Ursolic Acid Inhibit Mouse T Lymphocyte Activation Through NF-κB Signaling

Background and Object:Ursolic acid is a constituent of numerous medical plants which are having diversified phylogenetic origin and taxonomic position.Ursolic acid was revealed with many pharmacological effects.Although many scientists had studied the antitumor, anti-inflammatory and immunomodulatory activity,the effect of ursolic acid on T cell function remains uncertain.T cell is the mainly part of lymphocyte, which performs immunologic recognition and response,as well as immunoregulation in vivo.T cell plays a critical role in immune system and T cell activation is very important for normal or pathological immunological response.Rheumatoid arthritis(RA)is a kind of chronic multi-joint Synovium inflammation which destroy articular cartilage progressively,this commonly autoimmune disease is characteristic with infiltration by lymphocyte, macrophage,and plasma cell.Abnormal autoantigen presentation and autoreactivity T cell activation take part in the pathomechanism and etiopathogenisis of RA.It is believed that antigen induced T cell activation spur the T cell infiltrate into membrana synovialis,which cause inflammatory reaction,further induce synovialis hyperplasia,pannus formulation,and absorption of cartilage.RA is related with T cell excessive proliferation,lymphocyte activating markers as CD69,CD25,CD71, HLA-DR highly expressed in the joint Synovial fluid,Th1/Th2 cytokine in serum is obvious disequilibriurn.Although the pathogenesis of RA is unclear, study the lymphocyte activation and signaling,change the abnormal signal transmission,balance the pro and anti-inflammatory cytokine,further block the excessive lymphocyte activation,will provide new clue for the clinical diagnosis and treatment of RA.For this reason,use the mouse T cell model in vitro to screen active constituents from Chinese materia medica makes practical sense for RA treatment.T cell surface factors CD69,CD25,CD71 expression is associated with the cell proliferation and activation.IL-2 and IFN-γrepresent Th1 cytokines, and IL-4 represent Th2 cytokines.The level of IL-2,IFN-γand IL-4 could indicated the situation of Th1/Th2 banlance.NF-kB play an important role in the pathomechanism immunmodulation of inflammation and autoimmune disease,which lead to the progression of diseases such as rheumatic arthritis. Inhibitors of NF-kB might treat autoimmunity and inflammatory diseases. The decotion Bi Zhong Xiao is composed of Chinese herb including Herba Hedyotis Diffusae,Radix Actinidiae Chinensis,Caulis Trachelospermi et al, it is an effective decotion for RA treatment,ursolic acid is an active constituent exist in these herb.Previously research showed that ursolic acid has significant anti-inflammatory action,could ameliorate the symptom of collagen incuced mouse arthritis,but the mechanism remains unknown.The aim of this study is to investigate the effect of ursolic acid on T cell proliferation and activation,as well as NF-kB signaling in T cell.Our study may offer new data for the potentiality of ursolic acid in treatment of autoimmune and inflammation diseases.Methods:For investigate the immunomodulation effect of ursolic acid, we tested the effect of ursolic acid on mouse lymphocyte proliferation induced by ConA or PMA plus Ion by MTT assay at first.Secondly,we tested the effect of ursolic acid on PMA induced T cell superfic molecule CD69,CD25,CD71 expression by flow cytometry.Thirdly,we tested the effect of ursolic acid on PMA plus Ion induced Th1/Th2 cytokines IL-2,IFN-γ,IL-4mRNA expression use RT-PCR.At last,we investigated the effect of ursolic acid on PMA induced IkB-αphosphorylation and NF-kB P65 nuclear translocation in T cell by westem blotting.Results:(1)MTT assay shows ursolic acid(5-30μM/L)can inhibit the lymphocyte proliferation induced by ConA or PMA plus Ion in a dose dependent manner.(2)Flow cytometry shows ursolic acid(10-30μM/L)can decrease CD3~+CD69~+,CD3~+CD25~+,CD3~+CD71~+expression induced by PMA dose dependently.(3)RT-PCR assay shows ursolic acid(5-30μM/L) blocked the IL-2,IFN-γand IL-4mRNA expression induced by PMA plus Ion dose dependently.(4)ELISA assay shows ursolic acid(10-30μM/L) decreased the IL-2 secretion by T cell stimulated by PMA plus Ion in a dose dependent manner.(5)Western blotting assay shows ursolic acid(10-20μM/L) depressed PMA induced IkB-αphosphorylation,ursolic acid(20μM/L) blocked NF-IkB P65 nuclear translocation in T cell.Conlusion:(1)Ursolic acid could inhibit the lymphocyte activation and proliferation.(2)The inhibition effect of ursolic acid on T cell function is involved with NF-kB signaling pathway.