| Objective:Cordycepin has been proved to have a variety of pharmacological effects and biological activities,such as anti-inflammatory,anti-oxidation,antitumor,antifungi,antivirus,liver protection and kidney protection.In our previous study,we found that cordycepin can significantly inhibit the swelling of spleen and thymus in mice induced by complete Freund’s adjuvant(CFA).In addition,cordycepin can also inhibit the infiltration of T cells in the paw of mice with CFA induced inflammation.All these indicate that cordycepin is involved in the regulation of T cell activity.However,the molecular mechanism of the effect of cordycepin on T cell activity is not clear.In this study,human CD4~+T-cell leukemia cell line(Jurkat)was used as the model cells of T-cell study in vitro,and was pretreated with cordycepin and stimulated by Inducer to observe the effect of cordycepin on the activity of Jurkat T cells and explore the molecular mechanism of the effect.Methods:1.Detection of proliferation,apoptosis and cytokine secretion of Jurkat T cellsThe experiment was divided into four groups:normal control group(mock),CD3/CD28 antibody treatment group,CD3/CD28 antibody+cordycepin(50μg/m L)treatment group,CD3/CD28 antibody+cordycepin(100μg/m L)treatment group.Then Jurkat T cells in good growth condition were laid according to the grouping order and experimental requirements,and cultured for24 h.After 24 hours,cordycepin was pretreated with corresponding final concentration of cordycepin solution for 1 hour,and then anti-CD3 monoclonal antibody(anti-CD3),anti-CD28 monoclonal antibody(anti-CD28)and horseradish peroxidase labeled Goat anti mouse Ig G(HRP*Goat anti mouse Ig G(H+L))were added to incubate for 24 hours to establish the stereoscopic T cell model.CCK-8 kit was employd to detect the proliferation of Jurkat T cells in each group.Flow cytometry was used to detect the apoptosis of Jurkat T cells in each group.Enzyme linked immunosorbent assay(ELISA)was employd to detect the secretion of interleukin-2(IL-2),IL-6 and interferon-γ(IFN-γ)in the cell supernatant of Jurkat T cells in each group.2.Detection of ZAP70 phosphorylation and Erk phosphorylation in Jurkat T cellsThe experiment was divided into six groups:normal control group(mock),cordycepin(50μg/m L)treatment group,cordycepin(100μg/m L)treatment group,anti-CD3 treatment group,anti-CD3+cordycepin(50μg/m L)treatment group,anti-CD3+cordycepin(100μg/m L)treatment group.Jurkat T cells in good growth condition were prepared according to the grouping order and experimental requirements,and cultured for 24 h.After 24 hours,cordycepin solution of corresponding final concentration was pretreated for 1 hour according to grouping requirements,and then anti-CD3 was used to stimulate T cells for 30 minutes.Then the cells of each group were collected and the protein samples were prepared.The phosphorylation of ZAP70 and Erk and the expression of ZAP70 and Erk were detected by Western blot.3.Detection of NFAT1 translocation in Jurkat T cellsThe experiment was divided into four groups:normal control group(mock),cordycepin(50μg/m L)treatment group,anti-CD3 treatment group,anti-CD3+cordycepin(50μg/m L)treatment group.Jurkat T cells,which grow well and can express activated T-cell factor-1-fluorescent probe(NFAT1-m Cherry)fusion protein,were cultured for 24 hours with experimental requirements.After 24hours,cordycepin solution of corresponding final concentration was pretreated for 1 hour according to grouping requirements,and then stimulated with anti-CD3 to stimulate 15 min,30 min,2 h and 6 h.Then the cells in each group were fixed,stained,and transferred into the six hole glass plate.The glass bottom plate was observed under Leica Sp8 confocal microscope,and the image was obtained and analyzed by Leica Las AF software.Result:(1)CCK-8 assay showed that,compared with mock group,the proliferation activity of Jurkat T cells stimulated by CD3/CD28 antibody alone was significantly increased(P<0.01);the proliferation activity of Jurkat T cells pretreated with cordycepin was significantly lower than that stimulated alone(P<0.01),and the decrease was more obvious(P<0.01)with the increase of cordycepin dosage.These results suggest that cordycepin can significantly inhibit the proliferation of Jurkat T cells stimulated by CD3/CD28 antibody in a dose-dependent manner.(2)Flow cytometry showed that the apoptosis rate of Jurkat T cells stimulated by CD3/CD28 antibody alone was significantly higher than that of mock group(P<0.01);However,the apoptosis rate of Jurkat T cells treated with cordycepin(100μg/m L)was significantly higher than that stimulated by cordycepin alone(P<0.05).These results suggest that cordycepin can significantly promote the apoptosis of Jurkat T cells stimulated by CD3/CD28antibody.(3)The results of ELISA showed that the secretion of IL-2,IL-6 and IFN-γin Jurkat T cells stimulated by CD3/CD28 antibody was significantly increased(P<0.01);however,compared with that stimulated alone,the secretion of IL-2and IL-6 in Jurkat T cells pretreated with cordycepin(the concentration was 100μg/m L and 50μg/m L,respectively)was decreased(P<0.05).These results suggest that cordycepin can inhibit the secretion of IL-2 and IL-6 in Jurkat T cells.Cordycepin had no significant effect on IFN-γsecretion(P>0.05).(4)Western blot showed that the phosphorylation of ZAP70 protein was not detected with only cordycepin treatment;After anti-CD3 stimulation,the phosphorylation level of ZAP70 protein in Jurkat T cells was significantly increased,and the phosphorylation level of ZAP70 protein in Jurkat T cells pretreated with cordycepin was decreased in a dose-dependent manner compared with that in Jurkat T cells pretreated without cordycepin;In addition,cordycepin up-regulated the expression of ZAP70.These results suggest that cordycepin can inhibit the phosphorylation of ZAP70 in T cells in a dose-dependent manner.The phosphorylation signal of Erk was weak in Jurkat T cells without anti-CD3 stimulation,and cordycepin(100μg/m L)slightly attenuated the signal;the phosphorylation level of Erk in Jurkat T cells was up-regulated after anti-CD3 stimulation,and the phosphorylation level of Erk in Jurkat T cells pretreated with cordycepin was lower than that without cordycepin treatment.These results suggest that cordycepin can reduce the phosphorylation level of Erk protein in Jurkat T cells.(5)Fluorescence imaging showed that cordycepin alone did not induce NFAT1 translocation in Jurkat T cells,but slightly inhibited NFAT1translocation;After anti-CD3 stimulation,the accumulation of NFAT1 in the nucleus was increased significantly and reached the peak at 30 min,then gradually decreased to the normal level;The percentage of NFAT1 translocation cells in anti-CD3+cordycepin group was significantly lower than that in anti-CD3 group(15 min,30 min,2 h)(P<0.05).These results suggest that cordycepin can inhibit the nuclear translocation of NFAT1 in Jurkat T cells.Conclusion:(1)Cordycepin can inhibit the proliferation and induce the apoptosis of Jurkat T cells.(2)Cordycepin can inhibit the secretion of IL-2 and IL-6 in Jurkat T cells,but had no significant effect on IFN-γ.(3)Cordycepin can inhibit the phosphorylation of ZAP70 upstream of TCR signaling cascade and the phosphorylation of Erk in MAPK/Erk signaling pathway downstream of TCR signaling cascade.(4)Cordycepin can inhibit the nuclear translocation of NFAT1 in TCR signal cascade.In conclusion,cordycepin may regulate T cell activity by inhibiting ZAP70 phosphorylation,MAPK/Erk signaling pathway and NFAT signaling pathway in TCR signaling cascade. |
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