Glycyrrhizin protects mice from concanavalin A-induced hepatitis without affecting cytokine expression.

The administration of concanavalin A (Con A) to mice induces cytokine-dependent hepatitis. In the present study, the effect of glycyrrhizin on Con A-induced hepatitis was examined. Treatment of mice with Con A (0.2 mg/mouse, i.v.) induced elevation of the plasma transaminase activities at 24 h. Mice were treated with glycyrrhizin (100, 200 and 400 mg/kg, i.p.), and glycyrrhizin at the doses of 200 and 400 mg/kg inhibited the Con A-induced elevation of the plasma transaminase activities. At 1 h after Con A treatment, interferon-gamma, tumor necrosis factor-alpha, interleukin-2 and interleukin-6 proteins were released into the plasma. Although treatment with glycyrrhizin at 200 mg/kg inhibited Con A-induced hepatitis, it did not affect the release of any of these Con A-induced cytokines into the plasma. The present results clearly show that glycyrrhizin inhibited Con A-induced hepatitis without affecting cytokine expression.     

Development of anorexia in concanavalin A-induced hepatitis in mice

Anorexia that develops in chronic hepatitis is associated with cytokine expression in the brain. Treatment of mice with concanavalin A (12.5 mg/kg, i.v.) elevated the plasma alanine aminotransferase activity at 8.5 h after treatment. Tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta mRNA expression was induced at 6 and 24 h after concanavalin A treatment in both the liver and brain. Treatment of mice with concanavalin A reduced the body weight at 24 h after treatment and this decreased body weight was accompanied by a decreased food intake. Glycyrrhizin (200 mg/kg, i.p.) inhibited the concanavalin A-induced elevation of plasma alanine aminotransferase activity, however, it did not inhibit the concanavalin A-induced decreased body weight. The present results indicate that treatment of mice with concanavalin A caused the development of anorexia and that this anorexia might develop independently of the induction of hepatitis.     

Prevention of concanavalin A-induced mice hepatitis by molsidomine

Molsidomine is effective in reducing portal hypertension in cirrhosis, but its effect on hepatitis is not known. In the present study, the effect of molsidomine on hepatitis was examined using mouse concanavalin A (Con A)-induced hepatitis and mouse anti-Fas antibody-induced hepatitis. Treatment of mice with Con A caused elevation of plasma alanine aminotransferase (ALT) at 8 and 24 h (n=4). Pretreatment of mice with molsidomine (3, 10, 30 and 100 mg/kg, i.p.) prevented Con A-induced hepatitis. Treatment of mice with anti-Fas antibody (150 microg/kg, i.v.) caused elevation of plasma ALT at 3.5 h. Pretreatment mice with molsidomine (10 mg/kg, i.p.) showed only 47% inhibition of anti-Fas antibody caused elevation of plasma ALT. The present results showed effectiveness of molsidomine in preventing Con A-induced mice hepatitis.     

Synthetic derivatives of osthole for the prevention of hepatitis

Prevention of hepatitis is a worldwide issue. For most patients with liver disease, hepatoprotective drugs are required. But there are only a few hepatoprotective drugs available. Osthole is a coumarin compound and protects the liver from hepatitis by preventing the development of apoptosis. However, osthole exhibits low water solubility, and some structural modifications are required for sufficient hepatoprotection upon oral administration. We synthesized 28 osthole derivatives, and then studied their effects by using mice concanavalin A (Con A) -induced hepatitis. The osthole derivatives No.1, 9 and 19 showed stronger inhibition of Con A-induced elevation of plasma alanine aminotransferase (ALT). Oral administration of osthole at the dose of 100 mg/kg (n=10) inhibited 38.0% of the Con A-induced elevation of plasma ALT. In contrast, oral administration of Nos. 1, 9 and 19 at the dose of 100 mg/kg (n=5) caused 68.7%, 62.5% and 88.3% inhibition of the Con A-induced elevation of plasma ALT, respectively. These synthetic osthole derivatives could contribute to the development of hepatoprotective drugs effective for various types of liver diseases on oral administration.     

