Homoharringtonine Attenuates Cardiac Allograft Rejection In Mice And Its Mechanism

Background:Organ transplantation still faces concurrent allograft rejection,drug side effects,dose-limiting allograft toxicities and increased risk of infection and malignancy induced by current immunosuppressants.Homoharringtonine(HHT)is an effective anti-inflammatory,anti-viral and anti-tumor protein synthesis inhibitor that has been applied clinically.Here,we explored the therapeutic effect and mechanism of HHT with a mouse heart transplantation model.Methods:1.HHT-T cell crosstalk was modeled ex vivo to further verify the inhibitory effect of HHT on the activity and proliferation of T cells using CCK-8and flow experiments.2.Naive C57BL/6(B6)mice were used to observe the toxic effects of HHT on the liver,kidney and blood system by blood biochemistry and routine blood test,and the effects of HHT on the tissue structure of the heart,liver,kidney and lung were observed by Hematoxylin-eosin staining.3.A mouse heart transplantation model was constructed,and the potential mechanism of HHT prolonging allograft survival was evaluated by Kaplan-Meier analysis,Hematoxylin-eosin staining,immunostaining,enzyme linked immunosorbent assay and bulk RNA sequencing.4.HHT-T cell crosstalk was modeled ex vivo to further verify the molecular mechanism of HHT-induced Treg differentiation.Results:1.HHT inhibited the activation and proliferation of T cells and promoted their apoptosis ex vivo.2.Treatment of 0.5 mg/kg HHT for 10 days significantly prolonged the median survival time(MST)of the allografts from 7 days to 48 days(P<0.001)without nonimmune toxicity.3.The allografts had long-term survival after continuous HHT treatment for28 days.4.HHT significantly reduced lymphocyte infiltration in the graft,and IFN-γ-secreting CD4~+and CD8~+T cells in the spleen(P<0.01).5.HHT significantly increased the number of Tregs in spleen and lymph nodes(about 20%,P<0.001)and serum interleukin(IL)-10 levels.6.HHT down-regulated the expression of TCR signaling pathway-related genes(CD4,H2-Eb1,TRAT1,and CD74)and up-regulated the expression of IL-10 and TGF-βpathway-related genes and Treg signature genes(CTLA4,Foxp3,CD74,and ICOS).Conclusions:Mechanistically,HHT may promote the differentiation of T helper(Th)cells into Tregs by attenuating TCR signaling pathway,up-regulating the expression of Treg signature genes and IL-10 level,thereby prolonging the survival time of allografts.These findings may have therapeutic implications for organ transplant recipients,especially those with viral infections and malignancies that requires a more suitable anti-rejection medication.