南方医科大学学报

南方医科大学学报 ›› 2024, Vol. 44 ›› Issue (1): 25-35.doi: 10.12122/j.issn.1673-4254.2024.01.04

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沉默PDCD4表达可减轻脓毒症血管内皮细胞损伤：基于改善线粒体动力学

于佳池，李芮冰，夏 天，王佳楠，金家丞，袁漫秋，李绵洋   

  1. 解放军医学院，北京 100853；中国人民解放军总医院第一医学中心检验科，北京 100853；南开大学医学院，天津 300071
  • 发布日期:2024-01-24

PDCD4 knockdown ameliorates lipopolysaccharide- induced endothelial cell damage by improving mitochondrial dynamics

YU Jiachi, LI Ruibing, XIA Tian, WANG Jianan, JIN Jiacheng, YUAN Manqiu, LI Mianyang   

  1. Medical School of Chinese PLA, Beijing 100853, China; Department of Clinical Laboratory Medicine, First Medical Center, Chinese PLA General Hospital, Beijing 100853, China; Nankai University School of Medicine, Nankai University, Tianjin 300071 China
  • Published:2024-01-24

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摘要： 目的 探究程序性细胞死亡因子4（PDCD4）在缓解脓毒症血管内皮细胞损伤中的作用机制。方法 通过体外分别培养人脐静脉内皮细胞（HUVEC）和小鼠血管内皮细胞（C166）并给予脂多糖（LPS）处理，建立脓毒症血管内皮损伤细胞模型。分别设立对照组（NC）、LPS诱导组（NC+LPS）、si-PDCD4转染组（si-PDCD4）、si-PDCD4转染后LPS诱导组（si-PDCD4+LPS）、si-PDCD4转染后LPS诱导加线粒体分裂激动剂（FCCP）组（si-PDCD4+LPS+FCCP）。通过LC-MS/MS技术，分析PDCD4敲低前后蛋白组学变化。通过RT-PCR检测对照组和LPS组转染前后PDCD4、细胞炎症、凋亡以及线粒体分裂融合相关基因的mRNA表达量的变化；蛋白质印迹法检测线粒体分裂融合关键蛋白FIS1、DRP1和OPA1的表达水平；通过激光共聚焦技术观察JC-1、MitoSOX探针荧光强度以检测线粒体膜电位和线粒体活性氧变化。结果 与对照组相比，LPS组炎症相关指标白介素-6（IL-6）、肿瘤坏死因子（TNF-α）和单核细胞趋化蛋白1（MCP1）基因的mRNA表达升高（P<0.05）；PDCD4在脓毒症血管内皮细胞损伤中高表达（P<0.05）；蛋白组学分析结果表明，PDCD4敲低与线粒体动力学存在相关性；Western blot结果表明与对照组相比，LPS组诱导线粒体分裂蛋白FIS1、DRP1表达增加，融合蛋白OPA1减低（P<0.05）；MitoSOX和JC-1荧光探针结果提示LPS诱导下内皮细胞线粒体发生氧化应激，膜电位显著降低（P<0.05），敲减组的氧化应激和线粒体膜电位有所缓解。PDCD4敲减后的保护效应可被线粒体分裂激动剂FCCP逆转。敲减PDCD4后能够抑制LPS所引起的炎症和氧化应激水平，线粒体分裂和融合指标恢复平衡。结论 沉默PDCD4基因通过调控线粒体的动力学维持线粒体功能正常，减轻氧化应激，从而有利于缓解脓毒症血管内皮细胞损伤。

关键词: 脓毒症；血管内皮细胞；线粒体动力学；PDCD4；炎症

Abstract: Objective To elucidate the role of programmed cell death factor 4 (PDCD4) in mitochondrial dysfunction caused by sepsis-related vascular endothelial damage. Methods 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. Results 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 (P<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 (P<0.05), causing also mitochondrial oxidative stress and reduction of the mitochondrial membrane potential (P<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. Conclusion 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.

Key words: sepsis; vascular endothelial cells; mitochondrial dynamics; PDCD4; inflammation