A-induced Hepatic Injury In Mice Research Articles

Protective effects of hesperidin on concanavalin A-induced hepatic injury in mice.

Hesperidin (HDN) is a citrus bioflavonoid, which widely exists in many plants. Previous researches have proved that HDN has several functions such as anti-oxidant, anti-tumor, anti-inflammatory, immune regulation and so on. In the present study, we explored the protective effects of HDN on concanavalin A (Con A)-induced hepatic injury. Acute hepatic injury model was established successfully by intravenous administration of Con A (15 mg/kg) in male C57BL/6 mice, and HDN was pretreated for 10 days before Con A challenge. It was found that the hepatic injury was notably improved in HDN pretreated mice. Furthermore, hepatic oxidative stress and the production of proinflammatory cytokines including TNF-α and IFN-γ were decreased by HDN pretreatment. More importantly, compared with Con A-treated mice, the expression and releasing of HMGB1 and T-cell activation were markedly reduced in HDN pretreated mice. Thus, these results suggest that HDN protects mice from Con A-induced hepatic injury by suppressing hepatocyte oxidative stress, producing cytokines, expressing and releasing HMGB1 and activating T cells.

Epigallocatechin-3-gallate (EGCG) attenuates concanavalin A-induced hepatic injury in mice

(−)-Epigallocatechin-3-gallate (EGCG) is the most abundant polyphenolic compound present in green tea and has been shown to possess anti-inflammatory and anti-oxidative properties. In this study, we investigated the protective effects of EGCG against concanavalin A (ConA)-induced liver injury and the underlying mechanisms. EGCG (5mg/kg) was administered orally by gavage to mice twice daily for 10 days before an intravenous injection of ConA. We found that EGCG effectively rescued lethality, improved hepatic pathological damage, and decreased serum levels of alanine aminotransaminase (ALT) in ConA-challenged mice. Furthermore, EGCG also significantly prevented the release of tumor necrosis factor (TNF)-α, interferon (IFN)-γ, interleukin (IL)-4, and IL-6 in serum, reduced malondialdehyde (MDA) levels, and restored glutathione (GSH) content and superoxide dismutase (SOD) activity in liver tissues from ConA-challenged mice. Finally, nuclear factor (NF)-κB activation and expression levels of Toll-like receptor (TLR) 2, TLR4 and TLR9 protein in liver tissues were significantly inhibited by EGCG pretreatment. Taken together, our data suggest that EGCG possesses hepatoprotective properties against ConA-induced liver injury through its anti-inflammatory and anti-oxidant actions.

Kupffer cells contribute to concanavalin A-induced hepatic injury through a Th1 but not Th17 type response-dependent pathway in mice

Increasing evidence suggests that a close interaction of Kupffer cells with T cells plays a central role in concanavalin A-induced hepatic injury in mice, but the underlying mechanisms remain obscure. The present study aimed to determine the relative roles of Th1 and Th17 type responses in concanavalin A-induced hepatic injury in mice, and to investigate whether or not Kupffer cells contribute to hepatic injury via a Th1 or Th17 type response-dependent pathway. Immune-mediated hepatic injury was induced in C57BL/6 mice by intravenous injection of concanavalin A. Kupffer cells were inactivated by pretreatment with gadolinium chloride 24 hours before the concanavalin A injection. The interferon-gamma (IFN-gamma) and interleukin-17 (IL-17) pathways were blocked by specific neutralizing antibodies. Hepatic injury was assessed using serum transferase activity and pathological analysis. Expression of inflammatory cytokines within the liver was detected by real-time polymerase chain reaction and immunohistochemistry. Neutralization of IFN-gamma significantly attenuated concanavalin A-induced hepatic injury. However, neutralization of IL-17 failed to suppress the injury. Inactivation of Kupffer cells by gadolinium chloride pretreatment protected against concanavalin A-induced injury and significantly reduced hepatic cytokine levels including TNF-alpha, IL-6 and IFN-gamma but not IL-17. Our findings suggest that Kupffer cells contribute to concanavalin A-induced hepatic injury via a Th1 type response-dependent pathway and production of inflammatory cytokines including TNF-alpha, IL-6 and IFN-gamma.

