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.     

(-)-Epigallocatechin-3-gallate protects mice from concanavalin A-induced hepatitis through suppressing immune-mediated liver injury

(-)-Epigallocatechin-3-gallate (EGCG) is the major active component of green tea. Increasing evidence has suggested that EGCG exhibits anti-inflammatory, anti-oxidant and immunosuppressive effects. In this study, we investigated the effect of EGCG on concanavalin A (ConA)-induced hepatitis (CIH) in mice, a model of immune-mediated liver injury in humans. We pretreated mice with EGCG before ConA injection, and then measured alanine aminotransferase (ALT) levels in plasma, inflammatory infiltration and hepatocyte apoptosis in liver. Potential therapeutic mechanisms were elucidated further by measuring several inflammatory mediators. Mice pretreated with EGCG exhibited much less increased ALT levels in plasma, reduced inflammatory infiltration and hepatocyte apoptosis in liver compared with control mice pretreated with vehicle solutions. We further investigated the mechanisms of the protective effects of EGCG. In EGCG-pretreated mice, we found abrogated tumour necrosis factor (TNF)-alpha and interferon (IFN)-gamma at both protein levels in plasma and mRNA levels in liver. At the same time, the concentration of nitrite in plasma and inducible nitric oxide synthase production in liver were both down-regulated in these mice. Moreover, IFN-inducible protein-10 and macrophage inflammatory protein-1alpha expressions in liver were decreased significantly. Therefore, EGCG is capable of regulating immune-mediated liver injury in vivo. The protective effect depended on its suppressive effect on the production of important inflammatory mediators.     

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.     

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.     

A broad-spectrum caspase inhibitor blocks concanavalin A-induced hepatitis in mice

Fulminant hepatic failure (FHF) is a clinical syndrome resulting from massive death of liver cells or sudden and severe impairment of liver function. The causes of FHF are diverse and the overall mortality is very high. Recently, it became clear that apoptosis of hepatocytes is the critical cause of acute hepatic failure in FHF. It is well known that a family of cysteine proteases called caspase is one of the key mediators of the apoptotic pathway. Thus, caspases are attractive potential targets for the treatment of disorders resulting from excessive apoptosis. In this report, we examined the activity of a new caspase inhibitor, Xyz 033 mp. This nonpeptide inhibitor showed broad-spectrum caspase-inhibiting activity and protected primary rat hepatocytes from apoptotic death. In a mouse model of FHF induced by concavalin A (Con A), Xyz 033 mp suppressed elevated AST and ALT and specifically reduced IL-1 beta concentration. Also, Xyz 033 mp rescued mice from lethal experimental hepatitis induced by Con A. In addition, histological examinations indicated that Xyz 033 mp protected hepatocytes from the fatal apoptogenic effect of Con A. These results suggest that Xyz 033 mp may be a candidate therapeutic agent for FHF caused by massive apoptotic death of hepatocytes.     

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.     

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.     

NC-1500 prevents concanavalin A-induced mice hepatitis without affecting cytokine gene expression.

NC-1500 is a dihydropyridine type calcium channel blocker. The effect of NC-1500 on mice concanavalin A-induced hepatitis was examined. Treatment of mice with concanavalin A (Con A) caused elevation of plasma transaminase. Pretreatment of mice with NC-1500 (3, 10 and 30 mg/kg, p.o.) prevented this Con A-induced elevation of plasma transaminase. Treatment of mice with Con A induced tumor necrosis factor-alpha (TNF-alpha) mRNA expression in the liver. However, NC-1500 (30 mg/kg, p.o.) did not affect this Con A-induced TNF-alpha mRNA expression in the liver. The present results showed that NC-1500 inhibited Con A-induced hepatitis without affecting TNF-alpha mRNA expression in the liver.     

