Effects Of Anisomycin On The Allogeneic Skin Transplantation In Mice And Its Mechanism

Objective:To investigate the effects of Anisomycin on allogenetic skin transplantation in the mice and its mechanism.Methods:A model to evaluate lymphocyte proliferation stimulated with a polyclonal activator,concanavalin A(ConA),was established by vital dye carboxyl fluorescin diacetate succinmidyl ester(CFSE) labeling technique.Effects of the different doses of Anisomycin on the lymphocyte proliferation ability were estimated by flow cytometry.The percentage of the expression level of CD69 and CD25 on the in vivo lymphocytes was evaluated by fluorescin-conjugated monoclonal antibody double labeling technique.The percentage of the expression level of CD4 and CD25 on the in vivo lymphocytes was also evaluated by fluorescin-conjugated monoclonal antibody double labeling technique.To study the effect of Anisomycin on cytokines produced by the lymphocytes in vitro and in vivo, the expression of interleukin10(IL-10),interleukin2(IL-2),interleukin4(IL-4),interleukin12(IL-12), interleukin6(IL-6),transforming growth factorβ(TGF-β),immunoreactive fibronectinγ(IFN-γ),tumor necrosis factorα(TNF-α) in blood serum and IL-10,IL-2,IL-4,IFN-γ,TNF-αin supernatant of in vitro lymphocyte was detected by Luminex technique.The reactivity of the lymphocytes in unilateral or bilateral mixed lymphocyte reaction(MLR) and the reversible reactivity of the lymphocytes that stimulated by IL-2 in mixed lymphocyte reaction(MLR) were detected by MTT assay.A model of C57 mice→Balb/c mice allogenetic skin transplantation in mice was established to observe the effects of Anisomycin on survival time of transplanted skin compared with positive control of Cyclosporin A(CsA).Delayed type hypersensitivity(DTH) was used to detect the effect of Anisomycin on in vivo immune system.The effects of Anisomycin on micronuclei of bone marrow cells and sperm deformity in male mice under 5.0 mg/kg～60.0 mg/kg dosage groups was detected by microscopy.The chronic toxicity of Anisomycin to mice was evaluated by the paraffin section of heart,liver,spleen,lung and kidney from the mice treated by Anisomycin.Results:CFSE staining showed that the promoting effect of ConA on the lymphocyte proliferation was weaker and weaker with the increase of intraperitoneally injected Anisomucin dosage within 5.0 mg/kg～60.0 mg/kg.Under the dose of 5.0 mg/kg～60.0 mg/kg, intraperitoneal injection Anisomycin could definitely down-regulate the expression level of CD69 and CD25 on the lymphocyte surface in vivo(P＜0.05).Under the dose of 5.0 mg/kg～60.0 mg/kg,intraperitoneal injection Anisomycin could definitely down-regulate the expression level of CD4 and CD25 on the lymphocyte surface in vivo.At the same dose in vivo,the expression of IL-10,IL-4 and TGF-βrose up obviously(P＜0.05) while IL-12,IL-6 decrease rapidly(P＜0.01). The expression of IL-2,IFN-γand TNF-αhad no obvious change.In vitro,under the dose of 1.0 ng/ml～160.0 ng/ml,the expression of IL-10 and IL-4 rise up obviously(P＜0.05) while IL-2,IFN-γand TNF-αsharply decrease(P＜0.01).Under the dose of 10.0 ng/ml,Anisomycin could inhibit the reactivity of lymphocytes in both unilateral and bilateral MLR,and there was no difference of the inhibitory effect between Anisomycin and Dexamethasone(P＞0.05).The repressive effect of Anisomycin under the dose of 1.0 ng/ml～160.0 ng/ml could be reversed by IL-2 of 50.0 ng/ml.In vivo,12.5 mg/kg Anisomycin could obviously prolong the survival time of transplanted skin,and the effects of Anisomycin against allogeneic skin transplantation rejection were stronger than CsA(P＜0.01 ).Within the dose of 5.0 mg/kg～60.0 mg/kg,DTH in mice could be inhibited.Under the dose of 60.0 mg/kg,intraperitoneal injection Anisomycin could increase the micronuclei rate of bone marrow cell(P＜0.01).Under the same dose,Anisomycin could increase the aberration rate of sperm on male mice(P＜0.01).Within the dose of 5.0 mg/kg～60.0 mg/kg,no obviously chronic toxicity can be observed in the heart,liver,spleen, lung and kidney in mice.Conclusion:The results suggest that Anisomycin can inhibit lymphocyte behaviors such as proliferation,activation and reactivity;its effect can be reversed. Anisomycin can induce the up-regulation of CD4~+CD25~+ Treg cells.Anisomycin can increase the cytokines that up-regulate Th2 cells while reduce the expression of cytokines that up-regulate Thl cells. Anisomycin can inhibit the rejection of allogeneic skin transplantation in mice.Anisomycin has no heavy toxicity in vivo.Those results provide immportant theoretical and experimental evidence for the research and development of Anisomycin as a new immunosuppressant.