Zhizi-Chuanxiong herb pair alleviates atherosclerosis progression in ApoE-/- mice by promoting the methylation of FGFR3 to inhibit MAPK/ERK-mediated apoptosis
Zhizi-Chuanxiong herb pair alleviates atherosclerosis progression in ApoE-/- mice by promoting the methylation of FGFR3 to inhibit MAPK/ERK-mediated apoptosis
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DOI：
10.1016/j.jep.2023.117188
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发表时间：
2023-09-21
影响因子：
5.4
通讯作者：
Xu，Fengqin
中科院分区：
文献类型：
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作者：
Zhang，Yan;Li，Dandan;Xu，Fengqin
民族药理学相关性栀子（Gardenia jasminoidesEllis，Zhizi）和川芎（Ligusticum chuanxiongHort.，Chuanxiong）都是具有血管保护作用的中药，有解毒活血的作用，临床上用于治疗动脉粥样硬化（AS）。此前，栀子川芎在减缓家兔AS进展方面表现出良好的功效。然而其潜在机制尚不清楚。本研究旨在从DNA甲基化的角度探讨栀子-川芎药对（ZCHP）减缓AS进展的机制。材料与方法采用高脂饮食（HFD）喂养的ApoE−/−小鼠建立AS小鼠模型。使用自动生化分析仪、酶联免疫吸附测定、组织病理学、甲基捕获测序（MC-seq）、焦磷酸测序、定量逆转录聚合酶链反应（RT-qPCR）、蛋白质印迹和TUNEL染色来确定ZCHP治疗HFD诱导的AS的治疗效果和机制。结果ZCHP通过降低脂质水平、增强斑块稳定性和抗炎来减轻AS的发展。 MC-seq 和京都基因与基因组百科全书分析表明，ZCHP 纠正了异常低甲基化和高甲基化基因的表达，这些基因参与丝裂原激活蛋白激酶 (MAPK) 信号通路。蛋白-蛋白网络相互作用分析和分子对接表明，成纤维细胞生长因子3（FGFR3）和丝氨酸/苏氨酸蛋白激酶（AKT1）与ZCHP诱导的差异甲基化基因中的MAPK信号通路密切相关。此外，焦磷酸测序显示ZCHP可以诱导启动子区域的FGFR3高甲基化和AKT1低甲基化，这与MC-seq结果一致。分子对接表明ZCHP与FGFR3的对接更加紧密。此外，RT-qPCR 和蛋白质印迹显示，ZCHP 处理后 FGFR3 的 mRNA 和蛋白表达水平降低。最后，蛋白质印迹显示ZCHP抑制磷酸化MAPK和磷酸化细胞外信号调节激酶（ERK）的表达，TUNEL染色显示ZCHP治疗可以抑制AS细胞凋亡。结论我们的研究结果表明，ZCHP可以通过启动子区域的FGFR3高甲基化抑制MAPK/ERK信号介导的细胞凋亡，从而有效减弱AS进展。
Ethnopharmacological relevanceGardenia Fructus (Gardenia jasminoidesEllis, Zhizi) and Chuanxiong Rhizoma (Ligusticum chuanxiongHort., Chuanxiong) are both traditional Chinese medicines with vascular protective effects, which help detoxify and activate blood, and are clinically used to treat atherosclerosis (AS). Previously, Zhizi-Chuanxiong showed good efficacy in attenuating AS progression in rabbits. However, its potential mechanism is yet unclear.Aim of the studyThis study aimed to investigate the mechanism of the Zhizi-Chuanxiong herb pair (ZCHP) in attenuating AS progression from the perspective of DNA methylation.Materials and methodsAn AS mouse model was developed with ApoE−/−mice fed a high-fat diet (HFD). The therapeutic effects and mechanisms of ZCHP in treating HFD-induced AS were identified using an automated biochemical analyzer, enzyme-linked immunosorbent assays, histopathology, methyl-capture sequencing (MC-seq), pyrosequencing, quantitative reverse transcription-polymerase chain reaction (RT-qPCR), western blotting, and TUNEL staining.ResultsZCHP attenuated the development of AS by reducing lipid levels and enhancing the stability of plaques and via anti-inflammation. MC-seq and Kyoto Encyclopedia of Genes and Genomes analysis revealed that ZCHP corrected the expressions of both aberrant hypomethylated and hypermethylated genes, which are involved in the mitogen-activated protein kinase (MAPK) signaling pathway. Protein–protein network interaction analysis and molecular docking showed that fibroblast growth factor 3 (FGFR3) and serine/threonine protein kinase (AKT1) were closely related to the MAPK signaling pathway among differentially methylated genes induced by ZCHP. Furthermore, pyrosequencing showed that ZCHP could induce FGFR3 hypermethylation and AKT1 hypomethylation in the promoter region, which was consistent with the MC-seq results. Molecular docking showed that the ZCHP was more tightly docked to FGFR3. Furthermore, RT-qPCR and western blotting showed that the mRNA and protein expression levels of FGFR3 decreased after treatment with ZCHP. Finally, western blotting showed that ZCHP suppressed the expression of phosphorylated MAPK and phosphorylated extracellular signal-regulated kinase (ERK), and TUNEL staining showed that ZCHP treatment could inhibit apoptosis in AS.ConclusionOur findings suggest that ZCHP can effectively attenuate AS progression by inhibiting MAPK/ERK signaling–mediated apoptosis via FGFR3 hypermethylation in the promoter region.