PDF(Pubmed)
Abstract：
OBJECTIVE: To investigate the protective effect of dexmedetomidine (DEX) against erastin-induced ferroptosis in human renal tubular epithelial cells (HK-2 cells) and explore the underlying mechanism.
METHODS: HK-2 cells were treated with erastin alone or in combination with different concentrations (2.5, 5.0 and 10 μmol/L) of DEX, and the changes in cell viability were observed using CCK-8 assay. To explore the mechanism by which DEX inhibits erastin-induced ferroptosis, HK-2 cells were treated with erastin, erastin+10 μmol/L DEX, or erastin+10 μmol/L DEX+ML385 (a Nrf2 inhibitor), after which the cell viability was assessed. The level of intracellular Fe2+ was detected by cell ferrous iron colorimetric assay kit, and flow cytometry was performed to detect reactive oxygen species (ROS); MDA and reduced glutathione assay kits were used to detect the contents of MDA and GSH in the cells; The expressions of Nrf2, HO-1 and GPX4 proteins were detected by Western blotting.
RESULTS: Erastin treatment significantly inhibited the viability of the cells, decreased GSH content, and increased intracellular levels of Fe2+, ROS and MDA. The combined treatment with 10 μmol/L DEX markedly increased the viability of the cells, increased GSH content, reduced the levels of Fe2+, ROS and MDA, and upregulated the protein expressions of Nrf2, HO-1 and GPX4 in the cells. The application of ML385 obviously blocked the protective effect of DEX and caused significant inhibition of the Nrf2/HO-1/GPX4 pathway, decreased the cell viability and GSH content, and increased the levels of Fe2+, ROS and MDA in HK-2 cells.
CONCLUSIONS: The protective effect of DEX against erastin-induced ferroptosis of HK-2 cells is probably mediated by activation of the Nrf2/HO-1/GPX4 pathway to inhibit oxidative stress.
METHODS: HK-2 cells were treated with erastin alone or in combination with different concentrations (2.5, 5.0 and 10 μmol/L) of DEX, and the changes in cell viability were observed using CCK-8 assay. To explore the mechanism by which DEX inhibits erastin-induced ferroptosis, HK-2 cells were treated with erastin, erastin+10 μmol/L DEX, or erastin+10 μmol/L DEX+ML385 (a Nrf2 inhibitor), after which the cell viability was assessed. The level of intracellular Fe2+ was detected by cell ferrous iron colorimetric assay kit, and flow cytometry was performed to detect reactive oxygen species (ROS); MDA and reduced glutathione assay kits were used to detect the contents of MDA and GSH in the cells; The expressions of Nrf2, HO-1 and GPX4 proteins were detected by Western blotting.
RESULTS: Erastin treatment significantly inhibited the viability of the cells, decreased GSH content, and increased intracellular levels of Fe2+, ROS and MDA. The combined treatment with 10 μmol/L DEX markedly increased the viability of the cells, increased GSH content, reduced the levels of Fe2+, ROS and MDA, and upregulated the protein expressions of Nrf2, HO-1 and GPX4 in the cells. The application of ML385 obviously blocked the protective effect of DEX and caused significant inhibition of the Nrf2/HO-1/GPX4 pathway, decreased the cell viability and GSH content, and increased the levels of Fe2+, ROS and MDA in HK-2 cells.
CONCLUSIONS: The protective effect of DEX against erastin-induced ferroptosis of HK-2 cells is probably mediated by activation of the Nrf2/HO-1/GPX4 pathway to inhibit oxidative stress.
摘要：
目的：观察右美托咪定(DEX)对人肾小管上皮细胞(HK-2细胞)铁凋亡的保护作用并探讨其机制。
方法：HK-2细胞单独或与不同浓度（2.5、5.0和10μmol/L）的DEX联合使用，使用CCK-8测定观察细胞活力的变化。为了探讨DEX抑制擦除素诱导的铁细胞凋亡的机制，HK-2细胞用erastin处理，erastin+10μmol/LDEX,或erastin+10μmol/LDEX+ML385（一种Nrf2抑制剂），之后评估细胞活力。细胞亚铁比色法试剂盒检测细胞内Fe2+水平,流式细胞术检测活性氧（ROS）；MDA和还原型谷胱甘肽检测试剂盒检测细胞中MDA和GSH的含量；Westernblotting检测Nrf2，HO-1和GPX4蛋白的表达。
结果：Erastin处理显著抑制了细胞的活力，GSH含量降低，并增加了细胞内的Fe2+水平，ROS和MDA。10μmol/LDEX联合处理可显著提高细胞活力,GSH含量增加，降低了Fe2+的水平，ROS和MDA，并上调细胞中Nrf2、HO-1和GPX4的蛋白表达。ML385的应用明显阻断了DEX的保护作用,引起Nrf2/HO-1/GPX4通路的显著抑制,降低细胞活力和GSH含量，并增加了Fe2+的水平，HK-2细胞中的ROS和MDA。
结论：DEX对擦除素诱导的HK-2细胞铁凋亡的保护作用可能是通过激活Nrf2/HO-1/GPX4通路抑制氧化应激介导的。
方法：HK-2细胞单独或与不同浓度（2.5、5.0和10μmol/L）的DEX联合使用，使用CCK-8测定观察细胞活力的变化。为了探讨DEX抑制擦除素诱导的铁细胞凋亡的机制，HK-2细胞用erastin处理，erastin+10μmol/LDEX,或erastin+10μmol/LDEX+ML385（一种Nrf2抑制剂），之后评估细胞活力。细胞亚铁比色法试剂盒检测细胞内Fe2+水平,流式细胞术检测活性氧（ROS）；MDA和还原型谷胱甘肽检测试剂盒检测细胞中MDA和GSH的含量；Westernblotting检测Nrf2，HO-1和GPX4蛋白的表达。
结果：Erastin处理显著抑制了细胞的活力，GSH含量降低，并增加了细胞内的Fe2+水平，ROS和MDA。10μmol/LDEX联合处理可显著提高细胞活力,GSH含量增加，降低了Fe2+的水平，ROS和MDA，并上调细胞中Nrf2、HO-1和GPX4的蛋白表达。ML385的应用明显阻断了DEX的保护作用,引起Nrf2/HO-1/GPX4通路的显著抑制,降低细胞活力和GSH含量，并增加了Fe2+的水平，HK-2细胞中的ROS和MDA。
结论：DEX对擦除素诱导的HK-2细胞铁凋亡的保护作用可能是通过激活Nrf2/HO-1/GPX4通路抑制氧化应激介导的。
