臭灵丹中HTMF诱导Hep-2细胞凋亡

3,5-hydroxy-6,7,3′,4′-tetramethoxyflavone isolated from Laggera pterodonta induces Hep-2 cell apoptosis in vitro

目的:探讨中药臭灵丹中3,5-二羟基-6,7,3′,4′-四甲氧基黄酮(HTMF)诱导人喉癌Hep-2细胞凋亡的机制。方法:倒置显微镜观察细胞形态变化,Hoechst 33258染色,观察对Hep-2细胞的致凋亡能力;流式细胞仪检测HTMF对Hep-2细胞的凋亡率和线粒体跨膜电位(Δφm)的改变;Western blotting法检测凋亡蛋白caspase-3和caspase-9的变化。结果:倒置显微镜下Hep-2细胞随HTMF浓度加大而变小变圆,细胞存活数随浓度增加而减少。Hoechst 33258染色后高浓度组Hep-2细胞核固缩并出现呈致密浓染蓝白色颗粒状荧光的凋亡小体。流式结果显示HTMF对Hep-2细胞有促凋亡作用并呈明显的量效关系,线粒体跨膜电位(Δφm)随HTMF浓度的增加而下降。Western blotting结果显示HTMF可诱导Hep-2细胞中caspase-3和caspase-9蛋白活化,并呈浓度依赖性关系。结论:HTMF可通过线粒体途径诱导人喉癌Hep-2细胞凋亡。

Aim：To investigate the effect of 3,5-hydroxy-6,7,3′,4′-tetramethoxyflavone（HTMF） isolated from Laggera pterodonta on Hep-2 cell apoptosis and mechanism. Methods： Changes of the cell and nuclear morphological characteristics were observed under the inverted microscope and fluorescence microscope.The cell apoptosis was displayed by Hoechst 33258 staining and flow cytometry（FCM）.The mitochondrial inner membrane potential（Δφm） were analyzed by FCM.Western blotting was used to detect the expression of caspase-3 and caspase-9.Results： Cell morphological observation under the inverted microscope showed that the cell size and volume become smaller with increasing concentration of HTMF.There were some apoptotic bodies with thick white-blue fluorescence in Hoechst 33258 stained cells.Increased apoptosis of Hep-2 cells by FCM were observed in a dose depenetent manner,and decreased mitochondrial Δφm of Hep-2 cells by FCM were observed with increasing concentration of HTMF.Western blotting showed that HTMF dose-dependently increased the expressoin of caspase-3 and caspase-9 in Hep-2 cells.Conclusion：HTMF induced apoptosis in Hep-2 cells by the mitochondrial pathway.