Effects of blocking chemokine receptor CCR1 with BX471 in two models of fibrosis prevention and rescue in mice

BackgroundThe induction, progression and resolution of liver fibrosis are influenced by multiple chemokines. The inhibition of CCR1 signalling by a specific non-peptide inhibitor (BX471) reduces kidney fibrosis after unilateral ureteral obstruction via suppression of leukocyte recruitment in mice. However, it remains unclear whether selective CCR1 inhibition also affects hepatic fibrogenesis. Therefore we aimed to study the effect of this intervention on liver fibrosis in prevention (CCl₄ administration) and rescue (ABCB4-deficient mice) mouse models.MethodsIn the prevention model, hepatic fibrosis was induced by repeated injections of CCl₄. Additionally, the verum group was treated with subcutaneous injections of BX471, while controls received vehicle only. ABCB4 deficient mice (on the BALB/c-background) with sclerosing cholangitis and biliary fibrosis received BX471 or vehicle, respectively (rescue model). Liver histopathology was assessed after Sirius red staining of collagen, and hepatic collagen contents were measured. In addition, we performed gene expression analyses of fibrosis-related genes.ResultsBX471 injections were tolerated moderately well by all mice, and all mice developed hepatic fibrosis. Significant differences were neither observed in serum aminotransferase activities after 6 weeks of treatment between the two groups in the prevention nor in the rescue model. Interestingly, hepatic collagen contents were significantly higher in mice treated with BX471 in the prevention model as compared to controls but histological stages of liver sections did not differ. Of note, we observed only moderate effects on liver fibrosis in the ABCB4 knock-out model.ConclusionsOur data indicate that BX471 treatment did neither affect serum and tissue markers of liver injury and fibrosis in the CCl₄ model and only moderately in the Abcb4^-/- model of biliary fibrosis. The animal models indicate that treatment with BX471 alone is unlikely to exert major beneficial effects in chronic liver disease.     

Alterations of serum protein N-glycosylation in two mouse models of chronic liver disease are hepatocyte and not B cell driven

N-glycosylation of immunoglobulin G (IgG) has an important impact on the modification of the total serum N-glycome in chronic liver patients. Our aim was to determine the role and magnitude of the alterations in which hepatocytes and B cells are involved in two mouse models of chronic liver disease. Common bile duct ligation (CBDL) and subcutaneous injections with CCl(4) were induced in B cell-deficient and wild-type (WT) mice. IgG depletion was performed with beads covered with protein A/G and the depletions were evaluated by SDS-PAGE and Western blot analysis. N-glycan analysis was performed by improved DSA-FACE technology. Structural analysis of the mouse serum N-glycans was performed by exoglycosidase digests and MALDI-TOF mass spectrometry of permethylated glycans. The alterations seen in B cell-deficient mice closely resembled the alterations in WT mice, in both the CBDL and the CCl(4) models. N-glycan analysis of the IgG fraction in both mouse models revealed different changes compared with humans. Overall, the impact of IgG glycosylation on total serum glycosylation was marginal. Interestingly, the amount of fibrosis present in CBDL B cell-deficient mice was significantly increased compared with CBDL WT mice, whereas the opposite was true for the CCl(4) model as determined by Sirius red staining. However, this had no major effect on the alteration of N-glycosylation of serum proteins. Alterations of total serum N-glycome in mouse models of chronic liver disease are hepatocyte-driven. Undergalactosylation of IgG is not present in mouse models of chronic liver disease.     

Flk1+间充质干细胞减轻四氯化碳导致的肝纤维化的研究

许多慢性肝脏疾病都会发生肝纤维化,但是目前尚缺乏对肝纤维化切实有效的治疗手段。实验发现,Flk1(fetal liver kinase)阳性间充质干细胞(MSC)能够减轻四氯化碳(CCl4)所致小鼠肝纤维化。取雄性BALB／c小鼠骨髓,分离培养Flk1^ MSC,用CCl4制作雌性小鼠肝纤维化模型,在CCl4损伤后立即或1周后经尾静脉注射Flk1^ MSC,2或5周后检测受体小鼠肝脏的纤维化程度和供体细胞的植入。结果发现,CCl4损伤后立即注射Flk1^ MSC,可以使肝脏损伤程度明显减轻,减少胶原沉积,使肝脏羟脯氨酸含量及血清纤维化指标显下降;而损伤1周后注射细胞则无明显变化。免疫荧光、PCR和荧光原位杂交方法证实,在受体肝脏中有供体细胞植入,呈上皮细胞形态,并表达白蛋白,但是数量很少。因此,Flk1^ MSC具有潜在的植入肝组织的能力,并可能启动肝组织的内源性修复,减轻CCl4导致的肝纤维化。     

