Yu J, Li R, Xia T, Wang J, Jin J, Yuan M, Li M
Medical School of Chinese PLA, Beijing 100853, China.
Department of Clinical Laboratory Medicine, First Medical Center, Chinese PLA General Hospital, Beijing 100853, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2024 Jan 20;44(1):25-35. doi: 10.12122/j.issn.1673-4254.2024.01.04.
To elucidate the role of programmed cell death factor 4 (PDCD4) in mitochondrial dysfunction caused by sepsis-related vascular endothelial damage.
Cultured human umbilical vein endothelial cells (HUVECs) and mouse vascular endothelial cells (C166 cells) were transfected with a small interfering RNA targeting PDCD4 followed by treatment with lipopolysaccharide (LPS) alone or in combination with carbonyl cyanide 3-chlorophenylhydrazone (FCCP). The proteomic changes in the cells after PDCD4 knockdown were analyzed using LC-MS/MS technique. The mRNA expressions of PDCD4 and the genes associated with cell inflammation and apoptosis were detected with RT-PCR, and the expressions of FIS1, DRP1 and OPA1 proteins key to mitochondrial fission and fusion were determined using Western blotting. JC-1 and MitoSOX fluorescent probes were used to observe the changes in mitochondrial membrane potential and mitochondrial reactive oxygen species levels under by a laser confocal microscope.
LPS stimulation of the cells significantly increased the mRNA expressions of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and monocyte chemoattractant protein 1 (MCP1) and enhanced the cellular expression of PDCD4 ( < 0.05). Proteomic analysis suggested a correlation between PDCD4 knockdown and changes in mitochondrial dynamics in the cells. LPS treatment significantly increased the expressions of mitochondrial fission proteins FIS1 and DRP1 and lowered the expression of the fusion protein OPA1 in the cells ( < 0.05), causing also mitochondrial oxidative stress and reduction of the mitochondrial membrane potential ( < 0.05). In HUVECs, treatment with FCCP significantly attenuated the protective effect of PDCD4 knockdown, which inhibited LPS-induced inflammation and oxidative stress and restored the balance between mitochondrial fission and fusion.
PDCD4 knockdown protects vascular endothelial cells against LPS-induced damages by repressing mitochondrial fission and oxidative stress, promoting mitochondrial fusion, and maintaining normal mitochondrial function.
阐明程序性细胞死亡因子4（PDCD4）在脓毒症相关血管内皮损伤所致线粒体功能障碍中的作用。
用靶向PDCD4的小干扰RNA转染培养的人脐静脉内皮细胞（HUVECs）和小鼠血管内皮细胞（C166细胞），然后单独用脂多糖（LPS）或与羰基氰3-氯苯腙（FCCP）联合处理。使用液相色谱-串联质谱（LC-MS/MS）技术分析PDCD4基因敲低后细胞中的蛋白质组变化。用逆转录聚合酶链反应（RT-PCR）检测PDCD4以及与细胞炎症和凋亡相关基因的mRNA表达，并用蛋白质免疫印迹法测定线粒体分裂和融合关键蛋白FIS1、动力相关蛋白1（DRP1）和视神经萎缩蛋白1（OPA1）的表达。使用JC-1和MitoSOX荧光探针通过激光共聚焦显微镜观察线粒体膜电位和线粒体活性氧水平的变化。
LPS刺激细胞显著增加白细胞介素-6（IL-6）、肿瘤坏死因子-α（TNF-α）和单核细胞趋化蛋白1（MCP1）的mRNA表达，并增强细胞中PDCD4的表达（P<0.05）。蛋白质组学分析表明，PDCD4基因敲低与细胞中线粒体动力学变化相关。LPS处理显著增加细胞中线粒体分裂蛋白FIS1和DRP1的表达，并降低融合蛋白OPA1的表达（P<0.05），还导致线粒体氧化应激和线粒体膜电位降低（P<0.05）。在HUVECs中，FCCP处理显著减弱了PDCD4基因敲低的保护作用，PDCD4基因敲低可抑制LPS诱导的炎症和氧化应激，并恢复线粒体分裂与融合之间的平衡。
PDCD4基因敲低通过抑制线粒体分裂和氧化应激、促进线粒体融合以及维持正常线粒体功能，保护血管内皮细胞免受LPS诱导的损伤。
