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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 investigate the role of VK3-induced ROS in the lysosomal-mitochondrial cell death pathway in Hela cells by using oxidative stress model of vitamin K3 (VK3) replication. Methods Human cervical carcinoma Hela cells were divided into three groups: control group, VK3 group (30μmol / L), NAC group (80μmol / L) and VK3 combined with NAC group. MTT assay was used to detect the cell viability. The level of reactive oxygen species (ROS) was detected by DCFH-DA staining. The changes of lysosomal membrane permeability were observed by acridine orange (AO) staining. The cytoplasm p H changes, Rhodamine 123 staining was used to detect the changes of mitochondrial membrane permeability. Results Compared with the control group, NAC alone had no significant effect on Hela cells. VK3-treated Hela cells decreased (P <0.05), DCFH-DA staining increased (P <0.05) and ROS levels increased. AO staining increased the cytoplasmic green fluorescence of Hela cells after treated with VK3 for 3 h (P <0.05) (P <0.05), and increased the lysosome permeability. The ratio of FL1 / FL2 decreased after BCECF-AM staining and VK3 treatment for 6 h (P <0.05). The cells with acidic and acidic p H in cytoplasm (P <0.05). After Rhodamin staining and VK3 treatment for 6 h, the red fluorescence intensity in the cytoplasm decreased (P <0.05) and the mitochondrial membrane potential decreased. Compared with the VK3 alone group, VK3 combined with NAC increased the cell survival rate (P <0.05), reduced the ROS level (P <0.05), reduced the lysosomal permeability (P <0.05) / FL2 ratio increased (P <0.05). The number of acidic p H cells was 5.4% (P <0.05) and mitochondrial membrane potential increased (P <0.05). Conclusion VK3 induces the increase of lysosome and mitochondrial permeability through the ROS-dependent pathway during the oxidative stress-induced Hela cell injury. During this process, the cytoplasm undergoes acidification.