in Wistar rats using CCl₄ for 6 weeks. Meanwhile, two different doses of NIM811 (low‐dose 10 mg/kg and high‐dose 20 mg/kg) were given to the CCl₄‐treated rats. Liver fibrosis was then evaluated according to histopathological scoring and liver hydroxyproline content. Serum alanine aminotransferase, aspartate aminotransferase and albumin levels, expression of matrix metalloproteinase‐13, tissue inhibitor of metalloproteinase‐1, α‐smooth muscle actin and cyclophilin B and D in liver tissue were determined. Cyclophilin B and D were also studied in an hepatic stellate cell line. Results: Hydroxyproline content was decreased in both NIM811 groups compared with the model (P < 0.05). Liver necrosis and fibrosis were also attenuated in the NIM811 groups. NIM811 suppressed the expression of tissue inhibitor of metalloproteinase‐1, transforming growth factor beta mRNA and α‐smooth muscle actin protein in liver tissue. Expression of cyclophilin B in the fibrosis model was increased compared with the normal group (P < 0.05), and was decreased significantly in the low‐dose NIM811 treatment group (P < 0.05), which indicated that cyclophilin B might have a profibrotic effect. In vitro studies revealed that cyclophilin B and/or D knockout were associated with collagen inhibition. Conclusions: NIM811 attenuates liver fibrosis in a CCl₄‐induced rat liver fibrosis model, which may be related to binding with cyclophilin B and D.     

Adeno-associated virus vector-mediated production of hepatocyte growth factor attenuates liver fibrosis in mice

Purpose Adeno-associated virus (AAV) vectors can achieve long-term gene expression and are now feasible for use in human gene therapy. We constructed hepatocyte growth factor (HGF) expressing AAV (AAV5-HGF) and examined its effect in two mouse hepatic fibrosis models. Methods A model of hepatic fibrosis was established by carbon tetrachloride (CCl₄) administration in Balb/c mice. After the establishment of liver fibrosis, AAV5-HGF was injected once into the portal vein. Mice were killed 3, 6, 9, and 12 weeks after injection. Another model was established by bile duct ligation (BDL). Seven weeks after AAV5-HGF injection, mice underwent BDL, and were then killed 2 weeks after BDL. Results Mice that received AAV5-HGF achieved stable HGF expression both in the serum and liver for at least 12 weeks. In both models, significant improvement of the liver fibrosis was found in all mice receiving AAV5-HGF based on Azan-Mallory staining. Suppression of hepatic stellate cells (HSC) was confirmed by immunohistochemistry. Fibrogenic markers were significantly suppressed and collagenase activity increased in the livers of mice receiving AAV5-HGF. Conclusions A single injection of AAV vector containing HGF gene achieved long-term expression of HGF and resulted in resolution of mouse liver fibrosis. HGF gene therapy mediated by AAV is feasible for the treatment of liver fibrosis.     

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.     

Chymase inhibition attenuates tetrachloride‐induced liver fibrosis in hamsters

Aim: Chymase converts angiotensin I to angiotensin II, which may promote the development of liver fibrosis. In this study, whether a chymase inhibitor TY‐51469 attenuated tetrachloride (CCl₄)‐induced liver fibrosis was examined. Methods: Liver fibrosis was induced by the s.c. injection of 1 mL/kg of CCl₄ twice weekly for 8 weeks, and each hamster was given TY‐51469 (1 mg/kg per day) or placebo. Untreated hamsters were used as a control group. Results: Significant increases of serum alanine aminotransferase, total bilirubin and hyaluronic acid levels were observed in the placebo‐treated group compared with the control group, but these levels were significantly attenuated in the TY‐51469‐treated group. Liver chymase activity was significantly higher in the placebo‐treated group than in the control group, whereas the activity in the TY51469‐treated group was not. Total angiotensin II‐forming activity in the liver was also significantly higher in the placebo‐treatedgroup than in the control group or the TY‐51469‐treated group. The ratio of the fibrotic area to the total area in the liver was significantly higher in the placebo‐treated group than in the control group, but the ratio was significantly lower in the TY‐51469‐treated group than in the placebo‐treated group. A significant decrease in the number of α‐smooth muscle actin (SMA)‐positive cells was seen in the TY‐51469‐treated group compared to the placebo‐treated group. Conclusion: Significant correlations between the number of chymase‐positive cells and the degree of fibrosis and between the numbers of chymase‐positive cells and α‐SMA‐positive cells were observed. Thus, chymase inhibition may be a useful strategy for preventing liver fibrosis.     

Inhibitory effect of human interferon-beta-1a on activated rat and human hepatic stellate cells

Background and Aims: Hepatic stellate cells (HSC) are the primary cell type mediating hepatic fibrosis. Although known for its antiviral effects, the inhibitory effects of interferon‐beta (IFN‐β) on HSC treatment have not yet been established. Methods: Both human and rat activated HSC cell lines were incubated with increasing concentrations of recombinant human IFN‐β1a (rhIFN‐β1a) for 24, 48 or 72 h. The effects of rhIFN‐β1a on α‐smooth muscle actin (α‐SMA), collagen types I and III, transforming growth factor‐β1 (TGF‐β1), platelet‐derived growth factor‐BB (PDGF‐BB), and mothers against decapentaplegic homolog (Smad4, Smad7) expression in HSC were examined using Western blotting and immunocytochemistry. Proliferation of HSC was evaluated via bromodeoxyuridine assay. Results: rhIFN‐β1a treatment had a dose‐dependent, inhibitory effect on α‐SMA and collagen type I protein expression. In addition, rhIFN‐β1a decreased the expression of collagen type III, TGF‐β1, PDGF‐BB and Smad4 protein expression in HSC compared with untreated cells. We also observed increased Smad7 protein expression and decreased proliferation in rhIFN‐β1a‐treated HSC. Conclusions: Our data suggest that rhIFN‐β1a treatment decreased α‐SMA and collagen expression and inhibited the activation of HSC through the inhibition of the TGF‐β and PDGF pathways.     