High-mobility group box 1 exacerbates concanavalin A-induced hepatic injury in mice

High-mobility group box 1 (HMGB1) is a nuclear factor released extracellularly as an early endogenous alarmin of inflammation following injury and as a late mediator of lethality in sepsis. Although HMGB1 has been implicated in acute lung injury, rheumatoid arthritis, and allograft rejection, its role in T-cell mediated hepatitis remains obscure. Here, we investigated the role and the underlying mechanisms of HMGB1 in concanavalin A (Con A) induced hepatic injury. We demonstrate that high levels of HMGB1 were detected in the necrotic area and in the cytoplasm of hepatocytes after Con A treatment. Administration of exogenous recombinant HMGB1 enhanced Con A-induced hepatitis, while blockade of HMGB1 protected animals from T cell-mediated hepatitis as evidenced by decreased serum transaminase, associated with reduced hepatic necrosis and mortality. Blockade of HMGB1 by a neutralizing antibody inhibited proinflammatory cytokine production, NFκB activity, and the late stage of T/NKT cell activation. These finding thus suggest a pivotal factor of HMGB1 in Con A-induced hepatitis. Blockage of extracellular HMGB1 may represent a novel therapeutic strategy to prevent hepatic injury in T cell-mediated hepatitis.

Phosphoinositide 3-kinase γ inhibitor ameliorates concanavalin A-induced hepatic injury in mice

A pivotal role of phosphoinositide 3-kinase-γ (PI3Kγ) in inflammatory cell activation and recruitment makes it an attractive target for immunomodulatory therapy. In present study we investigated the therapeutic efficiency of AS605240, a selective PI3Kγ inhibitor, on hepatitis and liver fibrosis in murine models induced by concanavalin A (ConA). Orally administration of AS605240 significantly improved survival, decreased the serum levels of alanine aminotransaminase (ALT), prevented inflammatory infiltration to liver in ConA-induced hepatitis. TNF-α and IFN-γ at protein levels in serum and mRNA levels in liver were markedly reduced. Downregulated phospho-Akt level of inflammatory cells infiltrating the liver by AS605240 treatment was detected by immunohistochemistry analysis in liver and further confirmed by Western blotting analysis in splenocytes. In ConA-induced chronic liver fibrosis model, accumulation of smooth-muscle actin (SMA)-expressing cells was partially inhibited by AS605240 treatment. These observations suggest that AS605240 might be of therapeutic value for the treatment of ConA-induced hepatic injury.

Sphingosine-1-phosphate receptor agonists suppress concanavalin A-induced hepatic injury in mice

T cell-mediated immune responses play a critical role in a variety of liver injuries including autoimmune hepatitis. Injection of concanavalin A (Con A) into mice mimics the histological and pathological phenotype of T cell-mediated hepatitis. Recent advances in host immune control of organ transplantation include the development of sphingosine-1-phosphate (S1P) receptor agonists such as FTY720, which alter lymphocyte homing but do not suppress host general immunity. Herein we examined the effect of the new S1P receptor agonist KRP-203 on the Con A-induced liver damage model. In normal liver lymphocytes of BALB/c mice, both FTY720 and KRP203 promoted lymphocyte sequestering from the liver to secondary lymph nodes and significantly reduced the number of liver lymphocytes ( p < 0.05). Based on this observation, KRP203 was employed in the Con A-induced hepatitis model. KRP203 markedly reduced the number of CD4 + lymphocytes that infiltrate Con A-treated liver ( p < 0.05) and successfully reduced serum transaminase elevation ( p = 0.017), therefore protecting mice from Con A-induced liver injury. Interestingly this homing modulation less occurs in natural hepatic T cell homing through the chemokine receptor, CXCR4. Therefore, S1P receptor agonists preferentially target CXCR4 +CD4 + peripheral blood T lymphocytes and suppress the occurrence of Con A-induced hepatitis, suggesting their therapeutic usefulness against T cell-mediated hepatic injury.

Activated Kupffer cells play an important role in intra-hepatic Th1-associated necro-inflammation in Concanavalin A-induced hepatic injury in mice

Background/aims: To examine whether or not activated Kupffer cells play an important role in intra-hepatic Th1-associated necro-inflammation in Concanavalin A (Con A)-induced hepatic injury in mice. Methods: Con A was administered to Balb/c mice pretreated with or without gadolinium chloride (GdCl 3). Kupffer cell activation was evaluated by their ability to produce superoxide anions in situ under liver perfusion with nitro blue tetrazolium (NBT). Hepatic concentration of cytokines was measured by ELISA and the mRNA expression of CXC chemokine receptor 3 (CXCR3) was evaluated by RT-PCR. Immunohistochemical detection of CD4 positive lymphocytes in the liver was also performed. Results: GdCl 3-pretreatment significantly ( P<0.01) reduced the serum levels of alanine aminotransferase (ALT) in Con A-treated mice. Formazan deposition in Kupffer cells, the hepatic concentration of tumor necrosis factor-α and interferon-γ, the mRNA expression of CXCR3 and the CD4 positive lymphocytes in the liver were decreased in GdCl 3-pretreated mice as compared with those without GdCl 3-pretreatment ( P<0.05, respectively). Conclusions: Activated Kupffer cells, which produce superoxide anions, are involved in Con A-induced hepatic necro-inflammation in mice possibly through the activation of Th1-associated immune response mediated by CD4 and/or CXCR3 positive cells recruited into the liver.

