目的 探究人参皂苷Rb₃（ginsenoside Rb₃，Rb₃）抑制巨噬细胞脂质积累的分子机制。方法 采用氧化低密度脂蛋白（oxidized low-density lipoprotein，ox-LDL）诱导小鼠RAW264.7巨噬细胞建立脂质相关巨噬细胞（lipid-associated macrophages，LAMs）模型，给予Rb₃干预后，通过油红O染色及脂滴绿色荧光染色评估细胞内脂质积累情况；采用非靶向脂质组学分析脂质组成变化；分别进行转录组学与蛋白质组学检测，并对多组学结果进行生物信息学整合分析；利用Autodock对Rb₃与多组学交集分子进行分子对接；通过qRT-PCR与Western blotting验证关键分子的表达变化；通过细胞热迁移技术（cell thermal shift assay，CETSA）考察Rb₃与补体成分5a受体1（complement component 5a receptor 1，C5ar1）、白细胞分化抗原36（cluster of differentiation 36 antigen，Cd36）靶点的结合情况。结果 成功构建脂质富集的LAMs模型。Rb₃在无细胞毒性的剂量范围内呈剂量相关性地抑制细胞内脂质积累（P＜0.05、0.01、0.001）。脂质组学结果显示，模型组三酰甘油类脂质显著升高，而甘油磷脂类脂质降低；Rb₃处理后表现为三酰甘油类脂质下降、甘油磷脂类脂质升高，通路富集于甘油磷脂代谢通路。转录组学检测到1 291个差异表达基因，显著富集于过氧化物酶体增殖物激活受体（peroxisome proliferator-activated receptor，PPAR）信号通路。蛋白质组学检测到254个差异表达蛋白，通路富集于肌萎缩侧索硬化等相关信号通路。多组学交叉分析得到Cd36、C5ar1、三磷酸腺苷结合盒转运蛋白G1（adenosine triphosphate binding cassette transporter G1，Abcg1）等6个交集分子。分子对接预测Rb₃与Cd36、C5ar1和Abcg1具有良好的结合潜能。CETSA结果显示Rb₃可显著保护Cd36和C5ar1蛋白随温度提高的降解，表明Rb₃与Cd36、C5ar1蛋白存在结合作用。qRT-PCR与Western blotting结果显示，Rb₃可显著下调LAMs中Cd36和C5ar1表达（P＜0.05、0.01、0.001），同时上调PPARγ、Abca1和Abcg1的表达（P＜0.05）。结论 Rb₃通过激活PPAR信号通路和调控甘油磷脂代谢，靶向Cd36和C5ar1抑制其介导的脂质摄取，并促进Abca1与Abcg1介导的脂质外排，调控三酰甘油和甘油磷脂的胞内水平，从而发挥抑制巨噬细胞中脂质积累的作用。

Objective To investigate the molecular mechanisms by which ginsenoside Rb₃ (Rb₃) inhibits lipid accumulation in macrophages. Methods Oxidized low-density lipoprotein (ox-LDL) was used to induce lipid-associated macrophages (LAMs) model in murine RAW264.7 macrophages. After Rb₃ intervention, intracellular lipid accumulation was evaluated by oil red O staining and BODIPY-based neutral lipid fluorescence staining. Changes in lipid composition were analyzed using untargeted lipidomics. Transcriptomic and proteomic analyses were performed in parallel, followed by integrative multi-omics bioinformatic analysis. Molecular docking was conducted using Autodock to predict the binding potential between Rb₃ and candidate target molecules identified from multi-omics intersections. The expression levels of key genes and proteins were validated by qRT-PCR and Western blotting. Cell thermal shift assay (CETSA) was employed to evaluate the binding of Rb₃ to complement component 5a receptor 1 (C5ar1) and cluster of differentiation 36 antigen (Cd36) targets. Results A lipid-enriched LAMs model was successfully established. Within a non-cytotoxic concentration range, Rb₃ dose-dependently suppressed intracellular lipid accumulation (P < 0.05, 0.01, 0.001). Lipidomics revealed a marked elevation of triglycerides accompanied by a reduction in glycerophospholipids in model group, whereas Rb₃ treatment significantly reduced triglyceride levels and restored glycerophospholipids, with pathway enrichment mainly involving glycerophospholipid metabolism. Transcriptomic analysis identified 1 291 differentially expressed genes, which were significantly enriched in peroxisome proliferator-activated receptor (PPAR) signaling pathway. Proteomic analysis detected 254 differentially expressed proteins, with enrichment in pathways including amyotrophic lateral sclerosis-related signaling. Integrative multi-omics analysis identified six overlapping target molecules, including Cd36, C5ar1 and adenosine triphosphate binding cassette transporter G1 (Abcg1). Molecular docking predicted favorable binding affinities between Rb₃ and Cd36, C5ar1, Abcg1. CETSA results demonstrated that Rb₃ significantly protected Cd36 and C5ar1 proteins from temperature-dependent degradation, providing further evidence of direct binding between Rb₃ and these targets. qRT-PCR and Western blotting results showed that Rb₃ significantly down-regulated the expressions of Cd36 and C5ar1 in LAMs (P < 0.05, 0.01, 0.001), while up-regulated the expressions of PPARγ, Abca1 and Abcg1 (P < 0.05). Conclusion Rb₃ activates PPAR signaling pathway and regulates glycerophospholipid metabolism, targeting Cd36 and C5ar1 to inhibit their mediated lipid uptake and promote Abca1 and Abcg1 mediated lipid efflux, regulating the intracellular levels of triglycerides and glycerophospholipids, thereby exerting an inhibitory effect on lipid accumulation in macrophages.

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国家自然科学基金资助项目(82374116);国家自然科学基金资助项目(81773946);国家自然科学基金资助项目(81573673);国家自然科学基金资助项目(81001666)