The Effect Of Paeoniflorin On Activated Human T Lymphocytes

Eczema is a kind of skin inflammatory reaction caused by various kinds of internal and external factors.In the acute stage the skin lesion are mainly papulo-vesicle with obvious exudative tendency and lichenification are common in the chronic stage.Eczema can reoccur easily.Our previous studies found that Fu Fang Fu Ling Tang(FFFLT)has a significant inhibitory effect on allergic contact dermatitis mice,also it could treat eczema with good efficacy and mild side effect in clinical practise.Its efficacy superior to traditional antihistamines.The constituents of Fu Fang Fu Ling Tang were qualitatively and quantitatively analyzed by ultra performance liquid chromatography-diode array detector-electrospray ionization-mass spectrometry(UFLC-DAD-ESI-MS).Also the drug metabolism in vitro and anti-inflammatory effect of the main components were studied.It showed that Paeoniflorin(PF)was the main active ingredient of FFFLT and had good anti-inflammatory activity.It is reported that Paeoniflorin had pharmacological effects such as anti-inflammation and anti-allergy.T lymphocytes are the main components of lymphocytes,and they are the main cells in immune recognition,response and regulation of the body,which play a dominant role in the immune system.T lymphocyte is pivotal in eczema pathogenesis.Previous studies showed that PF inhibited activated human T lymphocytes proliferation,induced T lymphocytes apoptosis and inhibited IFN-? expression.The anti-inflamatory mechanism of PF in treating eczema need to be further studied.As a classical cell signal transduction pathway,NF-?B/MAPK signaling pathway plays an important role in inducing the expression,aggregation,activation and anti-inflammatory cytokines of inflammatory cytokines and anti-inflammatory cytokines.NF-?B is composed of Rel A(P65),Rel B,C-Rel and NF-?B2(P52).The encoded product of NF-?B consists of homodimers and various heterodimers,most of which are P50 and P65.They can participate in the downstream signal transduction pathway and activate a series of kinases by binding the inhibition of protein I?B,thereby regulate the expression of related genes.Mitogen-activated protein kinases(MAPKs)are a kind of Serine/Threonine protein kinases that exist widely in eukaryotic cells.Extracellular signals or stimuli such as inflammatory cytokines,growth factors,bacterial complexes can activate the MAPK signaling pathway.After activation,extracellular signals can be amplified and conducted to nucleus,then regulate the genes expression.The MAPKs family includes eight subfamilies,ERK,p38MAPK,ERK3,JNK(JNK),BMK1/ERK5,ERK27,ERK8 and NLK.Each pathway can activate their transcription factors and mediate biological effects after induced by different stimulus.Objective:To investigate the effect of PF on NF-?B p65,I?B-? phosphate,Phosphorylation of p38(p-p38),phosphorylation of ERK1/2(p-ERK1/2)and phosphorylation of JNK(p-JNK)in activated human peripheral blood T lymphocytes.To study the anti-inflammatory mechanism of PF.Methods:Human peripheral blood T lymphocytes were isolated and cultured in vitro.T lymphocytes were activated by CD3 combined with CD28 monoclonal antibody.Western blot was used to detect the effect of different concentrations of PF on activated T lymphocyte phosphorylation of I?B-? and NF-?B p65,phosphorylated p38(p-p38),Phosphorylation of ERK1/2(p-ERK1/2),phosphorylation of JNK(p-JNK)expression.Results:1.PF inhibits NF-?B p65 expression in activated T lymphocytes:Within 0-120 min,the expression of NF-?B p65 in activated T lymphocytes increased with time.The level of NF-?B p65 in CD3 + CD28 group was higher than that in blank control group(P<0.01).PF inhibited nuclear translocation of p65 in a concentration-dependent manner,and l0?M PF group inhibited NF-?B p65 expression significantly compared with CD3 CD28 group(P<0.05).2.PF inhibits NF-?B signaling pathway I?B-? phosphorylation in activated T lymphocytes:Within 0-120min,the level of I?B-? decreased with time and almost completely degraded at 60min.The level of P-I?B-? in activated T lymphocytes was significantly higher than that in the blank control group(P<0.05).Compared with the CD3 + CD28 group,1?M PF,5?M PF and 10?M PF groups could significantly inhibit the P-I?B-? expression(P<0.01).3.PF had no effect on MAPKs signaling pathway factors JNK and ERK phosphorylation in activated T lymphocytes:JNK and ERK phosphorylation was highest at lOmin within 0-120min.Compared with the CD3 + CD28 group,the expression level of MAPKs signaling pathway factors ERK and JNK were not affected in 1?M PF,5?M PF and 10?M PF groups(P>0.05).4.PF inhibits phosphorylation of MAPKs signaling pathway factor p38 in activated T lymphocytes:Within 0-120min,p38 phosphorylation was highest at 10min.P-p38 expression decreased with the increase of PF concentration.Compared with CD3+ CD28 group,10?M PF group could significantly inhibit the expression of P-p38 in T lymphocytes(P<0.01).Conclusion:1.PF inhibited NF-?B p65 and IKB-a phosphorylation in activated T lymphocytes in a concentration-dependent manner.PF may inhibit T lymphocytes activation by NF-?B signaling pathway.2.PF inhibited p3 8 kinase phosphorylation in activated T lymphocytes in a concentration-dependent manner.PF could not inhibit ERK and JNK phosphorylation.3.I?B?-NF-?B signaling pathways and p38 kinase phosphorylation are involved in PF-mediated immunosuppression in human T lymphocytes,suggestting that PF merits further study as a novel anti-inflammatory agent with a wide range of important applications.