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

张莉; 卢仁睿; 李静阳; 程申亮; 崔泷; 张文奇; 郑晓珂

doi:10.13748/j.cnki.issn1007-7693.20232981

褐色钟花树中Avellaneine D通过Nrf2/Keap1通路和线粒体凋亡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 Nrf2/Keap1 Pathway and Mitochondrial Apoptotic Pathway

摘要

摘要:
目的基于核因子E2相关因子2(nuclear factor erythroid 2 related factor 2，Nrf2)/Kelch样环氧氯丙烷相关蛋白1(Kelch-like epichlorohydrin-associated protein 1，Keap1)通路和线粒体凋亡通路探究褐色钟花树中Avellaneine D对阿霉素(doxorubicin，DOX)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⁻¹DOX诱导建立H9c2心肌细胞损伤模型，用Avellaneine D处理细胞24 h，检测细胞活力和乳酸脱氢酶(lactate dehydrogenase，LDH)水平，并检测白细胞介素-β(interleukin-1β，IL-1β)、白细胞介素-6(interleukin-6，IL-6)、肿瘤坏死因子α(tumor necrosis factor-α，TNF-α)以及超氧化物歧化酶(superoxide dismutase，SOD)、丙二醛(malondialdehyde，MDA)、谷胱甘肽过氧化物酶(glutathione peroxidase，GSH-Px)水平，用流式细胞技术检测活性氧(reactive oxygen species，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DOX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。流式细胞技术检测结果表明，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肿瘤抑制蛋白53(tumor protein p53，p53)、降低B淋巴细胞瘤-2相关X蛋白(B-cell lymphoma-2 associated X protein，Bax)/B淋巴细胞瘤-2 (B-cell lymphoma-2，Bcl-2)、抑制半胱氨酸天冬氨酸蛋白酶 9(cysteinyl aspartate specific proteinase 9，Caspase 9)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能改善DOX诱导的H9c2心肌细胞损伤，其机制可能与调节Nrf2/Keap1通路和线粒体凋亡通路有关。

Abstract:
OBJECTIVE To investigate the ameliorative effect of Avellaneine D from Tabebuia avellanedae on an myocardial injury induced by doxorubicin(DOX) and its mechanism of action based on the nuclear factor erythroid 2 related factor 2(Nrf2)/Kelch-like epichlorohydrin-associated protein 1(Keap1) and mitochondrial apoptotic pathways.
METHODS An H9c2 cardiomyocyte injury model was established using 1 μg·mL⁻¹ doxorubicin induction, and the cells were treated with Avellaneine D for 24 h. The cell viability and lactate dehydrogenase(LDH) level were measured, and the interleukin-1β(IL-1β), interleukin-6(IL-6), tumor necrosis factor-α(TNF-α), superoxide dismutase(SOD), malondialdehyde(MDA) and glutathione peroxidase(GSH-Px) levels were detected, the levels of reactive oxygen species(ROS), mitochondrial membrane potential and apoptosis rate were determined by flow cytometry assay, and the expression levels of key proteins in the mitochondrial apoptotic pathway and Nrf2/Keap1 pathway were examined via In-Cell-Western assay.
RESULTS Avellaneine D could effectively enhance cell viability and reduce the LDH level, and significantly inhibit the release of cardiomyocyte inflammatory factors of IL-6 and TNF-α, as well as improve oxidative stress, including reducing MDA level and enhancing GSH-px level. The results of flow cytometry assay showed that the Avellaneine D could 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 could inhibit the expression levesl of the key protein of the mitochondrial apoptosis pathway tumor protein p53(P53), reduce the expression level of B-cell lymphoma-2 associated X protein(Bax)/B-cell lymphoma-2(Bcl-2), inhibit the expression level of cysteinyl aspartate specific proteinase 9(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 ameliorate doxorubicin-induced H9c2 cardiomyocyte injury by regulation of the Nrf2/Keap1 and mitochondrial apoptotic the pathways.

HTML全文

参考文献(27)

施引文献

资源附件(0)