Objective Puerarin (PU) is a natural compound that exhibits limited oral bioavailability but has shown promise in the treatment of atherosclerosis (AS). However, the precise mechanisms underlying its therapeutic effects remain incompletely understood. This study aimed to investigate the effects of PU and its mechanisms in mitigating AS in both mice and humans.

Design The impact of PU on AS was examined in ApoE^-/- mice fed a high-fat diet (HFD) and in human patients with carotid artery plaque. To explore the causal link between PU-associated gut microbiota and AS, faecal microbiota transplantation (FMT) and mono-colonization of mice with Prevotella copri (P. copri) were employed.

Results PU alleviated AS by modulating the gut microbiota, as evidenced by alterations in gut microbiota composition and the amelioration of AS following FMT from PU-treated mice into ApoE^-/- mice fed HFD. Specifically, PU reduced the abundance of P. copri, which exacerbated AS by producing trimethylamine (TMA). Prolonged mono-colonization of P. copri undermines the beneficial effects of PU on AS. In clinical, the plaque scores of AS patients were positively correlated with the abundance of P. copri and plasma trimethylamine-N-oxide (TMAO) levels. A 1-week oral intervention with PU effectively decreased P. copri levels and reduced TMAO concentrations in patients with carotid artery plaque.

Conclusion PU may provide therapeutic benefits in combating AS by targeting P. copri and its production of TMA.

The RNA extraction, sequencing, and library construction were performed according to previously published methods with minor modifications.^[1,2] In brief, total RNA was extracted from samples using commercial kits (Guangdong Magigene Biotechnology Co., Ltd, China), and RNA quantity was measured using Qubit 3.0 (Thermo Fisher Scientific, MA, USA) and Nanodrop One (Thermo Fisher Scientific, MA, USA) instruments. Whole mRNAseq libraries were generated using the NEB Next® Ultra™ Nondirectional RNA Library Prep Kit for Illumina® (New England Biolabs, USA), following the manufacturer’s recommended protocol. The library was then sequenced using an Illumina NovaSeq 6000 platform. For mRNA analysis, raw data underwent quality control and the reads were mapped to a reference genome. After sequence alignment, gene expression levels were quantified and analyzed, and differentially expressed genes between two conditions/groups were identified using edgeR (version 3.16.5). The resulting p-values were adjusted using Benjamini and Hochberg’s approach to control the false discovery rate. Candidate genes were defined as those with FDR≤0.05 and |log2(fold change)|≥1, and these genes were subjected to enrichment analysis. Finally, differentially expressed genes were analyzed by GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis using the cluster Profiler (version 3.4.4) package. Enrichment analysis was considered significant when FDR≤0.05.

References

[1] Hao Y, Liao X, Wang X, Lao S, Liao W. The biological regulatory activities of Flammulina velutipes polysaccharide in mice intestinal microbiota, immune repertoire and heart transcriptome. Int J Biol Macromol. 2021;185:582-591.

[2] Lavelle A, Hoffmann TW, Pham HP, Langella P, Guédon E, Sokol H. Baseline microbiota composition modulates antibiotic-mediated effects on the gut microbiota and host. Microbiome. 2019;7(1):111.