Department of Cardiology, The First Affiliated Hospital of Soochow University, China.
Department of Cardiology, Affiliated Hospital of Jiangnan University, China.
J Toxicol Sci. 2023;48(8):469-479. doi: 10.2131/jts.48.469.
The use of doxorubicin (DOX) may contribute to cardiotoxicity, limiting its clinical application. Thiolutin (THL) has been found to exert protective roles in various biological activities, while its effects on DOX-induced cardiotoxicity are still uncovered. Cell counting kit 8 assay was utilized to detect cell viability and half maximal inhibitory concentration of THL in H9c2 cardiomyocytes. The level of lactate dehydrogenase (LDH), adenosine triphosphate (ATP), interleukin (IL)-18 and IL-1 beta (IL-1β) were measured using the corresponding detection kits, and flow cytometry determined cell apoptosis rate. The reactive oxygen species (ROS) accumulation was evaluated by utilizing immunofluorescence or flow cytometry assay. The protein levels of NLR family Pyrin domain 3 (NLRP3), pro-Caspase1, cleaved-Caspase1, gasdermin D (GSDMD) and cleaved-GSDMD (GSDMD-N) in H9c2 cells were detected by immunoblotting assay. The treatment of THL reduced H9c2 cell viability in a gradient-dependent manner. THL treatment reversed the DOX-induced inhibition of proliferation, decrease of ATP, up-regulation of LDH, IL-18, IL-1β and production of ROS, activation of NLRP3 and inflammasome-mediated pyroptosis in H9c2 cells. Additionally, NLRP3 knockdown abolished the effects of THL in DOX-treated H9c2 cells remarkably. This investigation proved that THL notably ameliorated DOX-induced apoptosis, oxidative stress, and pyroptosis in H9c2 cardiomyocytes. Besides, THL effectively inactivated DOX-induced NLRP3 inflammasome in H9c2 cells. These findings revealed a promising drug to assist DOX in its anti-cancer effects and protect the heart of patients.
阿霉素（DOX）的应用可能导致心脏毒性，限制了其临床应用。硫利度胺（THL）在各种生物活性中发挥保护作用，但其对 DOX 诱导的心脏毒性的影响尚不清楚。细胞计数试剂盒 8 检测法用于检测 H9c2 心肌细胞的细胞活力和 THL 的半最大抑制浓度。利用相应的检测试剂盒检测乳酸脱氢酶（LDH）、三磷酸腺苷（ATP）、白细胞介素（IL）-18 和白细胞介素-1β（IL-1β）水平，流式细胞术检测细胞凋亡率。利用免疫荧光或流式细胞术评估活性氧（ROS）积累。免疫印迹法检测 H9c2 细胞中 N 端衔接蛋白样受体家族含 pyrin 结构域 3（NLRP3）、原半胱天冬酶 1、裂解半胱天冬酶 1、gasdermin D（GSDMD）和裂解 GSDMD（GSDMD-N）的蛋白水平。THL 处理以梯度依赖的方式降低 H9c2 细胞活力。THL 处理逆转了 DOX 诱导的增殖抑制、ATP 减少、LDH、IL-18、IL-1β和 ROS 产生增加、NLRP3 激活和炎性体介导的细胞焦亡。此外，NLRP3 敲低显著消除了 THL 在 DOX 处理的 H9c2 细胞中的作用。这项研究证明，THL 显著改善了 DOX 诱导的 H9c2 心肌细胞凋亡、氧化应激和细胞焦亡。此外，THL 有效地使 DOX 诱导的 H9c2 细胞中 NLRP3 炎性体失活。这些发现为辅助 DOX 发挥抗癌作用并保护患者心脏提供了一种有前途的药物。