CD4+ T-cell dysfunctions through the impaired lipid rafts ameliorate concanavalin A-induced hepatitis in sphingomyelin synthase 1-knockout mice

Membrane microdomains consisting of sphingomyelin (SM) and cholesterol appear to be important for signal transduction in T-cell activation. The present study was designed to elucidate the role of membrane SM in vivo and in vitro using sphingomyelin synthase 1 (SMS1) knock out (SMS1(-/-)) mice and Concanavalin A (ConA)-induced hepatitis. After establishing SMS1(-/-) mice, we investigated CD4+ T-cell functions including proliferation, cytokine production and signal transduction in vivo. We also examined severity of hepatitis, cytokine production in serum and liver after ConA injection at a dose of 20 mg kg(-1). CD4+ T cells from SMS1(-/-) mice showed severe deficiency of membrane SM and several profound defects compared with wild-type controls as follows: (i) cellular proliferation and production of IL-2 and IFN-γ by co-cross-linking of CD3 and CD4; (ii) tyrosine phosphorylation of LAT and its association with ZAP-70; (iii) clustering and co-localization of TCR with lipid rafts. Consistent with these impaired CD4+ T-cell functions in vitro, SMS1(-/-) mice showed decreased serum levels of IL-6 and IFN-γ by ConA injection, which renders SMS1(-/-) mice less sensitive to ConA-induced hepatitis. These results indicated that the deficiency of membrane SM caused the CD4+ T-cell dysfunction through impaired lipid raft function contributed to protection of ConA-induced liver injury, suggesting that the membrane SM is critical for full T-cell activation both in vitro and in vivo.     

Suppressive role of hepatic dendritic cells in concanavalin A-induced hepatitis

Concanavalin A (Con A)‐induced hepatitis is a mouse model of acute autoimmune hepatitis. The aim of this study was to investigate the role of hepatic dendritic cells (DC) in the immune modulation of tissue damage. Almost all hepatic DC were plasmacytoid DC (CD11c⁺ I‐A^low B220⁺); however, conventional DC were CD11c⁺ I‐A^high B220^–. At an early stage (3–6 h) after Con A administration, the number of DC in both the liver and spleen decreased, increasing thereafter (12–24 h) in parallel with hepatic failure. The hepatic CD11c⁺ DC population contained many CD11b^‐ cells, while the majority of splenic CD11c⁺ DC were CD11b⁺. After Con A administration, the proportion of I‐A⁺ and CD11b⁺ cells within the CD11c⁺ DC population tended to increase in the liver, but not in the spleen. Similarly, expression of the activation markers CD80, CD86 and CD40 by CD11c⁺ DC increased in the liver, but not in the spleen. Next, adoptive transfer of DC isolated from the liver and spleen was performed 3 h after Con A administration to examine the immunomodulatory function of DC. Only hepatic DC had the ability to suppress hepatic failure. Analysis of cytokine production and subsequent identification of the effector cells showed that hepatic DC achieved this by suppressing the production of interleukin (IL)‐12 and IL‐2, rather than modulating effector cell function.     

低剂量Con A预刺激抑制足剂量Con A诱导的肝炎发生

目的:探讨低剂量刀豆蛋白A(ConA)预刺激对后续足剂量ConA诱导的小鼠肝炎的抑制作用。方法:足剂量ConA(15μg/g体重)静脉注射小鼠以诱导ConA肝炎模型。低剂量ConA预刺激组,在诱导ConA肝炎模型前12小时静脉注射低剂量ConA(3μg/g体重);PBS预处理对照组,在诱导ConA肝炎模型前12小时静脉注射PBS。观察两组血清转氨酶(ALT/AST)及肝脏病理变化,同时用流式细胞仪检测肝脏淋巴细胞亚群的比例及绝对数目变化。结果:与PBS预处理相比,低剂量ConA预刺激导致血清转氨酶(ALT/AST)水平显著降低(ALT由3433±788U/L降低到334±68U/L,AST由1358±297U/L降低到167±33U/L),肝脏病理切片显示由ConA引起的肝脏损伤明显减轻,同时伴随着肝脏内CD3+T淋巴细胞比例及绝对数目减少,并且其活化受到明显抑制。结论:低剂量ConA预刺激可以使小鼠抵抗后续的足剂量ConA诱导的肝脏损伤。这种现象与肝脏回流的T淋巴细胞减少及活化程度有关,其细胞与分子机制正在进一步探讨之中。     

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".     