Hepatoprotective effects of berberine on liver fibrosis via activation of AMP-activated protein kinase

Aims

We investigated the protective effect of berberine (BBR) on chronic liver fibrosis in mice and the potential mechanism underlying the activation of AMP-activated protein kinase (AMPK) pathway.

Main methods

CCl4-induced chronic liver fibrosis model in mice was established and activated rat hepatic stellate cell was treated with BBR. Cell viability was evaluated by SRB assay and protein expressions were detected by Western blot.

Key findings

Our results showed that BBR ameliorated the liver fibrosis in mice with CCl4-induced liver injury and inhibited the proliferation of hepatic stellate cell in dose- and time-dependent manner. BBR decreased the enzyme release of ALT, AST, and ALP in serum, elevated SOD and reduced MDA content of liver tissue in CCl4-induced liver fibrosis model. BBR delayed the formation of regenerative nodules and reduced necrotic areas compared to CCl4 group. Moreover, BBR treatment activated AMPK, decreased the protein expression of Nox4, TGF-β1 and the phosphorylated Akt. The expression of smooth muscle actin (α-SMA), the marker of activated hepatic stellate cell, was also reduced by BBR treatment.

Significance

Our studies firstly demonstrated that BBR exerted hepatoprotective effects possibly via activation of AMPK, blocking Nox4 and Akt expression. Our findings may benefit the development of new strategies in the prevention of chronic liver disease.     

Human amniotic epithelial cell transplantation induces markers of alternative macrophage activation and reduces established hepatic fibrosis

Chronic hepatic inflammation from multiple etiologies leads to a fibrogenic response that can progress to cirrhosis and liver failure. Transplantation of human amniotic epithelial cells (hAEC) from term delivered placenta has been shown to decrease mild to moderate hepatic fibrosis in a murine model. To model advanced human liver disease and assess the efficacy of hAEC therapy, we transplanted hAEC in mice with advanced hepatic fibrosis. Immunocompetent C57BL/6 mice were administered carbon tetrachloride (CCl(4)) twice weekly resulting in bridging fibrosis by 12 weeks. hAEC (2 × 10(6)) were infused via the tail vein at week 8 or weeks 8 and 10 (single and double dose, respectively). Human cells were detected in mouse liver four weeks after transplantation showing hAEC engraftment. CCl(4) treated mice receiving single or double hAEC doses showed a significant but similar decrease in liver fibrosis area associated with decreased activation of collagen-producing hepatic stellate cells and decreased hepatic protein levels of the pro-fibrogenic cytokine, transforming growth factor-beta1. CCl(4) administration caused hepatic T cell infiltration that decreased significantly following hAEC transplantation. Hepatic macrophages play a crucial role in both fibrogenesis and fibrosis resolution. Mice exposed to CCl(4) demonstrated increased numbers of hepatic macrophages compared to normal mice; the number of macrophages decreased significantly in CCl(4) treated mice given hAEC. These mice had significantly lower hepatic protein levels of the chemokine monocyte chemoattractant protein-1 than mice given CCl(4) alone. Alternatively activated M2 macrophages are associated with fibrosis resolution. CCl(4) treated mice given hAEC showed increased expression of genes associated with M2 macrophages including YM-1, IL-10 and CD206. We provide novel data showing that hAEC transplantation induces a wound healing M2 macrophage phenotype associated with reduction of established hepatic fibrosis that justifies further investigation of this potential cell-based therapy for advanced hepatic fibrosis.     

