The Effect Of Aerobic Glycolysis Pathway On Corneal Allograft Rejection

Objective:To investigate aerobic glycolysis activity during corneal tranplantation rejection,and further evaluate the effect and mechanism of aerobic glycolysis pathway on corneal allograft rejection.Methods:(1)The healthy mice were randomly divided into three groups,including a normal group,a syngeneic penetrating keratoplasty group,and an allogeneic penetrating keratoplasty group.The corneal transparency and immune rejection were observed by slit lamp.The mice were sacrificed around 18 days after transplantation,corneal tissues were isolated under microscope.To investigate aerobic glycolysis activity,Western blot and real-time PCR were performed to detect the expression of Glucose transporter-1(GLUT1),Fructose-2,6-bisphosphatase-3(PFKFB3),and Lactic Dehydrogenase(LDHA).The lactic acid content in corneal tissues was determined by lactic acid test kit.The expression and distribution of LDHA and c-Myc in corneal tissues were analyzed by immunofluorescence staining.(2)The healthy mice were randomly divided into three groups,including a syngeneic penetrating keratoplasty group,an allogeneic penetrating keratoplasty group,and an allogeneic penetrating keratoplasty treated with rapamycin(RAPA)nano-micelle ophthalmic solution.The effect of RAPA on corneal allograft rejection was evaluated by slit lamp.The mice were sacrificed around 18 days after transplantation,corneal tissues were isolated under microscope.The activity of mTOR signaling pathway and the expression of c-Myc in allografts were detected by Western blot.The expression of PFKFB3 and LDHA was analyzed by RT-PCR and Western blot.(3)The healthy mice were randomly divided into two groups,including an allogeneic penetrating keratoplasty group,and an allogeneic penetrating keratoplasty treated with3-PO(3-pyridinyl).-1-4-pyridinyl-2-propen-1-one)subconjunctivally.The effect of 3-PO on corneal allograft rejection was assessed by clinical slit lamp and the survival time was recorded.The corneal tissues were collected around 18 days after transplantation.The levels of lactic acid in cornea were quantified using lactic acid test kit.The transcriptional levels of Interleukin-1?(IL-1?),Tumor necrosis factor-alpha(TNF-?),Interleukin-17A(IL-17A)were analyzed by RT-PCR.The protein levels of IL-1?in corneal tissues were quantified by enzyme-linked immunosorbent assay(ELISA).Hematoxylin-eosin staining(HE)was used to examine the pathological changes of corneal grafts in each group.Results:(1)The results of Western Blot showed the content of PFKFB3 and LDHA in allogeneic corneal transplantation group was significantly increased,when compared with the control group.Compared with control group,the transcriptional levels of PFKFB3,LDHA and GLUT1 in allogeneic group were prouncedly elevated.The level of lactic acid was much higher in allogeneic group than in control groups.These results demonstrated the increased aerobic glycolysis in rejected corneal allografts.Immunofluorescence staining showed that the level of glycolytic-related proteins LDHA were significantly increased in rejected corneal allograft,amd mainly distributed in CD45+inflammatory cells.The expression of transcriptional factor c-Myc,which is regulated by mTOR signaling pathay,was also markedly elevated in rejected corneal allograft,amd mainly distributed in CD45~+inflammatory cells.(2)When compared with the control group without RAPA administration,RAPA treatment could prouncedly alleviated corneal allograft rejection,featured by longer survival time,higher corneal transparency and less neovascularization.Western blot revealed that RAPA treatment significantly reduced phosphorylation of 4E-BP1 and S6,which hinted decreased activity of mTOR signaling.As showed by Western blot and RT-PCR,RAPA treatment not only inhibited the expression of c-Myc,but also lowered the levels of glycolysis-related protein,including PFKFB3,LDHA and GLUT1.These results indicated that mTOR/c-Myc signaling would be involved in the regulation of aerobic glycolysis activity(3)Compared with the control group,the 3-PO administration could significantly reduced corneal allograft rejection,characterized by the longer survival time,transparency and less neovascularization.After treatment with 3-PO,the levels of lactic acid in corneal allografts were significantly decreased,when compared with untreated group,which hinted decreased aerobic glycolysis after 3-PO treatment.The expression levels of inflammatory cytokines IL-1?,TNF-?and IL-17 in corneal allografts of3-PO-treated group were much lower than those in untreated group.HE staining showed less inflammatory infiltration in the corneal stroma of 3-PO treated group than in untreated group.These observations suggested that blockade of aerobic glycolysis could significantly delayed corneal allograft rejection.Conclusion:(1)The rejected corneal allografts showed increased aerobic glycolysis.(2)The mTOR signaling pathway could regulate aerobic glycolysis through the key transcriptional factor c-Myc.(3)Blocking aerobic glycolysis could significantly delayed corneal allograft rejection.