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.     

Cycloastragenol mediates activation and proliferation suppression in concanavalin A-induced mouse lymphocyte pan-activation model

Context: Cycloastragenol (CAG) is a molecule isolated from various species in the genus Astragalus. Although the regulatory activity of Astragalus on immune system has been investigated, the effect of CAG on activated lymphocytes is poorly understood.

Objective: We aimed to biologically address the possible anti-inflammation potential of CAG on concanavalin A (Con A)-mediated mouse lymphocyte pan-activation model.

Materials and methods: Mouse lymphocytes were obtained from spleens and subjected to Con A for 24?h. Herein, the cells were treated with different concentrations of CAG. Cell viability was assayed by MTT. Pretreated by CAG and stimulated by Con A, the expression of CD69 and CD25, Th1/Th2/Th17 cytokines, cell cycle, proliferation and intracellular Ca²⁺ concentration ([Ca²⁺]_i) were analyzed by flow cytometry.

Results: The results declared that CAG significantly downregulated both CD69 and CD25 expressed on Con A activated CD3?+?T cells’ surface, as well as inhibiting proliferation of activated lymphocytes. In addition, CAG blocked the Con A-induced mitogenesis, exhibiting lymphocyte G₀/G₁-phase cell-cycle arrest with significant reduction of cells in S and G₂/M phases. Meanwhile, pretreated by CAG, a significant decline in [Ca²⁺]_i was observed. Furthermore, CAG significantly inhibited the production of Th1 cytokines IFN-γ, TNF, IL-2, Th2 cytokines IL-4, IL-6, IL-10 and Th17 cytokine IL-17?A on Con A-activated lymphocytes.

Conclusion: Our results reinforce that CAG has important anti-inflammatory activity by inhibiting lymphocytes activation, proliferation and cytokines expression, and shows, that this effect may be related to reduction of overall intracellular Ca²⁺ overload.     

Mice deficient in hepatocyte-specific IL-1Ra show delayed resolution of concanavalin A-induced hepatitis

Interleukin-1 receptor antagonist (IL-1Ra) is a specific IL-1 inhibitor that possesses anti-inflammatory activities. Several studies in human and mouse suggested a protective role for IL-1Ra in liver inflammation, and we previously demonstrated that hepatocytes produce high levels of IL-1Ra in response to inflammatory challenge in vitro and in vivo. In the present study, we investigated the production and the biological function of hepatocyte-derived IL-1Ra in concanavalin A (ConA)-induced hepatitis in mice. We show that the injured liver produces large amounts of IL-1Ra and that secreted and intracellular IL-1Ra isoforms are produced with different kinetics during the course of hepatitis. By using hepatocyte-specific IL-1Ra-deficient mice (IL-1Ra(ΔH)), we demonstrate that hepatocytes represent the major cellular source of local IL-1Ra. Most interestingly, hepatic necrosis and inflammation were increased in IL-1Ra(ΔH) as compared with wild-type mice during the late phase of the disease, leading to a delayed resolution of hepatitis in IL-1Ra(ΔH) mice. In conclusion, our results show that the local production of IL-1Ra by hepatocytes contributes to the resolution of hepatitis.     

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 (5 mg/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.     

