摘要
探讨黄皮果果核挥发油(essential oil of kernel,EOK)对诱导小鼠黑色素瘤细胞B16-F10增殖和凋亡的影响。3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐[3-(4,5-dimethl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide,MTT]法检测不同浓度的EOK处理B16-F10细胞24、48、72 h后的细胞存活率,并用不同浓度EOK处理B16-F10细胞24 h,荧光法检测线粒体膜电位(mitochondrial membrane potential,MMP)的变化、磷脂酰丝氨酸蛋白抗体/碘化丙啶(Annexin V-FITC/propidiun iodide,Annexin V-FITC/PI)染色检测细胞凋亡、蛋白免疫印迹法检测核转录因子-кB(nuclear factor-кB,NF-кB P65)及其磷酸化的蛋白(phosphorylate nuclear factor-кB,NF-кB P-P65)、Cleavedcaspase 3、Bax及Bcl-2蛋白表达的水平。结果显示,EOK各处理组均能明显抑制B16-F10细胞的增殖(P <0.05或P <0.01),其趋势呈浓度和时间依赖性。Annexin V-FITC/PI检测发现EOK可诱导B16-F10细胞凋亡,免疫印迹法也显示EOK可使B16-F10细胞NF-кB P65及其磷酸化的蛋白表达量明显降低,Cleaved-caspase 3的蛋白表达量增加,Bax/Bcl-2比值增大。结果表明,EOK可诱导B16-F10细胞凋亡,其机制与抑制胞内NF-кB P65蛋白表达,降低其磷酸化水平,激活Bcl-2/Bax/caspase 3信号通路有关。
The effect of the essential oil of kernel from wampee on proliferation and apoptosis in melanoma B16-F10 cells was investigated. The cell viability were detected by the 3-(4,5-dimethl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide(MTT)assay after treatment with various concentration EOK in 24 h,48 h,72 h.Then,the mitochondrial membrane potential(MMP),the apoptosis rate,and expression of nuclear factor-кB(NF-кB P65),phosphorylate nuclear factor-кB(NF-кB P-P65),Cleaved-caspase 3,Bax and Bcl-2 were measeured by fluorescence staining. Annexin V-FITC/propidiun iodide(Annexin V-FITC/PI)staining,and western blot after the cells treated with EOK for 24 h,respectively. Results showed that EOK could inhibit the proliferation activity of cells in a dose-and time dependent manner. The result of Annexin V-FITC/PI staining demonstrated that the apoptotic rate was significantly increase after treated with EOK. Western blot results demonstrated that EOK caused significantly decreased the expression of NF-кB P65 and NF-кB P-P65. However,the expression of Cleaved-caspase 3 and the ratio of Bax/Bcl-2 were significantly increased. In conclude,EOK induced B16-F10 cells apoptosis by down-regulating the expression of NF-кB,inhibiting its phosphorylation and the activation of Bcl-2/Bax/caspase 3 pathway.
引文
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