The Immunosuppressive Effect And Mechanism Of N,N'-dicyclohexyl-N-arachidonic Acid Ureide On Mice Allogeneic Heart Transplantation

Objectives:Existing immunosuppressive drugs,such as tacrolimus(FK506),have been widely used to inhibit immune rejection after organ transplantation.However,these agents are usually related to several side effects,such as nephrotoxicity and neurotoxicity,which affect the long-term survival of transplanted organs and reduces the quality of life.Therefore,it is urgent to develop novel immunosuppressive agents with high efficiency and low toxicity.Recently,traditional Chinese medicine-derived immunosuppressive drugs have attracted increasing attention owing to its low side effects and pluripotent medicinal value.N,N'-dicyclohexyl-N-arachidonic acid ureide(DCAAA)is a highly unsaturated fatty acid synthesized from "Banlangen-acid" which was isolated from traditional Chinese medicine,Radix Isatidis.In the present study,we used the mice allogeneic heart transplantation model and systematically investigated the toxicity,immunosuppressive effects and the underlying mechanism of DCAAA.Methods:First,we determined the toxic effects of DCAAA on mice using hemolysis test and acute toxicity test.Second,we assessed the effects of DCAAA on lymphocyte proliferation using lymphocyte transformation experiment and CFSE staining experiment.Next,we established a mice allogeneic heart transplantation model and investigated the effect after treatment with DCAAA alone or DCAAA in combination with FK506 on heart grafts survival time.On day 7 after heart transplantation,the graft,spleen,and blood were collected and used for pathological analysis,immunohistochemical staining,flow cytometry,qPCR,ELISA and mixed lymphocyte reaction experiments.Then,we systematically investigated the mechanism underlying DCAAA inhibition of lymphocyte proliferation in vitro using Annexin V-FITC/7AAD staining,clonal anergy,PI satining,transcriptome sequencing,and western blotting.Finally,we re-examined the molecular mechanism underlying DCAAA immunosuppressive effect in vivo.Results:We found that DCAAA treatment did not lead to red blood cell hemolysis and did not affect body and organ weights as well as liver and kidney function in mice.Lymphocyte transformation test and CFSE staining experiment showed that DCAAA treatment inhibited ConA-stimulated lymphocyte proliferation in a dose-dependent manner.In vivo experiments,the mean survival time of the heart graft in the control group was 6.8 ± 0.8 days.DCAAA treatment significantly prolonged graft survival time to 10.3 ± 1.8 days.Furthermore,compared to the FK506-treated group(12.3 ± 1.0 days),survival time in the DCAAA and FK506 combination group was significantly extended to 21±3.2 days.DCAAA treatment suppressed the immune response in recipients by reducing the infiltration of inflammatory cells,the mRNA level of inflammatory factors in the graft,the proportion of CD4+T cells in the spleen and graft,the concentration of interfron-y in serum.Moreover,DCAAA and FK506 showed a synergistic effect in the inhibition of allogeneic immune rejection.In vitro experiments,DCAAA treatment did not induce lymphocyte apoptosis and clonal anergy.DCAAA exerted its immunosuppressive effect by inhibiting activation of the T cell receptor-mediated phosphoinostide 3-kinase(PI3K)-protein kinase B(Akt)pathway,thereby arresting cell cycle transition from G1 to the S phase,and suppressing lymphocyte proliferation.Conclusions:In conclusion,our study reveals a novel,low-toxicity immunosuppressive agent that has the potential to reduce the toxic side effects of existing immunosuppressive agents when used in combination with them,which provides a theoretical basis for clinical application of organ transplants.