Avellaneine D from Tabebuia Avellanedae Ameliorates Doxorubicin-induced Damage in H9c2 Cardiomyocytes via Nrf2/Keap1 Pathway and Mitochondrial Apoptotic Pathway

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.