The Role And Mechanism Of HMGB1 Involved In Acute Rejection Of Liver Transplantation In Rats

Objectives:Immune rejection is inevitable in organ transplantation.Acute cellular rejection accounts for 26%-40%of the immune rejection after liver transplantation,which seriously threatens the survival and function of donor liver.Induction and maintenance of graft immune tolerance is the key to liver transplantation.Monocyte-derived dendritic cells act as antigen presenting cells to activate CD4~+T cells and play an important role in maintaining immune balance.Although high-mobility group box 1has been proved to be a key factor of ischemia-reperfusion injury,the specific role and mechanism of HMGB1 in immune rejection after liver transplantation have not been clarified.In this study,a stable rat liver transplantation acute rejection model was established,and DCs from bone marrow mononuclear source was induced and cultured in vitro to study the role of HMGB1 in rat liver transplantation acute rejection and related mechanisms,so as to induce and maintain long-term immune tolerance after liver transplantation to improve the quality and survival of patients.Method:1)The construction of a rat model of acute rejection after orthotopic liver transplantation:using improved Kamada’s"double cuff method"to establish the stable MHCⅡmismatch of orthotopic liver transplantation model,allograft transplantation group,Lewis rats as donors,Brown Norway rats as the receptors,the same gene transplantation group with the donor genome receptor adopts BN rats.Tissue specimen of liver were collected 5 and 10 days after the operation of the donor,and a stable transplantation model was screened by liver histopathological biopsy and immunohistochemistry.2)Expression of HMGB1 in rat liver transplantation acute rejection model:immunohistochemistry(IHC)was used to detect the expression of HMGB1 protein and the distribution of CD86~+DC cells in the grafts.ELISA was used to detect the expression of HMGB1 protein and the secretion of inflammatory factors(IL-6、TNF-α、IFN-γ)in serum.Real-time quantitative reverse transcription polymerase chain reaction(Q-PCR)was used to detect the expression of HMGB1 in grafts.3)In vitro induction and co-culture with HMGB1 Mo-DCs:flow cytometry was used to detect the maturation of Mo-DCs in vitro induction and co-culture with HMGB1.Flow cytometry combined with CFSE was used to detect the effect of Mo-DCs on the proliferation of CD4~+T lymphocytes.Flow cytometry was used to detect the expression of Th17/Treg-related characteristic cytokines in CD4~+T lymphocytes.Results:1)The model of orthotopic liver transplantation in rats with acute rejection,reap the same different genome liver tissue specimen is the same with the genome and the control group did not transplantation on histopathologic morphology characterized by typical acute rejection:collect abbacy mixed inflammatory cells infiltration,bile duct epithelial cells,inflammatory damage and veins lining under inflammatory cells invasion.In addition,immunohistochemistry showed strong positive CD3~+T lymphocytes in the allogeneic portal region.2)ELISA results showed that the levels of various inflammatory factors IL-6、TNF-α、IFN-γin the serum samples of the allogeneic transplantation group at 5 and 10days after the operation were higher than those of the same genome(P<0.05),and the expression levels of IL-6 and TNF-αwere higher than those of the same genome at 5days after the operation(P<0.05).The expression of HMGB1 protein and m RNA in homologous genome was higher than that in homologous genome(P<0.05).The immunohistochemical results of CD86 protein in the graft showed that the expression of homologous genome was significantly higher than that of homologous genome.3)Source of rat bone marrow dendritic cells under the stimulus of HMGB1protein were induced to mature,flow cytometry showed that dendritic cell surface markers CD80,CD86 and MHCⅡwere higher than the negative control group.CFSE results showed that the proliferation of CD4~+T lymphocytes was induced by HMGB1in mixed lymphocyte culture.In addition,intracellular cytokine detection showed that the secretion of IL-17 and IFN-γincreased in the HMGB1 stimulation group compared with the PBS group.Conclusions:1)A stable rat model of acute rejection of liver transplantation have been successfully constructed by Lewis rats and BN rats.2)HMGB1 is involved in the acute rejection of rat liver transplantation,which may promote the recruitment of Mo-DCs and T cells in the donor liver,and promote the maturation of Mo-DCs to promote donor-specific Th17 and Th1immune responses.