Protective role of estrogen-induced miRNA-29 expression in carbon tetrachloride-induced mouse liver injury

Previous studies have indicated that female animals are more resistant to carbon tetrachloride (CCl(4))-induced liver fibrosis than male animals, and that estradiol (E(2)) treatment can inhibit CCl(4)-induced animal hepatic fibrosis. The underlying mechanism governing these phenomena, however, has not been fully elucidated. Here we reported the role of estrogen-induced miRNA-29 (miR-29) expression in CCl(4)-induced mouse liver injury. Hepatic miR-29 levels were differentially regulated in female and male mice during CCl(4) treatment. Specifically, the levels of miR-29a and miR-29b expression were significantly decreased in the livers of male, but not female, mice following 4 weeks of CCl(4) treatment. The down-regulation of miR-29a and miR-29b in male mouse livers correlated with the early development of liver fibrosis, as indicated by increased expressions of fibrotic markers in male mice relative to female mice. In addition, E(2) was maintained at a higher level in female mice than in male mice. In contrast to TGF-β1 that decreased miR-29a/b expression in murine hepatoma IAR20 cells and normal hepatocytes, E(2) enhanced the expression of miR-29a/b through suppression of the nuclear factor-κB (NF-κB) signal pathway, which negatively regulates miR-29 expression. Furthermore, both E(2) treatment and intravenous injection of the recombinant adenovirus expressing miR-29a/b markedly increased the miR-29a/b level and attenuated the expression of fibrotic markers in mouse livers during CCl(4) treatment, supporting the protective role of E(2)-induced miR-29 in CCl(4)-induced hepatic injury. In conclusion, our results collectively demonstrate that estrogen can inhibit CCl(4)-induced hepatic injury through the induction of hepatic miR-29.     

Cholestatic liver fibrosis and toxin-induced fibrosis are exacerbated in matrix metalloproteinase-2 deficient mice

Matrix metalloproteinase (MMP) plays an important role in homeostatic regulation of the extracellular environment and degradation of matrix. During liver fibrosis, several MMPs, including MMP-2, are up-regulated in activated hepatic stellate cells, which are responsible for exacerbation of liver cirrhosis. However, it remains unclear how loss of MMP-2 influences molecular dynamics associated with fibrogenesis in the liver. To explore the role of MMP-2 in hepatic fibrogenesis, we employed two fibrosis models in mice; toxin (carbon tetrachloride, CCl4)-induced and cholestasis-induced fibrosis. In the chronic CCl4 administration model, MMP-2 deficient mice exhibited extensive liver fibrosis as compared with wild-type mice. Several molecules related to activation of hepatic stellate cells were up-regulated in MMP-2 deficient liver, suggesting that myofibroblastic change of hepatic stellate cells was promoted in MMP-2 deficient liver. In the cholestasis model, fibrosis in MMP-2 deficient liver was also accelerated as compared with wild type liver. Production of tissue inhibitor of metalloproteinase 1 increased in MMP-2 deficient liver in both models, while transforming growth factor β, platelet-derived growth factor receptor and MMP-14 were up-regulated only in the CCl4 model. Our study demonstrated, using 2 experimental murine models, that loss of MMP-2 exacerbates liver fibrosis, and suggested that MMP-2 suppresses tissue inhibitor of metalloproteinase 1 up-regulation during liver fibrosis.     

Effects of retinoic acid on the development of liver fibrosis produced by carbon tetrachloride in mice

The role of retinoic acid (RA) in liver fibrogenesis was previously studied in cultured hepatic stellate cells (HSCs). RA suppresses the expression of alpha2(I) collagen by means of the activities of specific nuclear receptors RARalpha, RXRbeta and their coregulators. In this study, the effects of RA in fibrogenesis were examined in carbon tetrachloride (CCl4) induced liver fibrosis in mice. Mice were treated with CCl4 or RA and CCl4, along side control groups, for 12weeks. RA reduced the amount of histologically detectable fibrosis produced by CCl4. This was accompanied by a attenuation of the CCl4 induced increase in alpha2(I) collagen mRNA and a lower (2-fold versus 3-fold) increase in liver hydroxyproline. Furthermore, RA reduced the levels of 3-nitrotyrosine (3-NT) protein adducts and thiobarbituric acid (TBA) reactive substance (TBARS) in the liver, which are formed as results of oxidative stress induced by CCl4 treatment. These in vivo findings support our previous in vitro studies in cultured HSC of the inhibitory effect of RA on type I collagen expression. The data also provide evidence that RA reduces CCl4 induced oxidative stress in liver, suggesting that the anti-fibrotic role of RA is not limited to the inhibition of type I collagen expression.     

