Abstract：
BACKGROUND: Atherosclerosis (AS) is a chronic disease characterized by lipid accumulation in the aortic wall and the formation of foam cells overloaded with large lipids inclusions. Currently, Western medicine is primarily used to improve lipid metabolism disorders and reduce inflammatory reactions to delay AS progression, but these medicines come with serious side effects and drug resistance. Gualou-Xiebai (GLXB) is a renowned herb pair that has been proven effective against AS. However, the potential molecular mechanism through which GLXB exerts the anti-atherosclerotic effects of increasing lipophagy in vascular smooth muscle cells (VSMCs) remains unknown.
OBJECTIVE: This study aims to explore the role of lipophagy and the therapeutic mechanism of GLXB in AS.
METHODS: UPLC-Q-TOF-MS for the determination of the main components of GLXB-containing serum. An AS mouse model was established by feeding a high-fat diet (HFD) to ApoE-/- mice for 12 weeks. Ultrasonography monitoring was used to confirm the successful establishment of the AS model. Plaque areas and lipid deposition were evaluated using HE staining and aorta imagingafter GLXB treatment. Immunofluorescence staining and Western blotting were utilized to observe the P2RY12 and lipophagy levels in AS mice. VSMCs were stimulated with oxidized low-density lipoprotein (ox-LDL) to induce foam cell formation. The degree of lipophagy and the related molecular mechanisms were assessed after treating the VSMCs with GLXB-containing serum or si-P2RY12 transfection. The active components of GLXB-containing serum that act on P2RY12 were screened and verified by molecular docking and dual-luciferase reporter assays.
RESULTS: Seventeen components of GLXB were identified in rat serum by UPLC-Q-TOF-MS. GLXB significantly reduced lipid deposition in HFD-fed ApoE-/- mice and ox-LDL-induced VSMCs. GLXB strikingly increased lipophagy levels by downregulating P2RY12, p62, and plin2, upregulating LC3Ⅱ protein expression, and increasing the number of autophagosomes. Notably, the lipophagy inhibitor CQ and the P2RY12 receptor agonist ADPβ abolished the GLXB-induced increase in lipophagy. Last, we confirmed that albiflorin, apigenin, luteolin, kaempferol, 7,8-dihydroxyflavone, and hesperetin from GLXB significantly inhibited P2RY12.
CONCLUSIONS: GLXB activates lipophagy and inhibits lipid accumulation-associated VSMC-derived foam cell formation through suppressing P2RY12 activation, resulting in anti-atherosclerotic effects. The GLXB components albiflorin, apigenin, luteolin, kaempferol, 7,8-dihydroxyflavone, and hesperetin are the potential active effectors against P2RY12.
OBJECTIVE: This study aims to explore the role of lipophagy and the therapeutic mechanism of GLXB in AS.
METHODS: UPLC-Q-TOF-MS for the determination of the main components of GLXB-containing serum. An AS mouse model was established by feeding a high-fat diet (HFD) to ApoE-/- mice for 12 weeks. Ultrasonography monitoring was used to confirm the successful establishment of the AS model. Plaque areas and lipid deposition were evaluated using HE staining and aorta imagingafter GLXB treatment. Immunofluorescence staining and Western blotting were utilized to observe the P2RY12 and lipophagy levels in AS mice. VSMCs were stimulated with oxidized low-density lipoprotein (ox-LDL) to induce foam cell formation. The degree of lipophagy and the related molecular mechanisms were assessed after treating the VSMCs with GLXB-containing serum or si-P2RY12 transfection. The active components of GLXB-containing serum that act on P2RY12 were screened and verified by molecular docking and dual-luciferase reporter assays.
RESULTS: Seventeen components of GLXB were identified in rat serum by UPLC-Q-TOF-MS. GLXB significantly reduced lipid deposition in HFD-fed ApoE-/- mice and ox-LDL-induced VSMCs. GLXB strikingly increased lipophagy levels by downregulating P2RY12, p62, and plin2, upregulating LC3Ⅱ protein expression, and increasing the number of autophagosomes. Notably, the lipophagy inhibitor CQ and the P2RY12 receptor agonist ADPβ abolished the GLXB-induced increase in lipophagy. Last, we confirmed that albiflorin, apigenin, luteolin, kaempferol, 7,8-dihydroxyflavone, and hesperetin from GLXB significantly inhibited P2RY12.
CONCLUSIONS: GLXB activates lipophagy and inhibits lipid accumulation-associated VSMC-derived foam cell formation through suppressing P2RY12 activation, resulting in anti-atherosclerotic effects. The GLXB components albiflorin, apigenin, luteolin, kaempferol, 7,8-dihydroxyflavone, and hesperetin are the potential active effectors against P2RY12.