Concanavalin A-induced hepatitis in mice is prevented by interleukin (IL)-10 and exacerbated by endogenous IL-10 deficiency

One single intra-venous (i.v.) injection of Concanavalin A (Con A) into mice provokes a cell-mediated immunoinflammatory hepatitis. We have presently evaluated the immunopharmacological effects of exogenous interleukin (IL)-10 and the role of endogenous IL-10 in this model by using exogenous IL-10, anti-IL-10 monoclonal antibody (mAb) and mice with disrupted IL-10 gene (IL-10 KO mice). Whilst exogenous IL-10 administered in a prophylactic (1 h prior to Con A) and even "early" therapeutic fashion (30 min after Con A) reduced the elevation of transaminase activities in plasma in a dose-dependent manner, observed in control mice, these biochemical markers of liver injury were significantly increased both in IL-10 KO mice as well as in those receiving anti-IL-10 mAb. Interestingly, doses of Con A lower than 20 mg/kg that were only capable of inducing slight serological signs of hepatitis in mice, exerted marked hepatitic effects when administered to either anti-IL-10 mAb-treated mice or to IL-10 KO mice. The disease modulating effects of exogenous IL-10 and either genetical or pharmacologically-induced IL-10 deficiency were associated with profound and opposite modifications of the Con A-induced increase in the circulating levels of IFN-gamma and TNF-alpha. Relative to control animals, the blood levels of these cytokines were diminished in IL-10-treated mice and augmented in both IL-10 KO mice and anti-IL-10 mAb-treated mice. These results prove the physiological antiinflammatory role of endogenous IL-10 in Con A induced hepatitis and the beneficial effects of IL-10 treatment to prevent this condition.     

Induction of thymocyte apoptosis by systemic administration of concanavalin A in mice: role of TNF-alpha, IFN-gamma and glucocorticoids

Administration of concanavalin A (Con A) is a well-established model of acute immune-mediated hepatitis. Here, we demonstrate that intravenous injection of Con A in mice induces profound thymic atrophy. Compared to liver damage, the kinetics of Con A-induced thymic atrophy is slower and more prolonged; the nadir in thymocyte number is reached 4 days after Con A injection, whereas peak transaminase levels are observed at 12-24 h. Marked alterations in the ratio of CD4+ and CD8+cells in the thymus and spleen and significantly increased rates of thymocyte and splenocyte apoptosis are observed. Neutralization of the cytokines TNF-alpha or IFN-gamma, which protects mice from Con A-induced hepatitis, prevents thymic atrophy as well as alterations in CD4+ and CD8+ cell numbers and apoptosis rates. However, neither TNF-alpha nor IFN-gamma are detectable in thymocyte lysates after Con A injection, whereas both cytokines are present in liver, spleen and serum. Administration of the glucocorticoid receptor antagonist mifepristone does not prevent thymic atrophy, thus ruling out a possible contribution of endogenous glucocorticoids. Con A-induced thymic atrophy is accompanied by down-regulation of Bcl-2 expression in the thymus, which is prevented by neutralization of TNF-alpha or IFN-gamma. These data demonstrate that the thymus is a critical target organ of Con A-induced inflammation; the effects of Con A on the thymus are mediated by extrathymic production of TNF-alpha and IFN-gamma, but not by glucocorticoids.     

Zaltoprofen inhibits concanavalin A-induced decrease of body weight in mice

Treatment of mice with concanavalin A (Con A) (12.5 mg/kg, i.v.) decreased the body weight at 24 h. This Con A-induced body weight decrease was accompanied by reduction in food and water intake. Zaltoprofen is a non-steroidal anti-inflammatory drug. The administration of Zaltoprofen (10 mg/kg) at 8 h after Con A treatment was found to inhibit the Con A-induced reduction in body weight. The food intake in mice treated with Con A plus Zaltoprofen (10 mg/kg) was four times greater than that in mice treated with only Con A. The present results showed inhibition of the Con A-induced body weight loss by Zaltoprofen and suggested its possible effectiveness for the treatment of "sickness behavior".     

