Loss of MMP 13 attenuates murine hepatic injury and fibrosis during cholestasis

Cholestasis occurs in a variety of clinical settings and often results in liver injury and secondary biliary fibrosis. Several matrix metalloproteinases (MMPs) are upregulated in the liver during cholestasis. The function of the major interstitial collagenase, MMP-13, in the initial phase of liver fibrosis is unknown. The aim of this study was to evaluate the role of MMP-13 during the development of cholestasis-induced liver fibrosis by comparing wild-type and MMP-13-deficient mice. Cholestasis was induced by bile duct ligation (BDL) for 5 days or 3 weeks. Activation and proliferation of hepatic stellate cells (HSCs) were detected by immunohistochemistry. Expression of MMP-13 mRNA increased significantly in BDL livers of WT mice. After BDL for 3 weeks liver fibrosis was suppressed in MMP-13-deficient mice versus WT animals. Activation and proliferation of HSCs were also suppressed in livers of MMP-13-deficient mice after BDL. To clarify the mechanism of this suppression, samples from 5-day BDL mice were used for evaluation of liver injury. Compared with those in WT animals, serum ALT and the number of hepatic neutrophils were reduced in MMP-13-deficient mice. Increased expression of the mRNA of inflammatory mediators such as tumor necrosis factor-alpha (TNF-alpha) was significantly suppressed in livers of MMP-13-deficient mice. Upregulation of fibrogenic markers, for example, transforming growth factor beta1 (TGF-beta1), was also significantly suppressed in livers of MMP-13-deficient mice versus in WT mice. In conclusion, distinct from the known function of interstitial collagenase to reduce liver fibrosis by degrading the extracellular matrix, MMP-13 contributes to accelerating fibrogenesis in cholestatic livers by mediating the initial inflammation of the liver.     

Matrix metalloproteinase inhibitor, CTS-1027, attenuates liver injury and fibrosis in the bile duct-ligated mouse

Aim:  Excessive matrix metalloproteinase (MMP) activity has been implicated in the pathogenesis of acute and chronic liver injury. CTS-1027 is an MMP inhibitor, which has previously been studied in humans as an anti-arthritic agent. Thus, our aim was to assess if CTS-1027 is hepato-protective and anti-fibrogenic during cholestatic liver injury.
Methods:  C57/BL6 mice were subjected to bile duct ligation (BDL) for 14 days. Either CTS-1027 or vehicle was administered by gavage.
Results:  BDL mice treated with CTS-1027 demonstrated a threefold reduction in hepatocyte apoptosis as assessed by the TUNEL assay or immunohistochemistry for caspase 3/7-positive cells as compared to vehicle-treated BDL animals ( P  < 0.01). A 70% reduction in bile infarcts, a histological indicator of liver injury, was also observed in CTS-1027-treated BDL animals. These differences could not be ascribed to differences in cholestasis as serum total bilirubin concentrations were nearly identical in the BDL groups of animals. Markers for stellate cell activation (α-smooth muscle actin) and hepatic fibrogenesis (collagen 1) were reduced in CTS-1027 versus vehicle-treated BDL animals ( P  < 0.05). Overall animal survival following 14 days of BDL was also improved in the group receiving the active drug ( P  < 0.05).
Conclusion:  The BDL mouse, liver injury and hepatic fibrosis are attenuated by treatment with the MMP inhibitor CTS-1027. This drug warrants further evaluation as an anti-fibrogenic drug in hepatic injury.     

Hepatocyte apoptosis after bile duct ligation in the mouse involves Fas.

