Objective: To investigate whether hesperetin (Hes) alleviates doxorubicin (DOX)-induced cardiomyocytotoxicity by reducing oxidative stress via regulating silent information regulator 1 (SIRT1)/nuclear transcription factor E2-related factor 2 (NRF2) signaling in H9c2 cells.
Methods: H9c2 cells were treated with DOX to establish the cardiotoxicity model and were randomly assigned to four groups, a control group (Control) and three treatment groups, receiving respectively DOX (the DOX group), Hes+DOX (the DOX+Hes group), and Hes+SIRT1 inhibitor EX527+DOX (the DOX+Hes+EX527 group). Cellular morphology was observed by the light microscope. Cell viability was evaluated by CCK-8. DOX-induced apoptosis in H9c2 cells was examined by flow cytometry. The levels of reactive oxygen species (ROS) in the H9c2 cells of the four groups were determied with 2'-7'-dichlorodihydrofluorescein diacetate (DCFH-DA) staining. The activities of lactate dehydrogenase (LDH), superoxide dismutase (SOD), catalase (CAT), and SIRT1 as well as the malondialdehyde (MDA) content were measured using ELISA kits. The expressions of cleaved caspase-3, cytochrome c, SIRT1, Ac-FOXO1, NRF2, and heme oxygenase 1 (HO-1) were determined by Western blot.
Results: Compared with the Control group, the DOX group showed swollen cellular morphology, decreased cell density and viability, and increased LDH activity in the medium ( P<0.01); both apoptosis and the expression of cleaved caspase-3 and cytochrome c increased ( P<0.01); the activities of CAT and SOD decreased while the contents of MDA and ROS increased ( P<0.01); the expression of SIRT1, NRF2, and HO-1 decreased, the activity of SIRT1 decreased, and the expression of Ac-FOXO1 increased ( P<0.01). Compared with the DOX group, the DOX+Hes group showed improved cellular morphology, increased cell density and viability, and decreased LDH activity in the medium ( P<0.01); the apoptosis and the expression of cleaved caspase-3 and cytochrome c decreased ( P<0.01); the activities of CAT and SOD increased while the levels of MDA and ROS decreased ( P<0.01); the expression of SIRT1, NRF2, and HO-1 increased, the activity of SIRT1 increased, and the expression of Ac-FOXO1 decreased ( P<0.01). Comparison of the findings for the DOX+Hes group and the DOX+Hes+EX527 group showed that EX527 could block the protective effects of Hes against DOX-induced cell injury, oxidative stress, and SIRT1/NRF2 signaling.
Conclusion: Hes inhibits oxidative stress and apoptosis via regulating SIRT1/NRF2 signaling, thereby reducing DOX-induced cardiotoxicity in H9c2 cells.
目的: 本研究旨在探讨橙皮素（hesperetin, Hes）能否通过调控沉默信息调节因子1（silent information regulator 1, SIRT1）/核转录因子E2相关因子2（nuclear transcription factor E2-related factor 2, NRF2）信号减轻氧化应激改善阿霉素（doxorubicin, DOX）诱导的H9c2心肌细胞毒性。
方法: 采用DOX诱导的H9c2心肌细胞毒性模型，随机分为4组：对照组（Control）、DOX处理组（DOX）、Hes加DOX处理组（DOX+Hes）以及Hes加SIRT1抑制剂EX527联合DOX处理组（DOX+Hes+EX527）。光镜下观察细胞形态，CCK-8检测各组细胞活力，流式细胞术检测各组细胞凋亡率，DCFH-DA染色观察各组ROS水平，ELISA试剂盒检测乳酸脱氢酶（lactic dehydrogenase, LDH）、超氧化物歧化酶（superoxide dismutase, SOD）、过氧化氢酶（catalase, CAT）和SIRT1活性以及丙二醛（malondialdehyde, MDA）含量，Western blot检测裂解的caspase-3（cleaved caspase-3）、细胞色素c（cytochrome c）、SIRT1、Ac-FOXO1、NRF2和血红素加氧酶1（heme oxygenase 1, HO-1）的表达。
结果: 和Control组相比，DOX组细胞形态肿胀，密度减小，细胞活力降低，培养基中LDH活性增加（P<0.01）；细胞凋亡增多，cleaved caspase-3和cytochrome c表达增加（P<0.01）；CAT和SOD活性降低，MDA含量和ROS水平增加（P<0.01）；SIRT1、NRF2和HO-1表达以及SIRT1活性降低，Ac-FOXO1表达增加（P<0.01）；与DOX组相比，DOX+Hes组细胞形态改善，密度和细胞活力增加，培养基中LDH活性降低（P<0.01）；细胞凋亡减少，cleaved caspase-3和cytochrome c表达降低（P<0.01）；CAT和SOD活性升高，MDA含量和ROS水平降低（P<0.01）；SIRT1、NRF2和HO-1表达以及SIRT1活性增加，Ac-FOXO1表达降低（P<0.01）；而与DOX+Hes组相比，EX527阻断Hes对DOX诱导的H9c2细胞损伤、氧化应激和SIRT1/NRF2信号的作用。
结论: Hes可以通过调控SIRT1/NRF2信号抑制氧化应激和细胞凋亡减轻DOX诱导的H9c2心肌细胞毒性。
Keywords: Doxorubicin; H9c2 cells; Hesperetin; Oxidative stress; SIRT1/NRF2.
Copyright© by Editorial Board of Journal of Sichuan University (Medical Sciences).