L-selectin and intercellular adhesion molecule-1 regulate the development of Concanavalin A-induced liver injury

Concanavalin A (Con A)-induced hepatitis is a model for human T cell-mediated hepatitis. We evaluated the role of L-selectin and intercellular adhesion molecule-1 (ICAM-1) in this model by injecting Con A intravenously in mice lacking L-selectin (L-selectin-/-), ICAM-1 (ICAM-1-/-), or both (L-selectin/ICAM-1-/-). Blood and liver samples were collected 0, 8, 24, and 48 h after Con A treatment. Increases in plasma transaminase levels, which peaked 8 h after injection, were reduced significantly in L-selectin-/-, ICAM-1-/-, and L-selectin/ICAM-1-/- mice compared with wild-type mice. Liver necrosis was more strongly inhibited in ICAM-1-/- mice than in L-selectin-/- mice but was most prominently reduced in L-selectin/ICAM-1-/- mice, in parallel with decreased plasma transaminase levels. The reduced severity of hepatitis in the mutant mice correlated with decreases in numbers of liver CD4+ T cells but not numbers of CD8+ T cells or neutrophils. Following Con A treatment, L-selectin deficiency reduced liver mRNA expression of tumor necrosis factor-alpha, and ICAM-1 deficiency reduced expression of interleukin-4. By contrast, reductions in liver macrophage inhibitor protein-1alpha mRNA occurred in all mutant mice. These results indicate that L-selectin and ICAM-1 contribute cooperatively to the development of Con A-induced hepatitis by regulating leukocyte infiltration and subsequent cytokine production.     

Leptin deficiency, not obesity, protects mice from Con A-induced hepatitis

Leptin-deficient ob/ob mice are protected from Con A-induced hepatitis. However, it is unclear whether leptin deficiency or obesity itself is responsible for this protection. To address this question, wild-type (WT) obese mice with high serum leptin levels were generated by injection of gold thioglucose (WT GTG). Both Con A-injected WT and WT GTG mice developed hepatitis, whereas no hepatic damage was observed in ob/ob mice. Moreover, TNF-alpha and IFN-gamma levels as well as expression of the activation marker CD69 were elevated in liver mononuclear cells of WT and WT GTG mice, but not in ob/ob mice following administration of Con A. The liver of WT and WT GTG mice had the same percentage of NK T cells, a lymphocyte population involved in Con A-induced hepatitis. This population decreased equally in both WT and WT GTG mice after Con A injection. In contrast, the liver of ob/ob mice contained 50% less NK T cells compared to WT and WT GTG mice. Furthermore, no decrease in NK T cells was observed in Con A-injected ob/ob mice. We conclude that leptin-deficiency, not obesity, is responsible for protection from Con A-induced hepatitis.     

Quercetin Protects Mice from ConA-Induced Hepatitis by Inhibiting HMGB1-TLR Expression and Down-Regulating the Nuclear Factor Kappa B Pathway

The dietary flavonoid quercetin has hepatoprotective effects. We analyzed the effects of quercetin on concanavalin A (ConA)-induced hepatitis in mice and its underlying molecular mechanisms of action. Mice were administered quercetin (50 mg/kg body weight, i.p.) or vehicle 30 min before intravenous administration of ConA. Quercetin pretreatment significantly reduced the ConA-induced elevations in plasma aminotransferase concentrations and liver necrosis, as well as reducing serum concentrations of the pro-inflammatory cytokines tumor necrosis factor (TNF)-α, interferon-γ, and interleukin-4. Quercetin pretreatment also reduced expression of high-mobility group box 1 protein (HMGB1) and toll-like receptor (TLR)-2 and TLR-4 messenger RNA (mRNA) and protein in liver tissues. Quercetin pretreatment significantly inhibited degradation of inhibitory kappa B alpha and modulated ConA-induced nuclear translocation in the liver of nuclear factor kappa B (NF-κB) p65. These results demonstrate that quercetin protects against ConA-mediated hepatitis in mice by attenuating the HMGB1–TLRs–NF-κB signaling pathway.     

