Department of Transfusion Medicine, Nanjing Drum Tower Hospital, Medical School of Nanjing University, Nanjing, China.
Department of Clinical Laboratory, Nanjing Drum Tower Hospital, Medical School of Nanjing University, Nanjing, China.
Immun Inflamm Dis. 2024 Nov;12(11):e70058. doi: 10.1002/iid3.70058.
Previous research has established that the oxidized low-density lipoprotein/β2-glycoprotein I/anti-β2-glycoprotein I antibody (oxLDL/β2GPI/anti-β2GPI) complex can stimulate macrophages to secrete molecules associated with atherosclerosis (AS), such as monocyte chemotactic protein 1 (MCP-1), tissue factor (TF), and tumor necrosis factor-α (TNF-α). This complex also enhances the uptake of oxLDL, thereby accelerating foam cell formation through the Toll-like receptor-4/nuclear factor kappa B (TLR4/NF-κB) pathway. Given the critical role of macrophage autophagy in the instability of vulnerable atherosclerotic plaques, it is imperative to investigate whether the oxLDL/β2GPI/anti-β2GPI complex influences macrophage autophagy in AS. This study aims to elucidate the effects and underlying mechanisms of the oxLDL/β2GPI/anti-β2GPI complex on macrophage autophagy in AS.
Experiments were conducted using murine macrophage RAW264.7 cells and the human monocytic cell line THP-1. Western blot analysis was employed to determine the expressions of autophagy-associated markers and signaling pathway proteins. Autophagosomes were detected through mRFP-GFP-LC3 adenoviral transfection and transmission electron microscopy (TEM).
Treatment of macrophages with the oxLDL/β2GPI/anti-β2GPI complex resulted in decreased expressions of Beclin1 and LC3 proteins, alongside an upregulation of SQSTM1/P62 protein expression. Additionally, there was a reduction in the number of autophagosomes and autolysosomes. An increase in the phosphorylation levels of phosphoinositide-3-kinase (PI3K), protein kinase B (AKT), and mammalian target of rapamycin (mTOR) was also observed. Notably, the expressions of autophagy-associated markers were partially restored when the TLR4/NF-κB and PI3K/AKT/mTOR pathways were inhibited by their respective inhibitors.
Our findings indicate that the oxLDL/β2GPI/anti-β2GPI complex inhibits macrophage autophagy in AS via the TLR4/NF-κB and PI3K/AKT/mTOR signaling pathways.
先前的研究已经证实，氧化型低密度脂蛋白/β2-糖蛋白 I/抗-β2-糖蛋白 I 抗体（oxLDL/β2GPI/抗-β2GPI）复合物可以刺激巨噬细胞分泌与动脉粥样硬化（AS）相关的分子，如单核细胞趋化蛋白 1（MCP-1）、组织因子（TF）和肿瘤坏死因子-α（TNF-α）。这种复合物还增强了 oxLDL 的摄取，从而通过 Toll 样受体 4/核因子 kappa B（TLR4/NF-κB）通路加速泡沫细胞的形成。鉴于巨噬细胞自噬在易损斑块不稳定性中的关键作用，有必要研究 oxLDL/β2GPI/抗-β2GPI 复合物是否影响 AS 中的巨噬细胞自噬。本研究旨在阐明 oxLDL/β2GPI/抗-β2GPI 复合物对 AS 中巨噬细胞自噬的影响及其潜在机制。
使用鼠巨噬细胞 RAW264.7 细胞和人单核细胞系 THP-1 进行实验。采用 Western blot 分析测定自噬相关标记物和信号通路蛋白的表达。通过 mRFP-GFP-LC3 腺病毒转染和透射电子显微镜（TEM）检测自噬体。
用 oxLDL/β2GPI/抗-β2GPI 复合物处理巨噬细胞，导致 Beclin1 和 LC3 蛋白表达减少，同时 SQSTM1/P62 蛋白表达上调。此外，自噬体和自溶体的数量减少。磷酸化的磷脂酰肌醇-3-激酶（PI3K）、蛋白激酶 B（AKT）和哺乳动物雷帕霉素靶蛋白（mTOR）的水平也升高。值得注意的是，当 TLR4/NF-κB 和 PI3K/AKT/mTOR 通路分别被其抑制剂抑制时，自噬相关标记物的表达部分恢复。
我们的研究结果表明，oxLDL/β2GPI/抗-β2GPI 复合物通过 TLR4/NF-κB 和 PI3K/AKT/mTOR 信号通路抑制 AS 中巨噬细胞自噬。
