Con A-induced Liver Injury In Mice Research Articles

The protective effect of cannabinoid type II receptor agonist AM1241 on ConA-induced liver injury in mice via mitogen-activated protein kinase signalling pathway.

IntroductionThe endocannabinoid system plays an important role in regulating the immune responses in inflammation. At present, there are no good clinical drugs for many immune liver diseases.MethodsWe explored the protective effect of the cannabinoid type II (CB2) receptor agonist AM1241 on the liver of mice with acute liver injury caused by concanavalin from the perspective of inflammation and immunity. Pathological evaluation in hepatic tissue was examined by haematoxylin and eosin (HE) staining and the levels of biochemical parameters in the serum were measured by automatic biochemical analysis. The content of inflammatory factors was measured by enzyme-linked immunosorbent assay and real-time quantitative reverse transcription polymerase chain reaction (real-time PCR). The liver apoptosis-related proteins were observed by immunohistochemistry. The expression of liver injury-related proteins was analysed by Western blot. Immune cells were isolated from the liver of mice and studied in vitro.ResultsReduced levels of alanine transaminase and aspartate transaminase were observed in ConA-induced liver injury mice treated with AM1241, together with attenuated liver damage evidenced by H&E staining. Moreover, AM1241 inhibited the protein and gene expression levels of TNF-α, IL-6 and IFN-γ in the livers of mice. The phosphorylation levels of p38, JNK, ERK1/2, P65 and cAMP response element-binding protein (CREB) in the mouse were significantly reduced in AM1241 pretreatment, while the level of p-JNK increased. In addition, the P/T-P65 and P/T-CREB of the AM1241 pretreatment group were significantly reduced. The results of immunohistochemistry measurement are consistent with those of Western blotting. The CB2-mediated effect is through macrophage-like Kupffer cells.ConclusionOur study suggests that the ConA-induced liver injury model in mice is protected by CB2 agonist AM1241 by modulation of CB2 receptor-rich immune cells, for example, Kupffer cells. Reduced inflammatory responses regulate apoptosis/cell death in the liver particularly hepatocytes and other parenchymal cells.

Hepatocyte-Derived Igκ Exerts a Protective Effect against ConA-Induced Acute Liver Injury.

Immunoglobulin (Igκ) has been reported to be expressed in sorted liver epithelial cells of μMT mice, and the sequence characteristics of hepatocyte-derived Igκ were different from those of classical B-cell-derived Igκ. However, the physiological function of hepatocyte-derived Igκ is still unclear. The expression of Igκ was firstly identified in primary hepatocytes and normal liver cell line (NCTC1469), and hepatocyte-derived Igκ expression was elevated and displayed unique localization in hepatocytes of concanavalin A (ConA)-induced hepatitis model. Moreover, Igκ knockout mice were more sensitive to ConA-induced hepatitis and had higher serum aspartate aminotransferase (AST) levels, more severe histological injury and a greater number of terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL)-positive cells as compared with littermate controls. Furthermore, knockdown of Igκ in primary hepatocytes and NCTC1469 cells led to accelerated activation of the mitochondrial death pathway and caspase-3 cleavage in vitro, which might be related to inhibition of NF-κB signaling pathway and activation of JNK via the cytoskeleton dynamics. Taken together, these results indicate that hepatocyte-derived Igκ mediates cellular resistance to ConA-induced liver injury by inhibiting activation of caspase-3 and the mitochondrial death pathway, suggesting that Igκ plays an important role in hepatocyte survival and exerts a protective effect against ConA-induced liver injury in mice.

Inhibition of microRNA-155 attenuates concanavalin-A-induced autoimmune hepatitis by regulating Treg/Th17 cell differentiation.

Autoimmune hepatitis (AIH) is a chronic progressive autoimmune disease characterized by hepatic inflammation. This study aimed to investigate the effect of antagomir-155 on concanavalin A (ConA)-induced AIH, and its possible mechanisms. According to the results, the expression of miR-155 was raised in liver tissues after 48 h exposure to ConA. Treatment with antagomir-155 attenuated ConA-induced liver injury in mice by reducing serum alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase levels. In addition, antagomir-155 significantly alleviated the differentiation of Treg/Th17 cells in the livers of AIH mice, and suppressed Th17-cells-mediated production of pro-inflammatory cytokines IL-17A, IL-23, but not Treg-cells-mediated production of anti-inflammatory cytokine IL-10. Finally, the beneficial effect of antagomir-155 on ConA-induced AIH was abolished by administration of recombinant IL-17A. Our data demonstrated that antagomir-155 treatment could prevent AIH via regulating the differentiation of Treg and Th17 cells, suggesting that microRNA-155 may be an intriguing therapeutic target of AIH.

