Liuwei Dihuang formula ameliorates perimenopausal atherosclerosis by modulating the microbiota-dependent TMA-TMAO metabolic axis

Background

Trimethylamine N-oxide (TMAO), a product of the gut microbiota, is essential to the pathophysiology of atherosclerotic cardiovascular disease (ASCVD). Although Liuwei Dihuang Formula (LWDH) can ameliorate perimenopausal atherosclerosis (AS) and modulate gut microbes, it is unknown how it regulates the trimethylamine (TMA)-TMAO pathway.

Purpose

This study aimed to examine whether LWDH could attenuates perimenopausal AS by modulating the gut microbiota-TMA-TMAO axis and to clarify the associated mechanisms, with an emphasis on its possible synergistic interaction with the probiotic Bifidobacterium animalis subsp. Lactis (B. lactis).

Methods

In ApoE knockout (ApoE^-/-) mice, a perimenopausal AS model was established via bilateral ovariectomy combined with a high-fat diet (HFD), followed by LWDH intervention. The TMAO and TMA levels in plasma and the liver were measured using targeted metabolomics. Microbial communities were analyzed using 16S rRNA sequencing. In vitro microbial culture experiments were carried out to validate the LWDH effect on the key bacterial populations and metabolic pathways. A combined intervention with LWDH and B. lactis was also performed to evaluate the potential synergistic effects.

Results

LWDH significantly reduced aortic plaque burden and decreased plasma and hepatic TMAO levels in perimenopausal AS mice. Mechanistic analyses revealed that LWDH remodeled the gut microbiota, suppressed TMA-producing bacteria, and enhanced TMA degradation, thereby reducing systemic TMAO accumulation. Notably, 16S rRNA sequencing revealed a close link between decreased Bifidobacterium abundance and elevated TMAO levels. In vitro assays confirmed that LWDH enhanced the TMA-degrading activity of B. lactis and downregulated the cutC gene in Escherichia coli, resulting in reduced TMA synthesis. Combined LWDH and B. lactis intervention led to greater reductions in plasma TMAO compared to B. lactis alone, accompanied by improved lipid metabolism and attenuation of systemic inflammation.

Conclusion

These findings are the first to show that LWDH mitigated perimenopausal AS progression by modulating the gut microbiota-dependent TMA-TMAO axis. The dual mechanisms of enhancing TMA degradation and inhibiting TMA production, together with the synergistic effects of LWDH and B. lactis, highlight a novel therapeutic strategy that integrates traditional Chinese medicine and probiotics for AS management.