国际麻醉学与复苏杂志   2023, Issue (4): 0-0
    
FOXO4通过抑制炎症反应减轻心肌细胞缺氧/复氧损伤
徐尚娴, 彭科, 单希胜, 孟晓文, 刘华跃, 稽富海, 陶文辉, 嵇富海1()
1.苏州大学附属第一医院
Overexpression of forkhead box protein O 4 protects H9c2 cardiomyocytes against hypoxia/reoxygenation injury through inhibiting inflammation
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摘要：

目的 探讨叉头框蛋白O（forkhead box protein O, FOXO）4在心肌细胞缺氧/复氧（hypoxia/reoxygenation， H/R）损伤中的作用及对炎症反应的影响。 方法 H9c2细胞体外培养至密度30%~50%，按随机数字表法分为6组（每组3孔）：对照组（Con组）、缺氧/复氧组（H/R组）、过表达FOXO4+H/R组（OF+H/R组）、过表达FOXO4阴性对照+H/R组（OF⁃NC+H/R组）、沉默FOXO4+H/R组（siFOXO4+H/R组）、沉默FOXO4阴性对照+H/R组（siRNA⁃NC+H/R组）。Con组细胞正常培养。H/R组细胞进行缺氧6 h复氧6 h处理。OF+H/R组、OF⁃NC+H/R组、siFOXO4+H/R组和siRNA⁃NC+H/R组细胞在质粒或RNAoligo转染5 h后正常培养48 h，再进行H/R处理。Cell Counting Kit‑8试剂盒检测细胞活性，乳酸脱氢酶（lactate dehydrogenase， LDH）试剂盒检测培养液中LDH含量，实时荧光定量聚合酶链反应（real⁃time fluorescence quantitative PCR， RT⁃qPCR）检测细胞内炎症因子TNF⁃α、IL⁃1β、IL⁃6 mRNA表达水平，Western blot检测细胞内FOXO4蛋白水平。 结果 与Con组比较，H/R组FOXO4蛋白水平下降（P<0.05），细胞活性降低（P<0.01），培养液中LDH水平升高（P<0.01），细胞中TNF⁃α、IL⁃1β、IL⁃6 mRNA表达水平升高（P<0.01）。与H/R组比较，OF+H/R组FOXO4蛋白水平升高（P<0.01），细胞活性升高（P<0.05），培养液中LDH水平下降（P<0.01），细胞中TNF⁃α、IL⁃1β、IL⁃6 mRNA表达水平降低（P<0.01或P<0.05）。与H/R组比较，siFOXO4+H/R组FOXO4蛋白水平降低（P<0.05），细胞活性降低（P<0.05），培养液中LDH水平升高（P<0.05），TNF⁃α、L⁃1β、IL⁃6 mRNA表达水平升高（P<0.05）。 结论 FOXO4可以减轻H9c2心肌细胞的H/R损伤，其机制与抑制炎症反应有关。
关键词： 叉头框蛋白O； H9c2细胞； 缺氧/复氧损伤； 炎症因子
Abstract:

Objective To investigate the effect of forkhead box protein O (FOXO) 4 on hypoxia/reoxygenation (H/R) injury and inflammation in cardiomyocytes. Methods H9c2 cells were cultured in vitro until the cells reached 30%‒50% confluence. According to the random number table method, the cells were randomly divided into six groups (n=3): a control (Con) group, a hypoxia/reoxygenation (H/R) group, an overexpression FOXO4+hypoxia/reoxygenation (OF+H/R) group, and an overexpression FOXO4 negative control+hypoxia/reoxygenation (OF‑NC+H/R) group, a siFOXO4+hypoxia/reoxygenation (siFOXO4+H/R) group and a siRNA negative control+hypoxia/reoxygenation (siRNA‑NC+H/R) group. The Con group were cultured without treatment. The H/R group was subjected to 6 h of hypoxia followed by reoxygenation for 6 h. The OF+H/R group, the OF‑NC+H/R group, the siFOXO4+H/R group and the siRNA‑NC+H/R group were transfected with plasmid or RNA oligo for 5 h followed by incubation with normal medium for 48 h, before H/R treatment. Then, the cell viability was detected by Cell Counting Kit‑8 assay. The content of lactate dehydrogenase (LDH) in the media was examined by LDH assay. The mRNA expression of inflammatory cytokines including tumor necrosis factor‑α (TNF‑α), interleukin (IL)‑1β, and IL‑6 was detected by real‑time fluorescence quantitative PCR (RT‑qPCR). The levels of FOXO4 were measured by Western blot. Results Compared with the Con group, the H/R group showed decreases in FOXO4 levels (P<0.05) and cell viability (P<0.01), as well as increases in LDH content released in the media (P<0.01) and the mRNA levels of TNF‑α, IL‑1β, and IL‑6 (P<0.01). Compared with the H/R group, the OF+H/R group presented increases in FOXO4 levels (P<0.01) and cell viability (P<0.05), as well as decreases in LDH content released in the media (P<0.01) and the mRNA levels of TNF‑α, IL‑1β, and IL‑6 (P<0.01 or P<0.05). Compared with the H/R group, the siFOXO4+H/R group showed decreases in FOXO4 levels (P<0.05) and cell viability (P<0.05), as well as increase in LDH content released in the media (P<0.05) and the mRNA levels of TNF‑α, IL‑1β, and IL‑6 (P<0.05). Conclusion FOXO4 alleviates H/R injury in H9c2 cardiomyocytes, which is associated with the inhibition of inflammation.
Key words: Forkhead box protein O; H9c2 cell; Hypoxia/reoxygenation injury; Inflammation