Arsenic trioxide (ATO) is among the first-line chemotherapeutic drugs for treating acute promyelocytic leukemia patients, but its clinical use is hampered due to cardiotoxicity. The present investigation unveils the mechanism underlying ATO-induced oxidative stress that promotes calcineurin (a ubiquitous Ca²⁺/calmodulin-dependent serine/threonine phosphatase expressed only during sustained Ca²⁺ elevation) expression, inflammatory cytokine release and apoptosis in H9c2 cardiomyoblasts, and its possible modulation with phloretin (PHL, an antioxidant polyphenol present in apple peel). ATO caused Ca²⁺ overload resulting in elevated expression of calcineurin and its downstream transcriptional effector NFATc causing the release of cytokines such as IL-2, IL-6, MCP-1, IFN-γ, and TNF-α in H9c2 cardiomyoblast. There was a visible increase in the nuclear fraction of NF-κB and ROS-mediated apoptotic cell death. The expression levels of cardiac-specific genes (troponin, desmin, and caveolin-3) and genes of the apoptotic signaling pathway (BCL-2, BAX, IGF1, AKT, ERK1, -2, RAF1, and JNK) in response to ATO and PHL were studied. The putative binding mode and the potential ligand–target interactions of PHL with calcineurin using docking software (Autodock and iGEMDOCKv2) showed the high binding affinity of PHL to calcineurin. PHL co-treatment significantly reduced Ca²⁺ influx and normalized the expression of calcineurin, NFATc, NF-κB, and other cytokines. PHL co-treatment resulted in activation of BCL-2, IGF1, AKT, RAF1, ERK1, and ERK2 and inhibition of BAX and JNK. Overall, these results revealed that PHL has a protective effect against ATO-induced apoptosis and we propose calcineurin as a druggable target for the interaction of PHL in ATO cardiotoxicity in H9c2 cells.

三氧化二砷（ATO）是治疗急性早幼粒细胞白血病患者的一线化疗药物之一，但其心脏毒性阻碍了其临床应用。本研究揭示了 ATO 诱导的氧化应激促进 H9c2 心肌细胞钙调磷酸酶（一种普遍存在的 Ca ²⁺ /钙调蛋白依赖性丝氨酸/苏氨酸磷酸酶，仅在持续 Ca ²⁺升高期间表达）表达、炎性细胞因子释放和细胞凋亡的潜在机制，以及它可能与根皮素（PHL，一种存在于苹果皮中的抗氧化多酚）进行调节。ATO引起Ca ²⁺过载导致钙调神经磷酸酶及其下游转录效应因子 NFATc 的表达升高，导致 H9c2 心肌细胞中细胞因子如 IL-2、IL-6、MCP-1、IFN-γ 和 TNF-α 的释放。NF-κB 的核部分和 ROS 介导的凋亡细胞死亡明显增加。心脏特异性基因（肌钙蛋白、结蛋白和caveolin-3）和凋亡信号通路基因（BCL-2、BAX、IGF1、AKT、ERK1、-2、RAF1和JNK ）的表达水平) 对 ATO 和 PHL 的反应进行了研究。使用对接软件（Autodock 和 iGEMDOCKv2）推测的 PHL 与钙调磷酸酶的结合模式和潜在的配体-靶标相互作用表明 PHL 对钙调神经磷酸酶具有高结合亲和力。PHL 联合治疗显着减少了 Ca ²⁺流入，并使神经钙蛋白、NFATc、NF-κB 和其他细胞因子的表达正常化。PHL 联合治疗导致BCL-2、IGF1、AKT、RAF1、ERK1和ERK2的激活以及BAX和JNK的抑制。总的来说，这些结果表明 PHL 对 ATO 诱导的细胞凋亡具有保护作用，我们建议神经钙蛋白作为 PHL 在 H9c2 细胞中 ATO 心脏毒性中相互作用的药物靶点。