Introduction

Iron retention is commonly observed in atherosclerotic plaques and is believed to be detrimental to atherosclerosis. Platelet P2Y12 is a target of antiplatelet therapy in preventing thrombotic complications of atherosclerosis. The protective effect of P2Y12 on hematopoiesis reported by our previous work implies the involvement of P2Y12 in iron metabolism.

Objectives

This study further investigated the role of P2Y12 in the iron metabolism of macrophages, the key player in systemic iron homeostasis and atherosclerosis.

Methods

The association between serum iron and the use of P2Y12 inhibitors was evaluated by a case-control study in human. Secondary iron overload and atherosclerosis animal models were established in P2Y12-deficient zebrafish to explore the role of P2Y12 in macrophage iron metabolism in vivo. Both iron-overloaded murine primary peritoneal macrophages (PMs) and ox-LDL–treated PMs with P2Y12 knockdown were used for in vitro studies. RNA sequencing and pharmacological approaches were performed to investigate the downstream mechanisms.

Results

Increased serum iron level was positively associated with P2Y12 inhibitor usage [odds ratio (OR) = 10.333 (1.281–83.370)]. Elevated serum iron level and transferrin saturation, reduced hepatic and splenic iron content, and decreased iron staining in macrophages were observed in secondary iron overload P2Y12-deficient zebrafish. Deficiency of P2Y12 in ApoEb^-/- zebrafish fed a high-fat diet reduced atherosclerosis progression and intraplaque iron retention. Furthermore, reduced ferritin, restored cell viability and expression of ferroptosis marker proteins, and decreased ROS formation and inflammatory cytokines were observed in both iron-overloaded and ox-LDL–treated PMs with P2Y12 knockdown in vitro, while reversed phenotypes were observed after agonist-induced P2Y12 activation. Mechanistically, P2Y12 inhibition in iron-overloaded or ox-LDL–treated PMs suppressed NF-κB p65 phosphorylation and hepcidin expression, both of which were reversed by P2Y12 activation.

Conclusion

P2Y12 inhibition decreased hepcidin autocrine through repressing NF-κB p65 phosphorylation in macrophages, preventing intracellular iron retention and atherosclerosis.

中文翻译：

---

巨噬细胞 P2Y12 调节铁转运，其抑制作用可防止动脉粥样硬化

 介绍

铁潴留常见于动脉粥样硬化斑块，被认为对动脉粥样硬化有害。血小板 P2Y12 是抗血小板治疗预防动脉粥样硬化血栓并发症的靶点。我们之前的工作报道的 P2Y12 对造血的保护作用意味着 P2Y12 参与铁代谢。

 目标

本研究进一步探讨了 P2Y12 在巨噬细胞铁代谢中的作用，巨噬细胞是系统性铁稳态和动脉粥样硬化的关键参与者。

 方法

通过一项人体病例对照研究评估血清铁与 P2Y12 抑制剂使用之间的关联。在 P2Y12 缺陷型斑马鱼中建立继发性铁过载和动脉粥样硬化动物模型，探讨 P2Y12 在体内巨噬细胞铁代谢中的作用。铁超负荷的小鼠原代腹膜巨噬细胞 （PM） 和 ox-LDL 处理的 P2Y12 敲低 PM 均用于体外研究。进行 RNA 测序和药理学方法以研究下游机制。

 结果

血清铁水平升高与 P2Y12 抑制剂的使用呈正相关 [比值比 （OR） = 10.333 （1.281–83.370）]。在继发性铁过载 P2Y12 缺陷型斑马鱼中观察到血清铁水平升高和转铁蛋白饱和度升高，肝脏和脾铁含量降低，以及巨噬细胞铁染色减少。喂食高脂饮食的 ApoEb^-/- 斑马鱼中 P2Y12 的缺乏减少了动脉粥样硬化的进展和斑块内铁滞留。此外，在体外敲低 P2Y12 的铁过载和 ox-LDL 处理的 PM 中观察到铁蛋白降低，恢复细胞活力和铁死亡标志物表达，减少 ROS 形成和炎性细胞因子，而在激动剂诱导的 P2Y12 激活后观察到相反的表型。从机制上讲，铁过载或 ox-LDL 处理的 PM 中的 P2Y12 抑制抑制了 NF-κB p65 磷酸化和铁调素表达，这两者都被 P2Y12 激活逆转。

 结论

P2Y12 抑制通过抑制巨噬细胞中 NF-κB p65 磷酸化来降低铁调素自分泌，防止细胞内铁滞留和动脉粥样硬化。