摘要：
背景：动脉粥样硬化（AS）是一种慢性疾病，其特征是脂质在主动脉壁中积聚，并形成了大量脂质包裹体的泡沫细胞。目前,西药主要用于改善脂质代谢紊乱，减轻炎症反应，延缓AS进展，但是这些药物有严重的副作用和耐药性。瓜楼-谢白（GLXB）是一种著名的草药对，已被证明对AS有效。然而,GLXB发挥抗动脉粥样硬化作用增加血管平滑肌细胞(VSMC)脂噬的潜在分子机制尚不清楚.
目的：本研究旨在探讨脂质吞噬在AS中的作用及GLXB的治疗机制。
方法：UPLC-Q-TOF-MS测定含GLXB血清的主要成分。通过给ApoE-/-小鼠喂食高脂饮食（HFD）12周来建立AS小鼠模型。超声监测用于确认AS模型的成功建立。GLXB治疗后，使用HE染色和主动脉成像评估斑块面积和脂质沉积。采用免疫荧光染色和免疫印迹法观察AS小鼠P2RY12和噬脂性水平。用氧化低密度脂蛋白（ox-LDL）刺激VSMC以诱导泡沫细胞形成。在用含GLXB的血清或si-P2RY12转染处理VSMC后，评估了脂质吞噬的程度和相关的分子机制。通过分子对接和双荧光素酶报告基因测定筛选和验证了作用于P2RY12的含GLXB血清的活性成分。
结果：通过UPLC-Q-TOF-MS在大鼠血清中鉴定出GLXB的17种成分。GLXB显著降低HFD喂养的ApoE-/-小鼠和ox-LDL诱导的VSMC的脂质沉积。GLXB通过下调P2RY12，p62和plin2，上调LC3Ⅱ蛋白表达，显著增加了吸脂水平，增加自噬体的数量。值得注意的是,吸脂性抑制剂CQ和P2RY12受体激动剂ADPβ消除了GLXB诱导的吸脂性增加.最后,我们确认了albiflorin,芹菜素,木犀草素,山奈酚,7,8-二羟基黄酮,来自GLXB的橙皮素显着抑制P2RY12。
结论：GLXB通过抑制P2RY12的激活激活脂肪吞噬并抑制脂质积累相关的VSMC衍生的泡沫细胞形成，产生抗动脉粥样硬化作用。GLXB成分albiflorin，芹菜素,木犀草素,山奈酚,7,8-二羟基黄酮,橙皮素是抗P2RY12的潜在活性效应物。
目的：本研究旨在探讨脂质吞噬在AS中的作用及GLXB的治疗机制。
方法：UPLC-Q-TOF-MS测定含GLXB血清的主要成分。通过给ApoE-/-小鼠喂食高脂饮食（HFD）12周来建立AS小鼠模型。超声监测用于确认AS模型的成功建立。GLXB治疗后，使用HE染色和主动脉成像评估斑块面积和脂质沉积。采用免疫荧光染色和免疫印迹法观察AS小鼠P2RY12和噬脂性水平。用氧化低密度脂蛋白（ox-LDL）刺激VSMC以诱导泡沫细胞形成。在用含GLXB的血清或si-P2RY12转染处理VSMC后，评估了脂质吞噬的程度和相关的分子机制。通过分子对接和双荧光素酶报告基因测定筛选和验证了作用于P2RY12的含GLXB血清的活性成分。
结果：通过UPLC-Q-TOF-MS在大鼠血清中鉴定出GLXB的17种成分。GLXB显著降低HFD喂养的ApoE-/-小鼠和ox-LDL诱导的VSMC的脂质沉积。GLXB通过下调P2RY12，p62和plin2，上调LC3Ⅱ蛋白表达，显著增加了吸脂水平，增加自噬体的数量。值得注意的是,吸脂性抑制剂CQ和P2RY12受体激动剂ADPβ消除了GLXB诱导的吸脂性增加.最后,我们确认了albiflorin,芹菜素,木犀草素,山奈酚,7,8-二羟基黄酮,来自GLXB的橙皮素显着抑制P2RY12。
结论：GLXB通过抑制P2RY12的激活激活脂肪吞噬并抑制脂质积累相关的VSMC衍生的泡沫细胞形成，产生抗动脉粥样硬化作用。GLXB成分albiflorin，芹菜素,木犀草素,山奈酚,7,8-二羟基黄酮,橙皮素是抗P2RY12的潜在活性效应物。
