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Background <p>Atherosclerosis is a chronic inflammatory disease. Inhibition of macrophage inflammatory secretion is the key to the prevention and treatment of atherosclerosis. Ershen Dan (ESD) has been shown to be effective in treating atherosclerosis; however, its pharmacological mechanisms remain unclear. This study elucidated the mechanism of action of ESD by investigating its relationship with macrophages.</p> Materials and methods <p>The chemical composition of ESD was analyzed using ultra performance liquid chromatography (UPLC)-Q-Exactive-tandem mass spectrometry (MS/MS). In the <i>in vivo</i>&#xa0;experiments, serum levels of low-density lipoprotein (LDL), triglycerides (TG), and total cholesterol (TC) in ApoE<sup>−/−</sup>&#xa0;mice were measured using a biochemical analyzer. The serum levels of key inflammatory factors were quantified using an enzyme-linked immunosorbent assay (ELISA). The aortic lipid plaque area was assessed using Oil Red O staining, while plaque characteristics were evaluated using hematoxylin and eosin (H&amp;E), Masson trichrome, and Sirius Red staining techniques. Network pharmacology analyses in conjunction with molecular dynamics simulations was used to screen the active components of ESD and their target proteins. By integrating database resources, the key target genes related to inflammatory responses in atherosclerosis were identified. The expression levels of neurogenic locus notch homolog protein 1 (NOTCH1), hairy and enhancer of split-1 (HES1), NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3), and nuclear factor (NF)-κB in mouse aortic tissue were detected using western blot analyses. CD86 levels in mouse aortas were quantitatively analyzed by immunohistochemistry. In the <i>in vitro</i>&#xa0;experiments, RAW264.7 cell viability was assessed using the Cell Counting Kit-8 (CCK-8) assay. Macrophage phenotypic changes were evaluated using immunofluorescence analyses. Intracellular levels of reactive oxygen species (ROS) were measured using dichloro-dihydro-fluorescein diacetate (DCFH-DA) probes. The expression levels of key proteins and genes were validated by western blotting and quantitative real-time polymerase chain reaction (qRT-PCR).</p> Results <p>Twenty bioactive chemical components were identified in ESD.&#xa0;<i>In vivo</i>&#xa0;studies demonstrated that ESD inhibits macrophage secretion of inflammatory factors through the NOTCH1/NF-κB/NLRP3&#xa0;signaling pathway, indicating its therapeutic potential for atherosclerosis.&#xa0;<i>In vitro</i>&#xa0;studies further revealed that ginsenoside Rg<sub>1</sub>&#xa0;and&#xa0;tanshinone II<sub>A</sub>, which are active constituents of ESD, exert anti-inflammatory effects by suppressing the NOTCH1/NF-κB/NLRP3&#xa0;pathway and reducing intracellular ROS levels in macrophages, supporting their role in atherosclerosis treatment.</p>

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Ershen Dan attenuates atherosclerosis by modulating the NOTCH1/NF-κB/NLRP3 signaling pathway to suppress M1 macrophage polarization

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年10月23日

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  • Wenjie Zhao,
  • Hui Wang,
  • Shijing Peng,
  • Qing Wei,
  • Lei Zhang,
  • Yunlun Li,
  • Wenqing Yang

摘要

Background

Atherosclerosis is a chronic inflammatory disease. Inhibition of macrophage inflammatory secretion is the key to the prevention and treatment of atherosclerosis. Ershen Dan (ESD) has been shown to be effective in treating atherosclerosis; however, its pharmacological mechanisms remain unclear. This study elucidated the mechanism of action of ESD by investigating its relationship with macrophages.

Materials and methods

The chemical composition of ESD was analyzed using ultra performance liquid chromatography (UPLC)-Q-Exactive-tandem mass spectrometry (MS/MS). In the in vivo experiments, serum levels of low-density lipoprotein (LDL), triglycerides (TG), and total cholesterol (TC) in ApoE−/− mice were measured using a biochemical analyzer. The serum levels of key inflammatory factors were quantified using an enzyme-linked immunosorbent assay (ELISA). The aortic lipid plaque area was assessed using Oil Red O staining, while plaque characteristics were evaluated using hematoxylin and eosin (H&E), Masson trichrome, and Sirius Red staining techniques. Network pharmacology analyses in conjunction with molecular dynamics simulations was used to screen the active components of ESD and their target proteins. By integrating database resources, the key target genes related to inflammatory responses in atherosclerosis were identified. The expression levels of neurogenic locus notch homolog protein 1 (NOTCH1), hairy and enhancer of split-1 (HES1), NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3), and nuclear factor (NF)-κB in mouse aortic tissue were detected using western blot analyses. CD86 levels in mouse aortas were quantitatively analyzed by immunohistochemistry. In the in vitro experiments, RAW264.7 cell viability was assessed using the Cell Counting Kit-8 (CCK-8) assay. Macrophage phenotypic changes were evaluated using immunofluorescence analyses. Intracellular levels of reactive oxygen species (ROS) were measured using dichloro-dihydro-fluorescein diacetate (DCFH-DA) probes. The expression levels of key proteins and genes were validated by western blotting and quantitative real-time polymerase chain reaction (qRT-PCR).

Results

Twenty bioactive chemical components were identified in ESD. In vivo studies demonstrated that ESD inhibits macrophage secretion of inflammatory factors through the NOTCH1/NF-κB/NLRP3 signaling pathway, indicating its therapeutic potential for atherosclerosis. In vitro studies further revealed that ginsenoside Rg1 and tanshinone IIA, which are active constituents of ESD, exert anti-inflammatory effects by suppressing the NOTCH1/NF-κB/NLRP3 pathway and reducing intracellular ROS levels in macrophages, supporting their role in atherosclerosis treatment.