右美托咪定通过激活Nrf2/HO-1/GPX4通路抑制肾小管上皮细胞的铁死亡

doi:10.12122/j.issn.1673-4254.2024.06.14

摘要/Abstract

摘要：

目的探讨右美托咪定（DEX）对Erastin诱导的人肾小管上皮细胞（HK-2）铁死亡的保护作用及其机制。方法构建Erastin诱导的HK-2细胞铁死亡模型。将HK-2细胞分为对照组、Erastin模型组、Erastin+2.5 μmol/L DEX组、Erastin+5 μmol/L DEX组、Erastin+10 μmol/L DEX组，采用CCK-8法检测细胞的存活率。将HK-2细胞分为对照组、Erastin模型组、Erastin+10 μmol/L DEX组、Erastin+10 μmol/L DEX+ML385（Nrf2抑制剂）组。采用CCK-8法检测细胞的存活率；使用细胞亚铁比色法测试盒检测细胞内Fe²⁺水平的变化；应用流式细胞术检测细胞内活性氧（ROS）的含量；使用丙二醛（MDA）和还原型谷胱甘肽（GSH）试剂盒检测细胞中MDA及GSH含量；Western blotting检测细胞中核因子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）蛋白的表达。结果与对照组相比，Erastin组的细胞存活率显著被抑制（P<0.001），同时细胞中GSH含量降低（P<0.001），Fe²⁺、ROS以及MDA含量升高（P<0.001）；与Erastin组相比，Erastin+10 μmol/L DEX组的细胞存活率明显升高（P<0.001），10 μmol/L DEX显著升高细胞中GSH含量（P<0.001），显著降低Fe²⁺、ROS以及MDA含量（P<0.001），并且显著上调细胞中Nrf2、HO-1和GPX4蛋白的表达（P<0.001）。使用ML385后，Nrf2/HO-1/GPX4通路被抑制（P<0.001），细胞存活率降低（P<0.001），GSH含量降低（P<0.001），而Fe²⁺、ROS以及MDA含量升高（P<0.05）。结论 DEX对Erastin诱导的HK-2细胞铁死亡具有保护作用，其机制可能是通过激活Nrf2/HO-1/GPX4通路实现抗氧化应激从而抑制铁死亡。

关键词: 右美托咪定, Erastin, 人肾小管上皮细胞, 铁死亡, Nrf2/HO-1/GPX4

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. Conclusion 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.

Key words: dexmedetomidine, erastin, HK-2 cells, ferroptosis, Nrf2/HO-1/GPX4 pathway

张方圆, 刘刚. 右美托咪定通过激活Nrf2/HO-1/GPX4通路抑制肾小管上皮细胞的铁死亡[J]. 南方医科大学学报, 2024, 44(6): 1135-1140.

Fangyuan ZHANG, Gang LIU. Dexmedetomidine inhibits ferroptosis of human renal tubular epithelial cells by activating the Nrf2/HO-1/GPX4 pathway[J]. Journal of Southern Medical University, 2024, 44(6): 1135-1140.

图/表 5

图1 DEX对Erastin处理后HK-2细胞存活率的影响

Fig.1 Effect of DEX on viability of HK-2 cells treated with erastin (Mean±SD, n=3). A: Effects of different concentrations of erastin on viability of HK-2 cells. ***P<0.001 vs 0 μmol/L. B: DEX at different concentrations increases viability of erastin-treated HK-2 cells. C: ML385 attenuates the protective effect of DEX in erastin-treated HK-2 cells. ***P<0.001 vs Control group; #P<0.05, ###P<0.001 vs Erastin group; &&&P<0.001 vs Erastin+DEX group.

图2 DEX对Erastin处理后HK-2细胞中Fe2+的影响

Fig.2 Effect of DEX on intracellular Fe2+ level in HK-2 cells treated with erastin (Mean±SD, n=3). ***P<0.001 vs Control group; ###P<0.001 vs Erastin group; &P<0.05 vs Erastin+DEX group.

图3 DEX对Erastin处理后HK-2细胞中Nrf2、HO-1和GPX4蛋白表达的影响

Fig.3 Effect of DEX on protein expressions of Nrf2, HO-1 and GPX4 in HK-2 cells treated with erastin (Mean±SD, n=3). A: Western blotting results. B-D: Expression levels of Nrf2, HO-1 and GPX4 proteins in HK-2 cells with different treatments. **P<0.01, ***P<0.001 vs Control group; ###P<0.001 vs Erastin group; &&&P<0.001 vs Erastin+DEX group.

图4 DEX对Erastin处理后HK-2细胞中ROS的影响

Fig.4 Effect of DEX on ROS level in HK-2 cells treated with erastin (Mean±SD, n=3). ***P<0.001 vs Control group; ###P<0.001 vs Erastin group; &&&P<0.001 vs Erastin+DEX group.

图5 DEX对Erastin处理后HK-2细胞中MDA和GSH的影响

Fig.5 Effect of DEX on MDA and GSH contents in HK-2 cells treated with Erastin (Mean±SD, n=3). A: MDA content in HK-2 cells with different treatment. B: GSH content in HK-2 cells with different treatment. ***P<0.001 vs Control group; ###P<0.001 vs Erastin group; &P<0.05, &&&P<0.001 vs Erastin+DEX group.

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