目的 探讨粉尘螨过敏原1（Der f 1）引发人气道上皮细胞炎症反应的作用机制。方法 将人支气管上皮细胞系BEAS-2B接种至96孔板中（5 × 10³个/孔），分为Der f 1组、Der f 1 + 铁抑素-1（Fer-1）组和阴性对照组，Der f 1组加40 μl Der f 1（100 μg/ml），Der f 1 + Fer-1组在加Der f 1前2 h加4 μl Fer-1（1 000 μmol/ml），阴性对照组加等量无酶水，培养24 h后，采用细胞计数试剂盒-8（CCK-8）进行细胞计数，ELISA测定3组细胞培养上清中的细胞因子白细胞介素-6（IL-6）、人胸腺基质淋巴细胞生成素（TSLP）、IL-33含量。将BEAS-2B细胞接种至6孔板中（5 × 10⁵个/孔），分为Der f 1组、Der f 1 + Fer-1组和阴性对照组，提取各组细胞总蛋白，十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）检测蛋白表达情况，以谷胱甘肽过氧化酶4（GPX4）抗体（1∶2 000）、长链脂酰辅酶A合成酶4（ACSL4）抗体（1∶2 000）为一抗，辣根过氧化物酶（HRP）标记的山羊抗兔IgG抗体为二抗（1∶10 000），蛋白质免疫印迹（Western blotting）检测细胞中GPX4和ACSL4蛋白表达水平。将BEAS-2B细胞接种至24孔板中（5 × 10⁴个/孔），分为Der f 1组、Der f 1 + Fer-1组和阴性对照组，荧光显微镜下观察各组细胞中亚铁离子（Fe²⁺）、活性氧（ROS）荧光强度，以及脂质过氧化物（LPO）染色后红色荧光与绿色荧光强度，并计算比值。将BEAS-2B细胞接种至6孔板中（5 × 10⁵个/孔），分为Der f 1组、Der f 1 + Fer-1组和阴性对照组，比色法检测各组细胞丙二醛（MDA）含量。将BEAS-2B细胞接种至6孔板中（5 × 10⁶个/孔），分为Der f 1组、Der f 1 + Fer-1组和阴性对照组，透射电子显微镜下观察各组细胞线粒体形态。采用GraphPad Prism 8.0.1软件进行统计学分析，组间比较采用单因素方差分析。结果 细胞活力检测结果显示，Der f 1组细胞活力为0.79 ± 0.03，低于阴性对照组（1.07 ± 0.08）（t = 6.663，P < 0.01）；Der f 1 + Fer-1组细胞活力为0.94 ± 0.03，高于Der f 1组（t = 6.694，P < 0.01）。ELISA结果显示，Der f 1组上清中IL-6含量为（117.30 ± 21.32）pg/ml，高于阴性对照组（50.07 ± 5.82）pg/ml（t = 5.279，P < 0.01），Der f 1 + Fer-1组上清中IL-6含量为（50.31 ± 12.28）pg/ml，低于Der f 1组（t = 4.721，P < 0.01）；Der f 1组上清中TSLP含量为（10.00 ± 2.37）pg/ml，高于阴性对照组（3.81 ± 0.92）pg/ml（t = 4.223，P < 0.05），Der f 1 + Fer-1组上清中TSLP含量为（4.41 ± 1.59）pg/ml，低于Der f 1组（t = 3.399，P < 0.05）；Der f 1组上清中IL-33含量为（24.18 ± 2.53）pg/ml，高于阴性对照组（12.09 ± 2.08）pg/ml（t = 6.39，P < 0.01），Der f 1 + Fer-1组上清中IL-33含量为（15.76 ± 1.39）pg/ml，低于Der f 1组（t = 5.045，P < 0.01）。Western blotting结果显示，Der f 1组GPX4蛋白相对表达量为0.38 ± 0.08，低于阴性对照组的1.00 ± 0.00（t = 13.21，P < 0.01），Der f 1 + Fer-1组相对表达量为0.72 ± 0.08，高于Der f 1组（t = 5.122，P < 0.01）；Der f 1组ACSL4蛋白相对表达量为1.74 ± 0.12，高于阴性对照组的1.00 ± 0.00（t = 10.65，P < 0.01）；Der f 1 + Fer-1组相对表达量为1.33 ± 0.13，低于Der f 1组（t = 4.094，P < 0.05）。荧光显微镜观察结果显示，Der f 1组细胞内Fe²⁺荧光强度为37.19 ± 5.42，高于阴性对照组的11.93 ± 0.54（t = 8.035，P < 0.01）；Der f 1 + Fer-1组为13.16 ± 1.89，低于Der f 1组（t = 7.253，P < 0.01）。Der f 1组细胞内ROS荧光强度为13.48 ± 3.36，高于阴性对照组的6.80 ± 0.60（t = 3.386，P < 0.05）；Der f 1 + Fer-1组ROS荧光强度为7.35 ± 0.42，低于Der f 1组（t = 3.134，P < 0.05）。Der f 1组细胞内LPO程度为3.91 ± 1.65，高于阴性对照组的0.31 ± 0.13（t = 3.775，P < 0.05）；Der f 1 + Fer-1组LPO程度为0.80 ± 0.15，低于Der f 1组（t = 3.262，P < 0.05）。比色法结果显示，Der f 1组细胞内MDA含量为（5.57 ± 1.66）nmol/mg，高于阴性对照组的（2.18 ± 0.51）nmol/mg（t = 3.393，P < 0.05），Der f 1 + Fer-1组MDA含量为（2.24 ± 0.38）nmol/mg，低于Der f 1组（t = 3.4，P < 0.05）。透射电子显微镜结果显示，阴性对照组细胞线粒体状态良好，线粒体边界清晰且线粒体嵴较多；Der f 1组与阴性对照组相比细胞线粒体体积减小、膜增厚并且线粒体嵴减少甚至消失；Der f 1 + Fer-1组与Der f 1组相比，细胞线粒体形态改变受到抑制，线粒体体积减小、膜增厚和线粒体嵴减少等形态变化减轻。结论 Der f 1可通过扰乱铁代谢、抑制抗氧化防御及促进脂质过氧化，诱导人气道上皮细胞发生铁死亡，进而增强IL-6、TSLP和IL-33等炎症因子的释放，促进气道炎症反应。

