Bezafibrate prevents hepatic stellate cell activation and fibrogenesis in a murine steatohepatitis model,and suppresses fibrogenic response induced by transforming growth factor‐β1 in a cultured stellate cell line

Aim: The aim of this study was to investigate the preventive actions of bezafibrate against non‐alcoholic steatohepatitis (NASH), the activation of hepatic stellate cells (HSC), and fibrogenesis by using a model of NASH and an in vitro model. Methods: Male KK‐A^y/TaJcl (KK‐A^y) mice were fed a methionine and choline‐deficient (MCD) diet or a MCD diet containing bezafibrate or pioglitazone for 7 weeks, after which biochemical parameters, pathological changes, and hepatic mRNA levels were assessed. An in vitro HSC model was designed by using a previously described RI‐T cell line stimulated by transforming growth factor‐β1 (TGF‐β1). Results: MCD diet‐fed KK‐A^y mice developed hepatic steatosis, oxidative stress, inflammation, and hepatic fibrosis. Bezafibrate markedly decreased the hepatic content of triglyceride accumulation of fatty droplets within hepatocytes, and increased the expression of hepatic fatty acid β‐oxidative genes in MCD diet‐fed KK‐A^y mice. Bezafibrate markedly inhibited the increases in the plasma alanine aminotransferase level and hepatic content of thiobarbituric acid‐reactive substances in this model. Moreover, it dramatically reduced hepatic inflammatory changes and fibrosis concomitantly with marked reductions in the mRNA levels for inflammatory cytokine, chemokine, and profibrogenic genes. Importantly, both bezafibrate and pioglitazone markedly reduced the mRNA levels of profibrogenic and fibrogenic genes in TGF‐β1‐stimulated cells. Conclusion: Bezafibrate improved hepatic steatosis and potently prevented inflammation, oxidative stress, HSC activation, and fibrogenesis in the liver. Moreover, this study was the first to demonstrate that bezafibrate directly inhibits hepatic fibrogenic response induced by TGF‐β1 in vitro. Hence bezafibrate may be a new therapeutic strategy against NASH and 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.     

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.     

Extracellular histones stimulate collagen expression in vitro and promote liver fibrogenesis in a mouse model via the TLR4-MyD88 signaling pathway

BACKGROUNDLiver fibrosis progressing to liver cirrhosis and hepatic carcinoma is very common and causes more than one million deaths annually. Fibrosis develops from recurrent liver injury but the molecular mechanisms are not fully understood. Recently, the TLR4-MyD88 signaling pathway has been reported to contribute to fibrosis. Extracellular histones are ligands of TLR4 but their roles in liver fibrosis have not been investigated.AIMTo investigate the roles and potential mechanisms of extracellular histones in liver fibrosis.METHODS In vitro, LX2 human hepatic stellate cells (HSCs) were treated with histones in the presence or absence of non-anticoagulant heparin (NAHP) for neutralizing histones or TLR4-blocking antibody. The resultant cellular expression of collagen I was detected using western blotting and immunofluorescent staining. In vivo, the CCl₄-induced liver fibrosis model was generated in male 6-week-old ICR mice and in TLR4 or MyD88 knockout and parental mice. Circulating histones were detected and the effect of NAHP was evaluated.RESULTSExtracellular histones strongly stimulated LX2 cells to produce collagen I. Histone-enhanced collagen expression was significantly reduced by NAHP and TLR4-blocking antibody. In CCl₄-treated wild type mice, circulating histones were dramatically increased and maintained high levels during the duration of fibrosis-induction. Injection of NAHP not only reduced alanine aminotransferase and liver injury scores, but also significantly reduced fibrogenesis. Since the TLR4-blocking antibody reduced histone-enhanced collagen I production in HSC, the CCl₄ model with TLR4 and MyD88 knockout mice was used to demonstrate the roles of the TLR4-MyD88 signaling pathway in CCl₄-induced liver fibrosis. The levels of liver fibrosis were indeed significantly reduced in knockout mice compared to wild type parental mice.CONCLUSIONExtracellular histones potentially enhance fibrogenesis via the TLR4–MyD88 signaling pathway and NAHP has therapeutic potential by detoxifying extracellular histones.     

