Recent evidence suggests that ClC-3, a member of the ClC family of Cl^- channels or Cl^-/H⁺ antiporters, plays a critical role in NADPH oxidase-derived reactive oxygen species (ROS) generation. However, the underling mechanisms remain unclear. In this study we investigated the effects and mechanisms of ClC-3 on NADPH oxidase activation and ROS generation in endothelial cells. Treatment with angiotensin II (Ang II, 1 μmol/L) significantly elevated ClC-3 expression in cultured human umbilical vein endothelial cells (HUVECs). Furthermore, Ang II treatment increased ROS production and NADPH oxidase activity, an effect that could be significantly inhibited by knockdown of ClC-3, and further enhanced by overexpression of ClC-3. SA-β-galactosidase staining showed that ClC-3 silencing abolished Ang II-induced HUVEC senescence, whereas ClC-3 overexpression caused the opposite effects. We further showed that Ang II treatment increased the translocation of p47phox and p67phox from the cytosol to membrane, accompanied by elevated Nox2 and p22phox expression, which was significantly attenuated by knockdown of ClC-3 and potentiated by overexpression of ClC-3. Moreover, overexpression of ClC-3 increased Ang II-induced phosphorylation of p47phox and p38 MAPK in HUVECs. Pretreatment with a p38 inhibitor SB203580 abolished ClC-3 overexpression-induced increase in p47phox phosphorylation, as well as NADPH oxidase activity and ROS generation. Our results demonstrate that ClC-3 acts as a positive regulator of Ang II-induced NADPH oxidase activation and ROS production in endothelial cells, possibly via promoting both Nox2/p22phox expression and p38 MAPK-dependent p47phox/p67phox membrane translocation, then increasing Nox2 NADPH oxidase complex formation.

中文翻译：

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ClC-3通过促进Nox2 NADPH氧化酶复合物的形成来促进血管紧张素II诱导的内皮细胞活性氧的产生。

最近的证据表明CLC-3，家庭的ClC氯中的一员^-通道或Cl ^- / H ⁺反转运蛋白，在NADPH氧化酶衍生的活性氧（ROS）产生中起关键作用。但是，底层机制仍然不清楚。在这项研究中，我们研究了ClC-3对内皮细胞中NADPH氧化酶激活和ROS生成的影响及其机制。血管紧张素II（Ang II，1μmol/ L）处理可显着提高培养的人脐静脉内皮细胞（HUVEC）中的ClC-3表达。此外，Ang II处理增加了ROS的产生和NADPH氧化酶的活性，这种作用可能会被ClC-3的敲低显着抑制，并通过ClC-3的过表达而进一步增强。SA-β-半乳糖苷酶染色显示，ClC-3沉默消除了Ang II诱导的HUVEC衰老，而ClC-3的过表达引起了相反的作用。我们进一步表明，Ang II处理增加了p47phox和p67phox从细胞质到膜的转运，并伴随着Nox2和p22phox表达的升高，这被ClC-3的敲低显着减弱，并且由于ClC-3的过表达而增强。此外，ClC-3的过表达增加了Ang II诱导的HUVEC中p47phox和p38 MAPK的磷酸化。用p38抑制剂SB203580进行的预处理消除了ClC-3过表达诱导的p47phox磷酸化增加以及NADPH氧化酶活性和ROS生成。我们的研究结果表明，ClC-3可能通过促进Nox2 / p22phox表达和p38 MAPK依赖的p47phox / p67phox膜易位，来作为Ang II诱导内皮细胞中NADPH氧化酶激活和ROS生成的正调节剂，