| With the development and application of various immunosuppressants,the incidence of rejection after transplantation decreased gradually.The immunosuppressive agents widely used in clinic are mainly T cell inhibitor,because the cellular rejection has been considered as the direct cause of graft rejection.However,clinical data shows that rejection occurs even after adequate T cell immunosuppressive therapy.Until then,it was found that donor-specific antibodies mediated the rejection and had an important effect on the loss of graft function and failure of transplantation.More and more studies have found that antibody-mediated rejection plays an important role in acute and chronic rejection after transplantation,which affects the survival rate and quality of life of recipients.There are many antibodies leading to antibody-mediated rejection,which mainly referred to the anti-donor HLA antibodies.However,increasing studies have found that "non-HLA antibodies" also play an important role in the damage of transplanted organs.Non-HLA antibodies generally refer to self-reactive antibodies and alloreactive antibodies in transplantation.Inflammatory,increased antigen expression,and the formation of new antigens through posttranslational modification all contribute to the formation of non-HLA antibodies that subsequently damage the graft.Ischemia-reperfusion(IR)injury is an important factor affecting the morbidity and mortality of recipients after organ transplantation,because ischemia-reperfusion injury is closely related to perioperative complications,including post-reperfusion Syndrome,graft delay,primary nonfunction,and acute rejection.Recently,it has been found that cells produce new antigen epitopes in the process of ischemia-reperfusion injury,which leads to acquired immunity.The harm of the ischemic injury related neo-epitopes may not be limited to ischemia-reperfusion injury itself,but may induce or aggravate the humoral rejection after organ transplantation.Part I.Generation of the ischemic injury related neo-epitopes.Renal ischemia-reperfusion injury model was performed in this part of the study(Single IR: the left kidney underwent sham surgery,and the right kidney underwent ischemia 100 days later;Double IR: left kidney underwent ischemia,followed by right kidney ischemia 100 days later).First of all,it was found that the right renal tissue after double IR was more serious than that of the right renal tissue after single IR,which indicated that the initial left renal ischemia had an effect on the right ischemic kidney later.Serum ANAX4 antibody levels increased significantly after double IR,suggesting that ANAX4 antibody may be associated with renal injury.The frequency of Tfh cells,Germinal Center B cells and memory B cells were increased after double IR,which indicated that double IR injury induced humoral immunity.The results of immunofluorescence showed that there was no significant difference in the expression of ANAX4 in each group,but the level of ANAX4 antibody in serum increased after double IR,which may be related to the activation of memory B cells to differentiate into antibody-producing plasma cells.FK506,a T cell immunosuppressant,was used during left kidney ischemia.The results showed that FK506 not only reduced the frequency of Tfh cells,Germinal Center B cells and memory B cells in spleen,it also reduced the level of ANAX4 antibody after double IR and improved the right renal function,demonstrating that the new epitope activates humoral immunity and does harm to the body.In addition,immunofluorescence results confirmed that C4 d and Ig G could infiltrate the ischemic kidney tissue,indicating that the complement system was also involved in humoral immune response.Part II.Effect and mechanism of ischemic injury-relatedneoepitopes in organ transplantationIn this part,we established an animal model of xenotransplantation after renal ischemia in mice(renal IR + transplantation),and FK506 were used during renal IR and/or renal transplantation to observe its effect on transplantation.The mice were randomly divided as follows: sham,transplantation + FK506,IR + transplantation,IR +transplantation + FK506,and IR + FK506+ transplantation + FK506.The results showed that the renal tissue damage was the most serious and the serum level of ANAX4 antibody was the highest in the IR + transplantation group.Compared with those in IR+transplantation +FK506 group,transplantation +FK506 group showed less pathological damage and lower level of ANAX4 antibody,which suggested that renal ischemia had adverse effects on the transplanted renal tissue later,and may be related to the generation of new epistopes.However,in the immunofluorescence of the transplanted kidney,there was no significant difference in the antigen epitope of ANAX4 among the groups,indicating that the ANAX4 antibody level was related to the humoral immune response after transplantation.We further tested the frequency of Tfh cells,Germinal Center B cells and memory B cells in spleen and lymph nodes,the results showed the frequency of three kinds of cells in the IR + transplantation + FK506 mice is higher in the transplantation +FK506 group,these results show that early renal ischemic injury are more likely to cause humoral immunity after transplantation,thus to transplant kidney damage.In the IR +FK506+ transplantation+ FK506 group,C4 d and Ig G deposition were decreased compared with those in the IR+ transplantation +FK506 group,and the proportion of the three kinds of immune cells was also decreased.Humoral immune activity after transplantation may indicate the antibody mediated rejection reaction,then we further tested donor specific antibody level,as expected,the renal ischemia can lead to the DSA-Ig G rise after renal transplantation which may cause renal allograft rejection.Part III.Prevention and treatment of humoral rejection aftertransplantation induced by neoepitopeIn the second part,it was confirmed that the ischemic injury related neo-epitopes induced humoral rejection after transplantation.In this part,we used cobra venom factor,CTLA4-Ig and/or FTY720 for the treatment of humoral rejection after transplantation.Cobra venom factor can reduce the pathological damage of transplanted kidney,the levels of ANAX4 antibody and DSA-Ig G in serum,improve renal function,especially in inhibiting complement deposition,but it has no effect on immune cells.In addition to improving the function and damage of transplanted kidney,CTLA4-Ig can also inhibit humoral rejection by inhibiting the activation and proliferation of Tfh cells,germinal center B cells and memory B cells,which were further enhanced when combined with FTY720.The combination of CTLA4-Ig and FTY720 is an effective method for the prevention and treatment of humoral rejection induced by ischemic injury associated epitopes after transplantation.Part ?.Effect and mechanism of melatonin on renal ischemiareperfusion injuryIn this part,renal IR was induced in C57BL/6 mice after melatonin administration.The experiment proved that melatonin preconditioning had a protective effect on the kidneys.Mice pretreated with melatonin had less tissue damage and no significant increase in serum creatinine and urea nitrogen levels even after experiencing ischemia reperfusion.The protective effect of melatonin was further confirmed by reducing oxidative stress,which was manifested as decreased lipid peroxidation and enhanced antioxidant capacity.Subsequent studies showed that melatonin preconditioning significantly limited the production of pro-inflammatory cytokines and infiltration of neutrophils and macrophages during renal ischemia injury.Further studies have shown that melatonin plays a protective role by down-regulating TLR4/My D88 to stimulate autophagy.In addition,down-regulation of the MEK/ERK/m TORC1 pathway is also necessary for melatonin-mediated autophagy.Collectively,our results provide novel evidence that antecedent melatonin treatment provides protection for the kidney against IR injury by enhancing autophagy,as regulated by the TLR4/My D88/MEK/ERK/m TORC1 signaling pathway. |
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