BACKGROUNS & AIMS: Cholestatic liver injury results from the intrahepatic accumulation of toxic bile salts. Toxic bile salt-induced hepatocyte apoptosis in vitro is Fas dependent. The aim of this study was to ascertain if hepatocyte apoptosis in vivo during cholestasis is Fas dependent. METHODS: Studies were performed in bile duct-ligated (BDL) Fas-deficient lpr (lymphoproliferation) and wild-type mice. RESULTS: Hepatocyte apoptosis was the predominant mechanism of cell death as determined by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling and trypan blue assays to quantitate apoptosis and necrosis. The mechanisms of hepatocyte apoptosis were dependent on the presence or absence of the Fas receptor and the duration of BDL. After BDL of 3 days' duration, increased hepatocyte apoptosis occurred only in wild-type but not lpr mice, indicating the apoptosis was Fas dependent. In contrast, after BDL of >/=7 days, hepatocyte apoptosis also occurred in lpr animals consistent with a Fas-independent mechanism of apoptosis. Hepatocyte apoptosis in BDL lpr mice was associated with an increase in Bax expression and Bax association with mitochondria. CONCLUSIONS: During extrahepatic cholestasis, hepatocyte apoptosis is mediated by Fas. However, in the absence of the Fas receptor, additional mechanisms of hepatocyte apoptosis occur. Inhibition of multiple apoptotic pathways is necessary to attenuate chronic cholestatic liver injury.     

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.     

Roles of AKT and sphingosine kinase in the antiapoptotic effects of bile duct ligation in mouse liver

Tumor necrosis factor (TNF) receptor- and Fas-mediated apoptosis are major death processes of hepatocytes in liver disease. Although antiapoptotic effects in the injured liver promote chronic hepatitis and carcinogenesis, scant information is known about these mechanisms. To explore this issue, we compared acute liver injury after TNF-alpha or anti-Fas antibody (Jo2) between livers from sham-operated mice and chronic injured liver via bile duct ligation (BDL). BDL inhibited hepatocyte apoptosis induced by TNF-alpha but not by Jo2. On the other hand, BDL inhibited the massive hemorrhage seen in livers treated with either TNF-alpha or Jo2. Inactivation of AKT blocked the antiapoptotic effect of BDL. Sphingosine kinase knockout mice also lost the antihemorrhagic effect of BDL and attenuated the antiapoptotic effects of BDL. In bile duct-ligated livers, hepatic stellate cells (HSCs) were activated and produced tissue inhibitor of metalloproteinase 1 in a sphingosine kinase (SphK)-1-dependent mechanism. In conclusion, BDL exerts antiapoptotic effects that appear to require activation of AKT in hepatocytes and SphK in HSCs.     

Liver fibrosis induced by hepatic overexpression of PDGF-B in transgenic mice

BACKGROUND/AIMS: In hepatic fibrogenesis, stellate cells are activated leading to production and deposition of extracellular matrix. To clarify the role of PDGF-B in liver fibrogenesis, we overexpressed PDGF-B in the liver of transgenic mice. METHODS: Transgenic mice for the conditional overexpression of PDGF-B in the liver under control of an albumin promoter were generated utilising the Cre/loxP system. Constitutive PDGF-B expression was achieved after breeding with mice expressing Cre-recombinase under actin promoter control. Tamoxifen inducible expression was achieved after breeding with mice expressing Cre under transthyretin receptor promoter control. Levels of fibrosis were assessed and the expression of regulators of matrix remodelling was measured. RESULTS: PDGF-B expression caused hepatic stellate cell and myofibroblast activation marked by alpha-smooth muscle actin and PDGFR-beta expression. Liver fibrosis was verified macroscopically, histologically and by collagen I mRNA quantification in 4-6 week-old animals. MMP-2, MMP-9 and TIMP-1 were upregulated whereas TGF-beta expression was unchanged. CONCLUSIONS: We identified PDGF-B as a proliferative and profibrogenic stimulus and potential inducer of stellate cell transdifferentiation in vivo. PDGF-B overexpression causes liver fibrosis without significantly upregulating TGF-beta1, suggesting a TGF-beta-independent mechanism. The established model provides a tool for testing anti-PDGF-B therapeutic strategies in liver fibrosis in vivo.     

