Inhibitory Effects And Mechanism Of Stephanthraniline A On T Cells Activation

ObjectiveStephanthraniline A(STA),a natural C21 steroid isolated from Stephanotis mucronata(Blanco)Merr.,was previously shown to have immunosuppressive activities.The purpose of this study was to further evaluate the inhibitory activities of STA on T cells immune responses and to elucidate its potential mechanisms.Methods1.The effects of STA on T cells activation and proliferation in vitro:T cells in single splenocytes were enriched by commercial nylon fiber column T and were stimulated by CD3 and CD28 cross-linking.After treatment with STA,T cells proliferation were determined by MTT assay;the cytokines(IL-2,IFN-y,IL-4 and IL-17)production and mRNA expression in activated T cells were measured by ELISA and RT-PCR method,respectively;T cell cycle progression was analyzed by flow cytometer.2.The effects of STA on CD4+T cells activation and proliferation in vitro:CD4+ T cells were isolated by negative selection with a Dynabeads(?)UntouchedTM mouse CD4 cells isolation kit and were stimulated by Concanavalin A(Con A).After treatment with STA,CD4+T cells proliferation were determined by CFDA SE labeled analysis;the surface expression levels of CD69 and CD25 were determined by flow cytometric analysis;the cytokines(IL-2 and IFN-y)production and mRNA expression in activated T cells were measured by ELISA and Real time-PCR method,respectively.3.The inhibitory mechanism of STA on T cells activation:RU486,a glucocorticoid receptor(GR)antagonist,was used to investigate if STA acts through activating the glucocorticoid receptor;the DNA binding activity of NFAT,NF?B and AP1 were assessed by EMSA;the expressions of NFAT1,p-IKBa,p-p38 MAPK,p-ZAP-70 and p-PKC? were assessed by Western blotting;the effects of STA on,Phorbol-12-myristate-13-acetate(PMA)plus ionomycin or CD28-stimulated T cells were determined to investigate whether STA effected T cell receptor(TCR)-mediated proximal signaling and Ca2+signaling;the molecular interaction of STA with PKC isoforms(??????????)were predicted by molecular docking studies;the effect of STA on PKC? activity was determined using ADP-GloTM Kinase Assay;Con A-induced T cells activation and proliferation were further investigate if the effects of STA could be reversed by the agonist of PKC?;the mRNA expressions of 84 key genes representative of 18 different signal transduction pathways were explored by using the mouse Signal Transduction Pathway Finder PCR arrays.4.The effects of STA on Con A-induced hepatitis in mice:C57BL/6 mice were intra-peritoneally given with STA 1 h prior to intravenous injection with Con A.After 8 h,the body weight were examined;serum was collected for assessment of enzyme activity of ALT and AST;slices of liver were stained with hematoxylin and eosin for histopathological analysis;slices of liver were stained with FITC-CD4 and PE-CD69 for Immunofluorescence assay,the concentrations of inflammatory cytokines(TNF-a and IL-1(3)and Th1/Th17 cytokines(IFN-y and IL-17)in serum were determined by ELISA.5.The synergetic effects of STA and Cyclosporine A(CsA)on T cells immune responses:The synergetic effects of STA and CsA were investigated in vitro on the cell proliferation,CD25 surface expression and cytokine IL-2 production of Con A-induced T cells by MTT method,flow cytometric analysis and ELISA,respectively;2,4-dinitrofluorobenzene(DNFB)-induced delayed-type hypersensitivity(DTH),a T cell-mediated response in mice was used to evaluate the synergetic effects of STA and CsA in vivo;the direct effect of STA on CYP3A4,which is the predominant enzyme for metabolism of CsA,was determined using P450-GIoTM CYP3A4 Screening System.Results1.STA has inhibitory effects on T cells activation and proliferation in vitro:STA significantly inhibited CD3/CD28 cross-linking-induced T cells proliferation,reduced the progression T cells into S/G2/M phases,and also suppressed the production and mRNA