Abstract

With the abundant production and wide application of zinc oxide nanoparticles (ZnONPs), the potential health risks of ZnONPs have raised serious concerns. Oxidative stress is recognized as the most important outcome of the toxicity induced by ZnONPs. The Nrf2-Keap1 system and its downstream antioxidative genes are the fundamental protective mechanisms for redox hemeostasis. However, the detailed mechanisms of Nrf2 activation in ZnONPs-treated endothelial cells and murine blood vessels have yet to be elucidated. Herein, we show that Nrf2 was activated and played a negative role in cell death induced by ZnONPs. Moreover, we demonstrate that HO-1 was the most extensively upregulated antioxidative gene-activated by Nrf2. Forced overexpression of HO-1, pharmacological activation of HO-1 with the agonists RTA-408 (omaveloxolone, an FDA-approved drug) and RTA-402 repressed cell death, and treatment with HO-1 antagonist SnPP exacerbated the cell death. Importantly, loss of HO-1 diminished the cytoprotective role induced by Nrf2 in ZnONPs-treated HUVEC cells, indicating that the Nrf2-HO-1 axis was the crucial regulatory mechanism for the antioxidative response in the context of ZnONPs-induced endothelial damage. Mechanistically, we demonstrate that the p62-Keap1 axis was not involved in the activation of Nrf2. Intriguingly, the degradation half-life of Nrf2 in HUVEC cells was increased from less than 1 h under quiescent conditions to approximately 6 h under ZnONPs treatment condition; moreover, ZnONPs treatment induced activation of Nrf2/HO-1 and accumulation of ubiquitin in the aorta ventralis of mouse, suggesting that the ubiquitin-proteasome system had been perturbed, which subsequently led to the stabilization of Nrf2 and activation of HO-1. This study might contribute to a better understanding of ZnONPs-associated toxicity.

摘要

随着氧化锌纳米粒子（ZnONPs）的大量生产和广泛应用，ZnONPs的潜在健康风险引起了人们的严重关注。氧化应激被认为是 ZnONPs 诱导毒性的最重要结果。Nrf2-Keap1 系统及其下游抗氧化基因是氧化还原止血的基本保护机制。然而，ZnONPs 处理的内皮细胞和鼠血管中 Nrf2 激活的详细机制尚未阐明。在此，我们表明 Nrf2 被激活并在 ZnONPs 诱导的细胞死亡中起负面作用。此外，我们证明 HO-1 是由 Nrf2 激活的最广泛上调的抗氧化基因。HO-1 的强制过度表达，HO-1 与激动剂 RTA-408（omaveloxolone，一种 FDA 批准的药物）和 RTA-402 抑制细胞死亡，用 HO-1 拮抗剂 SnPP 治疗会加剧细胞死亡。重要的是，HO-1 的缺失减弱了 Nrf2 在 ZnONPs 处理的 HUVEC 细胞中诱导的细胞保护作用，表明 Nrf2-HO-1 轴是 ZnONPs 诱导的内皮损伤背景下抗氧化反应的关键调节机制。从机制上讲，我们证明 p62-Keap1 轴不参与 Nrf2 的激活。有趣的是，Nrf2 在 HUVEC 细胞中的降解半衰期从静止条件下的不到 1 小时增加到 ZnONPs 处理条件下的约 6 小时；此外，ZnONPs 处理诱导了 Nrf2/HO-1 的激活和泛素在小鼠主动脉腹侧的积累，表明泛素-蛋白酶体系统已经受到干扰，这随后导致 Nrf2 的稳定和 HO-1 的激活。这项研究可能有助于更好地了解 ZnONPs 相关的毒性。