Proteomic analysis reveals upregulation of RKIP in S‐180 implanted BALB/C mouse after treatment with ascorbic acid

Tumor cells have an invasive and metastatic phenotype that is the main cause of death for cancer patients. Tumor establishment and penetration consists of a series of complex processes involving multiple changes in gene expression. In this study, intraperitoneal administration of a high concentration of ascorbic acid inhibited tumor establishment and increased survival of BALB/C mice implanted with S‐180 sarcoma cancer cells. To identify proteins involved in the ascorbic acid‐mediated inhibition of tumor progression, changes in the liver proteome associated with ascorbic acid treatment of BALB/C mice implanted with S‐180 were investigated using two‐dimensional gel electrophoresis and mass spectrometry. Eleven protein spots were identified whose expression was different between control and ascorbic acid treatment groups. In particular, Raf kinase inhibitory protein (RKIP) and annexin A5 expression were quantitatively up‐regulated. The increase in RKIP protein level was detected in the tumor tissue and accompanied by an increase in mRNA level. Our results suggest a possibility that these proteins are related to the ascorbic acid‐mediated suppression of tumor formation. J. Cell. Biochem. 106: 1136–1145, 2009. © 2009 Wiley‐Liss, Inc.     

S-adenosylmethionine blocks collagen I production by preventing transforming growth factor-beta induction of the COL1A2 promoter

To study the anti-fibrogenic mechanisms of S-adenosylmethionine (AdoMet), transgenic mice harboring the -17 kb to +54 bp of the collagen alpha2 (I) promoter (COL1A2) cloned upstream from the beta-gal reporter gene were injected with carbon tetrachloride (CCl4) to induce fibrosis and coadministered either AdoMet or saline. Control groups received AdoMet or mineral oil. AdoMet lowered the pathology in CCl4-treated mice as shown by transaminase levels, hematoxylin and eosin, Masson's trichrome staining, and collagen I expression. beta-Galactosidase activity indicated activation of the COL1A2 promoter in stellate cells from CCl4-treated mice and repression of such activation by AdoMet. Lipid peroxidation, transforming growth factor-beta (TGFbeta) expression, and decreases in glutathione levels were prevented by AdoMet. Incubation of primary stellate cells with AdoMet down-regulated basal and TGFbeta-induced collagen I and alpha-smooth muscle actin proteins. AdoMet metabolites down-regulated collagen I protein and mRNA levels. AdoMet repressed basal and TGFbeta-induced reporter activity in stellate cells transfected with COL1A2 promoter deletion constructs. AdoMet blocked TGFbeta induction of the -378 bp region of the COL1A2 promoter and prevented the phosphorylation of extracellular signal-regulated kinase 1/2 and the binding of Sp1 to the TGFbeta-responsive element. These observations unveil a novel mechanism by which AdoMet could ameliorate liver fibrosis.     

Multivariable difference gel electrophoresis and mass spectrometry: a case study on transforming growth factor-beta and ERBB2 signaling

Multivariable DIGE/MS was used to investigate proteins altered in expression and/or post-translational modification in response to activation of transforming growth factor (TGF)-beta receptors in MCF10A mammary epithelial cells overexpressing the HER2/Neu (ErbB2) oncogene. Proteome changes were monitored in response to exogenous TGF-beta over time (0, 8, 24, and 40 h), and proteins were resolved using medium range (pH 4-7) and narrow range (pH 5.3-6.5) isoelectric focusing combined with up to 2 mg of protein to allow inspection of lower abundance proteins. Triplicate samples were prepared independently and analyzed together across multiple DIGE gels using a pooled sample internal standard to quantify expression changes with statistical confidence. Unsupervised principle component analysis and hierarchical clustering of the individual DIGE proteome expression maps provided independent confirmation of distinct expression patterns from the individual experiments and demonstrated high reproducibility between replicate samples. Fifty-nine proteins (including some isoforms) that exhibited significant kinetic expression changes were identified using mass spectrometry and database interrogation and were mapped to existing biological networks involved in TGF-beta signaling. Several proteins with a potential role in breast cancer, such as maspin and cathepsin D, were identified as novel molecules associated with TGF-beta signaling.     

