紫花地丁止痒复方对RBL-2H3细胞脱颗粒的影响

Effect of Viola yedoensis Makino Antiitching Compound on RBL-2H3 cell degranulation and its relevant mechanisms

目的考察紫花地丁止痒复方(Viola yedoensis makino antiitching compound,VYAC)对RBL-2H3细胞脱颗粒的影响及机制。方法CCK-8检测VYAC对RBL-2H3细胞的毒性;C48/80诱导RBL-2H3细胞发生脱颗粒,台盼蓝染色、β-氨基己糖胺酶释放、组胺释放、细胞内Ca2+浓度,评价VYAC对C48/80诱导的RBL-2H3细胞脱颗粒情况;Western blot法检测相关蛋白(Syk、p-Syk、PI3K、Akt、p-Akt)的表达。结果100 mg·L^-1的C48/80能够明显刺激RBL-2H3细胞发生脱颗粒,脱颗粒率达74%(P<0.05);VYAC呈剂量依赖性抑制β-氨基己糖胺酶和组胺的释放(P<0.05),且剂量在800 mg·L^-1以下时不影响RBL-2H3细胞存活;VYAC能够明显减弱细胞内荧光强度,降低细胞内Ca2+浓度;VYAC(25、100 mg·L^-1)明显抑制PI3K蛋白表达、抑制Syk、Akt蛋白的磷酸化(P<0.05)。结论VYAC能够抑制过敏性皮炎中肥大细胞脱颗粒,抑制Ca2+内流,其机制可能是抑制Syk/PI3K/Akt活化。

Aim To explore the effect of Viola yedoen-sis Makino Antiitching Compound(VYAC)on mast cell degranulation in allergic dermatitis and the rele-vant mechanisms.Methods RBL-2H3 cells were in-duced with C48/80 to establish degranulation cell mod-el;Trypan blue staining,β-hexosaminase,histamine,and intracellular Ca^2+concentration were used to eval-uate the degranulation effect of VYAC on RBL-2H3 cells induced by C48/80;CCK-8 assay was used to measure the cell cytotoxicity of VYAC;Western blot was used to detect the effect of VYAC on(Syk,p-Syk,PI3K,Akt and p-Akt)protein expressions.Re-sults C48/80(100 mg·L^-1)significantly stimula-ted the degranulation of RBL-2H3 cells,with a degran-ulation rate of 74%(P<0.05);VYAC suppressed the release ofβ-hexosaminase and histamine in a dose-dependent manner(P<0.05),and without cytotoxici-ty at doses below 800 mg·L^-1;VYAC reduced the intracellular fluorescence intensity,which represented Ca^2+concentration in intracellular;VYAC(25,100 mg·L^-1)decreased(Syk,p-Syk,PI3K,Akt and p-Akt)protein expressions(P<0.05).Conclusions VYAC can inhibit mast cell degranulation in allergic dermatitis,which may be through Syk/PI3K/Akt path-way and inhibiting Ca^2+influx.