Blood lipid levels and body weight of AS mice.

The aim of this study was to elucidate the specific mechanism through which 7-difluoromethoxy-5,4’-dimethoxygenistein (DFMG) inhibits angiogenesis in atherosclerosis (AS) plaques, given its previously observed but poorly understood inhibitory effects. In vitro, a model using Human Umbilical Vein Endothelial (HUVEC-12) cells simulated the initial lesion in the atherosclerotic pathological process, specifically oxidative stress injury, by exposing cells to 30 μmol/L LPC. Additionally, an AS mouse model was developed in ApoE knockout mice through a 16-week period of high-fat feeding. DFMG demonstrated a reduction in tubule quantities in the tube formation assay and neovascularization induced by oxidative stress-damaged endothelial cells in the chicken embryo chorioallantoic membrane assay. Furthermore, DFMG decreased lipid levels in the blood of ApoE knockout mice with AS, along with a decrease in atherosclerotic plaques and neovascularizations in the aortic arch and descending aorta of AS animal models. DFMG treatment upregulated microRNA140 (miR-140) expression and suppressed VEGF secretion in HUVEC-12 cells. These effects were counteracted by Toll-like receptor 4 (TLR4) overexpression in HUVEC-12 cells subjected to oxidative injury or in a mouse model of AS. Dual-luciferase reporter assays demonstrated that miR-140 directly targeted TLR4. Immunohistochemical assay findings indicated a significant inverse relationship between miR-140 expression and TLR4 expression in ApoE knockout mice subjected to a high-fat diet. The study observed a close association between DFMG inhibitory effects on angiogenesis and plaque stability in AS, and the inhibition of the TLR4/NF-κB/VEGF signaling pathway, negatively regulated by miR-140.

本研究旨在阐明7-二氟甲氧基-5,4’-二甲氧基染料木黄酮（7-difluoromethoxy-5,4’-dimethoxygenistein，DFMG）抑制动脉粥样硬化（atherosclerosis，AS）斑块血管生成的具体机制——此前虽已观察到其抑制效应，但相关机制尚未明确。体外实验中，研究采用人脐静脉内皮（Human Umbilical Vein Endothelial，HUVEC-12）细胞模型，通过30 μmol/L溶血磷脂酰胆碱（Lysophosphatidylcholine，LPC）处理细胞，模拟动脉粥样硬化病理进程中的初始损伤：氧化应激损伤。此外，通过16周高脂喂养，构建载脂蛋白E（ApoE）基因敲除小鼠动脉粥样硬化模型。管形成实验结果显示，DFMG可减少氧化应激损伤内皮细胞诱导的小管生成数量；鸡胚尿囊膜实验也证实其可抑制新生血管生成。进一步实验表明，DFMG可降低动脉粥样硬化模型ApoE敲除小鼠的血脂水平，同时减少其主动脉弓及降主动脉中的动脉粥样硬化斑块与新生血管。在HUVEC-12细胞中，DFMG处理可上调微小RNA140（microRNA140，miR-140）的表达，并抑制血管内皮生长因子（vascular endothelial growth factor，VEGF）的分泌。当氧化损伤的HUVEC-12细胞或动脉粥样硬化小鼠模型中过表达Toll样受体4（Toll-like receptor 4，TLR4）时，上述效应会被抵消。双荧光素酶报告基因实验证实，miR-140可直接靶向TLR4。免疫组化实验结果显示，高脂喂养的ApoE敲除小鼠体内，miR-140的表达与TLR4的表达呈显著负相关。本研究发现，DFMG对血管生成的抑制效应与动脉粥样硬化斑块稳定性密切相关，其通过miR-140负调控TLR4/核因子κB（NF-κB）/VEGF信号通路发挥作用。