Department of Pharmacology, College of Medicine, Institute of Health Sciences, Gyeongsang National University, Jinju, Republic of Korea; Department of Convergence Medical Science (BK21 Plus), Gyeongsang National University, Jinju, Republic of Korea.
College of Pharmacy, Kangwon National University, Chuncheon, Republic of Korea.
Int J Biochem Cell Biol. 2018 Jun;99:100-108. doi: 10.1016/j.biocel.2018.04.006. Epub 2018 Apr 5.
Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key transcription factor that responds to oxidative stress and xenobiotics. Multiple lines of evidence suggest that Nrf2 activation protects against aging, inflammation, and many diseases, including cancer. Nrf2 activators derived from natural sources have been widely studied. In this study, we investigated the effect of amentoflavone (AFN), a biflavonoid found in many plants, on Nrf2 signaling in human keratinocytes (HaCaT cells). AFN significantly increased ARE luciferase activity by Nrf2 accumulation in the nucleus. Subsequently, the levels of a Nrf2 target protein, NQO-1, were significantly increased by AFN in a dose- and time-dependent manner. To verify the mechanism of AFN-induced activation of Nrf2 signaling, we measured generation of reactive oxygen species (ROS). Interestingly AFN triggered mild ROS production. Additionally, AFN-induced Nrf2 activation was inhibited by N-acetyl cysteine. Therefore, we studied the effect of ROS-related signaling on Nrf2 by measuring the activation of AKT and members of the mitogen-activated protein kinase family, such as extracellular signal-regulated kinase (ERK1/2) and p38. The results showed that the pharmacological inhibitor of PI3K/AKT (LY294002) or p38 (SB 203580), but not ERK1/2 (U0126), abrogated AFN-induced Nrf2 activation. Subsequently, we found that silencing or inhibition of p38 resulted in decrease of AKT phosphorylation as well as inhibition of Nrf2 accumulation. Furthermore, we found that AFN stabilized Nrf2 by inhibiting its ubiquitination. Taken together, our results suggest that AFN contributes to Nrf2 activation through ROS-mediated activation of the p38-AKT pathway in HaCaT cells.
核因子红细胞 2 相关因子 2 (Nrf2) 是一种关键的转录因子，可响应氧化应激和外源性物质。有多项证据表明，Nrf2 的激活可预防衰老、炎症和许多疾病，包括癌症。从天然来源中提取的 Nrf2 激活剂已得到广泛研究。在这项研究中，我们研究了来源于多种植物的双黄酮amentoflavone (AFN) 对人角质形成细胞 (HaCaT 细胞) 中 Nrf2 信号的影响。AFN 通过 Nrf2 在核内的积累，显著增加了 ARE 荧光素酶的活性。随后，AFN 以剂量和时间依赖的方式显著增加了 Nrf2 靶蛋白 NQO-1 的水平。为了验证 AFN 诱导 Nrf2 信号激活的机制，我们测量了活性氧物种 (ROS) 的产生。有趣的是，AFN 触发了轻度的 ROS 产生。此外，N-乙酰半胱氨酸抑制了 AFN 诱导的 Nrf2 激活。因此，我们通过测量 AKT 和丝裂原活化蛋白激酶家族成员（如细胞外信号调节激酶 (ERK1/2) 和 p38）的激活来研究 ROS 相关信号对 Nrf2 的影响。结果表明，PI3K/AKT 的药理学抑制剂 (LY294002) 或 p38 (SB 203580)，但不是 ERK1/2 (U0126)，可阻断 AFN 诱导的 Nrf2 激活。随后，我们发现沉默或抑制 p38 导致 AKT 磷酸化减少以及 Nrf2 积累减少。此外，我们发现 AFN 通过抑制其泛素化来稳定 Nrf2。总之，我们的结果表明，AFN 通过 ROS 介导的 p38-AKT 通路在 HaCaT 细胞中激活 Nrf2。