expression of cytokines(IL-2,IFN-y,IL-4 and IL-17).2.STA has inhibitory effects on CD4+T cells activation and proliferation in vitro:STA significantly inhibited the proliferation of CD3+ CD4+ T cells,but not that of CD3+ CD8+ T cells.STA significantly inhibited CD69 and CD25 expressions,and the production and mRNA expression levels of IL-2 and IFN-y in Con A-stimulated CD4+ T cells.3.The inhibitory mechanism of STA on T cells activation:The inhibitory effect of STA was not reverted by the addition of RU486.STA did not increase the apoptotic rates of CD3/CD28 cross-linking-stimulated T cells,and didn't change the level of Bcl-2 and Bax mRNA expression and Bcl-2/Bax ratio.STA decreased the DNA-bindirng activities of NFAT,NF?B or AP-1,and reduced NFAT dephosphorylation,I?B-? and p38 phosphorylation in CD4+ T cells.STA didn't effect the phosphorylation of Zap70 in activated CD4+ T cells,but inhibited PMA plus ionomycin or CD28 activated T cells proliferation and IL-2 production.STA fitted tightly into the ATP pocket of PKC? via five hydrogens,inhibited PKC? activity,and significantly blocked phosphorylation of PKC? in CD4+ T cells.The inhibition of STA on the proliferation and IL-2 production of Con A-stimulated T cells wasn't reversed by PKC? angonist.STA regulated mRNA expression of the genes related to 10 signaling pathways in CD3/CD28 crosslinking-stimulated T cells.4.STA pretreatment attenuated Con A-induced hepatitis,and the activation and aggregation of CD4+ T cells in hepatic tissue in mice:STA significantly inhibited serum ALT and AST activities in Con A-treated mice,and restored the body weight change induced by Con A.STA prevented the development of histopathological changes,and significantly inhibited TNF-a and IL-1? levels induced by Con A.STA also decreased the number of CD4+CD69+ T cells in the liver of Con A-treated mice and reduced the level of IFN-y and IL-17 in serum.5.STA and CsA synergize to inhibit T cell response in vitro and in vivo:Con A-induced T cells proliferation,IL-2 production and CD25 expression weren't inhibited by low-dose STA or CsA alone,but was significantly reduced by the combination of STA and CsA.DNFB-induced mice ear swelling,hyperplasia and infiltration of inflammatory cells were also significantly diminished by the combined treatment with nontherapeutic dose of STA and CsA.In addition,STA significantly decreased CYP3A4 activity.Conclusions1.STA has inhibitory effects on T cells activation and proliferation in vitro,and CD4+ T cells were more sensitive to inhibitory effects of STA.2.STA inhibited T cells proliferation by promoting cell cycle arrest without inducing apoptosis.3.Distinct from glucocorticoids,the inhibitory effects of STA on T cells activation were not dependent on glucocorticoid receptor.4.Distinct from calcineur ininhibitors(CNIs),STA inhibited T cells activation and proliferation through proximal TCR signaling-and Ca2+ signaling-independent way.5.STA may targeted PKC?,inhibited its phosphorylation and catalytic activation,subsequently induced the arrest of downstream NFAT,NF?B and MAPK signaling cascades,which leading to inhibition of T cells activation?6.The inhibitory mechanism of STA on T cells was also correlated with the regulation of multi-signal transduction pathways.7.STA could used in the treatment of T cell-mediated inflammatory and autoimmune diseases by intra-peritoneally or orally administration.STA reduced Con A-induced CD4+ T cells activation and aggregation in hepatic tissue,and then attenuated hepatitis in mice.8.STA synergized the inhibitory effects of CsA on T cell response in vitro and in vivo.These effects were attributable to its different and complementary molecular mechanisms,and may partly due to its increasing of the bioavailability of CsA via inhibiting CYP3A4.These results highlight the potential of STA as an effective leading compound for use in the treatment of T cell-mediated inflammatory and autoimmune diseases.