Inhibition of VDAC1 prevents Ca2+-mediated oxidative stress and apoptosis induced by 5-aminolevulinic acid mediated sonodynamic therapy in THP-1 macrophages
Inhibition of VDAC1 prevents Ca2+-mediated oxidative stress and apoptosis induced by 5-aminolevulinic acid mediated sonodynamic therapy in THP-1 macrophages
复制标题
抑制 VDAC1 可防止 THP-1 巨噬细胞中由 5-氨基乙酰丙酸介导的声动力疗法诱导的 Ca2 介导的氧化应激和细胞凋亡
DOI：
10.1007/s10495-014-1045-5
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发表时间：
2014-12-01
期刊：
影响因子：
7.2
通讯作者：
Tian, Ye
中科院分区：
文献类型：
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作者：
Chen, Haibo;Gao, Weiwei;Tian, Ye
已知超声与来源于5-氨基乙酰丙酸（ALA-SDT）的内源性原卟啉IX组合诱导多种癌细胞和巨噬细胞的凋亡。巨噬细胞在斑块中的持续滞留与动脉粥样硬化的病理生理学和进展有关。本文研究了电压依赖性阴离子通道1（VDAC 1）的抑制对ALA-SDT诱导的THP-1巨噬细胞凋亡的影响。用VDAC 1抑制剂4，4 '-二异硫氰基芪-2，2'-二磺酸（DIDS）二钠盐预处理细胞1小时或通过小干扰RNA下调VDAC 1表达并暴露于超声。MTT法检测细胞活力，Hoechst 33342/碘化丙啶染色和流式细胞术检测细胞凋亡沿着坏死。通过共聚焦显微镜和Western印迹评估细胞色素c释放水平。通过Western印迹分析全长半胱天冬酶、半胱天冬酶活化和VDAC同种型的水平。用荧光探针测定细胞内活性氧产生、线粒体膜通透性和细胞内Ca 2 + [Ca 2 +]（i）水平。我们证实DIDS对VDAC 1的药理学抑制显著阻止ALA-SDT诱导的THP-1巨噬细胞的细胞凋亡。此外，DIDS显着抑制细胞内ROS的产生和凋亡的生化变化，如线粒体内膜透化，线粒体膜电位的损失，细胞色素c的释放和激活的caspase-3和caspase-9。ALA-SDT可升高[Ca ~（2+）]（i）水平，DIDS可显著降低[Ca ~（2+）]（i）水平。钙离子螯合剂BAPTA-AM能显著抑制细胞内ROS的产生和细胞凋亡。有趣的是，ALA治疗显着增加VDAC 1蛋白水平排他，和VDAC 1表达的下调特异性siRNA也显着消除细胞凋亡。总之，这些结果表明VDAC 1在ALA-SDT诱导的THP-1巨噬细胞凋亡中起着至关重要的作用，靶向VDAC 1是调节巨噬细胞凋亡的潜在方式，这一发现可能与动脉粥样硬化的治疗策略相关。
Ultrasound combined with endogenous protoporphyrin IX derived from 5-aminolevulinic acid (ALA-SDT) is known to induce apoptosis in multiple cancer cells and macrophages. Persistent retention of macrophages in the plaque has been implicated in the pathophysiology and progression of atherosclerosis. Here we investigated the effects of inhibition of voltage-dependent anion channel 1 (VDAC1) on ALA-SDT-induced THP-1 macrophages apoptosis. Cells were pre-treated with VDAC1 inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) disodium salt for 1 h or downregulated VDAC1 expression by small interfering RNA and exposed to ultrasound. Cell viability was assessed by MTT assay, and cell apoptosis along with necrosis was evaluated by Hoechst 33342/propidium iodide staining and flow cytometry. Levels of cytochrome c release was assessed by confocal microscope and Western blot. The levels of full length caspases, caspase activation, and VDAC isoforms were analyzed by Western blot. Intracellular reactive oxygen species generation, mitochondrial membrane permeability, and intracellular Ca2+ [Ca2+](i) levels were measured with fluorescent probes. We confirmed that the pharmacological inhibition of VDAC1 by DIDS notably prevented ALA-SDT-induced cell apoptosis in THP-1 macrophages. Additionally, DIDS significantly inhibited intracellular ROS generation and apoptotic biochemical changes such as inner mitochondrial membrane permeabilization, loss of mitochondrial membrane potential, cytochrome c release and activation of caspase-3 and caspase-9. Moreover, ALA-SDT elevated the [Ca2+](i) levels and it was also notably reduced by DIDS. Furthermore, both of intracellular ROS generation and cell apoptosis were predominately inhibited by Ca2+ chelating reagent BAPTA-AM. Intriguingly, ALA-treatment markedly augmented VDAC1 protein levels exclusively, and the downregulation of VDAC1 expression by specific siRNA also significantly abolished cell apoptosis. Altogether, these results suggest that VDAC1 plays a crucial role in ALA-SDT-induced THP-1 macrophages apoptosis, and targeting VDAC1 is a potential way regulating macrophages apoptosis, a finding that may be relevant to therapeutic strategies against atherosclerosis.