Department of Emergency Medicine, Central People's Hospital of Zhanjiang, Zhanjiang, Guangdong, China.
J Physiol Investig. 2024 Jul 1;67(4):174-186. doi: 10.4103/ejpi.EJPI-D-24-00021. Epub 2024 Aug 8.
Sepsis is a syndrome of systemic inflammatory response resulting from infection, which can lead to severe lung injury. Histone deacetylase 4 (HDAC4) is a key protein known to regulate a wide range of cellular processes. This study was designed to investigate the role of HDAC4 in lipopolysaccharide (LPS)-induced alveolar epithelial cell injury as well as to disclose its potential molecular mechanisms. The alveolar epithelial cell injury model was established by inducing A549 cells with LPS. A549 cell viability was detected by cell counting kit-8 assay and the transfection efficiency of small interfering RNA targeting HDAC4 was appraised utilizing Western blot. The levels of inflammatory cytokines and oxidative stress markers were detected using corresponding assay kits. Dichloro-dihydro-fluorescein diacetate assay was used for the measurement of reactive oxygen species (ROS) content. Flow cytometry, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolyl-carbocyanine iodide-1 staining, adenosine triphosphate (ATP) assay kits, and MitoSOX Red assay kits were employed to estimate cell apoptosis, mitochondrial membrane potential, ATP level, and mitochondrial ROS level, respectively. The oxygen consumption rate of A549 cells was evaluated with XF96 extracellular flux analyzer. Western blot was applied for the evaluation of HDAC4, apoptosis- and c-Jun N-terminal kinase (JNK)/activating protein-1 (AP-1) signaling pathway-related proteins. HDAC4 expression was found to be increased in LPS-induced A549 cells and HDAC4 silence inhibited inflammatory damage, repressed oxidative stress, alleviated cell apoptosis, improved mitochondrial function, and blocked JNK/AP-1 signaling in A549 cells stimulated by LPS, which were all reversed by JNK activator anisomycin. Collectively, the interference with HDAC4 could ameliorate LPS-induced alveolar epithelial cell injury, and such protective effect may be potentially mediated through the JNK/AP-1 signaling pathway.
脓毒症是一种由感染引起的全身炎症反应综合征，可导致严重的肺损伤。组蛋白去乙酰化酶 4（HDAC4）是一种已知调节广泛细胞过程的关键蛋白。本研究旨在探讨 HDAC4 在脂多糖（LPS）诱导的肺泡上皮细胞损伤中的作用，并揭示其潜在的分子机制。通过诱导 A549 细胞用 LPS 建立肺泡上皮细胞损伤模型。通过细胞计数试剂盒-8 测定 A549 细胞活力，并通过 Western blot 评估靶向 HDAC4 的小干扰 RNA 的转染效率。使用相应的测定试剂盒检测炎症细胞因子和氧化应激标志物的水平。二氯二氢荧光素二乙酸酯测定法用于测定活性氧（ROS）含量。流式细胞术、5,5',6,6'-四氯-1,1',3,3'-四乙基苯并咪唑基-碳氰化碘-1 染色、三磷酸腺苷（ATP）测定试剂盒和 MitoSOX Red 测定试剂盒分别用于估计细胞凋亡、线粒体膜电位、ATP 水平和线粒体 ROS 水平。使用 XF96 细胞外通量分析仪评估 A549 细胞的耗氧率。Western blot 用于评估 HDAC4、凋亡和 c-Jun N-末端激酶（JNK）/激活蛋白-1（AP-1）信号通路相关蛋白。LPS 诱导的 A549 细胞中 HDAC4 表达增加，HDAC4 沉默抑制炎症损伤，抑制氧化应激，减轻细胞凋亡，改善线粒体功能，并阻断 LPS 刺激的 A549 细胞中 JNK/AP-1 信号通路，这些作用均被 JNK 激活剂 anisomycin 逆转。总之，干扰 HDAC4 可以改善 LPS 诱导的肺泡上皮细胞损伤，这种保护作用可能是通过 JNK/AP-1 信号通路介导的。