Bone Marrow-Derived Mesenchymal Stem Cells Overexpressing Akt1 Protect Against ConA-Induced Liver Injury in Mice

Liver injury associated with veno-occlusive disease and graft-versus-host disease (GVHD) is a frequent and severe complication of hematopoietic stem cell transplantation, and remains an important cause of transplant-related mortality. Bone marrow derived mesenchymal stem cells (MSCs) have been evaluated for the prevention and treatment of refractory GVHD. However, poor cell viability has limited the therapeutic capacity of mesenchymal stromal cell therapy in vivo. In this study, we genetically engineered C57BL/6 mouse bone marrow MSCs using ex vivo retroviral transduction to overexpress Akt1, a serine threonine kinase and pro-survival signal protein, and tested the hypothesis that Akt1-expressing MSCs (Akt1-MSCs) are more resistant to apoptosis and can ameliorate acute liver injury induced by concanavalin A (ConA) in BALB/c mice. Cell proliferation and apoptosis analyses showed that, under both regular culture and high concentration IFN-γ (100 ng/mL) stimulation conditions, Akt1-GFP-MSCs had proliferation and survival (anti-apoptotic) advantages with down-regulated apoptosis pathways, compared to control GFP-MSCs. Twenty-four hours after receiving lethal dose of ConA (40 mg/kg, intravenous) (N=10 each group), no mouse survived, with or without 1x106 Akt1-MSCs or GFP-MSCs administration (intravenous); however, 3 and 1 survived in the 5×106 Akt1-MSCs group and 5×106 GFP-MSCs groups, respectively. In subsequent sub-lethal dose ConA (20 mg/kg) experiments, compared to GFP-MSCs, mice received Akt1-MSCs administration had significantly lower serum AST, ALT, TNF-α and IFN-γ levels and less histopathological abnormalities. In addition, Akt1-MSCs treated mice had significantly higher serum concentrations of IL-10, vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF). In vivo imaging showed that, hepatic fluorescence signal in sub-lethal ConA+Akt1-MSCs group was significantly stronger than ConA+GFP-MSCs group on day 0, and persisted up to 14 days, whereas the signal in ConA+GFP-MSCs, Akt1-MSCs and GFP-MSCs groups was negligible on both day 7 and day 14. Thus, bone marrow derived MSCs genetically enhanced with Akt1 had survival advantage in vitro and in vivo, and have the potential to be a potent therapy for prevention and amelioration of GVHD-associated liver impairment. Further translational pre-clinical studies are ongoing to further determine the efficacy, dosage and timing of administration of Akt1-MSCs in animal models. DisclosuresNo relevant conflicts of interest to declare.

Differential Roles of JNK in ConA/GalN and ConA-Induced Liver Injury in Mice

Tumor necrosis factor-alpha-mediated liver injury can be induced by several different means; however, the signaling events and mechanisms of cell death are likely different. We investigated the mechanism of both apoptotic and necrotic hepatocyte cell death as well as the role of c-Jun NH2-terminal kinase (JNK) in the ConA and ConA/D-galactosamine (GalN) models of murine liver injury. ConA alone induced primarily necrotic cell death with no caspase activation, whereas ConA/GalN induced apoptosis in addition to necrotic cell death. The bi-modal death pattern in the ConA/GalN model was confirmed by the use of transgenic mice expressing a dominant-negative form of Fas-associated death domain in which the mice were resistant to apoptotic but not necrotic cell death. JNK1 and, more significantly, JNK2 participated in the induction of hepatocyte apoptosis in response to ConA/GalN. Deletion of JNK led to the stabilization of FLIP L, reduced caspase-8 activation, decreased Bid cleavage, and inhibition of the mitochondrial apoptosis pathway. In contrast, JNK did not participate in necrotic death induced by ConA either alone or in combination with GalN. As such, JNK-deficient mice remained susceptible to necrotic liver injury in both model systems. Thus, ConA and ConA/GalN mouse models induce liver injury with different mechanisms of cell death, and JNK contributes to apoptotic but not necrotic cell death. These findings further elucidate the specific pathways involved in tumor necrosis factor-alpha-mediated liver injury.