Establishing And Mechanism Study Of A New Autoimmune Hepatitits-Like (AIH-Like) Mouse Model

Autoimmune hepatitis（AIH）is a serious liver injury disease which is induced by the disorder of immune system and attack of hepatocytes direct or indirect by immune cells.However,the pathogenesis of AIH is not fully understood till now.Immunosuppressive treatment is the major therapy of AIH at present.When the condition worsened,liver transplant is the only choice.80%patients can achieve remission by immunosuppressive treatment,but always have serious side effects and easy to relapse after medicine withdrawal.Other 15%～20%patients are resistant to immunosuppressants and the therapeutic outcome is hard to control.The existing animal models are sensitive to immunosuppressive treatment.In this thesis,we developed a new AIH-like mouse model which was resistant to immunosuppressants and maybe meanful for the pathogenesis study and drug screening of AIH.The main research contents are as follows:1.AAGL-induced hepatitis was a new AIH-like mouse model which was different from the classical ConA modelAn acute hepatitis was induced by i.v.injection of agrocybe aegerita galectin（AAGL,also called AAL）.AAGL-induced hepatitis was in a dose and time-dependent manner.AAGL had no lethal effect on primary hepatocytes and could not induce hepatitis in nude mice,which indicated that AAGL-induced hepatitis was immune-related.Immunosuppressants prednisolone or cyclosporine A（Cs A）could inhibit ConA-induced hepatitis,but couldn’t ameliorate AAGL-induced hepatitis,which indicated that AAGL-induced hepatitis was a new AIH-like mouse model.2.Mutiple cells participated in AAGL-induced hepatitis,but there was no single dominant cell subgroupFlow cytometry analysis of lymphocytes in mouse liver revealed that T cells were the first to infiltrate to liver after AAGL injection and then NKT,NK cells also accumulated in liver.Meanwhile,activated lymphocytes（IFN-γ⁺cells）also increased.The pretreatment of anti-CD4 or anti-CD8 antibody couldn’t ameliorate but even worsened AAGL-induced hepatitis.AAGL could still induce hepatitis in CD1d^-/-mice.Anti-ASGM1 antibody which inhibited NK cells could slightly alleviate but couldn’t effectively inhibit AAGL-induced hepatitis.All these results indicated that multiple cells（Teff,Tregs,NKT,NK）were involved in AAGL-induced hepatitis,but the single main dominant cell subgroup hadn’t been indentified.The pathogenesis of AAGL-induced hepatitis was more complex than ConA model.3.Cytokines IL-1βand IL-10 played important roles in AAGL-induced hepatitisWe used the high-throughput sequencing technology to detect the genes expression profiles of liver and found that the expression of IL-1βand IL-10 were much higher in AAGL than ConA group.The pretreatment of anti-IL-1βantibody could alleviate AAGL-induced hepatitis.Immunohistochemistry revealed that the pretreatment of anti-IL-1βantibody dramatically decreased the infiltration of T/NKT cells in liver after AAGL treatment,which indicated that IL-1βpromoted liver injury through recruiting T/NKT cells into liver.Flow cytometry results revealed that the percentage of IL-10⁺cells in liver and spleen lymphocytes increased after AAGL treatment.Western bolt showed that the expression of Foxp3 increased both in liver and spleen,which indicated Tregs were activated and participated in inhibition of AAGL-induced hepatitis.4.Glatiramer acetate（GA）could ameliorate AAGL-induced hepatitisThe pretreatment of GA could ameliorate AAGL-induced hepatitis.Flow cytometry results showed that GA could increase the percentage of Tregs in mouse spleen after AAGL administration.q PCR results revealed that GA could also significantly increase the expression of IL-1Ra in mouse liver after AAGL treatment and the infiltration of T/NKT cells decreased,which indicated that GA might alleviate AAGL-induced hepatitis by increasing the ratio of Tregs or inducing the expression of IL-1Ra which could inhibit the activity of IL-1β.All these data demonstrated that the increase of intrahepatic Tregs may be the future therapy strategy of AIH patients who are resistant to immunosuppressive treatment and GA may be the candidate medicine for the therapy of these patients.5.Identification of glycoprotein receptors on lymphocytes membrane bound by AAGL/ConAWhen the CRD of AAGL was blocked by lactose,it couldn’t induce hepatitis.Recombinant AAGL could still induce hepatitis,but the mutant AAGL-63 which completely lost carbohydrate recognition activity couldn’t induce hepatitis,which indicated that AAGL-induced hepatitis was related to its carbohydrate recognition activity.To indentify the glycoprotein receptors on lymphocytes membrane bound by AAGL/ConA,we isolated the lymphocytes membrane proteins which were incubated with AAGL/ConA and obtained the interacting proteins which were detected by MS.These proteins were analyzed with GO annotation and the highly specific-binding proteins were analyzed with PANTHER pathway.It was found to be different between AAGL and ConA,which needed to be further confirmed.The membrane receptors identification of AAGL is the basis for the further study of the pathogenesis of AAGL-induced AIH-like.