背景:脓毒症诱导的急性肾损伤(AKI)是一种异常严重且危及生命的综合征。其发病机制与炎症反应密切相关,凋亡,氧化应激,和铁中毒。阳离子转运调节因子样蛋白1(CHAC1),作为促凋亡因子,可能参与细胞凋亡,氧化应激,和铁中毒。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细胞(HK-2)中的作用。
方法:用LPS攻击HK-2细胞,构建脓毒症诱导的AKI体外模型。CHAC1在LPS诱导的HK-2细胞中的作用采用Westernblot分析,细胞计数试剂盒-8(CCK-8),流式细胞术,和比色测定。此外,N-乙酰半胱氨酸(NAC)与HK-2细胞孵育,以深入定义氧化应激与细胞凋亡或铁凋亡之间的关系。
结果:CHAC1在脓毒症诱导的多器官功能障碍综合征(MODS)小鼠肾组织中的表达增强,通过基因表达综合数据库(GSE60088微阵列数据集),在LPS诱导的HK-2细胞中。LPS处理可显著降低细胞活力,通过转染siCHAC1#1和siCHAC1#2而不是siNC至少部分恢复。此外,CHAC1的下调抵消了LPS诱导的活性氧水平和丙二醛浓度,同时恢复了LPS诱导的谷胱甘肽浓度。同时,干扰CHAC1中和LPS诱导的细胞凋亡率,和切割的聚(ADP-核糖)聚合酶(PARP)/PARP的相对水平,和裂解的caspase-3/caspase-3。此外,CHAC1的沉默恢复了LPS诱导的谷胱甘肽过氧化物酶4(GPx4)的蛋白水平增强,而拮抗了LPS诱导的ACSL4和铁的相对蛋白水平。此外,应用NAC逆转CHAC1对HK-2细胞凋亡和铁凋亡的影响。
结论:CHAC1通过增强LPS诱导的HK-2细胞的氧化应激而加剧了铁凋亡和凋亡。
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 in vitro. 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.
CONCLUSIONS: CHAC1 exacerbated ferroptosis and apoptosis by enhancing oxidative stress in LPS-induced HK-2 cells.