沉默FABP4通过调节Nrf2/GPX4轴减轻缺氧/复氧诱导的肾小管上皮细胞铁死亡

Silencing FABP4 alleviated ferroptosis of renal tubular epithelial cells induced by hypoxia/reoxygenation through regulating Nrf2/GPX4 axis

  • 摘要:
    目的  探讨脂肪酸结合蛋白4(FABP4)对缺氧/复氧(H/R)处理的人肾小管上皮细胞(HK-2)铁死亡的影响及作用机制。
    方法  体外培养HK-2细胞,经缺氧处理24 h后再复氧不同时间(1、3、6 h),实时荧光定量聚合酶链反应和蛋白质印迹法检测各时间点HK-2细胞中脂肪酸结合蛋白4(FABP4)信使RNA(mRNA)和蛋白水平。采用小干扰RNA技术沉默HK-2细胞中FABP4基因表达,再进行H/R(缺氧24 h复氧6 h)处理,或联合Nrf2抑制剂ML385处理。采用细胞计数试剂盒-8(CCK-8)检测各组细胞增殖活性,酶联免疫吸附试验(ELISA)检测乳酸脱氢酶(LDH)水平,生化法检测丙二醛(MDA)、谷胱甘肽(GSH)和亚铁离子(Fe2+)水平,2',7'-二氯二氢荧光素二乙酸酯荧光探针法检测活性氧簇(ROS)水平,蛋白质印迹法检测FABP4、核因子E2相关因子2(Nrf2)、血红素加氧酶-1(HO-1)、谷胱甘肽过氧化物酶4(GPX4)和溶质载体家族7成员11(SLC7A11)蛋白表达水平。
    结果  随着复氧时间的延长,HK-2细胞中FABP4 mRNA和蛋白水平均升高(均为P<0.05)。H/R处理可降低细胞增殖活性,升高细胞上清液中LDH水平,同时升高HK-2细胞中MDA、Fe2+及ROS水平,降低GSH水平,降低Nrf2、HO-1、GPX4、SLC7A11蛋白水平(均为P<0.05)。沉默FABP4可升高H/R处理的HK-2细胞增殖活性(P<0.05),降低细胞中MDA、Fe2+及ROS水平,升高GSH水平,同时升高Nrf2、HO-1、GPX4、SLC7A11蛋白水平(均为P<0.05)。而联合ML385处理后可逆转FABP4基因沉默对H/R处理的HK-2细胞铁死亡的改善作用。
    结论  沉默FABP4可减轻H/R诱导的HK-2细胞铁死亡,其机制可能与激活Nrf2/GPX4轴有关。

     

    Abstract:
    Objective  To investigate the effects and mechanisms of fatty acid binding protein 4 (FABP4) on ferroptosis in human renal tubular epithelial cells (HK-2) treated with hypoxia/reoxygenation (H/R).
    Methods  HK-2 cells were cultured in vitro and subjected to hypoxia for 24 hours followed by reoxygenation for different durations (1, 3, 6 hours). The messenger RNA (mRNA) and protein levels of FABP4 in HK-2 cells were detected at each time point using real-time fluorescent quantitative polymerase chain reaction and Western blotting. Small interfering RNA (siRNA) technology was used to silence the expression of FABP4 gene in HK-2 cells, which were then treated with H/R (24 hours of hypoxia and 6 hours of reoxygenation) or treated with the Nrf2 inhibitor ML385. Cell proliferation activity was assessed using cell counting kit-8 (CCK-8). Lactate dehydrogenase (LDH) levels were measured by enzyme-linked immune absorbent assay (ELISA). Malondialdehyde (MDA), glutathione (GSH) and ferrous ion (Fe2+) levels were determined by biochemical technology. Reactive oxygen species (ROS) levels were detected using the 2',7'-dichlorodihydrofluorescein diacetate fluorescence probe. Protein expression levels of FABP4, nuclear factor E2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11 (SLC7A11) were measured by Western blotting.
    Results  The mRNA and protein levels of FABP4 in HK-2 cells increased with prolonged reoxygenation time (all P<0.05). H/R treatment reduced cell proliferation activity, increased LDH levels in the cell supernatant, and elevated MDA, Fe2+ and ROS levels in HK-2 cells while decreasing GSH levels and the protein levels of Nrf2, HO-1, GPX4 and SLC7A11 (all P<0.05). Silencing FABP4 enhanced the proliferation activity of H/R-treated HK-2 cells (P<0.05), reduced MDA, Fe2+ and ROS levels, increased GSH levels, and elevated the protein levels of Nrf2, HO-1, GPX4 and SLC7A11 (all P<0.05). However, these beneficial effects of FABP4 silencing on H/R-induced ferroptosis in HK-2 cells were reversed by co-treatment with ML385.
    Conclusions  Silencing FABP4 alleviated H/R-induced ferroptosis in HK-2 cells, possibly by activating the Nrf2/GPX4 axis.

     

/

返回文章
返回