Monocyte-derived fibrocytes elimination had little contribution on liver fibrosis

BackgroundMonocyte-derived fibrocytes play an important role in the progression of fibrosis in the skin, lungs, heart and kidney. However, the contribution of fibrocytes to liver fibrosis is unclear. The aim of this study was to investigate whether fibrocytes contributed to fibrosis progression in the livers of carbon tetrachloride (CCl₄)-treated mice.MethodsC57BL/6J mice were divided into 4 groups: normal control group, CCl₄-treated group, CCl₄ + control liposome-treated group, and CCl₄ + clodronate liposome-treated group. For the elimination of systemic monocyte and monocyte-derived fibrocyte, one group was treated with clodronate liposome, and another group with control liposome as a control. After 4 weeks of treatment, hepatic mononuclear cells were subjected to immunofluorescent (IF) staining and fluorescence-activated cell sorter (FACS) analysis to detect fibrocytes. Measurement of collagen-positive Sirius red stained area and collagen-I mRNA expression in the liver were performed to evaluate the degree of liver fibrosis quantitatively.ResultsIn the liver of the CCl₄-treated and CCl₄ + control liposome-treated groups, the number of fibrocytes, the area positive for Sirius red staining and collagen-I mRNA expression significantly increased compared with those in the normal control group. In the liver of the CCl₄ + clodronate liposome-treated group, few fibrocytes was observed as in the normal control group, but Sirius red staining positive area and collagen-I mRNA expression were increased and equivalent to the CCl₄-treated and CCl₄ + control liposome-treated groups.ConclusionMonocyte-derived fibrocytes play a minimal role in CCl₄-induced liver fibrosis. Cells other than fibrocytes such as hepatic stellate cells play a central role in liver fibrosis.     

(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.     

Hepatocyte growth factor protects against Fas‐mediated liver apoptosis in transgenic mice

Background: Apoptosis via the Fas/Fas ligand signalling system plays an important role in the development of various liver diseases. The administration of an agonistic anti‐Fas antibody to mice causes massive hepatic apoptosis and fulminant hepatic failure. Several growth factors including hepatocyte growth factor (HGF) have been found to prevent apoptosis. Methods: In this study, we demonstrated the overexpression of HGF to have a protective effect on Fas‐mediated hepatic apoptosis using a transgenic mice (Tg mice) model. Results: In HGF Tg mice, the elevation of alanine aminotransferase was dramatically inhibited at 12 and 24 h after the administration of 0.15 mg/kg anti‐Fas antibody. HGF Tg mice showed a significantly lower number of apoptotic hepatocytes at 12 h compared with wild‐type (WT) mice. Furthermore, 85% (six of seven) HGF Tg mice were able to survive after the administration of 0.3 mg/kg anti‐Fas antibody, while none of the WT mice survived. The Bcl‐xL expression was increased in HGF Tg mice, while there was no difference in the expression of Bax, Bid, Mcl‐1 and bcl‐2 between WT mice and HGF Tg mice. In addition, the HGF Tg mice showed more Akt phosphorylation than the WT mice both before and after the anti‐Fas antibody injection. Conclusions: Taken together, our findings suggest that HGF protects against Fas‐mediated liver apoptosis in vivo, and the upregulation of Bcl‐xL via Akt activation may also play a role in the protective effects of HGF.     

Glycyrrhizic acid inhibits apoptosis and fibrosis in carbontetrachloride-induced rat liver injury

AIM:To investigate anti-apoptotic effects of glycyrrhizic acid(GA) against fibrosis in carbon tetrachloride(CCl4)-induced liver injury and its contributing factors.METHODS:Liver fibrosis was induced by administration of CCl4 for 8 wk.Pathological changes in the liver of rats were examined by hematoxylin-eosin staining.Collagen fibers were detected by Sirius red staining.Hepatocyte apoptosis was determined by TUNEL assay and the expression levels of cleaved caspase-3,Bax,α-SMA,connective tissue growth factor(CTGF),matrix metalloproteinase(MMP) 2 and MMP9 proteins were evaluated by western blot analysis,and α-SMA m RNA,collagen type Ⅰ and Ⅲ m RNA were estimated by real-time PCR.RESULTS:Treatment with GA significantly improved the pathological changes in the liver and markedly decreased the positive area of Sirius red compared with rats in the CCl4-treated group.TUNEL assay showed that GA significantly reduced the number of TUNEL-positive cells compared with the CCl4-treated group.The expression levels of cleaved caspase-3,Bax,α-SMA,CTGF,MMP2 and MMP9 proteins,and α-SMA m RNA,collagen type Ⅰ and Ⅲ m RNA were also significantly reduced by GA compared with the CCl4-treated group(P 0.05).CONCLUSION:GA treatment can ameliorate CCl4-induced liver fibrosis by inhibiting hepatocyte apoptosis and hepatic stellate cell activation.     

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.