摘要
旨在研究电压依赖性阴离子通道(voltage dependent anion channel1,VDAC1)对卡介苗(Bacillus Calmette-Guerin, BCG)诱导的小鼠巨噬细胞RAW264.7凋亡的调控作用及其机制。设计并合成VDAC1的小干扰RNA(si-VDAC1)转染小鼠巨噬细胞RAW264.7后,再用BCG感染。后续采用MTT法检测RAW264.7细胞存活率,流式细胞仪检测细胞凋亡率、线粒体膜电位及细胞内ROS水平,进而通过实时荧光定量PCR和Western blot检测凋亡相关因子Cyt C、Caspase-3、PARP1、Caspase-9基因(mRNA)及其蛋白的表达。随后,通过使用VDAC1寡聚抑制剂VBIT-4对RAW264.7进行孵育2 h后感染BCG,并采用免疫荧光染色技术观察Caspase-3和Western blot检测凋亡相关因子Cyt C、Cleaved-Caspase 3、Cleaved-PARP1、Cleaved-Caspase 9的含量变化。BCG感染巨噬细胞12 h后,与对照相比,BCG感染后小鼠巨噬细胞存活率约下降至50%,而凋亡率显著上调到52.12%(P<0.001),线粒体膜电位降低(P<0.001),细胞内ROS水平升高(P<0.001),并且凋亡关键调控因子Cyt C、Caspase-3、PARP1、Caspase-9的mRNA及蛋白表达水平显著上调(P<0.001);当用si-VDAC1和BCG共同处理小鼠巨噬细胞时,细胞存活率、细胞凋亡率、线粒体膜电位水平、细胞内ROS水平及凋亡关键调控因子均显著得到了恢复(P<0.001);使用VDAC1寡聚抑制剂VBIT-4处理后同BCG处理组相比,VBIT-4显著抑制了VDAC1寡聚体的形成和凋亡相关因子Cleaved-Caspase 3、Cleaved-PARP1、Cleaved-Caspase 9剪切体的含量(P<0.001)。结果表明,VDAC1可通过寡聚形成大的通道影响线粒体外膜通透性,通过Cyt C等凋亡蛋白释放的途径参与调控BCG感染RAW264.7诱导的细胞凋亡。
The objective of this study was to investigate the regulatory effect and its mechanism of VDAC1 on the apoptosis of RAW264.7 induced by Bacillus calmette-Guerin(BCG).Small interfering RNAs(si-VDAC1) of VDAC1 were designed and synthesized and transfected into RAW264.7,which was then infected with BCG(MOI 1∶10).Subsequently, MTT assay was used to detect survival rate of RAW264.7 cell, and flow cytometry was used to detect cell apoptosis rate, mitochondrial membrane potential and intracellular ROS levels.Then the expression of apoptosis-related factors CytC,Caspase-3,PARP1,Caspase-9 were detected by real-time quantitative PCR and Western blot, respectively.Meanwhile, RAW264.7 was infected with BCG after two hour-incubation with VDAC1 oligomer inhibitor VBIT-4.Immunofluorescent staining technique was use to observe Caspase-3,and Western blot was used to detect apoptosis-related factors CytC,cleaved Caspase-3,cleaved PARP1,cleaved Caspase-9.After BCG infection for 12 h, compared with the control, the survival rate of RAW264.7 infected with BCG decreased to 50%,while the apoptosis rate was significantly increased to 52.12%(P<0.001),mitochondrial membrane potential decreased(P<0.001),intracellular ROS levels increased(P<0.001),and the mRNA and protein expressions of key apoptosis regulators CytC,Caspase-3,PARP1,Caspase-9were significantly upregulated(P<0.001).When RAW264.7was treated with si-VDAC1and BCG,the survival rate,apoptosis rate,mitochondrial membrane potential level,intracellular ROS level and key regulatory factors of apoptosis were significantly restored(P<0.001).In addition,VBIT-4significantly inhibited VDAC1 oligomer formation and contents of cleaved Caspase-3,cleaved PARP1,cleaved Caspase-9cleavages(P<0.001)compared with BCG treatment group.The study demonstrated VDAC1can affect the permeability of mitochondrial outer membrane by forming large channels through oligomerization,and VDAC1can also participate in regulating apoptosis of RAW264.7inducted by BCG infection through the release of apoptotic protei
作者
宋阿北
李瑞乾
缪西鹏
谢玉杰
高瑞
许立华
SONG Abei;LI Ruiqian;MIAO Xipeng;XIE Yujie;GAO Rui;XU Lihua(School of Agriculture,Ningria University,Yinchuan 750021,China)
出处
《中国兽医学报》
CAS
CSCD
北大核心
2022年第9期1878-1888,共11页
Chinese Journal of Veterinary Science
基金
国家自然科学基金资助项目(31760722,31560687)。
