Two sesquiterpene aminoquinones protect against oxidative injury in HaCaT keratinocytes via activation of AMPKα/ERK-Nrf2/ARE/HO-1 signaling
Two sesquiterpene aminoquinones protect against oxidative injury in HaCaT keratinocytes via activation of AMPKα/ERK-Nrf2/ARE/HO-1 signaling
复制标题
两种倍半萜氨基醌通过激活 AMPK α/ERK-Nrf2/ARE/HO-1 信号传导防止 HaCaT 角质形成细胞的氧化损伤
DOI：
10.1016/j.biopha.2018.02.034
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发表时间：
2018-04-01
影响因子：
7.5
通讯作者：
Lin, Hou-Wen
中科院分区：
文献类型：
--
作者：
Liu, Li;Wu, Wei;Lin, Hou-Wen
目的：研究从脆弱海绵中分离得到的两种倍半萜氨基醌类化合物--二倍半萜氨基醌H(DA8)和3‘-甲氨基香豆酮(DA14)对过氧化氢(H_2O_2)诱导的人角质形成细胞氧化损伤的保护作用，并探讨其可能的作用机制。2，7-二氯二氢化荧光素二乙酸酯(DCFH-DA)荧光法测定细胞内活性氧(ROS)生成。用实时定量聚合酶链式反应和蛋白印迹分析检测信使RNA和蛋白的表达。免疫细胞化学方法检测核因子红系P45相关因子2(Nrf2)在细胞内的定位。采用抗氧化剂反应元件(ARE)-荧光素酶报告基因分析和RNA干扰技术确定ARE和Nrf2的作用。关键发现：DA8和DA14(DAS)通过抑制ROS的积累来拮抗H(2O)O(2)诱导的细胞存活率下降。同时，DAS增加HO-1的表达和ARE活性，诱导Nrf2的表达，以及Nrf2在细胞核内的积聚。然而，沉默Nrf2可抑制DAS诱导的HO-1的表达，并激活荧光素酶。此外，DAS还可诱导细胞内环磷酸腺苷活化蛋白激酶α(AMPKα)和细胞外信号调节激酶(ERK)的磷酸化，而AMPKα和ERK的特异性抑制剂可抑制HO1的上调和NRF2的激活。意义：DAS通过AMPKα和ERK的磷酸化激活NRF2/ARE/HO-1通路，对过氧化氢诱导的细胞毒性具有保护作用。提示DA8和DA14可能是治疗氧化损伤所致皮肤病的候选药物。
Aims: To investigate the cytoprotective effects of two sesquiterpene aminoquinones isolated from the marine sponge Dysidea fragilis, Dysidaminone H (DA8) and 3'-methylamino-avarone (DA14), we examined their effects against hydrogen peroxide (H2O2)-induced oxidative injury in human keratinocyte cell line and elucidated the underlying mechanisms.Main methods: Cell viability was detected using a CCK-8 assay kit. Intracellular reactive oxygen species (ROS) production was measured by fluorescence of 2, 7-Dichlorodi-hydrofluorescein diacetate (DCFH-DA). Messenger RNA and protein expression were measured by real-time quantitative PCR and western blotting analysis. Immunocytochemistry was performed to determine the intracellular location of nuclear factorerythroid 2 p45 related factor 2 (Nrf2). The antioxidant response element (ARE)-luciferase reporter gene assay and RNA interference were used to establish the role of ARE and Nrf2.Key findings: DA8 and DA14 (DAs) resisted H(2)O(2)induced decline of cell viability by inhibiting the accumulation of ROS. Meanwhile, DAs increased HO-1 expression and ARE activity and induced Nrf2 expression, as well as the accumulation of Nrf2 in the cell nucleus. However, silencing of Nrf2 abolished DAs-induced HO-1 expression and ARE luciferase activation. In addition, DAs induced the phosphorylation of both cyclic AMP-activated protein kinase-alpha (AMPK alpha) and extracellular signal-regulated kinase (ERK), while specific inhibitors of AMPK alpha and ERK abrogated HO1 upregulation and Nrf2 activation.Significance: DAs provided cytoprotective effects against H2O2-induced cytotoxicity by activation of the Nrf2/ARE/HO-1 pathway via phosphorylation of AMPK alpha and ERK. The findings suggested that DA8 and DA14 might be the candidate therapeutic agents for skin diseases caused by oxidative injury.