目的探究艾司氯胺酮是否通过下调Krüppel样因子6（KLF6）的表达，减轻缺氧/复氧（H/R）诱导的H9c2心肌细胞损伤。方法利用H9c2细胞建立H/R模型，设置对照组、模型组及不同质量浓度（1、2、4、8 μg·mL^-1）艾司氯胺酮组。通过转染sh-KLF6或oe-KLF6质粒调控KLF6表达，观察艾司氯胺酮对转染细胞的影响。采用CCK-8法检测细胞活性，流式细胞术检测细胞凋亡率，实时荧光定量PCR（qRT-PCR）和Western blotting检测KLF6基因及KLF6、Bax、GPX4、FTH1蛋白表达，试剂盒及ELISA法检测丙二醛（MDA）、活性氧（ROS）、超氧化物歧化酶（SOD）及炎症因子水平。结果与对照组相比，模型组细胞活性显著降低，KLF6表达上调，细胞凋亡率和Bcl-2相关X蛋白（Bax）蛋白表达明显升高，MDA和ROS含量显著升高，GPX4和FTH1蛋白表达及SOD含量降低，肿瘤坏死因子-α（TNF-α）、白细胞介素（IL）-6和IL-1β含量明显升高（P＜0.05）。与模型组相比，8 μg·mL^-1艾司氯胺酮处理显著提高了细胞活性，降低了细胞凋亡率、Bax蛋白表达、MDA和ROS含量，同时升高了GPX4、FTH1蛋白及SOD含量，并抑制了TNF-α、IL-6和IL-1β释放（P＜0.05）。在基因调控实验中，抑制KLF6表达（转染质粒sh-KLF6）可显著降低细胞凋亡率、Bax蛋白表达、MDA和ROS含量，升高GPX4、FTH1蛋白及SOD含量，并减少炎症因子释放，从而减轻H/R诱导的细胞损伤；而在艾司氯胺酮处理基础上过表达KLF6（转染质粒oe-KLF6）则逆转了艾司氯胺酮的保护作用，导致细胞凋亡率、Bax蛋白表达、MDA和ROS含量升高，GPX4、FTH1蛋白及SOD含量降低，炎症因子释放增加。结论艾司氯胺酮通过下调KLF6表达，抑制氧化应激、铁死亡及炎症反应，从而减轻H/R诱导的心肌细胞损伤。

Objective To investigate whether esketamine (ESK) alleviates hypoxia/reoxygenation (H/R)-induced injury in H9c2 cardiomyocytes by downregulating the expression of Krüppel-like factor 6 (KLF6). Methods An H/R model was established using H9c2 cells, with untreated cells serving as the control group. The model group was treated with different concentrations of ESK (1, 2, 4, and 8 μg·mL^-1). To regulate KLF6 expression, plasmids sh-KLF6 (silencing) or oe-KLF6 (overexpression) were transfected into the cells to observe the effects of ESK on transfected cells. Cell viability was assessed using the CCK-8 assay, and apoptosis rates were detected by flow cytometry. The expression of KLF6 gene and proteins (KLF6, Bax, GPX4, FTH1) was measured by quantitative realtime PCR (qRT-PCR) and Western blotting. Levels of malondialdehyde (MDA), reactive oxygen species (ROS), superoxide dismutase (SOD), and inflammatory factors [tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-1β] were detected using kits and ELISA. Results Compared with the control group, the model group showed significantly reduced cell viability, upregulated KLF6 expression, increased apoptosis rate and Bax protein expression, elevated MDA and ROS levels, decreased GPX4 and FTH1 protein expression and SOD content, and increased levels of TNF-α, IL-6, and IL-1β (P < 0.05). Compared with the model group, treatment with 8 μg·mL^-1 ESK significantly improved cell viability, reduced apoptosis rate, Bax protein expression, MDA, and ROS levels, while increasing GPX4, FTH1 protein expression, and SOD content, and inhibiting the release of TNF-α, IL-6, and IL-1β (P < 0.05). In the gene regulation experiments, silencing KLF6 (transfection with sh-KLF6 plasmid) significantly reduced apoptosis rate, Bax protein expression, MDA and ROS levels, and increased GPX4, FTH1 protein expression, and SOD content, thereby alleviating H/R-induced cell injury. Conversely, overexpressing KLF6 (transfection with oe-KLF6 plasmid) in the presence of ESK reversed its protective effects, leading to increased apoptosis rate, Bax protein expression, MDA and ROS levels, decreased GPX4, FTH1 protein expression, and SOD content, and increased inflammatory factor release. Conclusion Esketamine alleviates H/R-induced cardiomyocyte injury by downregulating KLF6 expression, thereby inhibiting oxidative stress, ferroptosis, and inflammatory responses.

R972

河南省医学科技攻关计划项目（ LHG120230886）