CHAC1 通过促进氧化应激加剧 LPS 诱导的 HK-2 细胞铁死亡和凋亡。
CHAC1 exacerbates LPS-induced ferroptosis and apoptosis in HK-2 cells by promoting oxidative stress.
机构信息
Department of Critical Care Medicine, Chongqing General Hospital, Chongqing, China.
Department of ICU, Affiliated Hospital of Hebei University, Baoding, Hebei, China;
出版信息
Allergol Immunopathol (Madr). 2023 Mar 1;51(2):99-110. doi: 10.15586/aei.v51i2.760. eCollection 2023.
BACKGROUND
Sepsis-induced acute kidney injury (AKI) is a singularly grievous and life-threatening syndrome. Its pathogenesis is closely related to inflammatory response, apoptosis, oxidative stress, and ferroptosis. Cation transport regulator-like protein 1 (CHAC1), as a proapoptic factor, may be involved in apoptosis, oxidative stress, and ferroptosis. This study aimed to explore the role of CHAC1 in the lipopolysaccharide (LPS)-induced the human renal proximal tubular epithelial (HK-2) cells.
METHODS
HK-2 cells were challenged with LPS to construct a model of sepsis-induced AKI . The role of CHAC1 in the LPS-induced HK-2 cells was explored using Western blot assay, cell counting kit-8 (CCK-8), flow cytometry, and colorimetric assays. Additionally, N-acetyl cysteine (NAC) was incubated with HK-2 cells to define deeply the relation between oxidative stress and apoptosis or ferroptosis.
RESULTS
The expression of CHAC1 was enhanced in the kidney tissues of mice with sepsis--induced multiple organ dysfunction syndrome (MODS), through the Gene Expression Omnibus database (GSE60088 microarray dataset), and in the LPS-induced HK-2 cells. The cell viability was significantly reduced by LPS treatment, which was at least partly restored by the transfection of siCHAC1#1 and siCHAC1#2 but not siNC. In addition, down-regulation of CHAC1 counteracted the LPS-induced reactive oxygen species level and malonaldehyde concentrations while restored the LPS-induced glutathione concentrations. Meanwhile, interference of CHAC1 neutralized LPS-induced apoptosis rate, and the relative level of cleaved poly(ADP-ribose) polymerase (PARP)/PARP, and cleaved caspase-3/caspase-3. In addition, silencing of CHAC1 recovered the LPS-induced enhanced protein level of glutathione peroxidase 4 (GPx4) whereas antagonized the LPS-induced relative protein level of ACSL4 and that of iron. Moreover, application of NAC inverted the effect of CHAC1 on apoptosis and ferroptosis in HK-2 cells.
CONCLUSION
CHAC1 exacerbated ferroptosis and apoptosis by enhancing oxidative stress in LPS-induced HK-2 cells.
背景
脓毒症诱导的急性肾损伤（AKI）是一种严重且危及生命的综合征。其发病机制与炎症反应、细胞凋亡、氧化应激和铁死亡密切相关。阳离子转运调节蛋白样蛋白 1（CHAC1）作为一种促凋亡因子，可能参与细胞凋亡、氧化应激和铁死亡。本研究旨在探讨 CHAC1 在脂多糖（LPS）诱导的人肾近端肾小管上皮（HK-2）细胞中的作用。
方法
用 LPS 刺激 HK-2 细胞构建脓毒症诱导的 AKI 模型。用 Western blot 检测、细胞计数试剂盒-8（CCK-8）、流式细胞术和比色法探讨 CHAC1 在 LPS 诱导的 HK-2 细胞中的作用。此外，用 N-乙酰半胱氨酸（NAC）孵育 HK-2 细胞，以深入了解氧化应激与细胞凋亡或铁死亡之间的关系。
结果
通过基因表达综合数据库（GSE60088 微阵列数据集）和 LPS 诱导的 HK-2 细胞，发现脓毒症诱导的多器官功能障碍综合征（MODS）小鼠肾脏组织中 CHAC1 的表达增强。LPS 处理后细胞活力明显降低，siCHAC1#1 和 siCHAC1#2 转染至少部分恢复了细胞活力，但 siNC 没有恢复。此外，下调 CHAC1 可拮抗 LPS 诱导的活性氧水平和丙二醛浓度，同时恢复 LPS 诱导的谷胱甘肽浓度。同时，干扰 CHAC1 可中和 LPS 诱导的细胞凋亡率，以及裂解多聚（ADP-核糖）聚合酶（PARP）/PARP 和裂解半胱天冬酶-3/半胱天冬酶-3 的相对水平。此外，沉默 CHAC1 恢复了 LPS 诱导的谷胱甘肽过氧化物酶 4（GPx4）蛋白水平升高，拮抗了 LPS 诱导的 ACSL4 和铁相对蛋白水平升高。此外，NAC 的应用逆转了 CHAC1 对 LPS 诱导的 HK-2 细胞凋亡和铁死亡的影响。
结论
CHAC1 通过增强 LPS 诱导的 HK-2 细胞中的氧化应激，加重铁死亡和细胞凋亡。
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