College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, People's Republic of China.
Divison of Cardiology, Affiliated Hospital of Nanjing University of Chinese Medicine, Jiangsu Province Hospital of Chinese Medicine, Nanjing, People's Republic of China.
J Ethnopharmacol. 2024 Nov 15;334:118582. doi: 10.1016/j.jep.2024.118582. Epub 2024 Jul 14.
Radiation-induced heart disease (RIHD) is one of the most serious complications in patients receiving chest radiotherapy, partially offsetting its benefits. At present, there is a lack of effective treatments for RIHD. Ferroptosis is a newly discovered type of cell death that results from iron-dependent lipid peroxide accumulation. It was recently shown that irradiation generates severe ferroptosis, providing new insights for the treatment of RIHD. Abelmoschus manihot (L.) possesses excellent pharmacological properties and is widely used in treating various ischemic heart and brain diseases; however, its efficacy and mechanism in treating RIHD are unknown.
This study aimed to investigate the efficacy and mechanism of total extracts from A. manihot (L.) (TEA) in treating RIHD.
C57BL/6 mice and H9C2 cells were exposed to irradiation to induce RIHD in vivo and in vitro, respectively. In vivo, we evaluated the protective effects of TEA (150 and 300 mg/kg) on RIHD. Body and heart weight changes of mice were calculated in each group, and malondialdehyde (MDA) level, glutathione/oxidized glutathione (GSH/GSSH) and nicotinamide adenine dinucleotide phosphate (NADPH/NADP) ratios, western blot, heart histology, and immunohistochemistry were used to evaluate TEA effectiveness. We identified the potential mechanism of radiation-induced cardiomyocyte injury in H9C2 cells treated with small interfering RNA. We determined the effective dose of TEA (0.6 mg/mL) using a Cell Counting Kit-8 assay. Intracellular Fe and lipid peroxidation levels were detected by Phen Green™ SK diacetate probe, BODIPY 581/591 C11 staining, and MDA, GSH, and NADPH kits, and the level of target protein was evaluated by immunofluorescence and western blot.
Radiation inhibited system Xc-cystine (xCT)/glutathione peroxidase 4 (GPX4) expression and activity in cardiomyocytes in a time and dose-dependent manner. After silencing xCT/GPX4, MDA significantly increased and GSH/GSSH and NADPH/NADP ratios were reduced. xCT/GPX4 inhibition drove ferroptosis in radiation-induced H9C2 injury. Oxidative stress in H9C2 was significantly enhanced by irradiation, which also significantly increased NADPH oxidase 4 (NOX4) expression and inhibited nuclear factor E2-related factor 2 (Nrf2) expression in vivo and in vitro. Inhibition of xCT/GPX4 drove ferroptosis in radiation-induced H9C2 injury, which was aggravated by inactivation of Nrf2 and alleviated by inhibition of NOX4. Compared with the ionizing radiation-only group, TEA improved body weight loss, MDA levels, and histological changes induced by irradiation in mice hearts, and increased the ratio of GSH/GSSH and NADPH/NADPin vivo; it also reduced lipid peroxidation and intracellular Fe accumulation, restored MDA levels, and elevated the ratios of GSH/GSSH and NADPH/NADP in irradiation-injured H9C2 cells. TEA up-regulated Nrf2, xCT, and GPX4 expression and inhibited NOX4 expression in vivo and in vitro.
Ferroptosis induced by redox imbalance mediated through the NOX4/xCT/GPX4 axis is a potential mechanism behind radiation-induced cardiomyocyte injury, and can be prevented by TEA.
放射性心脏病（RIHD）是接受胸部放射治疗的患者最严重的并发症之一，部分抵消了放射治疗的益处。目前，RIHD 缺乏有效的治疗方法。铁死亡是一种新发现的细胞死亡类型，是由铁依赖性脂质过氧化物积累引起的。最近的研究表明，照射会产生严重的铁死亡，为 RIHD 的治疗提供了新的思路。黄蜀葵（Abelmoschus manihot (L.)）具有极好的药理学特性，广泛用于治疗各种缺血性心脏和脑部疾病；然而，其治疗 RIHD 的疗效和机制尚不清楚。
本研究旨在探讨黄蜀葵总提取物（TEA）治疗 RIHD 的疗效和机制。
采用 C57BL/6 小鼠和 H9C2 细胞分别进行体内和体外照射，诱导 RIHD。体内实验中，我们评估了 TEA（150 和 300mg/kg）对 RIHD 的保护作用。计算各组小鼠的体重和心脏重量变化，采用丙二醛（MDA）水平、谷胱甘肽/氧化型谷胱甘肽（GSH/GSSH）和烟酰胺腺嘌呤二核苷酸磷酸（NADPH/NADP）比值、western blot、心脏组织学和免疫组织化学等方法评估 TEA 的效果。我们在接受小干扰 RNA 处理的 H9C2 细胞中鉴定了辐射诱导心肌细胞损伤的潜在机制。通过细胞计数试剂盒-8 实验确定 TEA（0.6mg/mL）的有效剂量。通过 Phen Green™ SK 二乙酸酯探针、BODIPY 581/591 C11 染色、MDA、GSH 和 NADPH 试剂盒检测细胞内铁和脂质过氧化水平，并通过免疫荧光和 western blot 评估靶蛋白水平。
辐射以时间和剂量依赖的方式抑制了心肌细胞中系统 Xc-胱氨酸（xCT）/谷胱甘肽过氧化物酶 4（GPX4）的表达和活性。沉默 xCT/GPX4 后，MDA 明显增加，GSH/GSSH 和 NADPH/NADP 比值降低。xCT/GPX4 抑制导致辐射诱导的 H9C2 损伤中的铁死亡。照射显著增强了 H9C2 中的氧化应激，这也显著增加了 NADPH 氧化酶 4（NOX4）的表达，并抑制了体内和体外的核因子 E2 相关因子 2（Nrf2）的表达。xCT/GPX4 抑制驱动辐射诱导的 H9C2 损伤中的铁死亡，这种作用被 Nrf2 的失活加重，被 NOX4 的抑制减轻。与单纯离子照射组相比，TEA 改善了照射诱导的小鼠心脏体重减轻、MDA 水平和组织学变化，并增加了体内 GSH/GSSH 和 NADPH/NADP 的比值；它还减少了脂质过氧化和细胞内铁的积累，恢复了 MDA 水平，并提高了照射损伤的 H9C2 细胞中 GSH/GSSH 和 NADPH/NADP 的比值。TEA 在体内和体外均上调了 Nrf2、xCT 和 GPX4 的表达，并抑制了 NOX4 的表达。
NOX4/xCT/GPX4 轴介导的氧化还原失衡诱导的铁死亡是辐射诱导心肌细胞损伤的潜在机制，可被 TEA 预防。