Time Course Profile and Cell-Type-Specific Production of Monokine Induced by Interferon-γ in Concanavalin A-Induced Hepatic Injury in Mice: Comparative Study with Interferon-Inducible Protein-10

Background: We have previously shown that interferon-inducible protein-10 (IP-10), a chemokine for activated lymphocytes, was specifically induced in the liver of Concanavalin A (Con A)-treated mice. The aim of this study was to investigate the time course profile and cell-type-specific hepatic production of monokine induced by interferon- n (MIG), a chemokine which shares its receptor and most of its activity with IP-10, in Con A-treated mice and to compare them with those of IP-10. Methods: Hepatic mRNA expression of MIG and IP-10 was studied by means of Northern blot analysis and in situ hybridization in Con A-treated mice. The levels of MIG and IP-10 in the serum and culture supernatants of murine hepatoma-, hepatic sinusoidal endothelial cell-, hepatic stellate cell- and macrophage-derived cell lines were determined by means of specific enzyme-linked immunosorbent assays. Results: The serum level of MIG slowly reached a maximum at 12 h after Con A injection and remained elevated for a long time, whereas that of IP-10 reached a maximum at 3 h and declined quickly, a finding supported by Northern blot analysis. Using in situ hybridization, the mRNA of MIG as well as IP-10 was found to be expressed in hepatocytes and hepatic non-parenchymal cells. Similar to IP-10, MIG was produced by hepatoma-, hepatic sinusoidal endothelial cell-, hepatic stellate cell- and macrophage-derived cell lines in vitro. Conclusions: Although both MIG and IP-10 were produced by hepatocytes and hepatic nonparenchymal cells in Con A-treated mice, the time course profile of MIG was distinguishable from that of IP-10. The fact that hepatic MIG and IP-10 were produced sequentially in this hepatitis model may suggest that a non-redundant role is played by these two chemokines in the process of hepatic necroinflammation.

Involvement of intrasinusoidal hemostasis in the development of concanavalin A-induced hepatic injury in mice.

Concanavalin A (Con A) stimulation induces T-cell activation-associated hepatic injury. This study is designed to show the involvement of microcirculatory disturbance in the pathogenesis. Con A administration led to prominent intrasinusoidal hemostasis, which consisted of erythrocyte agglutination, lymphocyte/neutrophil sticking to endothelial cells, and platelet aggregation and degranulation, resulting in a marked decrease in the intrahepatic blood flow and elevation of portal perfusion pressure. After hemostasis, confluent hepatic necrosis occurred within the congested area of liver parenchyma. Reduction in the extent of hemostasis by the treatment with heparin (thrombin inhibitor) or cyproheptadine (serotonin inhibitor) decreased hepatic injury. Pretreatment with either anti-tumor necrosis factor alfa (TNF-alpha) or anti-interferon gamma (IFN-gamma) monoclonal antibody (MAb) moderately decreased hemostasis and hepatic injury, whereas combined use of two MAbs almost perfectly protected mice from these disorders. Complete obliteration of hemostasis and hepatic injury was also accomplished by the pretreatment with FK506 which suppressed TNF-alpha and IFN-gamma production. Intrasinusoidal accumulation of leukocytes and platelets was, however, not blocked by FK506, indicating that Con A activities other than the stimulation of cytokine production are responsible for this event. The administration of anti-CD3 MAb, a T-cell stimulant without agglutination activities, which elevated plasma cytokine levels in a comparable degree without inducing prominent leukocyte infiltration, did not induce hepatic congestion and injury. These findings indicate that the agglutination activities of Con A and T-cell activation mediated TNF-alpha/IFN-gamma production are both required for the induction of intrasinusoidal hemostasis, which is indispensable for the development of hepatic injury.

Requirement of IL-4 and liver NK1+ T cells for concanavalin A-induced hepatic injury in mice.

Con A-induced hepatic injury of mice accompanied by elevated transaminase was inhibited after in vivo depletion of liver NK cells and NK1+ T cells with intermediate TCR by anti-NK1 Ab or anti-IL-2Rbeta Ab. However, depletion of liver NK cells alone by anti-asialo-GM1 Ab did not inhibit hepatic injury. Although depletion of NK1+ T cells inhibited Con A-induced IL-2R expression of CD4+ high TCR (TCRhigh) cells and IL-4 mRNA expression of hepatic mononuclear cells, exogenous IL-4 engendered Con A-induced hepatic injury and endowed the expression of IL-2R of CD4+ TCRhigh cells. It was also found that in vivo treatment with anti-IL-4 Ab before Con A administration inhibited Con A-induced hepatic injury. In addition, although Con A did not induce hepatic injury in MHC class I-deficient mice, exogenous IL-4 again engendered severe hepatic injury in these mice. Further, while serum TNF-alpha levels induced by Con A were greatly decreased in NK1+ T cell-depleted mice and class I-deficient mice, TNF-alpha levels were recovered by exogenous IL-4. These findings reveal that although CD4+ TCRhigh cells in the liver and their production of TNF-alpha are the direct effectors of Con A-induced hepatic injury, liver NK1+ T cells also play an important role in this hepatitis model. Con A hepatitis may serve as an experimental model for human autoimmune hepatitis.

