The Role And Mechanism Of Liraglutdie, A Glucagon-like Peptide-1 Receptor Agonist, In The Chronic Rejection Of Murine Heart Transplantation

Part I GLP-1 receptor agonist liraglutide plays a role in chronic rejection and regulates the expression of GLP-1 receptors in graftsObjective: To investigate the role and mechanism of liraglutdie in chronic cardiac rejection,and explore the effect of liraglutide in the reglulation of GLP-1 receptors expression in grafts.Methods: Heterotopic murine cardiac transplantation was performed in a complete major histocompatibility complex class?MHC?-mismatched model?BALB/c into B6?.The recipient mice were subcutaneously administered the vehicle?0.9% saline solution?or liraglutide?300?g-1kg-112h-1?from the day of transplantation.the weight and blood glucose changes of each group mice were observed and recorded.Allografts were harvested at 8 weeks and histologically analyzed.EVG staining and Masson staining were used to analyze the pathogenesis of chronic allograft vasculopathy?CAV?and interstitial fibrosis of each group allografts.The expression and distribution of GLP-1 receptor in each group were analyzed by immunofluorescence and Western Blot.Results: With liraglutide treatment,liraglutide ameliorated CAV?luminal occlusion: liraglutide,32.28±6.28%;vehicle,63.39±7.78%,p < 0.05?and cardiac fibrosis?liraglutide,13.76±1.49%;vehicle,22.82±2.67%,p < 0.05?.Western Blot showed that the expression of glp-1 receptor in the graft was up-regulated compared with the control group?p <0.05?.immunofluorescence analysis of the graft sections by co-labeling the cells with an endothelial marker(CD³¹)and GLP-1R.The results showed that the ratio of double-positive cells(CD³¹+GLP-1R⁺ cells)to all GLP-1R⁺ cells was ⁶0.3% in the vehicle group and ⁷2.4% in the liraglutide group?p > 0.05?respectively,which suggested that the majority of GLP-1Rs was distributed on the vascular endothelium.In addition,the percentage of double-labeled cells(CD³¹+GLP-1R⁺ cells)in all CD31⁺ cells was higher in the liraglutide-treated group in comparison with the vehicle-treated group?liraglutide,48.18±5.96%;vehicle,20.40±3.0%;p < 0.05;Figure 2D?.These data suggest that liraglutide treatment enhance the upregulation of GLP-1R after cardiac transplantation.Conclusion: GLP-1 receptor agonist\liraglutide inhibits the development of chronic rejection,and GLP-1 receptors in graft endothelial cells may play an important role in chronic cardiac rejection.Part ? GLP-1 receptor agonist liraglutide has an effect on infiltration of inflammatory cells in chronic cardiac rejectionObjective: To investigate the effect of liraglutdie on infiltration of inflammatory cells in chronic cardiac rejection.Methods: Heterotopic murine cardiac transplantation was performed in a complete histocompatibility complex class?MHC?-mismatched model?BALB/c into B6?,The recipient mice were subcutaneously administered the vehicle?0.9% saline solution?or liraglutide?300?g-1kg-112h-1?from the day of transplantation.Allografts were harvested at 2 or 8 weeks and histologically analyzed,and inflammatory infiltrates were measured using immunohistochemistry.Flow cytometry was used to analyze the proportion,activation and differentiation of T cell subgroups in each group.Immunohistochemistry was used to analyze the expression of chemokines and adhesion molecules in the allografts at 2 week.In vitro,human coronary artery endothelial cells?HCAECs?induced with TNF-? were used to evaluate the expression of adhesion moleculesResults: The immunohistochemical analysis showed that the percentage of CD4⁺,CD8⁺,CD11b⁺ cells infiltrated in allografts was decreased after the liraglutide treatment at week 2 or 8.Flow cytometry analysis showed that liralgutide had no effect on the proportion,activation and differentiation of spleen T cells.Moreover,the inhibition of MCP-1 and the adhesion molecule VCAM-1 were also detected in the allografts of the liraglutide-treated group at week 2.In addition,TNF-? induced VCAM-1 m RNA expression in HCAECs at 16 h.This effect was significantly inhibited by the addition of liraglutide.Conclusion: Liraglutde attenuated chronic cardiac rejection by reducing the infiltration of inflammatory cells and preventing endothelial dysfunction.Part ? The role and mechanism of GLP-1 receptor agonist liraglutide in End MT in chronic cardiac rejectionObjective: To investigate the role and mechanism of GLP-1 receptor agonist Liraglutide in End MT in chronic cardiac rejection.Methods: Heterotopic murine cardiac transplantation was performed in a complete histocompatibility complex class?MHC?-mismatched model?BALB/c into B6?,The recipient mice were subcutaneously administered the vehicle?0.9% saline solution?or liraglutide?300?g-1kg-112h-1?from the day of transplantation.Allografts were harvested at 8 weeks.immunofluorescence and Western Blot were used to analyze endothelial-to-mesenchymal transition?End MT?in cardiac allografts.In vitro,human coronary artery endothelial cells?HCAECs?challenged with TGF-?1 were used to evaluate the GLP-1R expression and explore liraglutide's effects on End MT and its possible molecular mechanisms using western blotting.Results: Immunofluorescence double-labeling with the cell markers CD³¹ and ?-SMA was used to identify End MT.The quantitation analysis of CD³¹+?-SMA+ cells in all CD³¹+ cells,which was regarded as the ratio of endothelial cells undergoing End MT,indicated a decreased ratio in the liraglutide group?liraglutide,15.80±2.75;vehicle,35.04±6.05,p < 0.05?.Additionally,approximately 37.2%(the percentage of CD³¹+?-SMA+ cells in all ?-SMA+ cells)fibroblasts developed in the process of chronic rejection were endothelial origin.Liraglutide significantly ameliorated this pathological process?liraglutide,21.95±2.21;vehicle,37.25±6.42,p < 0.05?.Moreover,the western blot analysis demonstrated that liraglutide downregulated the expression of ?-SMA and maintained that of the CD³¹ marker.Western blot analysi also indicated that liraglutide directly inhibited the TGF-?1-induced phosphorylation of Smad3 and AKT in a GLP-1R-dependent mannerConclusion: Liraglutide inhibits cardiac allograft End MT in vivo and vitro.