Protection by a cyclic AMP-specific phosphodiesterase inhibitor, rolipram, and dibutyryl cyclic AMP against Propionibacterium acnes and lipopolysaccharide-induced mouse hepatitis

OBJECTIVE AND DESIGN: To study the effect of cellular cAMP-increasing agents on Propionibacterium acnes (P. acnes) and lipopolysaccharide (LPS)-induced mouse hepatitis. MATERIAL: Male BALB/c mice were used. Macrophages/Kupffer cells isolated from P. acnes-primed murine liver were used for the in vitro study. TREATMENT: Type IV phosphodiesterase (PDE)-specific inhibitor, rolipram, was administered (10, 30 mg/kg, p. o.). Dibutyryl cyclic AMP (dbcAMP) was injected (10, 100 mg/kg, i.p.) into the mice. METHOD: Plasma TNFalpha estimated by the use of an L-929 cell cytotoxic assay and plasma transaminase activities were measured for the in vivo study. The LPS-induced production of TNFalpha in vitro from the cultured macrophage/Kupffer cells was determined by ELISA. RESULTS: Rolipram suppressed the elevation of plasma transaminases induced by injection of LPS, and dbcAMP had a tendency to suppress them. Both agents attenuated the LPS-induced release of TNFalpha in vivo, and suppressed the TNFalpha production from the cultured macrophage/Kupffer cells. CONCLUSIONS: These results suggest that rolipram and dbcAMP have potential to inhibit TNFalpha production from activated macrophage/Kupffer cells, and it may be partially involved in the protecting effect in the P. acnes/LPS hepatitis model.     

Blockade of IL-33 ameliorates Con A-induced hepatic injury by reducing NKT cell activation and IFN-γ production in mice

IL-33, a recently described member of the IL-1 family, has been identified as a cytokine endowed with pro-Th2 type functions. To date, there are only limited data on its role in physiological and pathological hepatic immune responses. In this study, we examined the role of IL-33 in immune-mediated liver injury by exploring the model of concanavalin A (Con A)-induced hepatitis. We observed that the level of IL-33 expression in the liver was dramatically increased at 12?h after Con A injection. Meanwhile, ST2L, the receptor of IL-33, was significantly up-regulated in lymphocytes including T and natural killer T (NKT) cells, especially in NKT cells. Moreover, administration of recombinant IL-33 exacerbated Con A-induced hepatitis, while pretreatment of IL-33-blocking antibody or psST2-Fc plasmids showed a protective effect probably by inhibiting the activation of late stage of T cells and NKT cells and also decreasing the production of the cytokine IFN-??. Furthermore, depletion of NKT cells abolished the protective effect of IL-33-blocking antibody, and IL-33 failed to exacerbate Con A-induced hepatitis in IFN-??^?/? mice. These data suggested the critical roles of NKT cells and IFN-?? in the involvement of IL-33 in Con A-induced hepatitis. Blockade of IL-33 may represent a novel therapeutic strategy through IL-33/ST2L signal to prevent immune-mediated liver injury.     

The pathogenic roles of tumor necrosis factor receptor p55 in acetaminophen-induced liver injury in mice

Acetaminophen (APAP) causes a massive production of intrahepatic tumor necrosis factor alpha (TNF-alpha). However, it still remains elusive regarding the roles of TNF-alpha in APAP-induced liver injury. Hence, we examined pathogenic roles of the TNF-alpha-TNF receptor with a molecular weight of 55 kDa (TNF-Rp55) axis in APAP-induced hepatotoxicity using TNF-Rp55-deficient [TNF-Rp55-knockout (KO)] mice. When wild-type (WT) BALB/c and TNF-Rp55-KO mice were intraperitoneally injected with a lethal dose of APAP (750 mg/kg), the mortality of TNF-Rp55-KO mice was marginally but significantly reduced compared with WT mice. Upon treatment with a nonlethal dose (600 mg/kg), WT mice exhibited an increase in serum transaminase levels. Histopathologically, centrilobular hepatic necrosis with leukocyte infiltration was evident at 10 and 24 h after APAP challenge. Moreover, mRNA expression of adhesion molecules, several chemokines, interferon-gamma (IFN-gamma), and inducible nitric oxide synthase (iNOS) was enhanced in the liver. On the contrary, serum transaminase elevation and histopathological changes were attenuated in TNF-Rp55-KO mice injected with APAP (600 mg/kg). The gene expression of all molecules except for IFN-gamma and iNOS was significantly attenuated in TNF-Rp55-KO mice. Moreover, anti-TNF-alpha neutralizing antibodies alleviated liver injury when administered at 2 or 8 h after but not at 1 h before APAP challenge to WT mice. Collectively, the TNF-alpha-TNF-Rp55 axis has pathogenic roles in APAP-induced liver failure.     

