Li Xinhua, Yan Aili, Chang Jinrui, Li Fen, Zhu Juanxia
Department of Pharmacology and Toxicology, Xi'an Medical University, Xi'an 710021, China.
Department of Pathology, Xi'an Medical University, Xi'an 710021, China.
Sichuan Da Xue Xue Bao Yi Xue Ban. 2023 Sep;54(5):947-953. doi: 10.12182/20230960207.
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.
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.
Compared with the Control group, the DOX group showed swollen cellular morphology, decreased cell density and viability, and increased LDH activity in the medium ( <0.01); both apoptosis and the expression of cleaved caspase-3 and cytochrome c increased ( <0.01); the activities of CAT and SOD decreased while the contents of MDA and ROS increased ( <0.01); the expression of SIRT1, NRF2, and HO-1 decreased, the activity of SIRT1 decreased, and the expression of Ac-FOXO1 increased ( <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 ( <0.01); the apoptosis and the expression of cleaved caspase-3 and cytochrome c decreased ( <0.01); the activities of CAT and SOD increased while the levels of MDA and ROS decreased ( <0.01); the expression of SIRT1, NRF2, and HO-1 increased, the activity of SIRT1 increased, and the expression of Ac-FOXO1 decreased ( <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.
Hes inhibits oxidative stress and apoptosis via regulating SIRT1/NRF2 signaling, thereby reducing DOX-induced cardiotoxicity in H9c2 cells.
研究橙皮素（Hes）是否通过调节沉默信息调节因子1（SIRT1）/核转录因子E2相关因子2（NRF2）信号通路减轻阿霉素（DOX）诱导的H9c2细胞心肌细胞毒性。
用DOX处理H9c2细胞建立心脏毒性模型，并随机分为四组，即对照组（Control）和三个处理组，分别接受DOX（DOX组）、Hes+DOX（DOX+Hes组）、Hes+SIRT1抑制剂EX527+DOX（DOX+Hes+EX527组）。通过光学显微镜观察细胞形态。用CCK-8法评估细胞活力。采用流式细胞术检测DOX诱导的H9c2细胞凋亡。用2′,7′-二氯二氢荧光素二乙酸酯（DCFH-DA）染色法测定四组H9c2细胞中活性氧（ROS）水平。使用ELISA试剂盒检测乳酸脱氢酶（LDH）、超氧化物歧化酶（SOD）、过氧化氢酶（CAT）和SIRT1的活性以及丙二醛（MDA）含量。通过蛋白质免疫印迹法检测裂解的半胱天冬酶-3、细胞色素c、SIRT1、乙酰化FOXO1、NRF2和血红素加氧酶1（HO-1）的表达。
与对照组相比，DOX组细胞形态肿胀，细胞密度和活力降低，培养基中LDH活性升高（<0.01）；细胞凋亡以及裂解的半胱天冬酶-3和细胞色素c的表达增加（<0.01）；CAT和SOD活性降低，而MDA和ROS含量增加（<0.01）；SIRT1、NRF2和HO-1的表达降低，SIRT1活性降低，乙酰化FOXO1的表达增加（<0.01）。与DOX组相比，DOX+Hes组细胞形态改善，细胞密度和活力增加，培养基中LDH活性降低（<0.01）；细胞凋亡以及裂解的半胱天冬酶-3和细胞色素c的表达降低（<0.01）；CAT和SOD活性增加，而MDA和ROS水平降低（<0.01）；SIRT1、NRF2和HO-1的表达增加，SIRT1活性增加，乙酰化FOXO1的表达降低（<0.01）。DOX+Hes组和DOX+Hes+EX527组结果比较显示，EX527可阻断Hes对DOX诱导的细胞损伤、氧化应激和SIRT1/NRF2信号通路的保护作用。
Hes通过调节SIRT1/NRF2信号通路抑制氧化应激和细胞凋亡，从而减轻DOX诱导的H9c2细胞心脏毒性。
