Function And Mechanism Of GHSR In Acute Transplant Rejection Of Allograft

Backgroud:For many solid organ diseases and end-stage tumors, hematological system diseases, organ transplantation and hematopoietic stem cell transplantation has become an important means of treatment. With the invention and widespread use of immunosuppressive agents, most transplant rejection has been effectively controlled, survival of graft has been significantly improved. But there is still a considerable number of transplant rejection controlled ineffective, leading to graft disfunction or failure, those severe may even lead to a second transplant or death. Therefore, the study of transplant rejection is still in full swing.Acute transplant rejection is the most common type of rejection in allogeneic transplantation, usually happens in a few days to two weeks after transplantation, about 80-90% of rejection occur within one month after surgery. There are also a number of transplant recipients with relapse for a long period of time. An accepted fact in transplant rejection is T cell immune responses play an important role in the world. Wherein, in acute rejection, CD4 + T cell-mediated delayed-type hypersensitivity is a major mechanism of injury. T cells can identify the alloantigens by direct and indirect recognition. The passenger leukocytes in graft(primarily are mature DC and macrophages and other APC) can move into the circulation and lymph tissue, present the antigens to T cells of host in peripheral lymphoid tissues when contacting with the T cell of host, leading to transplant rejection(direct identification). Meanwhile, exfoliated cells or MHC antigens in graft may also be presented to T cells after being processed by APC of host(indirect identification).The growth hormone secretagogue receptor(GHSR), also known as the receptor of ghrelin, was first identified as the target of the growth hormone secretagogue(GHS), and then was cloned in human pituitary and hypothalamus. Zigman JM et al found that GHSR were widely expressed in the central and peripheral tissues. Its endogenous ligand is ghrelin. GHSR is a heterotrimeric G protein-coupled receptor(GPCR) which has two subtype : 1a and 1b, while GHSR-1a is identified as active form of GHSR-1a. GHSR-1a contains 366 amino acids, and its molecular mass of about 41 k Da. As a member of GPCR, GHSR-1a contains the typical seven transmembrane α-helix hydrophobic domains connected by three loops intracellular and extracellular.GHSR has a various of biological effects, including:(1) stimulating the release of various hormones such as growth hormone(GH), adrenocorticotrophic hormone(ACTH), cortisol and so on;(2)regulating food intake and energy metabolism;(3) Affecting glucose and lipid metabolism;(4) Regulating gastrointestinal motility and secretion and pancreatic function;(5)regulating cell proliferation and survival;(6) playing an important role in attenuation of proinflammatory cascades and regulation of the aging and gastrointestinal homeostasis;(7) protecting the nervous and cardiovascular systems.However, the relationship between GHSR and transplantation immunology has been poorly studied. We intend to use mouse cervical heterotopic heart transplantation model to investigate its function and mechanism in transplant rejection.Part I: mouse cervical heterotopic heart transplantation model and the function of GHSR in actue transplant rejectionObjective: To construct mouse cervical heterotopic heart transplantation model and study the function of GHSR in actue transplant rejection.Methods: 1)、Male Balb/c mice aged 6 weeks were chosen as donor, male GHSR-/-(experimental group) and C57BL/6(control group)aged 8 weeks were chosen as recipient. They underwent cervical heterotopic heart transplantation. 2)、Observed Graft survival time. 3)、Took pathological examination(HE staining) at the fifth day after the sugury. 4)、Detected the variation of the expression of m RNA of inflammatory cytokines IL-1, 6, 17, TNF-a in graft by Real-time PCR. 5)、Analyzed the variation of T cell subsets in spleen and lymph nodes in recipient mice by FCM.Result: We confirmed that GHSR defect could significantly prolong allograft survival time and alleviate the graft rejection after transplantation in pathology. GHSR defect alsocould reduce the expression of inflammatory cytokines in the graft and reduce the proportion of CD4 + T cells, increase the proportion of Treg cells.Conclusion: GHSR defect might reduce acute transplant rejection, playing a protective role.Part Ⅱ:The study of mechanism of GHSR in acute transplant rejectionObjective: To study the mechanism of GHSR in acute transplant rejection.Meyhods: 1)、Analized infiltrating cells in graft by immunohistochemical. 2)、 Detected the variation of the expression of m RNA of cell-surface molecules in graft by RT-PCR. 3)、Detected T cell proliferation capacity by MLR.4)、Assay ZAP-70, IKKβ, p65 protein phosphorylation levels by western blot. 5)、Detected the changes of NF-AT, AP-1 and Foxp3 protein by western blot.Result: Immunohistochemistry showed infiltrating cells were mainly T cell, but it was less than control group in quantities; GHSR defect could reduce T cell proliferation ability and affect NF-κB pathway; Due to the increase of NF-AT and AP-1, the expression of Foxp3 in GHSR-/- mice was higher than that in control group.Conclusion: These results indicate that GHSR play an important role in transplant rejection by affecting T cell proliferation、differentiation、activation and other functions.