线粒体裂变因子对OGD/R诱导的PC12细胞线粒体凋亡通路的影响
Effect of Mitochondrial Fission Factor on Mitochondrial Apoptosis Pathway in OGD/R-stimulated PC12 Cells
钱旭东 1李国芸 2卜一 1王红梅 1张硕 1窦志杰1
作者信息
- 1. 承德医学院附属医院神经内科,承德 067000
- 2. 承德医学院附属医院呼吸内科,承德 067000
- 折叠
摘要
目的 探究线粒体裂变因子(mitochondrial fission factor,MFF)在氧糖剥夺/复氧(oxygen glucose deprivation/re-oxygenation,OGD/R)处理的PC12细胞中的表达及其对线粒体凋亡通路的影响.方法 选用PC12细胞系构建体外缺血性神经元损伤模型.利用实时荧光定量PCR(qRT-PCR)和蛋白质免疫印迹(Western blot)实验明确MFF mRNA和蛋白在不同处理时间点的表达情况.通过RNA干扰技术沉默MFF基因表达,利用qRT-PCR、Western blot检测MFF mRNA和蛋白的表达情况;CCK-8检测沉默MFF对OGD/R处理的PC12细胞活力的影响;JC-1和试剂盒检测线粒体膜电位、腺昔三磷酸(adenosine triphosphate,ATP)和活性氧(reactive oxygen species,ROS)水平变化;Western blot检测细胞色素C(cytochrome C,Cyt-C)、Bax和Bcl2的表达;酶联免疫吸附法(ELISA)检测Caspase-3和Caspase-9的活性变化.结果 MFF在体外O GD/R诱导的PC12细胞中的mRNA和蛋白含量明显升高.MFF沉默增强了 O GD/R损伤后PC12细胞的活力,降低了细胞凋亡率,并促进Bcl2和抑制Bax蛋白的表达.敲低MFF明显提高了 OGD/R处理后PC12细胞的线粒体膜电位,促进ATP合成,降低ROS含量,抑制线粒体细胞色素C释放和Caspase-3和Caspase-9的活性.结论 沉默MFF基因显著提高了 OGD/R处理后PC12细胞的存活率,改善了线粒体功能.
Abstract
Objective To explore the expression level and effects of mitochondrial fission factor(MFF)on mitochondrial function and apoptosis of MFF in oxygen glucose deprivation/re-oxygenation(OGD/R)-stimulated PC12 cells.Methods PC12 cells were used to establish ischemic neuron injury model in vitro.First,quantitative real time-PCR(qRT-PCR)and Western blot assays were performed to determine the mRNA and protein levels of MFF at different time points after OGD/R treatment.Then,MFF was knocked down by RNA interference technology,the mRNA and protein levels were measured by qRT-PCR and Western blot assay.Cell counting kit-8(CCK-8)and TdT-mediated dUTP nick end labeling(TUNEL)assays were used to determine the effect of MFF silencing on cell survival.The levels of mitochondrial membrane potential,adenosine triphosphate(ATP)and reactive oxygen species(ROS)were detected by JC-1 and corresponding kits,respectively.The cellular localization of cytochrome C(Cyt-C)and protein levels of Bax and Bc12 were quantified by Western blotting.The activities of Caspase-3 and Caspase-9 were assessed by using enzyme linked immunosorbent assay(ELISA)assay.Results Both the mRNA and protein levels of MFF in OGD/R-stimulated PC12 cells were significantly increased.Further experimental data showed that silence of MFF obviously enhanced the cell viability and inhibited cell apoptosis in OGD/R-induced PC12 cells,along with in-creased Bcl2 protein level and reduced Bax protein level.After MFF knockdown,mitochondrial membrane potential and ATP synthesis were greatly elevated,while ROS generation,Cyt-C release and the activities of Caspase-3 and Caspase-9 were reduced in OGD/R-stimulated PC12 cells.Conclusion Silencing of MFF increases cell survival and improves mitochondrial function.
关键词
线粒体凋亡/氧糖剥夺/复氧模型/PC12细胞/线粒体裂变因子Key words
mitochondrial apoptosis/oxygen glucose deprivation/re-oxygenation/PC12 cell/mitochondrial fission factor引用本文复制引用
基金项目
河北省医学科学研究项目(20220005)
出版年
2024
NETL
NSTL
万方数据