背景:山楂叶是玫瑰科植物的干叶的组合,即,CrataeguspinnatifidaBge。或者CrataeguspinnatifidaBge.var.少校N.E.Br.,主要在东亚种植,北美,和欧洲。山楂叶黄酮(HLF)是提取的主要成分。HLF已经证明了预防高血压的潜力,炎症,高脂血症,和动脉粥样硬化。然而,其抗动脉粥样硬化作用背后的潜在药理机制尚待探索.
目的:研究了HLF对脂质介导的泡沫细胞形成的体内和体外作用,特别关注巨噬细胞中分泌的IIA型磷脂酶A2(sPLA2-IIA)的水平。
方法:使用超高效液相色谱法和液相色谱-串联质谱法分析了HLF的主要成分。在体内,HLF,浓度为5mg/kg时,20mg/kg,和40毫克/千克,给予高脂饮食(HFD)喂养的载脂蛋白E基因敲除小鼠(ApoE-/-)16周。收集主动脉和血清样品,通过质谱分析鉴定病变区域和脂质,以解剖病理过程。RAW264.7细胞与氧化低密度脂蛋白(ox-LDL)单独孵育,或ox-LDL与不同剂量的HLF(100、50和25μg/ml)联合使用,或ox-LDL加24小时sPLA2-IIA抑制剂,用于细胞生物学分析。使用生化分析仪和ELISA检测血脂和炎性细胞因子,通过HE和主动脉Masson染色评估斑块大小和斑块胶原含量。通过油红O染色观察巨噬细胞中的脂质沉积。通过RT-qPCR和Western印迹分析确定sPLA2-IIA和SCAP-SREBP2-LDLR的表达。
结果:使用UPLC-Q-TOF-MS/MS研究了HLF的化学概况,允许初步鉴定20种化合物,包含1种酚酸,9个黄酮醇和10个黄酮,包括Isovitexin,玻璃化蛋白-4″-O-葡萄糖苷,槲皮素-3-O-robibioside,芦丁,vitexin-2″-O-鼠李糖苷,槲皮素,等。HLF降低总胆固醇(TC),甘油三酯(TG),低密度脂蛋白胆固醇(LDL-C),ApoE-/-小鼠的非高密度脂蛋白胆固醇(non-HDL-C)水平(P<0.05),降低ox-LDL摄取,抑制炎症因子水平,如IL-6,IL-8,TNF-α,和IL-1(P<0.001),和减轻主动脉斑块与较厚的纤维帽。HLF有效减弱ox-LDL处理的RAW264.7巨噬细胞中的泡沫细胞形成,细胞内TC水平降低,游离胆固醇(FC),胆固醇酯(CE),IL-6,TNF-α,和IL-1β(P<0.001)。在体内和体外实验中,HLF显著下调sPLA2-IIA的表达,SCAP,SREBP2,LDLR,HMGCR,和LOX-1(P<0.05)。此外,sPLA2-IIA抑制剂可有效减轻RAW264.7巨噬细胞的炎症释放,并通过抑制sPLA2-IIA分泌调节SCAP-SREBP2-LDLR信号通路(P<0.05)。
结论:HLF通过抑制sPLA2-IIA减弱SCAP-SREBP2-LDLR信号通路对动脉粥样硬化有保护作用,为了减少LDL吸收导致的泡沫细胞形成,并减缓小鼠动脉粥样硬化的进展。
BACKGROUND: Hawthorn leaves are a combination of the dried leaves of the Rosaceae plants, i.e., Crataegus pinnatifida Bge. or Crataegus pinnatifida Bge. var. major N. E. Br., is primarily cultivated in East Asia, North America, and Europe. hawthorn leaf flavonoids (HLF) are the main part of extraction. The HLF have demonstrated potential in preventing hypertension, inflammation, hyperlipidemia, and atherosclerosis. However, the potential pharmacological mechanism behind its anti-atherosclerotic effect has yet to be explored.
OBJECTIVE: The in vivo and in vitro effects of HLF on lipid-mediated foam cell formation were investigated, with a specific focus on the levels of secreted phospholipase A2 type IIA (sPLA2-II A) in macrophage cells.
METHODS: The primary constituents of HLF were analyzed using ultra-high performance liquid chromatography and liquid chromatography-tandem mass spectrometry. In vivo, HLF, at concentrations of 5 mg/kg, 20 mg/kg, and 40 mg/kg, were administered to apolipoprotein E knockout mice (ApoE-/-) fed by high-fat diet (HFD) for 16 weeks. Aorta and serum samples were collected to identify lesion areas and lipids through mass spectrometry analysis to dissect the pathological process. RAW264.7 cells were incubated with oxidized low-density lipoprotein (ox-LDL) alone, or ox-LDL combined with different doses of HLF (100, 50, and 25 μg/ml), or ox-LDL plus 24-h sPLA2-IIA inhibitors, for cell biology analysis. Lipids and inflammatory cytokines were detected using biochemical analyzers and ELISA, while plaque size and collagen content of plaque were assessed by HE and the Masson staining of the aorta. The lipid deposition in macrophages was observed by Oil Red O staining. The expression of sPLA2-IIA and SCAP-SREBP2-LDLR was determined by RT-qPCR and Western blot analysis.
RESULTS: The chemical profile of HLF was studied using UPLC-Q-TOF-MS/MS, allowing the tentative identification of 20 compounds, comprising 1 phenolic acid, 9 flavonols and 10 flavones, including isovitexin, vitexin-4″-O-glucoside, quercetin-3-O-robibioside, rutin, vitexin-2″-O-rhamnoside, quercetin, etc. HLF decreased total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and non-high-density lipoprotein cholesterol (non-HDL-C) levels in ApoE-/- mice (P < 0.05), reduced ox-LDL uptake, inhibited level of inflammatory factors, such as IL-6, IL-8, TNF-α, and IL-1ꞵ (P < 0.001), and alleviated aortic plaques with a thicker fibrous cap. HLF effectively attenuated foam cell formation in ox-LDL-treated RAW264.7 macrophages, and reduced levels of intracellular TC, free cholesterol (FC), cholesteryl ester (CE), IL-6, TNF-α, and IL-1β (P < 0.001). In both in vivo and in vitro experiments, HLF significantly downregulated the expression of sPLA2-IIA, SCAP, SREBP2, LDLR, HMGCR, and LOX-1 (P < 0.05). Furthermore, sPLA2-IIA inhibitor effectively mitigated inflammatory release in RAW264.7 macrophages and regulated SCAP-SREBP2-LDLR signaling pathway by inhibiting sPLA2-IIA secretion (P < 0.05).
CONCLUSIONS: HLF exerted a protective effect against atherosclerosis through inhibiting sPLA2-IIA to diminish SCAP-SREBP2-LDLR signaling pathway, to reduce LDL uptake caused foam cell formation, and to slow down the progression of atherosclerosis in mice.