Abstract

P-glycoprotein (Pgp) is an ATP-dependent transmembrane protein involved in the efflux of lipophilic substances. The aim of this study was to evaluate the effect of oxidative stress on the transport of a Pgp substrate through the monolayer of Caco-2 cells overexpressing this transport protein. Oxidative stress was modeled by incubating the cells with H₂O₂. Exposure to H₂O₂ at concentrations of 10 and 50 μM for 3 h reduced the Pgp activity but not the content of Pgp, while the integrity of the cell monolayer did not change. The increase of the prooxidant concentration to 100 μM reduced the content of Pgp, violated the integrity of the cell monolayer, and increased the transcellular and paracellular transport of fexofenadine. A 24-h exposure to 0.1–1 µM H₂O₂ resulted in an increase in the content of Pgp mediated by the Nrf2 transcription factor, while the activity of the transport protein remained unchanged. At a prooxidant concentration of 10 µM, the Pgp activity decreased and the cell membrane permeability increased, while at concentrations of 50–100 µM, the content (100 µM) and activity of Pgp decreased, and the paracellular and transcellular permeability of the cell monolayer increased for fexofenadine, a substrate of the transport protein. Thus, H₂O₂ increased the transport of the Pgp substrate fexofenadine through the cell monolayer by inhibiting the activity of the transport protein, reducing its content, as well as violating the integrity of the cell membrane and intercellular contacts. The cells can adapt to these effects by increasing the content of Pgp.

中文翻译：

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氧化应激对 P-糖蛋白底物通过细胞单层转运的影响

摘要

P-糖蛋白 (Pgp) 是一种 ATP 依赖性跨膜蛋白，参与亲脂性物质的流出。本研究的目的是评估氧化应激对 Pgp 底物通过过表达这种转运蛋白的 Caco-2 细胞单层转运的影响。通过用H ₂ O ₂孵育细胞来模拟氧化应激。暴露于 H ₂ O ₂在 10 和 50 μM 浓度下 3 小时降低 Pgp 活性但不降低 Pgp 含量，而细胞单层的完整性没有改变。促氧化剂浓度增加到 100 μM 会降低 Pgp 的含量，破坏细胞单层的完整性，并增加非索非那定的跨细胞和细胞旁转运。24 小时暴露于 0.1–1 µM H ₂ O ₂导致由 Nrf2 转录因子介导的 Pgp 含量增加，而转运蛋白的活性保持不变。促氧化剂浓度为 10 µM 时，Pgp 活性降低，细胞膜通透性增加，而浓度为 50-100 µM 时，Pgp 的含量（100 µM）和活性降低，细胞单层的细胞旁和跨细胞通透性降低非索非那定（转运蛋白的底物）增加。因此，H ₂ O ₂通过抑制转运蛋白的活性、降低其含量以及破坏细胞膜和细胞间接触的完整性，增加了 Pgp 底物非索非那定通过细胞单层的转运。细胞可以通过增加 Pgp 的含量来适应这些影响。