Bone marrow-derived fibrocytes participate in pathogenesis of liver fibrosis

BACKGROUND/AIMS: Hepatic stellate cells (HSCs) play a key role in hepatic fibrogenesis. However, their origin is still unknown. We tested the hypothesis that bone marrow (BM) contributes to the population of HSCs. METHODS: Chimeric mice transplanted with donor BM from collagen alpha1(I)-GFP+ reporter mice were subjected to the bile duct ligation (BDL)-induced liver injury. RESULTS: In response to injury, BM-derived collagen-expressing GFP+ cells were detected in liver tissues of chimeric mice. However, these cells were not activated HSCs in that they did not express alpha-smooth muscle actin or desmin and could not be isolated with the HSC fraction. Meanwhile, the majority of these BM-derived cells co-expressed collagen-GFP+ and CD45+, suggesting that these cells represent a unique population of fibrocytes. Consistent with their lymphoid origin, the number of GFP+CD45+ fibrocytes found in BM and spleen of chimeric mice increased in response to injury. Fibrocytes cultured in the presence of TGF-beta1 differentiated into SMA+desmin+ collagen-producing myofibroblasts, potentially contributing to liver fibrosis. CONCLUSIONS: In response to the BDL-induced liver injury: (i) HSCs do not originate in the BM; (ii) collagen-producing fibrocytes are recruited from the BM to damaged liver.     

Alpha-1 antitrypsin Z protein (PiZ) increases hepatic fibrosis in a murine model of cholestasis

Alpha-1 antitrypsin (alpha1-AT) deficiency is the most common genetic cause of liver disease in children. The homozygous alpha1-ATZ mutation (PiZZ) results in significant liver disease in 10% of all affected patients. The alpha1-ATZ mutation also may lead to worse liver injury in the setting of other liver diseases such as cystic fibrosis, nonalcoholic fatty liver disease, and hepatitis C. Although cholestatic injury is common to many forms of liver disease, its effect on the PiZZ phenotype is unknown. To elucidate the interplay of cholestasis and the PiZZ phenotype, we performed bile duct ligation (BDL) on C57BL/6 mice possessing a transgenic alpha1-ATZ mutation and littermate controls. PiZ transgenic mice undergoing BDL developed more liver fibrosis by quantification of Sirius red staining (P = 0.0003) and hydroxyproline (P = 0.007) than wild-type mice after BDL. More activated hepatic stellate cells (HSCs) and apoptotic cells also were observed in the PiZ BDL model. Quantitative real time polymerase chain reaction (PCR) of the endoplasmic reticulum (ER) stress markers CHOP and GRP78 were 4-fold and 2-fold more up-regulated, respectively, in PiZ BDL mice when compared with wild-type BDL mice (P = 0.02, P = 0.02). Increased apoptosis was also noted in PiZ BDL mice by terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) and cleaved caspase-3 histological staining. CONCLUSION: PiZ transgenic mice are more susceptible to liver fibrosis induced by cholestasis from BDL. Cholestasis therefore may lead to increased fibrosis in alpha1-AT deficiency, and the alpha1-ATZ mutation may act as a modifier gene in patients with concurrent cholestatic liver diseases such as cystic fibrosis.     

Infiltrating neutrophils in bile duct-ligated livers do not promote hepatic fibrosis

Bile duct obstruction causes rapid infiltration of neutrophils into the liver and leads ultimately to hepatic fibrosis. In this study, we assessed whether neutrophils play an active role in the pathogenesis of hepatic fibrosis under conditions of biliary obstruction. We performed bile duct ligation (BDL) on rats, some of which were depleted of neutrophils by means of an anti-neutrophil antiserum. Rats treated with the antiserum had 48% fewer neutrophils than control rats. Despite this, they showed no difference in either bile duct proliferation or hepatic fibrogenesis after BDL compared with control rats. In a second set of experiments, we performed BDL on mice with an underlying defect in neutrophil function due to transgenic expression of interleukin-8. Mice with neutrophil dysfunction deposited less (−22%) collagen in their livers after BDL than wild-type mice, but the difference was not statistically significant. In summary, data from two independent rodent models indicate that infiltrating neutrophils do not influence hepatic fibrogenesis following bile duct obstruction. The findings suggest that neutrophils play little if any role in the immunomodulation of liver fibrosis.     