Bimodal role of endogenous interleukin-6 in concanavalin A-induced hepatitis in mice

Acute concanavalin A (Con A)-induced hepatitis in mice is an animal model for hepatic injury induced by activated T cells. The evolution of hepatic involvement can be followed from hour to hour by measuring serum transaminase levels. We investigated the possible role of endogenous interleukin-6 (IL-6) in this model. We found serum IL-6 levels and splenic IL-6 mRNA during Con A-induced hepatitis to be significantly lower in interferon-gamma (IFN-gamma)-deficient mice, which are resistant against the Con A-induced syndrome, than in wild-type ones, suggesting that systemic IL-6 production favors development of hepatic injury. However, IL-6-deficient mice proved to be more susceptible to the disease than wild-type mice, indicating that endogenous IL-6 plays a predominantly hepatoprotective role. Experiments in which wild-type mice were treated with anti-IL-6 antibodies, before or after Con A challenge, allowed us to reconcile these contrasting observations. The antibody injections resulted in a biphasic alteration of serum IL-6 levels, initial neutralization being followed by rebound increased levels due to accumulation of IL-6 in the form of antigen-antibody complexes. The effect of antibody on disease severity differed depending on the time of injection. Antibody injection at 2.5 h post Con A resulted in delayed disease manifestation, whereas treatment initiated before Con A resulted in accelerated disease. We conclude that endogenous IL-6 plays a bimodal role. IL-6 present before Con A challenge as well as that induced in the very early phase after Con A injection triggers hepatoprotective pathways. Continuation of IL-6 production beyond this early phase, by some other pathway, seems to be harmful to hepatocytes.     

A critical involvement of oxidative stress in acute alcohol-induced hepatic TNF-alpha production

Tumor necrosis factor-alpha (TNF-alpha) production is a critical factor in the pathogenesis of alcoholic liver injury. Both oxidative stress and endotoxin have been implicated in the process of alcohol-induced TNF-alpha production. However, a cause-and-effect relationship between these factors has not been fully defined. The present study was undertaken to determine the mediators of acute alcohol-induced TNF-alpha production using a mouse model of acute alcohol hepatotoxicity. Alcohol administration via gavage at a dose of 6 g/kg to 129/Sv mice induced hepatic TNF-alpha production in Kupffer cells as demonstrated by measuring protein levels, immunohistochemical localization, and mRNA expression. Alcohol intoxication caused liver injury in association with increases in plasma endotoxin and hepatic lipid peroxidation. Treatment with an endotoxin neutralizing protein significantly suppressed alcohol-induced elevation of plasma endotoxin, hepatic lipid peroxidation, and inhibited TNF-alpha production. Treatment with antioxidants, N-ACETYL-L-CYSTEINE, or dimethylsulfoxide, failed to attenuate plasma endotoxin elevation, but significantly inhibited alcohol-induced hepatic lipid peroxidation, TNF-alpha production and steatosis. All treatments prevented alcohol-induced necrotic cell death in the liver. This study thus systemically dissected the relationship among plasma endotoxin elevation, hepatic oxidative stress, and TNF-alpha production following acute alcohol administration, and the results demonstrate that oxidative stress mediates endotoxin-induced hepatic TNF-alpha production in acute alcohol intoxication.     

Protection from concanavalin A (Con A)-induced T cell-dependent hepatic lesions and modulation of cytokine release in mice by sodium fusidate

The immunomodulatory effects of the antibiotic sodium fusidate (SF) were tested in a model of T cell-dependent hepatic injury that can be induced in normal mice by a single i.v. injection of Con A. Signs of hepatitis with elevated transaminase activities in plasma, severe infiltration of the liver by neutrophil granulocytes, lymphocytes and monocytes, and necrotic areas were observed in control mice treated intraperitoneally with PBS 24 h and 1 h before Con A challenge. T cell- and macrophage-derived cytokines (IL-2, interferon-gamma (IFN-γ), tumour necrosis factor-alpha (TNF-α), IL-1β, IL-6) were released with different kinetics in the circulation of these mice. SF, 20, 40 or 80 mg/kg, administered 24 h and 1 h before Con A challenge, protected the mice against the hepatitic effects of Con A. The protective effects of SF were dose-dependent and accompanied by profound modifications of blood levels of cytokines induced by Con A, so that, relative to control mice, SF (80 mg/kg)-treated animals showed markedly diminished plasma levels of IL-2, IFN-γ and TNF-α, along with augmented levels of IL-6. These results suggest that SF might be useful in the treatment of immunoinflammatory liver diseases in humans.     