Objective To decipher the mechanisms underlying human airway epithelial cell inflammation induced by the major allergen Der f 1 of dust mite. Methdos BEAS-2B cells were seeded onto 96-well plates at a density of 5 × 10³ cells per well and divided into three groups, including the Der f 1 group, Der f 1 + ferrostatin-1 (Fer-1) group, and negative control group. Cells in the Der f 1 group were treated with 40 μl of Der f 1 (100 μg/ml), and cells in the Der f 1 + Fer-1 group was pretreated with 4 μl Fer-1 (1 000 μmol/ml) 2 hours prior to Der f 1 treatment, while cells in the negative control group were given an equivalent volume of nuclease-free water. After 24 hours of incubation, cell viability was assessed in the Der f 1 group, Der f 1 + Fer-1 group and negative control group using the CCK-8 assay, and concentrations of interleukin-6 (IL-6), thymic stromal lymphopoietin (TSLP), and IL-33 were measured in the cell culture supernatants with enzyme-linked immunosorbent assay (ELISA). For Western blotting assay, BEAS-2B cells were seeded onto 6-well plates at a density of 5 × 10⁵ cells per well, and assigned into three groups, including the Der f 1 group, Der f 1 + Fer-1 group, and negative control group. Total protein was extracted from BEAS-2B cells, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and probed with primary antibodies against glutathione peroxidase 4 (GPX4, 1︰2 000 dilution) and acyl-CoA synthetase long-chain family member 4 (ACSL4, 1︰2 000 dilution), followed by incubation into horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibody (1︰10 000 dilution). The GPX4 and ACSL4 protein expression was determined in cells using Western blotting assay. To assess ferroptosis-related indicators, BEAS-2B cells were seeded onto 24-well plates at a density of 5 × 10⁴ cells per well and assigned into three groups, including the Der f 1 group, Der f 1 + Fer-1 group, and negative control group. Intracellular fluorescence intensity of Fe²⁺ and reactive oxygen species (ROS) were measured under a fluorescence microscope, and the ratio of red to green fluorescence intensity was estimated following lipid peroxidation (LPO) staining. For colorimetric assay, BEAS-2B cells were seeded onto 6-well plates at a density of 5 × 10⁵ cells per well and assigned into three groups, including the Der f 1 group, Der f 1 + Fer-1 group, and negative control group, and malondialdehyde (MDA) content was measured using a colorimetric assay. In addition, BEAS-2B cells were seeded onto 6-well plates at a density of 5 × 10⁶ cells per well and assigned into three groups, including the Der f 1 group, Der f 1 + Fer-1 group, and negative control group, and mitochondrial morphology was examined under a transmission electron microscope. All statistical analyses were performed using the software GraphPad Prism 8.0.1, and differences of means among three groups were tested for statistical significance with one-way analysis of variance (ANOVA). Results The cell viability was significantly lower in the Der f 1 group (0.79 ± 0.03) than in the negative control (1.07 ± 0.08) (t = 6.663, P < 0.01), and co-treatment with Fer-1 restored cell viability to 0.94 ± 0.03 (t = 6.694, P < 0.01). ELISA showed that the IL-6 concentration in the supernatant of the Der f 1 group was (117.30 ± 21.32) pg/ml, which was higher than that of the negative control group (50.07 ± 5.82) pg/ml (t = 5.279, P < 0.01). The IL-6 concentration in the supernatant of the Der f 1 + Fer-1 group was (50.31 ± 12.28) pg/ml, which was lower than that of the Der f 1 group (t = 4.721, P < 0.01). The TSLP concentration in the