Transcriptional profiling after bile duct ligation identifies PAI-1 as a contributor to cholestatic injury in mice

Extrahepatic cholestasis leads to complex injury and repair processes that result in bile infarct formation, neutrophil infiltration, cholangiocyte and hepatocyte proliferation, extracellular matrix remodeling, and fibrosis. To identify early molecular mechanisms of injury and repair after bile duct obstruction, microarray analysis was performed on liver tissue 24 hours after bile duct ligation (BDL) or sham surgery. The most upregulated gene identified encodes plasminogen activator inhibitor 1 (PAI-1, Serpine 1), a protease inhibitor that blocks urokinase plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) activity. Because PAI-1, uPA, and tPA influence growth factor and cytokine processing as well as extracellular matrix remodeling, we evaluated the role of PAI-1 in cholestatic liver injury by comparing the injury and repair processes in wild-type (WT) and PAI-1-deficient (PAI-1-/-) mice after BDL. PAI-1-/- mice had fewer and smaller bile infarcts, less neutrophil infiltration, and higher levels of cholangiocyte and hepatocyte proliferation than WT animals after BDL. Furthermore, PAI-1-/- mice had higher levels of tPA activation and mature hepatocyte growth factor (HGF) after BDL than WT mice, suggesting that PAI-1 effects on HGF activation critically influence cholestatic liver injury. This was further supported by elevated levels of c-Met and Akt phosphorylation in PAI-1-/- mice after BDL. In conclusion, PAI-1 deficiency reduces liver injury after BDL in mice. These data suggest that inhibiting PAI-1 might attenuate liver injury in cholestatic liver diseases.     

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.     

TRAIL mediates liver injury by the innate immune system in the bile duct-ligated mouse

The contribution of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a death ligand expressed by cells of the innate immune system, to cholestatic liver injury has not been explored. Our aim was to ascertain if TRAIL contributes to liver injury in the bile duct-ligated (BDL) mouse. C57/BL6 wild-type (wt), TRAIL heterozygote (TRAIL(+/-)), and TRAIL knockout (TRAIL(-/-)) mice were used for these studies. Liver injury and fibrosis were examined 7 and 14 days after BDL, respectively. Hepatic TRAIL messenger RNA (mRNA) was 6-fold greater in BDL animals versus sham-operated wt animals (P < 0.01). The increased hepatic TRAIL expression was accompanied by an increase in liver accumulation of natural killer 1.1 (NK 1.1)-positive NK and natural killer T (NKT) cells, the predominant cell types expressing TRAIL. Depletion of NK 1.1-positive cells reduced hepatic TRAIL mRNA expression and serum alanine aminotransferase (ALT) values. Consistent with a role for NK/NKT cells in this model of liver injury, stress ligands necessary for their recognition of target cells were also up-regulated in hepatocytes following BDL. Compared to sham-operated wt mice, BDL mice displayed a 13-fold increase in terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and an 11-fold increase in caspase 3/7-positive hepatocytes (P < 0.01). The number of TUNEL and caspase 3/7-positive cells was reduced by >80% in BDL TRAIL knockout animals (P < 0.05). Likewise, liver histology, number of bile infarcts, serum ALT values, hepatic fibrosis, and animal survival were also improved in BDL TRAIL(-/-) animals as compared to wt animals. Conclusion: These observations support a pivotal role for TRAIL in cholestatic liver injury mediated by NK 1.1-positive NK/NKT cells.     

In vivo inhibition of rat stellate cell activation by soluble transforming growth factor β type II receptor: A potential new therapy for hepatic fibrosis

