Mechanism Of All-trans Retinoic Acid-induced Immune Tolerance In Corneal Transplantation Via Autophagy/NLRP3 Pathway

Background:When there is inflammation and vasculogenic changes in the bed,the original "immune amnesty" mechanism of the cornea is destroyed.At this time,the corneal transplantation is called high-risk corneal transplantation.For high-risk patients,the incidence of immune rejection after transplantation is significantly higher than that of low-risk patients,which is still the greatest threat to the success rate of corneal transplantation.Traditional immunosuppressants have some side effects,such as short duration,susceptibility to infection and metabolic disorder,which can not be ignored.It is necessary to investigate the mechanism of corneal allograft rejection and develop new therapeutic methods.Corneal allograft rejection is a complex immune response in which different types of immune cells are involved in different parts,and recent studies have shown that,after transplantation,the immune response can develop either in the direction of Cell-mediated immunity tolerance or in the direction of immune rejection mediated by T helper cells,and the fate of corneal grafts,is determined by the predisposition between the two reactions.It also provides a new direction and thought for us to explore the mechanism of corneal transplantation rejection.This study investigated the role of T helper cell 17(Th17)/Regulatory T lymphocytes(Treg)balance in rat corneal allograft rejection,to investigate the role of all-trans retinoic acid in corneal transplantation and to explore its molecular mechanism of regulating Th17/Treg balance through autophagy NLRP3 inflammatory corpuscle pathway.Methods:The rat penetrating keratoplasty model was established with SD rat as donor and Wistar rat as recipient.The rats were randomly divided into control group C,Autotransplantation Group I,Allotransplantation Group A+R+MA,group C,Group I and Group A were given intraperitoneal injection of DMSO blank solvent.Group R was given intraperitoneal injection of all-trans-retinoic acid(10mg·Kg1·d-1),in group MA,all-trans-retinoic acid and autophagy inhibitor(3MA)(10mg·Kg-1·d-1)were injected intraperitoneally,14 days of continuous medication.According to the rejection scoring system,the graft rejection of each group was evaluated and recorded,and the average survival time and graft survival rate were compared.On the 12th day after operation,3 corneas of each group were histopathologically sectioned,and the ratio of Treg to Th17 cells in peripheral blood was measured by flow cytometry in 5 rats of C/I/A/R Group,the expression of IL17A,IL-6,TGF-β and IL-10 in peripheral blood plasma was detected by ELISA,the mRNA expression of TGF-β,IL-10,Foxp3,ROR-γt,IL-17A,IL-6,NLRP3,IL-1βand P62 were detected by fluorescence quantitative PCR.And ATRA treatment after anti-CD3,Anti-cd28 and Transforming growth factor-β activation of initial CD4+T cells,intracellular expression of transcription factors Foxp3 and interleukin IL-17A in activated CD4+T cells was assessed by Flow cytometry.Results:The mean graft survival time was 26.9±1.87 days in the all-transretinoic acid group and 11.5± 1.70 days in Group A,respectively,there was a significant difference between the two gro(p<0.01).The survival rate of autograft group was 100%.The mean survival time of corneal grafts in the all-trans-retinoic acid and autophagy group was 9.8±1.54 days,which was significantly different from that in the all-trans-retinoic acid group(p<0.01).Histopathologic examination showed a lot of lymphocyte infiltration in Group A,normal corneal structure in Group I and R,no obvious cell infiltration in Group A,edema and inflammatory cell infiltration in group MA.The results of flow cytometry showed that the downregulation of the ratio of Treg cells in group A was significantly different from that in Group R and a,and the expression of IL-17A and IL-6 protein in Group A was significantly higher than that in Group R,in RA Group,the expression of IL-10 and TGF-β in peripheral blood was significantly increased.RT-qPCR results showed that the expression of NLRP3,IL-1β,P62 decreased in all-trans-retinoic acid R group,while the expression of NLRP3,IL-1β,P62 increased in Group A,and the mRNA expression of NLRP3,IL-1β,P62 increased in MA Group,the mRNA expression of Foxp3,IL-10 and TGF-β,which are specific transcription factors of regulatory T cells,were significantly increased in R group and decreased in A and MA group.The mRNA expression of the Th17 cell specific transcription factor ROR-γt and the related functional protein IL-17A was also decreased in Group R,but increased in Group A and group MA.Conclusion:It was preliminarily proved that intraperitoneal injection of alltrans-retinoic acid could induce autophagy,inhibit the expression of inflammatory bodies,to promote the differentiation of Treg cells and inhibit the expression of Th17 cells,thus delay the rejection of corneal transplantation.