Protective effect of L-theanine on carbon tetrachloride-induced acute liver injury in mice

We studied effects of L-theanine, a unique amino acid in tea, on carbon tetrachloride (CCl(4))-induced liver injury in mice. The mice were pre-treated orally with L-theanine (50, 100 or 200 mg/kg) once daily for seven days before CCl(4) (10 ml/kg of 0.2% CCl(4) solution in olive oil) injection. L-theanine dose-dependently suppressed the increase of serum activity of ALT and AST and bilirubin level as well as liver histopathological changes induced by CCl(4) in mice. L-theanine significantly prevented CCl(4)-induced production of lipid peroxidation and decrease of hepatic GSH content and antioxidant enzymes activities. Our further studies demonstrated that L-theanine inhibited metabolic activation of CCl(4) through down-regulating cytochrome P450 2E1 (CYP2E1). As a consequence, L-theanine inhibited oxidative stress-mediated inflammatory response which included the increase of TNF-α and IL-1β in sera, and expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in livers. CCl(4)-induced activation of apoptotic related proteins including caspase-3 and PARP in mouse livers was also prevented by L-theanine treatment. In summary, L-theanine protects mice against CCl(4)-induced acute liver injury through inhibiting metabolic activation of CCl(4) and preventing CCl(4)-induced reduction of anti-oxidant capacity in mouse livers to relieve inflammatory response and hepatocyte apoptosis.     

Sphingosine 1-phosphate regulates regeneration and fibrosis after liver injury via sphingosine 1-phosphate receptor 2

Sphingosine 1-phosphate (S1P), a bioactive lipid mediator, stimulates proliferation and contractility in hepatic stellate cells, the principal matrix-producing cells in the liver, and inhibits proliferation via S1P receptor 2 (S1P(2)) in hepatocytes in rats in vitro. A potential role of S1P and S1P(2) in liver regeneration and fibrosis was examined in S1P(2)-deficient mice. Nuclear 5-bromo-2'-deoxy-uridine labeling, proliferating cell nuclear antigen (PCNA) staining in hepatocytes, and the ratio of liver weight to body weight were enhanced at 48 h in S1P(2)-deficient mice after a single carbon tetrachloride (CCl(4)) injection. After dimethylnitrosamine (DMN) administration with a lethal dose, PCNA staining in hepatocytes was enhanced at 48 h and survival rate was higher in S1P(2)-deficient mice. Serum aminotransferase level was unaltered in those mice compared with wild-type mice in both CCl(4)- and DMN-induced liver injury, suggesting that S1P(2) inactivation accelerated regeneration not as a response to enhanced liver damage. After chronic CCl(4) administration, fibrosis was less apparent, with reduced expression of smooth-muscle alpha-actin-positive cells in the livers of S1P(2)-deficient mice, suggesting that S1P(2) inactivation ameliorated CCl(4)-induced fibrosis due to the decreased accumulation of hepatic stellate cells. Thus, S1P plays a significant role in regeneration and fibrosis after liver injury via S1P(2).     

Dynamic proteomic analysis of protein expression profiles in whole brain of Balb/C mice subjected to unpredictable chronic mild stress: implications for depressive disorders and future therapies

