褐色钟花树中Avellaneine D通过线粒体凋亡通路和Nrf2/Keap1通路改善阿霉素诱导的H9c2心肌细胞损伤

Avellaneine D from Tabebuia avellanedae ameliorates doxorubicin-induced damage in H9c2 cardiomyocytes via mitochondrial apoptotic pathway and Nrf2/Keap1 pathway

关键词:  褐色钟花树  Avellaneine D  心肌损伤  线粒体凋亡通路  Nrf2/Keap1通路  氧化应激  炎症

Key words:  Tabebuia avellanedae  Avellaneine D  cardiomyocyte injury  mitochondrial apoptosis pathway  Nrf2/Keap1 pathway  oxidative stress  inflammation

摘要:

目的 探究褐色钟花树中Avellaneine D对阿霉素诱导的心肌细胞损伤的改善作用及其作用机制。方法 采用1 μg·mL- 1阿霉素诱导建立H9c2心肌细胞损伤模型，用Avellaneine D处理细胞6 h，检测细胞活力和LDH水平，并检测IL-1β、IL-6、TNF-α以及SOD、MDA、GSH-Px水平，用流式细胞技术检测ROS水平、线粒体膜电位水平和细胞凋亡水平，用In-Cell-Western法检测细胞中线粒体凋亡通路和Nrf2/Keap1通路中关键蛋白表达水平。结果 Avellaneine D有效提升细胞活力，降低LDH水平，有效抑制阿霉素诱导损伤后的心肌细胞炎症因子IL-6和TNF-α水平释放，降低MDA，提高GSH-px，改善氧化应激。流式结果表明，Avellaneine D降低细胞ROS水平，提高线粒体膜电位水平，抑制心肌细胞凋亡率。In-Cell-Western检测结果表明，Avellaneine D抑制线粒体凋亡通路关键蛋白P53、降低Bax/Bcl-2、抑制Caspase9表达水平，提高氧化应激通路关键蛋白Nrf2表达水平，降低Keap1表达水平。结论 Avellaneine D能提高阿霉素诱导损伤的心肌细胞活力，降低LDH和炎症水平，其机制可能与调节线粒体凋亡通路和Nrf2/Keap1通路有关。

Abstract:

OBJECTIVE To investigate the ameliorative effect of Avellaneine D from Tabebuia avellanedae on an in vitro myocardial injury model induced by doxorubicin(Dox) and its mechanism of action. METHODS An H9c2 cardiomyocyte injury model was established using 1 μg·mL- 1 doxorubicin induction, and the cells were treated with Avellaneine D for 6 h. The cell viability and LDH level were measured, and measured IL-1β, IL-6, TNF-α, and SOD, MDA, GSH-Px levels, the levels of ROS, mitochondrial membrane potential and apoptosis rate were detected by flow cytometry assay, and In-Cell-Western assay was used to detect the expression levels of key proteins in the mitochondrial apoptotic pathway and Nrf2/Keap1 pathway in cells. RESULTS The Avellaneine D could effectively enhance cell viability, reduce LDH level, and effectively inhibit the release of cardiomyocyte inflammatory factors IL-6 and TNF-α levels after doxorubicin-induced injury, as well as reduce MDA level, enhance GSH-px level, improve oxidative stress. The results of flow cytometry assay showed that the Avellaneine D can decrease cell ROS level, enhance mitochondrial membrane potential level, and inhibit apoptosis rate of cardiomyocytes. The results of In-Cell-Western assay showed that the Avellaneine D can inhibit the expression level of the key protein of the mitochondrial apoptosis pathway P53, reduce the expression level of Bax/Bcl-2, inhibit the expression level of Caspase9, and increase the expression level of the key protein of the oxidative stress pathway Nrf2, reduce the expression level of Keap1. CONCLUSION Avellaneine D can enhance cardiomyocyte viability and reduce the levels of LDH and inflammation in doxorubicin-induced injury, the mechanism may be related to the regulation of the mitochondrial apoptotic pathway and the Nrf2/Keap1 pathway.