Transforming growth factor beta (TGF-beta) is a well characterized cytokine that appears to play a major role in directing the cellular response to injury, driving fibrogenesis, and, thus, potentially underlying the progression of chronic injury to fibrosis. In this study, we report the use of a novel TGF-beta receptor antagonist to block fibrogenesis induced by ligation of the common bile duct in rats. The antagonist consisted of a chimeric IgG containing the extracellular portion of the TGF-beta type II receptor. This "soluble receptor" was infused at the time of injury; in some experiments it was given at 4 days after injury, as a test of its ability to reverse fibrogenesis. The latter was assessed by expression of collagen, both as the mRNA in stellate cells isolated from control or injured liver and also by quantitative histochemistry of tissue sections. When the soluble receptor was administered at the time of injury, collagen I mRNA in stellate cells from the injured liver was 26% of that from animals receiving control IgG (P < 0.0002); when soluble receptor was given after injury induction, collagen I expression was 35% of that in control stellate cells (P < 0.0001). By quantitative histochemistry, hepatic fibrosis in treated animals was 55% of that in controls. We conclude that soluble TGF-beta receptor is an effective inhibitor of experimental fibrogenesis in vivo and merits clinical evaluation as a novel agent for controlling hepatic fibrosis in chronic liver injury.     

Immune stimulation of hepatic fibrogenesis by CD8 cells and attenuation by transgenic interleukin-10 from hepatocytes

BACKGROUNDS & AIMS: Immunomodulatory cytokines, including interleukin-10 (IL-10), may mediate hepatic fibrosis. METHODS: We generated transgenic (TG) mice with hepatocyte expression of rat IL-10 (rIL-10) to assess its impact on lymphocyte subsets and activation of hepatic stellate cells following liver injury from carbon tetrachloride (CCl 4 ) or thioacetamide (TAA). RESULTS: Fibrosis was reduced in the TG animals in both models, which was not explained solely by differences in liver injury. By fluorescence-activated cell sorter (FACS), there were less CD4+ T cells in naive TG mice, and, following fibrosis induction, CD4+ T cells decreased only in wild-type (WT) mice, whereas increases in CD8+ T cells seen in WT animals were significantly attenuated in TG mice. Subtotal irradiation diminished fibrosis equally in both WT and TG groups, suggesting that rIL-10's antifibrotic effect was lymphocyte mediated. To assess the role of lymphocytes on stellate cell activation, either whole splenic lymphocytes, CD4+, or CD8+ T-cell subsets from WT animals with CCl 4 fibrosis were adoptively transferred to severe combined immunodeficiency (SCID) recipients, which led to stellate cell activation and fibrogenic stimulation as assessed by expression of transforming growth factor (TGF)-beta1 and collagen I messenger RNA (mRNA) and by immunoblot of alpha-smooth muscle actin. Moreover, serum aminotransferase levels and stellate cell activation mRNA were significantly higher among the CD8+ T-cell recipients. CONCLUSIONS: Transgenic expression of rIL-10 in liver leads to reduced fibrosis and alterations in liver lymphocyte subsets both in untreated liver and following fibrosis induction. In this model, fibrosis may be a CD8+ T-cell-mediated disease that is attenuated by rIL-10.     

Kupffer cell engulfment of apoptotic bodies stimulates death ligand and cytokine expression

Hepatocyte apoptosis by death receptors, hepatic inflammation, and fibrosis are prominent features of liver diseases. However, the link between these processes remains unclear. Our aim was to ascertain whether engulfment of apoptotic bodies by Kupffer cells promotes hepatic inflammation and fibrosis. Isolated murine Kupffer cells efficiently engulfed apoptotic bodies generated from UV-treated mouse hepatocytes. Engulfment of the apoptotic bodies, but not latex beads, stimulated Kupffer cell generation of death ligands, including Fas ligand, and tumor necrosis factor alpha (TNF-alpha). Both apoptotic body phagocytosis and death ligand generation were attenuated by gadolinium chloride, a Kupffer cell toxicant. Kupffer cells isolated from 3-day bile duct-ligated (BDL) mice were phenotypically similar to apoptotic body-"fed" Kupffer cells with enhanced death ligand expression; inhibition of hepatocyte apoptosis with a caspase inhibitor prevented this Kupffer cell activation. Consistent with a role for Kupffer cells in liver inflammation and fibrosis, gadolinium chloride attenuated neutrophil infiltration and markers for stellate cell activation. In conclusion, these findings support a model of cholestatic liver injury where Kupffer cell engulfment of apoptotic bodies promotes inflammation and fibrogenesis.     