The etiology and pathophysiology of depression remain unknown. Previous works were mostly performed on single observation time-point which might be insufficiently to reveal the molecular events changed during the disease development. Adult BALB/c mice were exposed to unpredictable chronic mild stress (UCMS) for different periods and differential 2D gel electrophoresis (DIGE) approach was employed to the brain tissue to explore the molecular disease signatures. Sustained elevation of corticosterone level was observed, suggesting the hyperactivity of hypothalamic-pituitary-adrenal (HPA) axis when the mice were subjected to the stressful situation. The behavioral results indicated the depressive alterations of the mice exposing to UCMS. The altered proteins identified by proteomics showed that abnormal energy mobilization under stress condition was accompanied by overproduction of reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress. Cytoskeleton protein and anti-oxidant enzymes were also changed by UCMS treatment. The results of biochemical and immunohistochemical assay confirmed the changes identified by DIGE analysis. These results indicated that the insufficiency of ATP synthesis, overwhelming ROS production and ER stress subsequently contributed to the cytoskeletal damage and inhibition to expression of some anti-oxidant proteins, which might ultimately bring functional neuron to apoptosis or death. Proteins whose expression is affected may provide tools for potential treatment strategies.     

Delta-like ligand 4/DLL4 regulates the capillarization of liver sinusoidal endothelial cell and liver fibrogenesis

Liver sinusoidal endothelial cells (LSECs) undergo capillarization, or loss of fenestrae, and produce basement membrane during liver fibrotic progression. DLL4, a ligand of the Notch signaling pathway, is predominantly expressed in endothelial cells and maintains liver sinusoidal homeostasis. The aim of this study was to explore the role of DLL4 in LSEC capillarization. The expression levels of DLL4 and the related genes, capillarization markers and basement membrane proteins were assessed by immunohistochemistry, immunofluorescence, RT-PCR and immunoblotting as appropriate. Fenestrae and basement membrane formation were examined by electron microscopy. We found DLL4 was up-regulated in the LSECs of human and CCl4-induced murine fibrotic liver, consistent with LSEC capillarization and liver fibrosis. Primary murine LSECs also underwent capillarization in vitro, with concomitant DLL4 overexpression. Bioinformatics analysis confirmed that DLL4 induced the production of basement membrane proteins in LSECs, which were also increased in the LSECs from 4 and 6-week CCl4-treated mice. DLL4 overexpression also increased the coverage of liver sinusoids by hepatic stellate cells (HSCs) through endothelin-1 (ET-1) synthesis. The hypoxic conditions that was instrumental in driving DLL4 overexpression in the LSECs. Consistent with the above findings, DLL4 silencing in vivo alleviated LSEC capillarization and CCl4-induced liver fibrosis. In conclusion, DLL4 mediates LSEC capillarization and the vicious circle between fibrosis and pathological sinusoidal remodeling.     

Measurement of liver collagen synthesis by heavy water labeling: effects of profibrotic toxicants and antifibrotic interventions

Enhanced production of collagen is central to fibrotic disorders such as hepatic cirrhosis and pulmonary fibrosis. We describe a sensitive, quantitative, and high-throughput technique for measuring hepatic collagen synthesis in vivo through metabolic labeling with heavy water ((2)H(2)O). Rats and mice received (2)H(2)O in drinking water for up to 35 days. Deuterium incorporation into collagen-bound amino acids (AA) alanine and hydroxyproline (OHP) was measured by gas chromatography-mass spectrometry. A threefold stimulation of collagen fractional synthesis was observed under the maximum dosage of carbon tetrachloride (CCl(4); 1.67 ml/kg). Deuterium enrichment was systematically 20% higher in AA from monomeric collagen relative to dimeric collagen, consistent with slower turnover of the latter. Administration of 1% griseofulvin to mice resulted in a significant, threefold increase in liver collagen synthesis, observable within 12 days and consistent with predicted interstrain differences (C57/Bl6J > BALB/c). Deuterium enrichments of OHP from total liver proteins correlated well with alanine or OHP from isolated collagen. Fibrogenesis subsided after withdrawal of CCl(4) exposure and was reduced to various degrees by coadministration of interferon-gamma, rosiglitazone, atorvastatin, or enalapril. Changes in isotopically measured collagen synthesis correlated with, but were more sensitive and reproducible than, standard histological staining (trichrome) for fibrosis. In summary, liver collagen synthesis can be measured sensitively and with high precision over a short time period, without radioactivity, thereby providing a relatively high-throughput in vivo strategy for rapidly measuring profibrotic activities of suspected hepatotoxicants and antifibrotic activities of drug candidates.