Immunopathogenesis of hepatic fibrosis in chronic liver injury induced by repeatedly administered concanavalin A.

Liver fibrosis is commonly observed in chronic liver disease. However, the immunological mechanisms underlying hepatic fibrosis due to chronic inflammation are not well defined, mainly because suitable experimental models have not been established. We have found that weekly i.v. administration of concanavalin A (Con A) in BALB/c mice brought about a striking alanine aminotransferase increase, resulting in piecemeal necrosis with bridging fibrosis in the parenchyma. Using this fibrosis model, we demonstrated the kinetics of cytokine mRNA expression in liver. Transforming growth factor (TGF)-beta1, TGF-alpha, basic fibroblast growth factor (bFGF) and hepatocyte growth factor mRNAs were up-regulated after each Con A administration. Furthermore, either anti-IFN-gamma, anti-tumor necrosis factor (TNF)-alpha or anti-TGF-beta mAb given together with Con A markedly inhibited the development of hepatic fibrosis. Treatment with either anti-IFN-gamma or anti-TNF-alpha mAb also completely prevented hepatic injury; in contrast, treatment with anti-TGF-beta mAb did not. The treatment with anti-TGF-beta mAb did not affect the levels of hepatic mRNAs for either IFN-gamma or TNF-alpha after Con A injection. Treatment with either anti-IFN-gamma or anti-TNF-alpha did not affect the expression levels of TGF-beta in the liver. In conclusion, the continuous presence of both severe liver damage and up-regulation of TGF-beta synthesis is necessary to induce hepatic fibrosis in this model.     

经肝动脉同种异体脂肪干细胞移植治疗小鼠自身免疫性肝炎的实验研究

目的: 探讨同种异体脂肪干细胞经肝动脉移植对刀豆素A(Con A)诱导的小鼠自身免疫性肝炎的治疗价值。方法: Con A注射法建立小鼠自身免疫性肝炎模型。将40只小鼠随机分为4组:阴性对照组、经肝动脉移植组、经尾静脉移植组(移植脂肪干细胞2×10⁶)和阳性对照组。阳性对照组应用环磷酰胺腹腔注射。观察移植前后肝脏组织病理病变情况、血清炎症指标和肝功能指标的改变。结果: 经肝动脉移植组和经尾静脉移植组行脂肪干细胞移植后全部存活,未发生明显并发症;经肝动脉移植组血清炎症指标(IFN-γ、IL-4和IL-5)及血清天冬氨酸氨基转移酶、丙氨酸氨基转移酶及碱性磷酸酶明显下降(P<0.05);经尾静脉移植组血清炎症指标及血清肝功能指标有所下降,但无显著差异。经肝动脉移植组及阳性对照组病理学上改善最明显,肝内炎症细胞浸润及肝细胞坏死均明显减少。结论: 同种异体脂肪干细胞移植有助于改善Con A介导的小鼠自身免疫性肝炎,经肝动脉途径可提高脂肪干细胞肝移植的效能。     

Pentoxifylline inhibits anti-Fas antibody-induced hepatitis by affecting downstream of CPP32-like activity in mice

The effect of pentoxifylline on anti-Fas antibody-induced hepatitis was studied. The administration of anti-Fas antibodies (250 microg/kg, i.v.) to mice elevated plasma alanine aminotransferase (ALT) activity at 3 h. This anti-Fas antibody-induced elevation of ALT was inhibited by treatment with pentoxifylline at the doses of 10 and 100 mg/kg (i.p.). Anti-Fas antibody administration also elevated the CPP32-like protease activity in the liver at 3 h. Although pentoxifylline at 100 mg/kg, i.p., inhibited the anti-Fas antibody-induced elevation of plasma ALT, this treatment did not significantly inhibit the anti-Fas antibody-induced elevation of CPP32-like activity. The present results clearly showed that treatment with pentoxifylline inhibited anti-Fas antibody-induced hepatitis, at least in part, by affecting a reaction downstream of CPP32-like protease activation.