背景:Kamebakaurin是日本花盘和花盘的活性成分,在中医中用于改善过敏患者的症状。我们使用BMMC研究了kamebakaurin抗过敏作用的分子机制。
方法:脱粒率,组胺释放,和白细胞介素(IL)-4,白三烯B4(LTB4),研究了半胱氨酰白三烯在抗原触发的BMMC上的产生。此外,通过Westernblot检查kamebakaurin对信号转导蛋白的影响,并计算与Syk和Lyn激酶结构域的结合。使用小鼠模型研究了kamebakaurin对抗原诱导的高通透性的影响。
结果:在10μm时,kamebakaurin部分抑制脱颗粒,组胺释放,和IL-4的生产。在30μm时,kamebakaurin部分减少LTB4和半胱氨酰白三烯的产生并抑制脱粒,组胺释放,和IL-4的生产。SykY519/520及其下游蛋白的磷酸化,Gab2,被kamebakaurin还原,用30μmkamebakaurin观察到完全抑制。相比之下,Erk的磷酸化仅被部分抑制,即使存在30μmkamebakaurin。SykY519/520已知通过存在于其自身ATP结合位点的分子内ATP自动磷酸化,这种自动磷酸化引发脱粒,组胺释放,和IL-4的生产。对接模拟研究表明,kamebakaurin阻断了ATP与Syk中ATP结合位点的结合。因此,kamebakaurin与SykATP结合位点结合对Syk自磷酸化的抑制似乎导致组胺释放和IL-4产生的减少。Kamebakaurin以剂量依赖性方式抑制抗原诱导的血管通透性过高,但并未降低组胺诱导的血管通透性过高。
结论:Kamebakaurin通过抑制Syk磷酸化改善过敏症状;因此,kamebakaurin可能是新型抗过敏药物的先导化合物。
BACKGROUND: Kamebakaurin is an active constituent of both Rabdosia japonica and Rabdosia excisa, which are utilized in Chinese traditional medicine for improving symptoms in patients with allergies. We investigated the molecular mechanisms of the anti-allergic effects of kamebakaurin using BMMCs.
METHODS: The degranulation ratio, histamine release, and the interleukin (IL)-4, leukotriene B4 (LTB4), and cysteinyl leukotriene productions on antigen-triggered BMMC were investigated. Additionally, the effects of kamebakaurin on signal transduction proteins were examined by Western blot and binding to the Syk and Lyn kinase domain was calculated. The effects of kamebakaurin on antigen-induced hyperpermeability were investigated using mouse model.
RESULTS: At 10 μ
m, kamebakaurin partially inhibited degranulation, histamine release, and IL-4 production. At 30 μ
m, kamebakaurin partially reduced LTB4 and cysteinyl leukotriene productions and suppressed degranulation, histamine release, and IL-4 production. Phosphorylation of both Syk Y519/520 and its downstream protein, Gab2, was reduced by kamebakaurin, and complete inhibition was observed with 30 μ
m kamebakaurin. In contrast, phosphorylation of Erk was only partially inhibited, even in the presence of 30 μ
m kamebakaurin. Syk Y519/520 is known to be auto-phosphorylated via intramolecular ATP present in its own ATP-binding site, and this auto-phosphorylation triggers degranulation, histamine release, and IL-4 production. Docking simulation study indicated kamebakaurin blocked ATP binding to the ATP-binding site in Syk. Therefore, inhibition of Syk auto-phosphorylation by kamebakaurin binding to the Syk ATP-binding site appeared to cause a reduction of histamine release and IL-4 production. Kamebakaurin inhibited antigen-induced vascular hyperpermeability in a dose-dependent fashion but did not reduce histamine-induced vascular hyperpermeability.
CONCLUSIONS: Kamebakaurin ameliorates allergic symptoms via inhibition of Syk phosphorylation; thus, kamebakaurin could be a lead compound for the new anti-allergic drug.