The Effect Of Corneal Graft Senescence After Corneal Transplantation On Allograft Rejection And Its Mechanism

Objective:To investigate the effect and mechanism of corenal cellular senescence on corenal allograft rejection.Methods:1.Cellular senescence analysis during corneal allograft rejection.Penetrating keratoplasty was performed between fully mismatched C57BL/6(donor)and BALB/C(recipient)mice,and the recipient mice were randomly divided into normal group,syngeneic group and allogeneic group.The corneal grafts were collected,and used to evaluate the cellular senescent phenotype.The corneal cellular senescence was examined by SA-?-Gal staining,and the distribution of cellular senescence in corneal grafts was evaluated using frozen sections of corneal grafts.The expression of p16,p21 and other senescence-related proteins in corneal grafts were detected by Western blot.2.The effect of donor cornea with p16 knockout on immune rejection.Corneal transplantation was performed using wild-type or p16 knockout C57BL/6 mice as corneal donors,and BALB/C as corneal receptor.The corneal grafts were examined by slit lamp,and inflammatory infiltration of corneal allografts was examined through immunofluorescence.3.The effect of senescent clearance by Navitoclax(ABT-263)on allograft rejection.The corneal transplantation was operated between fully mismatched C57BL/6(donor)and BALB/C(recipient)mice.The recipient mice were randomly divided into allogeneic group and allogeneic group with ABT-263 treatment.The corneal allografts were examined using slit lamp.Quantitative RT-PCR was employed to determine the expression of pro-inflammatory cytokines(such as interleukin-1?(IL-1?),IL-12,IL-17A,tumor necrosis factor-?(TNF-?)and interferon-?(IFN-?)).Moreover,SA-?-Gal staining and quantitative RT-PCR were employed to assess the effe of ABT-263 on senescent clearance.4.The influence of cellular senescence on antigen-presenting cell(APC)activation.Corneal transplantation was performed,and the recipient mice were randomly separated into allogeneic group and allogeneic group with ABT-263 intervention.The grafts were collected after treatment with ABT-263 on postoperative day 10,and then the expression of pro-inflammatory cytokines(TNF-?,IL-1?)and co-stimulatory molecules(CD80,CD86)in corneal grafts and iris samples were determined to evaluate the effect of cellular senescence on APC activation by quantitative RT-PCRResults:1.Corneal senescence occurred during corneal allograft rejection.Compared with normal group,the corneal grafts both in syngeneic and allogeneic group showed cellular senescence in the stroma and endothelium during corenal allograft rejection,but with more cellular senescence in allogeneic group.Western blot revealed significantly increased protein levels of p16 and p21 in allogeneic group,when compared with normal and syngeneic group.2.Donor corena with p16 knockout significantly alleviated corneal allograft rejection Compared with wild-type group,p16 knockout group showed delayed corneal allograft rejection,characterized by prolonged survival time and reduced CD45~+inflammatory cell infiltration.These results demonstrated that corneal cellular senescence contributed to exacerbated allograft rejection.3.Pharmcologically clearning senescent cell pronouncedly prolonged corneal survival.Using pharmacological approach,we found that clearance of senescent cell by ABT-263significantly alleviated corneal allograft rejection,along with longer survival time and less expression of pro-inflammatory cytokines.Moreover,we also validated that ABT-263 could also siginificantly remove senescent cells in corneal allografts.Collectively,these results indiactaed that senescenct clearance could markedly alleviate allograft rejection.4.Cellular senescence probably contributed to corneal allograft rejection through APC activation.Compared with the allogeneic group,the proinflammatory cytokines and co-stimulatory molecules,which were indicative of APC activation,in corneal allografts and iris tissues were significantly downregulated after treatment with ABT-263 on postoperative day10.These results suggested that cellular senescence exacerbated cornmeal allograft rejection probably through APC activation.Conclusion:1.During corneal allograft rejection,cellular senescence occurred in the stromal cell and endothelial cell of corneal grafts.Blocking cellular senescence or clearing senescent cell delayed corneal allograft rejection.2.Cellular senescence exacerbated corneal allograft rejection probably by activating APC.