TPN10466 ameliorates Concanavalin A-induced autoimmune hepatitis in mice via inhibiting ERK/JNK/p38 signaling pathway

Autoimmune hepatitis (AIH) eventually progresses to liver fibrosis, cirrhosis, and even hepatocellular carcinoma, causing irreversible damage to the liver. Concanavalin A-induced hepatitis in mice is a well-established model with pathophysiology similar to that of immune-mediated liver injury in human viral and autoimmune hepatitis, and it has been widely used to explore the pathogenesis and clinical treatment of human immune hepatitis. Artemisinin has been shown to exhibit anti-inflammatory effects through unclear mechanisms. In this study, we aimed to assess the effect of the artemisinin derivative TPN10466 on AIH. In vitro studies showed that TPN10466 dose dependently inhibited the percentage of IFN-γ-producing T cells. Further studies showed that TPN10466 attenuated the disease severity of AIH by downregulating the ability of lymphocytes to secrete IFN-γ and by reducing lymphocyte number in the liver. In addition, we found that TPN10466 treatment reduced T-cell responses by inhibiting JNK, ERK, and p38 pathways. In conclusion, our work suggests that TPN10466 provides protection against the autoimmune disease AIH by suppressing the inflammatory response of T cells, suggesting that TPN10466 may be a promising potential agent for the treatment of AIH.     

Glucocorticoids enhance concanavalin A-induced mitogenic response through the inhibition of nitric oxide production.

Glucocorticoids (GC) are known to inhibit mitogen-induced proliferation of T cells. In this study we show two experimental situations where the addition of GC increases lymphocyte proliferation. It has been reported by different authors that rat spleen (SPL) cells proliferate poorly after concanavalin A (Con A) activation. These poor responses have been related to the suppressor activity of macrophages. Similarly, it is known that T-cell proliferation is depressed in the presence of an excess of macrophages in the culture. Here we show that in both experimental situations, the inclusion of dexamethasone (DEX), a synthetic glucocorticoid, in the culture medium enhances the Con A-stimulated proliferation. We provide evidence that this effect is a consequence of the inhibition of nitric oxide (NO) synthesis by the hormone. Furthermore, we also demonstrate that rat SPL cells are inefficient antigen-presenting cells (APC) because of their spontaneous high production of NO. Taken together our results suggest that the effects of GC on T-cell activation may be to promote or inhibit proliferation depending on the level of endogenous NO synthesis. The possible significance of these results is briefly discussed.     

Magnolol suppresses NF-kappaB activation and NF-kappaB regulated gene expression through inhibition of IkappaB kinase activation

The mis-regulation of nuclear factor-kappa B (NF-kappaB) signal pathway is involved in a variety of inflammatory diseases that leds to the production of inflammatory mediators. Our studies using human U937 promonocytes cells suggested that magnolol, a low molecular weight lignan isolated from the medicinal plant Magnolia officinalis, differentially down-regulated the pharmacologically induced expression of NF-kappaB-regulated inflammatory gene products MMP-9, IL-8, MCP-1, MIP-1alpha, TNF-alpha. Pre-treatment of magnolol blocked TNF-alpha-induced NF-kappaB activation in different cell types as evidenced by EMSA. Magnolol did not directly affect the binding of p65/p50 heterodimer to DNA. Immunoblot analysis demonstrated that magnolol inhibited the TNF-alpha-stimulated phosphorylation and degradation of the cytosolic NF-kappaB inhibitor IkappaBalpha and the effects were dose-dependent. Mechanistically, a non-radioactive IkappaB kinases (IKK) assay using immunoprecipitated IKKs protein demonstrated that magnolol inhibited both intrinsic and TNF-alpha-stimulated IKK activity, thus suggesting a critical role of magnolol in abrogating the phosphorylation and degradation of IkappaBalpha. The involvement of IKK was further verified in a HeLa cell NF-kappaB-dependent luciferase reporter system. In this system magnolol suppressed luciferase expression stimulated by TNF-alpha and by the transient transfection and expression of NIK (NF-kappaB-inducing kinase), wild type IKKbeta, constitutively active IKKalpha and IKKbeta, or the p65 subunit. Magnolol was also found to inhibit the nuclear translocation and phosphorylation of p65 subunit of NF-kappaB. In line with the observation that NF-kappaB activation may up-regulate anti-apoptotic genes, it was shown in U937 cells that magnolol enhanced TNF-alpha-induced apoptotic cell death. Our results suggest that magnolol or its derivatives may have potential anti-inflammatory actions through IKK inactivation.