Effect of losartan,an angiotensin II antagonist,on secondary biliary cirrhosis

BACKGROUND/AIMS: Chronic bile duct obstruction leads to biliary cirrhosis and portal hypertension. The hepatic stellate cells are involved in this process and can be activated by angiotensin II. The aim of the present study was to determine the effect of losartan, an angiotensin II antagonist, on experimental biliary cirrhosis. METHODOLOGY: Wistar rats were allocated to one of three groups: bile duct ligation (BDL), bile duct ligation and losartan treatment (BDLL), and sham-operated animals (SHAM). After 28 days, liver and spleen weight, hepatic volume, portal flow, and hepatocytes, bile ducts, hepatic stellate cell population and collagen IV volume fraction were evaluated. RESULTS: The portal flow was lower in the BDL group than in the BDLL group, and lower in both groups than in the SHAM group. Hepatocyte volume fraction was higher in the BDLL group than in the BDL group, and lower in both groups than in the SHAM group. Liver and spleen weight, hepatic volume, hepatic stellate cells population and collagen IV were higher in the BDL group than in the BDLL group, and higher in both groups than in the SHAM group. CONCLUSIONS: These results suggest that losartan can inhibit both the liver fibrosis and portal hypertension occurring in secondary biliary cirrhosis.     

Effect of selective cyclooxygenase-2 inhibitor meloxicam on liver fibrosis in rats with ligated common bile ducts.

Aim: Cholestasis triggers fibrogenesis in the liver. Hepatic cyclooxygenase-2 (COX-2) expression increases in various chronic liver diseases caused either by viruses or toxins. We hypothesized that selective COX-2 inhibitor meloxicam could suppress inflammation and fibrogenesis in a rat model of cholestasis induced by bile duct ligation (BDL). Methods: Forty-three Sprague-Dawley rats were assigned to one of four treatment groups (sham-operation, BDL, daily meloxicam injections following BDL, and daily meloxicam injection without BDL). Liver histopathology was analyzed with hematoxylin-eosin and Masson's trichrome staining. The expression of alpha-smooth muscle actin (alpha-SMA), transforming growth factor-beta1 (TGF-beta1), and COX-2 were measured with immunohistochemical staining. The levels of COX-2, TGF-beta1, and matrix metalloproteinase-9 (MMP-9) production were measured with the Western blot method and an enzyme immunoassay. Results: Meloxicam treatment attenuated the expression of alpha-SMA, TGF-beta1, and COX-2 in rats that were treated with BDL for 3 weeks. This was associated with a marked reduction in collagen accumulation and histological improvement. In addition, meloxicam treatment was found to downregulate the levels of hepatic COX-2, TGF-beta1, and MMP-9 production. Conclusion: Cholestasis in BDL rats induces hepatic COX-2 expression. Selective COX-2 inhibitor meloxicam reduces BDL-induced hepatic fibrosis, and this is associated with reduced hepatic TGF-beta1 expression as well as decreased cyclooxygenase activity in the liver.     

Overexpression of Mcl-1 Attenuates Liver Injury and Fibrosis in the Bile Duct–Ligated Mouse

Hepatocyte apoptosis contributes to liver injury and fibrosis after cholestatic injury. Our aim was to ascertain if the anti-apoptotic protein Mcl-1 alters liver injury or fibrosis in the bile duct–ligated mouse. Markers of apoptosis and fibrosis were compared in wild-type and transgenic mice expressing human Mcl-1 after bile duct ligation. Compared to hMcl-1 transgenic animals, ligated wild-type mice displayed a significant increase in TUNEL-positive cells and in caspase 3/7-positive hepatocytes. Consistent with apoptotic injury, the pro-apoptotic protein Bak underwent a conformational change to an activated form upon cholestatic injury, a change mitigated by hMcl-1 overexpression. Likewise, liver histology, number of bile infarcts, serum ALT values, markers of hepatic fibrosis, and animal survival were improved in bile duct–ligated mice transgenic for hMcl-1 as compared to wild-type mice. In conclusion, increased Mcl-1 expression plays a role in hepatoprotection upon cholestatic liver injury.