Altered factor VII activating protease expression in murine hepatic fibrosis and its influence on hepatic stellate cells

Background: Platelet‐derived growth factor‐BB (PDGF‐BB) is a profibrotic factor in liver fibrosis through its ability to stimulate hepatic stellate cells (HSC). The liver‐derived serine protease factor VII activating protease (FSAP) regulates the activities of PDGF‐BB in a cell‐specific manner. Aims: Our aim was to determine the influence of FSAP on the activation of HSC and to analyse the regulation of FSAP in hepatic fibrogenesis. Methods: The effect of FSAP on PDGF‐stimulated p42/p44 mitogen‐activated protein kinase (MAPK) activation in primary rat HSC was determined by Western blotting. Migration and proliferation of HSC was evaluated in Boyden chamber experiments and ³H‐thymidine incorporation assays respectively. Expression of FSAP was analysed in a CCl₄ mouse model of liver fibrosis by Western blot, quantitative real‐time polymerase chain reaction and immunohistochemistry. Results: FSAP inhibited PDGF‐BB‐stimulated p42/p44 MAPK phosphorylation, proliferation and migration of HSC. FSAP mRNA expression level was increased 3 h after CCl₄ application and decreased after 18 h and, in established fibrosis, after chronic CCl₄ administration. In parallel, there was a decrease in the circulating FSAP protein in chronic fibrosis. Concurrently, the homogenous hepatic expression pattern of FSAP was disturbed. Immunohistochemistry revealed a decrease of FSAP in hepatocytes in inflammatory and fibrotic lesions. Conclusions: Our results demonstrate an inhibitory effect of FSAP on PDGF‐mediated activation of HSC. In addition, FSAP expression is transiently increased in acute‐phase reaction but decreased during chronic fibrogenesis, which in turn may influence PDGF‐BB availability and myofibroblast activity.     

(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.     

Loss of Matrix Metalloproteinase-2 Amplifies Murine Toxin-Induced Liver Fibrosis by Upregulating Collagen?I Expression

Background and Aims

Matrix metalloproteinase-2 (MMP-2), a type IV collagenase secreted by activated hepatic stellate cells (HSCs), is upregulated in chronic liver disease and is considered a profibrotic mediator due to its proliferative effect on cultured HSCs and ability to degrade normal liver matrix. Although associative studies and cell culture findings suggest that MMP-2 promotes hepatic fibrogenesis, no in vivo model has definitively established a pathologic role for MMP-2 in the development and progression of liver fibrosis. We therefore examined the impact of MMP-2 deficiency on liver fibrosis development during chronic CCl₄ liver injury and explored the effect of MMP-2 deficiency and overexpression on collagen I expression.

Methods

Following chronic CCl₄ administration, liver fibrosis was analyzed using Sirius Red staining with quantitative morphometry and real-time polymerase chain reaction (PCR) in MMP-2?/? mice and age-matched MMP-2+/+ controls. These studies were complemented by analyses of cultured human stellate cells.

Results

MMP-2?/? mice demonstrated an almost twofold increase in fibrosis which was not secondary to significant differences in hepatocellular injury, HSC activation or type I collagenase activity; however, type I collagen messenger RNA (mRNA) expression was increased threefold in the MMP-2?/? group by real-time PCR. Furthermore, targeted reduction of MMP-2 in cultured HSCs using RNA interference significantly increased collagen I mRNA and protein, while overexpression of MMP-2 resulted in decreased collagen I mRNA.

Conclusions

These findings suggest that increased MMP-2 during the progression of liver fibrosis may be an important mechanism for inhibiting type I collagen synthesis by activated HSCs, thereby providing a protective rather than pathologic role.     

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.     

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.     

