Alisol B 23-Acetate Inhibits IgE/Ag-mediated Mast Cell Activation And Allergic Reaction

Objective: In the previous study we investigated the anti-inflammatory effects and mechanisms of triterpenoid Alisol B 23-Acetate(AB23A)in mouse bone marrow-derived mast cells(BMMCs),rat basophilic leukemia(RBL-2H3)and human mast cells(HMC-1)in vitro and in vivo,providing a theory for the development of new anti-inflammatory drugs basis and experimental basis.Methods: 1.MTT assay was used to detect non-cytotoxicity range of AB23 A in BMMCs,RBL-2H3 and HMC-1.2.The enzyme-linked immunosorbent assay(ELISA)was used to determine the release of histamine from Ig E/Ag-activated BMMCs,leukotriene C4(LTC4)synthesis,and interleukin-6(IL-6)production by AB23 A.The effect of AB23 A on the activation of Ca2+ mobilization in BMMCs was determined by immunofluorescence.3.Use Western Blot assay to determin the effects of AB23 A on Ig E/Ag-activated BMMCs,RBL-2H3 and Phorbol 12-Myristate 13-Acetate(PMA)and calcium ionophore(A23187)-activated HMC-1 related proteins expression or phosphorylation levels,including phospholipase Cγ(PLCγ),serine-threonine protein kinase/inhibitor of nuclear factor kappa-B kinase/nuclear factor kappa-B,Akt/IKK/NF-κB),mitogen-activated protein kinases/cytosolic phospholipase A2(MAPKs/c PLA2),cyclooxygenase-2(COX-2),etc.4.Immunoprecipitation(IP)was used to detect the effect of AB23 A on the expression and phosphorylation of spleen tyrosine kinase(Syk)and Src family kinases Lyn and Fyn in Ig E/Ag-activated BMMCs.5.Passive cutaneous anaphylaxis(PCA)was used to study the effect of AB23 A on Ig E/Ag-mediated anti-inflammatory effects in mice,detected the exudation of Evans blue in mouse ears and the effect of AB23 A on the degree of ear swelling and the number of mast cells in mice was studied by toluidine blue staining.Results:1.MTT results showed that the AB23 A concentrations at 2,5,10 and 20 μM had no significant cytotoxicity in BMMCs,RBL-2H3 and HMC-1.2.AB23 A inhibited histamine release,LTC4 synthesis,IL-6 production and calcium mobilization in Ig E/Ag-mediated BMMCs.3.AB23 A inhibited the phosphorylation levels of PLCγ,p38,ERK1/2,c PLA2,Akt,IKKα/β,and nuclear translocation of c PLA2 and NF-κB in Ig E/Ag-mediated BMMCs.4.AB23 A inhibited the phosphorylation level of Syk and had no effect on the phosphorylation and expression of Lyn and Fyn in Ig E/Ag-mediated BMMCs.5.AB23 A inhibited Ig E/Ag-mediated RBL-2H3 and PMA and A23187-mediated phosphorylation levels of PLCγ,p38,ERK1/2,Akt,IKKα/β,and nuclear translocation of NF-κB in HMC-1.6.AB23 A has suppressed Ig E/Ag mediated PCA response,AB23 A inhibited Evans blue exudation,attenuated ear thickness without change of mast cell number.Conclusions: 1.AB23 A inhibited Ca2+ mobilization on Ig E/Ag-mediated phosphorylation of PLCγ in BMMCs;inhibited LTC4 synthesis by inhibiting Ig E/Ag-mediated phosphorylation of p38,ERK1/2 c PLA2 and nuclear translocation of c PLA2 in BMMCs;suppressed IL-6 production and COX-2 expression by attenuating Akt/IKK/NF-κB pathway in Ig E/Ag-mediated BMMCs;These results may be due to AB23 A inhibition of Ig E/Ag-mediated phosphorylation of Syk in BMMCs.AB23 A inhibited Ig E/Ag-mediated RBL-2H3 and PMA and A23187-mediated phosphorylation or expression of related protein in HMC-1,which is consistent with the effect of AB23 A on BMMCs.2.AB23 A also inhibited Ig E/Ag-mediated PCA response through inhibition of mast cell activation in vivo.It is suggested that AB23 A has a good anti-inflammatory effect in vitro and in vivo,and has the potential of anti-inflammatory lead compounds.Our group first systematically reported the anti-inflammatory effect and mechanism of AB23 A.