维生素K3通过ROS介导细胞质酸化诱导Hela细胞凋亡

Apoptosis of He La cells induced by vitamin K_3 through ROS-mediated cytosolic acidification

目的利用维生素K3(VK3)复制氧化应激模型,探讨VK3诱导的ROS在Hela细胞溶酶体-线粒体细胞死亡途径过程中的作用及机制。方法以人宫颈癌Hela细胞为研究对象,实验分为:对照组、VK3组(30μmol/L)、NAC组(80μmol/L)和VK3与NAC联合作用组。MTT法检测细胞生存率,DCFH-DA染色观察细胞内活性氧簇(ROS)水平,吖啶橙(AO)染色观察溶酶体膜通透性变化,BCECF-AM染色流式细胞术检测细胞质p H变化,罗丹明123(Rhodamine 123)染色检测线粒体膜通透性变化。结果与对照组相比,单独应用NAC对Hela细胞无明显影响。VK3作用组Hela细胞生存率降低(P<0.05),DCFH-DA染色荧光增强(P<0.05),ROS水平增加;AO染色在VK3作用3 h后Hela细胞的细胞质绿色荧光增强(P<0.05),红色颗粒状荧光减弱(P<0.05),溶酶体通透性增加;BCECF-AM染色,VK3作用6 h后FL1/FL2比值降低(P<0.05),细胞质呈酸性,酸性p H的细胞数为13.6%(P<0.05);Rhodamin染色,VK3作用6 h后细胞质内红色荧光强度降低(P<0.05),线粒体膜电势降低。与单独应用VK3组比较,VK3与NAC联合作用于Hela细胞时,细胞生存率增加(P<0.05),ROS水平降低(P<0.05),溶酶体通透性降低(P<0.05),FL1/FL2比值增加(P<0.05),酸性p H的细胞数为5.4%(P<0.05),线粒体膜电势增加(P<0.05)。结论在氧化应激诱导Hela细胞损伤过程中,VK3通过ROS依赖途径,诱导先后发生溶酶体及线粒体通透性增加,在这个过程中细胞质发生酸化。

Objective To explore the effect and mechanism of vitamin K_3- induced reactive oxygen species（ ROS） in lysosomal- mitochondrial cell death pathway of He La cells through establishing oxidative stress model of cervical carcinoma He La cells by vitamin K_3.Methods Human cervical carcinoma He La cells were selected and divided into control group,vitamin K_3group（ 30 μmol / L）,NAC group（ 80 μmol/L）,and vitamin K_3＋ NAC group. MTT assay was used to detect the survival rate of human cervical carcinoma He La cells;DCFH- DA staining was used to observe ROS level; acridine orange（ AO） staining was used to observe the change of lysosome membrane permeability; BCECF- AM flow cytometry was used to detect the change of cytoplasmic p H value; Rhodamine 123 staining was used to detect the change of mitochondrial membrane permeability. Results Compared with control group,NAC had no significant impact on He La cells. The survival rate of He La cells in vitamin K_3 group decreased significantly（ P〈0. 05）,fluorescence of DCFH- DA staining enhanced（ P〈0. 05）,ROS level increased; AO staining showed that cytoplasmic green fluorescence of He La cells enhanced after treated by vitamin K_3 for three hours（ P〈0. 05）,red fluorescence weakened（ P〈0. 05）,lysosome membrane permeability increased; BCECF- AM staining showed that FL1 / FL2 ratio decreased after treated by vitamin K_3 for six hours（ P〈0. 05）,cytoplasmic acidification was found,the number of acidification cells was 13. 6%（ P〈0. 05）; Rhodamin staining showed that cytoplasmic red fluorescence of He La cells weakened after treated by vitamin K_3 for six hours（ P〈0. 05）,potential of mitochondrial membrane decreased. Compared with vitamin K_3 group,the survival rate of He La cells increased significantly in vitamin K_3＋ NAC group（ P〈0. 05）,ROS level decreased significantly（ P〈0. 05）,lysosome membrane permeability decreased significantly（ P〈0. 05）,FL1 / FL2 ratio increased significantly（ P〈0. 05）,the number of acidification cells was 5. 4%（ P〈0. 05）,potential of mitochondrial membrane increased significantly（ P〈0. 05）. Conclusion During the course of He La cells damage induced by oxidative stress,vitamin K_3 can induce the increase of lysosome membrane permeability and mitochondrial membrane permeability through ROS- dependent pathway,during which cytoplasmic acidification occurs.