Thymosinβ4 alleviates cholestatic liver fibrosis in mice through downregulating PDGF/PDGFR and TGFβ/Smad pathways

Liver fibrosis is an important health problem without adequate and effective therapeutics. In this study, effects of thymosinβ4 (Tβ4) on hepatic fibrogenesis and the underlying molecular mechanisms were explored in bile duct ligation (BDL)-induced mice cholestatic liver fibrosis model. Results showed exogenous Tβ4 significantly reduced the mortality and liver/body weight ratio in BDL mice. Histological examinations and biochemical analyses demonstrated that BDL induced evident portal fibrosis and a significant increase in hepatic collagen contents. However, these changes were significantly attenuated by exogenous Tβ4. Quantitative real-time PCR assays showed that Tβ4 suppressed BDL-induced increases in many fibrotic genes expression including α-smooth muscle actin (α-SMA), collagen I, III and fibronectin, TGFβ1, TGFβR II, Smad2, Smad3, and PDGFRβ. Results from immunohistochemistry and Western blots also showed that Tβ4 reduced TGFβ1 and PDGFRβ protein levels in the liver tissues of BDL mice. In vitro studies using LX-2 cells demonstrated that Tβ4 could decrease PDGFRβ and TGFβR II levels in hepatic stellate cells. Taken together, findings in our present studies suggested that exogenous Tβ4 alleviated BDL-induced cholestatic liver fibrosis through downregulating PDGF/PDGFR and TGFβ/Smad pathways.     

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.     

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.     

Fenretinide stimulates the apoptosis of hepatic stellate cells and ameliorates hepatic fibrosis in mice

Aim: To investigate whether fenretinide, a clinically proved apoptosis‐inducing chemopreventive agent in tumor cells, can induce apoptosis in hepatic stellate cells (HSCs) and resolve hepatic fibrosis. Methods: CCl₄‐induced liver fibrosis in mice and rat activated hepatic stellate cells (HSC‐T6) as well as hepatocytes (BRL‐3A) were studied. Results: The duplex staining of proliferating cell nuclear antigen and α‐ smooth muscle actin or terminal deoxynucleotidyl transferase‐mediated deoxyuridine triphosphate nick‐end labeling and α‐ smooth muscle actin demonstrated that fenretinide executed its anti‐fibrosis effect in liver by inducing apoptosis rather than inhibiting proliferation of HSCs, while it had no apparently apoptotic effect on hepatocytes. Fenretinide could elicit apoptosis of HSC‐T6 in vitro at the concentration range from 0.5 to 5 µM, but at higher concentrations ≥5 µM was required to induce apoptosis in hepatocytes (BRL‐3A). Conclusion: Further studies using malondialdehyde measurement, Western blot, antioxidant, inhibitors for p53, caspase 8 and 9 – as well as anti‐Fas neutralizing antibody – have shown that in HSC‐T6, fenretinide‐induced apoptosis involves a reactive oxygen species (ROS)‐generated, P53‐independent, mitochondria‐associated intrinsic pathway, whereas in hepatocytes (BRL‐3A), a ROS‐generated, P53‐dependent, Fas‐related extrinsic pathway is triggered only at high concentration.     

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.     

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.     

Anti‐PDGF‐B monoclonal antibody reduces liver fibrosis development

Aim: To evaluate the usefulness of a platelet‐derived growth factor (PDGF)‐B specific monoclonal antibody (mAb) as a therapeutic agent to treat chronic liver fibrosis. Methods: Liver fibrosis was induced in ICR mice by bile duct ligation (BDL) or BALB/c mice by weekly injection of concanavalin A (ConA) for 4 or 8 weeks. A mAb specific for mouse and human PDGF‐B chain, AbyD3263, was generated, tested in vitro and administered twice a week throughout the experimental period. Results: AbyD3263 showed neutralizing activity against mouse and human PDGF‐B chain in cell‐based assays, as measured in vitro by inhibition of phosphorylation of PDGF receptor β and proliferation of hepatic stellate cells induced by PDGF‐BB. The half life of AbyD3263 in mice exceeded 7 days and dosing of animals twice a week resulted in constant plasma levels of the mAb. Induction of liver fibrosis by BDL and ConA resulted in elevated levels of alanine aminotransferase (ALT) in plasma and hydroxyproline in the liver. Treatment with AbyD3263 did not modify ALT levels, but significantly reduced hydroxyproline content in the liver with a maximum reduction of 39% and 54% in the BDL and ConA models, respectively, compared to controls. Conclusios: Consistent with the notion that PDGF‐BB plays an important role in the progression of liver fibrosis, AbyD3263 exhibits efficacy in pre‐clinical disease models suggesting that pharmacological inhibition of PDGF‐B chain may be a therapeutic approach to treat liver fibrosis.