卫生研究

目的探讨白藜芦醇(RES)与三氧化二砷(As2O3)联合处理诱导肺癌细胞凋亡的作用及机制。方法以人肺腺癌A549细胞为研究对象,按处理方式分为对照组、单独RES或As2O3处理组及二者联合组,观察RES和As2O3联合处理对细胞存活率、集落形成率、活性氧(ROS)、谷胱甘肽(GSH)、线粒体膜电位、细胞凋亡等的影响;同时,加入谷胱甘肽合成酶抑制剂L-丁硫氨酸亚砜胺(L-BSO),观察RES是否通过GSH耗竭导致细胞内ROS蓄积,进而增强As2O3诱导的细胞凋亡。结果RES单独作用于细胞时,在浓度大于20μmol/L时,A549细胞存活率随浓度的增加而下降。与As2O3单独处理相比,加入60μmol/L RES组的集落形成率、细胞凋亡率、ROS水平、GSH含量、线粒体膜电位的变化及细胞色素c和Cas-3水平的差异均有统计学意义(P<0.05)。经谷胱甘肽合成酶抑制剂BSO预处理后,RES和As2O3诱导的细胞凋亡由30.0%增加至77.7%,同时细胞内GSH含量锐减而ROS大量蓄积。结论 RES与As2O3联合处理与RES和As2O3单独处理相比,可诱导更多的A549细胞凋亡,其机制可能是RES和As2O3联合处理可降低GSH含量,诱导ROS产生与蓄积,使线粒体膜电位下降和细胞色素c释放入细胞质,活化Cas-3,最终诱导细胞凋亡。

Objective To explore the synergistically effects and mechanisms of resveratrol( RES) enhanced the oxidative stress and apoptotic cell death induced by As2O3( arsenic trioxide). Methods According to the result of MTT assay,human lung adenocarcinoma A549 cells were divided into four treatment groups as follow: control group,single RES or As2O3 treated group and the group treated with RES and As2O3.Then the differences of cell viability,colony formation,level of ROS,GSH content,mitochondrial membrane potential and apoptosis rate were compared with single or combined treatment. In addition,pre-treatment with L-buthionine sulfoximine( L-BSO),the inhibitory of GSH synthesis,was used to identify the role of GSH in synergisticallyapoptosis induced by RES and As2O3. Results The detected results demonstrated that RES could effectively inhibited the growth of A549 cells when its concentration above 20μmol /L,and inhibition effects was concentration dependent manner. The rate of colony formation, GSH content, mitochondrial membrane potential and apoptosis rate in combination group were significantly lower than that of single RES or As2O3 treatment group( P < 0. 05),whereas,RES markedly increased the level of ROS,the expression of cytochrome c and caspase 3 induced by As2O3. When pre-treatment with BSO before RES and As2O3 combination incubation,beside the apoptosis rate was increased from 30. 0% to77. 7%,the GSH content was sharply depleted while ROS massively accumulated in intracellular. Conclusion RES could significantly intensified the effects of As2O3 in inhibiting the proliferation,depleting GSH content,ROS accumulation,mitochondrial membrane potential decline and cytochrome c releasing,thus leading to cells apoptosis via Cas-3 activation in a mitochondria-dependent pathway.