Rotection Of Spred-2 In The ConA-induced Liver Injury

Background: T cells play central roles in the pathogenesis of liver diseases, including viral hepatitis, autoimmune hepatitis, alcoholic liver disease and fatty liver disease. Although T cell response is crucial in the liver pathology, the underlying regulatory mechanism by cytokine signaling remains unclear. There are two different processes for acute hepatitis B virus (HBV) clearance and liver cell damage, the clearance of HBV is completed before the liver cell injury, and the lack of specific CTL activity could not make HBV replication under control, it could cause a large number of nonspecific inflammatory cells, particularly non-specific CD8+T cells chemotactic cytokines under the action of a large invasion, which cause extensive damage in liver tissue.To address this, studies with animal models of hepatitis are necessary. Concanavalin A(ConA)-induced hepatitis is a well-established animal model of hepatitis and its histological features resemble those of viral-, autoimmune- or druginduced hepatitis in humans. ConA-induced hepatitis, mainly through the rapid activation of polyclonal T cells were mainly CD4+T cells, CD8+T cells and NKT cells to realize their own immune injury. Characterized by the rapid serum alanine aminotransferase levels increased and serum contains large amounts of cytokines, leukocyte infiltration in the liver, liver cell necrosis and apoptosis. In that ConA induced by the excessive activation of the immune system caused by acute liver injury model, As hepatic T cells are rapidly activated following ConA injection and mice lacking T cells were resistant to the hepatotoxicity. Activation of the cells is dependent on Th1 cytokine IFNγand its signaling pathway.Evidence indicates that MAP kinase cascade plays critical roles in the liver innate system during antiviral defense, acute phase response, hepatic injury and regeneration. Suppressor of cytokine signaling(Spred)proteins are intracellular inhibitors of cytokine signaling pathways, mainly MAP kinase cascade. Spred-1 Spred-2 and Spred-3 could inhibit growth factor induced-ERK activated and downregulate Ras-ERK single, Considering the involvement of MAPK cascade in the liver biology, it is reasonable to speculate that Spred proteins may play a role in T-cell-mediated liver injury. In the present study, we have focused on Spred-2 in T cells and investigated the regulatory mechanism behind T-cell-mediated liver injury.We here showed that forced expression of Spred-2 in T cells protected mice from ConA induced liver injury by inhibiting the Th1 responses. Our findings suggest that Spred-2 delivery in T cells may be therapeutic in T-cell-mediated hepatitis.Objective: In the present study, we explored the role of Spred-2 in T cells in concanavalin A(ConA)-induced hepatitis.Methods: Mice were given a single intravenous injection of ConA at 15 mg/kg of body weight otherwise noted. 6h, 12h, 24h after ConA injection, mice were anesthetized, bled and sacrificed. Aliquots of plasma was plunged directly into liquid nitrogen and subsequently stored at-80℃until assay. Livers were perfused with saline, excised, snap frozen in liquid nitrogen, a part of the livers was fixed in 10% formalin, and another part of liver digested with 0.25% collagenase solution. Serum levels of alanine aminotransferase ( ALT) were measured using standardized techniques. For the measurement of caspase-3,-8,-9, colorimetric assay ki(tMBL)for each caspase was used and ELISA for IFNγactivities. Flow cytometry for CD8+T cell, CD4+T cell and NKT cell. Real time PCR for CXCL9/10, CXCR3 receptor, perforin and Fas,TNG-αand IL-17.Results: Liver injury at 12 h after ConA injection in Spred-2KO mice was much severer than that in the WT mice, as evidenced by increased serum levels of ALT and exacerbated injured area relative to the WT mice ( 7476.20±1387.54 IU/L vs 3752.63±803.36 IU/L, P < 0.05). Activities of caspase-3,-8, and-9 were significantly elevated in Spred-2KO liver(63.78±11.90 vs. 17.35±4.48, P < 0.05; 48.21±7.62 vs. 8.89±1.92, P < 0.05; 48.07±7.39 vs. 8.23±2.36, P < 0.05). In Spred-2KO mice, systemic levels of cytokines such as sera IFNγand liver IFNγat 12h after ConA-injection were higher than those in WT mice(1.89±0.18 ng/ml vs 0.81±0.22 ng/ml, P < 0.05; 2.57±0.49 vs. 1.40±0.25, P < 0.05). FACS analyses demonstrated that the numbers of hepatic CD8+T cells in Spred-2KO liver were significantly higher than those in the WT control(8.41±2.13 105/ml vs. 5.15±1.18 105/ml, P < 0.05), CD4 + T cells and NKT cells in mice with no significant difference between WT. The mRNA level of CXCL9/10, CXCR3 receptor, perforin and Fas were also elevated.Conclusions: 1,In the process of acute hepatitis, WT mice reduce the degree of liver cell injury and cell apoptosis, Spred-2 gene may play a protective role in liver cells.2, In the process of acute hepatitis, WT mice may via inhibit Th1 cell responses, reduce inflammation and the level of chemokine liver cells to play a protective role.3, In ConA-induced hepatitis, Spred-2 gene play a protective role and maybe a new therapeutic target of T cells induced liver injury.