supernatant of the Der f 1 group was (10.00 ± 2.37) pg/ml, which was higher than that of the negative control group (3.81 ± 0.92) pg/ml (t = 4.223, P < 0.05). The TSLP concentration in the supernatant of the Der f 1 + Fer-1 group was (4.41 ± 1.59) pg/ml, which was lower than that of the Der f 1 group (t = 3.399, P < 0.05). The IL-33 concentration in the supernatant of the Der f 1 group was (24.18 ± 2.53) pg/ml, which was higher than that of the negative control group (12.09 ± 2.08) pg/ml (t = 6.39, P < 0.01). The IL-33 concentration in the supernatant of the Der f 1 + Fer-1 group was (15.76 ± 1.39) pg/ml, which was lower than that of the Der f 1 group (t = 5.045, P < 0.01). Western blotting determined lower relative GPX4 protein expression in the Der f 1 group (0.38 ± 0.08) than in the negative control group (1.00 ± 0.00) (t = 13.21, P < 0.01), and higher GPX4 protein expression in the Der f 1 + Fer-1 group (0.72 ± 0.08) than in the Der f 1 group (t = 5.122, P < 0.01). Conversely, ACSL4 protein expression was upregulated in the Der f 1 group (1.74 ± 0.12 vs. 1.00 ± 0.00; t = 10.65, P < 0.01) and reduced by Fer-1 treatment (1.33 ± 0.13, t = 4.094, P < 0.05). Fluorescence microscopy showed increased intracellular fluorescence intensity of Fe²⁺ in the Der f 1 group (37.19 ± 5.42 vs. 11.93 ± 0.54; t = 8.035, P < 0.01), which was significantly lowered by Fer-1 treatment (13.16 ± 1.89, t = 7.253, P < 0.01). The intracellular ROS fluorescence intensity in the Der f 1 group was 13.48 ± 3.36, which was higher than that in the negative control group (6.80 ± 0.60, t = 3.386, P < 0.05). The fluorescence intensity in the Der f 1 + Fer-1 group was 7.35 ± 0.42, which was lower than that in the Der f 1 group (t = 3.134, P < 0.05). The intracellular LPO fluorescence intensity in the Der f 1 group was 3.91 ± 1.65, which was higher than that in the negative control group (0.31 ± 0.13, t = 3.775, P < 0.05). The LPO fluorescence intensity in the Der f 1 + Fer-1 group was 0.80 ± 0.15, which was lower than that in the Der f 1 group (t = 3.262, P < 0.05). The colorimetric assay results showed that the intracellular MDA content in the Der f 1 group was (5.57 ± 1.66) nmol/mg, which was higher than that in the negative control group (2.18 ± 0.51) nmol/mg (t = 3.393, P < 0.05). The MDA content in the Der f 1 + Fer-1 group was (2.24 ± 0.38) nmol/mg, which was lower than that in the Der f 1 group (t = 3.4, P < 0.05). Transmission electron microscopy displayed well mitochondrial status, clear mitochondrial boundary and many mitochondrial cristae in the negative control group, and Der f 1 exposure led to mitochondrial shrinkage, membrane thickening, and reduction or loss of cristae. These structural alterations were markedly attenuated in the Der f 1 + Fer-1 group, including weakening of mitochondrial shrinkage, membrane thickening and reduced numbers of mitochondrial cristae. Conclusion Der f 1 induces ferroptosis in human airway epithelial cells through disrupting iron homeostasis, suppressing antioxidant defense, and promoting lipid peroxidation, thereby potentiating the release of IL-6, TSLP, and IL-33 to exacerbate airway inflammation.