Objective

Efferocytosis dysfunction contributes to the progression and rupture of atherosclerotic plaques. Efferocytosis is crucially modulated by intracytoplasmic Ca²⁺, and mitochondrial calcium uniporter (MCU) complex proteins serve as key channels for regulating Ca²⁺ concentration. Therefore, it was speculated that MCU may affect the development of atherosclerosis (AS) by regulating efferocytosis. In the present study, we aimed to investigate whether MCU could affect foam cell formation by regulating efferocytosis.

Methods

We stimulated primary macrophages (Møs) using oxidized low-density lipoprotein (ox-LDL) to mimic the atherosclerotic microenvironment and treated them with Ru360, an MCU-specific inhibitor, and UNC1062, an inhibitor of efferocytosis. Additionally, we conducted double staining to determine the Mø efferocytosis rate. We measured the expression of MCU complexes and efferocytosis-associated proteins using western blotting (WB) and real-time quantitative polymerase chain reaction (RT-qPCR), respectively. In addition, we separately detected the Ca²⁺ level in the cytoplasm and mitochondria (MT) using Fluo-4 AM and Rhod-2 methods. We separately determined the reactive oxygen species (ROS) level in cytoplasm and MT using dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescent probing method and Mito-SOXTM superoxide indicator staining. Additionally, we conducted the enzyme-linked immunosorbent assay (ELISA) to detect the production of interleukin-6 (IL-6), interleukin-18 (IL-18), interleukin-1β (IL-1β), and tumor necrosis factor-alpha (TNF-α). Oil Red O staining was performed to measure cytoplasmic lipid levels.

Results

Ru360 attenuated ox-LDL-induced efferocytosis dysfunction, and attenuated the upregulation of MCU and MCUR1 induced by ox-LDL, and meanwhile attenuated the downregulation of MCUb induced by ox-LDL. Ru360 attenuated the decrease of intracytoplasmic Ca²⁺ concentration induced by ox- LDL, Ru360 also attenuated the ROS production induced by ox- LDL, attenuated the release of IL-6, IL-18, IL-1β, and TNF-α induced by ox- LDL, and attenuated the increase of intracytoplasmic lipid content induced by ox-LDL. UNC1062 attenuated the effects of Ru360 in reducing inflammatory cytokines and intracytoplasmic lipid content.

Conclusions

In this study, we found that MCU inhibition modulated intracytoplasmic Ca²⁺ concentration, improved impaired Mø efferocytosis, and reduced ROS generation. Macrophage efferocytosis removed apoptotic cells and prevented the release of inflammatory factor and foam cell formation, and this can be a potential new therapeutic target for alleviating atherosclerosis.

中文翻译：

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小鼠模型中线粒体钙单向转运蛋白对氧化低密度脂蛋白影响的巨噬细胞胞吞作用的调节

客观的

胞吞功能障碍导致动脉粥样硬化斑块的进展和破裂。胞质作用主要受胞质内 Ca ²⁺调节，线粒体钙单向转运蛋白 (MCU) 复合体蛋白是调节 Ca ²⁺浓度的关键通道。因此推测MCU可能通过调节胞吞作用影响动脉粥样硬化(AS)的发生发展。在本研究中，我们旨在研究 MCU 是否可以通过调节胞吞作用来影响泡沫细胞的形成。

方法

我们使用氧化低密度脂蛋白 (ox-LDL) 刺激初级巨噬细胞 (Møs) 来模拟动脉粥样硬化微环境，并用 MCU 特异性抑制剂 Ru360 和胞吞作用抑制剂 UNC1062 对其进行处理。此外，我们进行了双重染色以确定 Mø 胞吞率。我们分别使用蛋白质印迹 (WB) 和实时定量聚合酶链反应 (RT-qPCR) 测量了 MCU 复合物和胞吞作用相关蛋白的表达。此外，我们使用Fluo-4 AM和Rhod-2方法分别检测了细胞质和线粒体（MT）中的Ca ^2+水平。我们使用二氯二氢荧光素二乙酸酯（DCFH-DA）荧光探测方法和Mito-SOXTM超氧化物指示剂染色分别测定细胞质和MT中的活性氧（ROS）水平。此外，我们还进行了酶联免疫吸附测定（ELISA）来检测白细胞介素6（IL-6）、白细胞介素18（IL-18）、白细胞介素1β（IL-1β）和肿瘤坏死因子的产生- α（TNF-α）。进行油红O染色来测量细胞质脂质水平。

结果

Ru360减弱了ox-LDL诱导的胞吞功能障碍，减弱了ox-LDL诱导的MCU和MCUR1的上调，同时减弱了ox-LDL诱导的MCUb的下调。^{Ru360 减弱 ox- LDL 诱导的细胞质内 Ca 2+}浓度的降低，Ru360 还减弱 ox- LDL 诱导的 ROS 产生，减弱 ox- LDL 诱导的 IL-6、IL-18、IL-1β 和 TNF-α 的释放。 ox-LDL，并减弱ox-LDL引起的细胞质内脂质含量的增加。UNC1062 减弱了 Ru360 在减少炎症细胞因子和胞质内脂质含量方面的作用。

结论

在这项研究中，我们发现 MCU 抑制调节胞浆内 Ca ²⁺浓度，改善受损的 Mø 胞吞作用，并减少 ROS 生成。巨噬细胞胞吞作用清除了凋亡细胞，阻止了炎症因子的释放和泡沫细胞的形成，这可能成为缓解动脉粥样硬化的潜在新治疗靶点。