Role of sinusoidal endothelial cells of the liver in concanavalin A-induced hepatic injury in mice

CD4+ T lymphocytes have been identified as being responsible for organ damage in the murine model of experimental liver injury induced by intravenous injection of concanavalin A (Con A). Liver sinusoidal endothelial cells (SEC) and Kupffer's cells (KC) are among the first cells that come into contact with lymphocytes in the liver sinusoid. We aimed to investigate the respective role of these cell populations in the initial steps of T-cell-mediated liver injury in Con A-induced hepatitis. By electron microscopy, we could show that intravenously applied Con A bound predominantly to SEC but not to KC. KC depletion by gadolinium chloride treatment of mice did not result in protection from liver injury, indicating that KCs are not primarily involved in the generation of liver injury. We could show that a CD4+ T-cell line (LNC.2) displayed selective cytotoxicity toward SEC (>50%) but not KC (12%) or fibroblasts (5%) in the presence of Con A in vitro. Microscopic observation revealed that the SEC monolayer was rapidly destroyed by LnC2 in the presence of Con A. Specificity of the Con A-induced cytotoxicity was shown by the ability of a competitive ligand, methyl-alpha-D-mannopyranoside, to reduce T-cell-mediated cytotoxicity to SEC by more than 50%. Tumor necrosis factor alpha (TNF-alpha) was produced by LnC2 in high amounts after Con A stimulation (>6 ng/mL), but antiserum to TNF-alpha did not reduce LnC2-mediated cytotoxicity toward SEC. In conclusion, we could show for the first time that liver SECs have accessory function and are selectively destroyed by CD4+ T lymphocytes in the presence of Con A. We speculate that SEC damage is an early event in T-cell-mediated liver injury recruiting T lymphocytes from the sinusoidal circulation. Loss of the SEC barrier function then exposes underlying hepatocytes to further attack by activated T lymphocytes. These results offer a model of initiating events in T-cell-mediated liver diseases, such as viral or autoimmune hepatitis, and suggest an important role for sinusoidal endothelial cells.

Role of sinusoidal endothelial cells of the liver in concanavalin A-induced hepatic injury in mice

CD4+ T lymphocytes have been identified as being responsible for organ damage in the murine model of experimental liver injury induced by intravenous injection of concanavalin A (Con A). Liver sinusoidal endothelial cells (SEC) and Kupffer's cells (KC) are among the first cells that come into contact with lymphocytes in the liver sinusoid. We aimed to investigate the respective role of these cell populations in the initial steps of T-cell-mediated liver injury in Con A-induced hepatitis. By electron microscopy, we could show that intravenously applied Con A bound predominantly to SEC but not to KC. KC depletion by gadolinium chloride treatment of mice did not result in protection from liver injury, indicating that KCs are not primarily involved in the generation of liver injury. We could show that a CD4+ T-cell line (LNC.2) displayed selective cytotoxicity toward SEC (>50%) but not KC (12%) or fibroblasts (5%) in the presence of Con A in vitro. Microscopic observation revealed that the SEC monolayer was rapidly destroyed by LnC2 in the presence of Con A. Specificity of the Con A-induced cytotoxicity was shown by the ability of a competitive ligand, methyl-alpha-D-mannopyranoside, to reduce T-cell-mediated cytotoxicity to SEC by more than 50%. Tumor necrosis factor alpha (TNF-alpha) was produced by LnC2 in high amounts after Con A stimulation (>6 ng/mL), but antiserum to TNF-alpha did not reduce LnC2-mediated cytotoxicity toward SEC. In conclusion, we could show for the first time that liver SECs have accessory function and are selectively destroyed by CD4+ T lymphocytes in the presence of Con A. We speculate that SEC damage is an early event in T-cell-mediated liver injury recruiting T lymphocytes from the sinusoidal circulation. Loss of the SEC barrier function then exposes underlying hepatocytes to further attack by activated T lymphocytes. These results offer a model of initiating events in T-cell- mediated liver diseases, such as viral or autoimmune hepatitis, and suggest an important role for sinusoidal endothelial cells. (Hepatology 1996 Oct;24(4):824-9)