Zhizi-Chuanxiong herb pair alleviates atherosclerosis progression in ApoE-/- mice by promoting the methylation of FGFR3 to inhibit MAPK/ERK-mediated apoptosis

Abstract

Ethnopharmacological relevance: Gardenia Fructus (Gardenia jasminoides Ellis, Zhizi) and Chuanxiong Rhizoma (Ligusticum chuanxiong Hort., 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 study: This 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 methods: An 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.

Results: ZCHP 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.

Conclusion: Our findings suggest that ZCHP can effectively attenuate AS progression by inhibiting MAPK/ERK signaling-mediated apoptosis via FGFR3 hypermethylation in the promoter region.

Keywords: Apoptosis; Atherosclerosis; DNA methylation; MAPK/ERK signaling pathway; Zhizi-chuanxiong herb pair.