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Abstract：
BACKGROUND: Micheliolide (MCL), which is the active metabolite of parthenolide, has demonstrated promising clinical application potential. However, the effects and underlying mechanisms of MCL on atherosclerosis are still unclear.
METHODS: ApoE-/- mice were fed with high fat diet, with or without MCL oral administration, then the plaque area, lipid deposition and collagen content were determined. In vitro, MCL was used to pretreat macrophages combined by ox-LDL, the levels of ferroptosis related proteins, NRF2 activation, mitochondrial function and oxidative stress were detected.
RESULTS: MCL administration significantly attenuated atherosclerotic plaque progress, which characteristics with decreased plaque area, less lipid deposition and increased collagen. Compared with HD group, the level of GPX4 and xCT in atherosclerotic root macrophages were increased in MCL group obviously. In vitro experiment demonstrated that MCL increased GPX4 and xCT level, improved mitochondrial function, attenuated oxidative stress and inhibited lipid peroxidation to suppress macrophage ferroptosis induced with ox-LDL. Moreover, MCL inhibited KEAP1/NRF2 complex formation and enhanced NRF2 nucleus translocation, while the protective effect of MCL on macrophage ferroptosis was abolished by NRF2 inhibition. Additionally, molecular docking suggests that MCL may bind to the Arg483 site of KEAP1, which also contributes to KEAP1/NRF2 binding. Furthermore, Transfection Arg483 (KEAP1-R483S) mutant plasmid can abrogate the anti-ferroptosis and anti-oxidative effects of MC in macrophages. KEAP1-R483S mutation also limited the protective effect of MCL on atherosclerosis progress and macrophage ferroptosis in ApoE-/- mice.
CONCLUSIONS: MCL suppressed atherosclerosis by inhibiting macrophage ferroptosis via activating NRF2 pathway, the related mechanism is through binding to the Arg483 site of KEAP1 competitively.
METHODS: ApoE-/- mice were fed with high fat diet, with or without MCL oral administration, then the plaque area, lipid deposition and collagen content were determined. In vitro, MCL was used to pretreat macrophages combined by ox-LDL, the levels of ferroptosis related proteins, NRF2 activation, mitochondrial function and oxidative stress were detected.
RESULTS: MCL administration significantly attenuated atherosclerotic plaque progress, which characteristics with decreased plaque area, less lipid deposition and increased collagen. Compared with HD group, the level of GPX4 and xCT in atherosclerotic root macrophages were increased in MCL group obviously. In vitro experiment demonstrated that MCL increased GPX4 and xCT level, improved mitochondrial function, attenuated oxidative stress and inhibited lipid peroxidation to suppress macrophage ferroptosis induced with ox-LDL. Moreover, MCL inhibited KEAP1/NRF2 complex formation and enhanced NRF2 nucleus translocation, while the protective effect of MCL on macrophage ferroptosis was abolished by NRF2 inhibition. Additionally, molecular docking suggests that MCL may bind to the Arg483 site of KEAP1, which also contributes to KEAP1/NRF2 binding. Furthermore, Transfection Arg483 (KEAP1-R483S) mutant plasmid can abrogate the anti-ferroptosis and anti-oxidative effects of MC in macrophages. KEAP1-R483S mutation also limited the protective effect of MCL on atherosclerosis progress and macrophage ferroptosis in ApoE-/- mice.
CONCLUSIONS: MCL suppressed atherosclerosis by inhibiting macrophage ferroptosis via activating NRF2 pathway, the related mechanism is through binding to the Arg483 site of KEAP1 competitively.
摘要：
背景：米氏内酯（MCL），它是小白菊内酯的活性代谢产物，已显示出有希望的临床应用潜力。然而,MCL对动脉粥样硬化的影响和潜在机制尚不清楚。
方法：ApoE-/-小鼠饲喂高脂饮食，有或没有MCL口服给药，然后是斑块区域，测定脂质沉积和胶原含量。体外，MCL用于预处理与ox-LDL结合的巨噬细胞，铁凋亡相关蛋白的水平，NRF2激活，检测线粒体功能和氧化应激。
结果：MCL给药显著减弱动脉粥样硬化斑块进展，斑块面积减少的特征，减少脂质沉积和增加胶原蛋白。与HD组相比,MCL组动脉粥样硬化根巨噬细胞GPX4和xCT水平明显升高。体外实验表明，MCL提高了GPX4和xCT水平，改善线粒体功能，减轻氧化应激并抑制脂质过氧化以抑制ox-LDL诱导的巨噬细胞铁凋亡。此外，MCL抑制KEAP1/NRF2复合物的形成,增强NRF2核易位,而NRF2抑制可以消除MCL对巨噬细胞铁凋亡的保护作用。此外,分子对接表明MCL可能与KEAP1的Arg483位点结合，这也有助于KEAP1/NRF2的结合。此外，转染Arg483（KEAP1-R483S）突变质粒可以消除巨噬细胞中MC的抗铁凋亡和抗氧化作用。KEAP1-R483S突变也限制了MCL对ApoE-/-小鼠动脉粥样硬化进展和巨噬细胞铁凋亡的保护作用。
结论：MCL通过激活NRF2通路抑制巨噬细胞铁凋亡，从而抑制动脉粥样硬化，相关机制是通过竞争性结合KEAP1的Arg483位点。
方法：ApoE-/-小鼠饲喂高脂饮食，有或没有MCL口服给药，然后是斑块区域，测定脂质沉积和胶原含量。体外，MCL用于预处理与ox-LDL结合的巨噬细胞，铁凋亡相关蛋白的水平，NRF2激活，检测线粒体功能和氧化应激。
结果：MCL给药显著减弱动脉粥样硬化斑块进展，斑块面积减少的特征，减少脂质沉积和增加胶原蛋白。与HD组相比,MCL组动脉粥样硬化根巨噬细胞GPX4和xCT水平明显升高。体外实验表明，MCL提高了GPX4和xCT水平，改善线粒体功能，减轻氧化应激并抑制脂质过氧化以抑制ox-LDL诱导的巨噬细胞铁凋亡。此外，MCL抑制KEAP1/NRF2复合物的形成,增强NRF2核易位,而NRF2抑制可以消除MCL对巨噬细胞铁凋亡的保护作用。此外,分子对接表明MCL可能与KEAP1的Arg483位点结合，这也有助于KEAP1/NRF2的结合。此外，转染Arg483（KEAP1-R483S）突变质粒可以消除巨噬细胞中MC的抗铁凋亡和抗氧化作用。KEAP1-R483S突变也限制了MCL对ApoE-/-小鼠动脉粥样硬化进展和巨噬细胞铁凋亡的保护作用。
结论：MCL通过激活NRF2通路抑制巨噬细胞铁凋亡，从而抑制动脉粥样硬化，相关机制是通过竞争性结合KEAP1的Arg483位点。
