Integrating 1H NMR-Based Fecal Metabolomics with Gut Microbiota-Metabolite Correlation Analysis and Network Pharmacology to Explore Anti-Hyperlipidemic Mechanisms of Red Clover in High-Fat Diet-Induced Mice

Introduction:: Hyperlipidemia (HLP) is a major risk factor for cardiovascular disease, and red clover (RC) extract has shown potential anti-hyperlipidemic effects in mice fed a high-fat diet (HFD). However, the underlying mechanisms remain poorly understood. This study aimed to elucidate the anti-hyperlipidemic mechanisms of RC by integrating 1H NMR-based fecal metabolomics, gut microbiota-metabolite correlation analysis, and network pharmacology. materials and methods: Fecal metabolic profiles of HFD mice before and after RC intervention were analyzed by 1H NMR metabolomics to identify differential biomarkers and associated metabolic pathways. Gut microbiota-metabolite interactions were assessed using linear discriminant analysis effect size analysis and Spearman correlation analysis. Network pharmacology was applied to identify key bioactive components and their targets. Methods:: The fecal metabolic profiles of HFD-induced mice treated with or without RC extract were analyzed using 1H NMR metabolomics to identify differential metabolites and pathways. Gut microbiota composition was assessed using linear discriminant analysis effect size (LEfSe), and Spearman correlation analysis was employed to explore microbiota-metabolite-lipid interactions. Network pharmacology and molecular docking were used to identify the bioactive RC isoflavonoids, their in vivo metabolites, and key molecular targets. results: Fecal metabolomics revealed that the anti-hyperlipidemic activity RC was mainly associated with amino acid metabolism, urea cycle modulation, oxidative stress reduction and glycolysis regulation. RC treatment effectively reversed HFD-induced dysbiosis. Enrichment of beneficial genera including Muribaculaceae, Akkermansia, Alloprevotella and Alistipes was associated with increased production of short-chain fatty acids and decreased low-density lipoprotein cholesterol levels, whereas reduction of harmful genera Dubosiella, Erysipelotrichaceae, Streptococcus and Faecalibaculum was inversely correlated. Three RC isoflavonoids and their 31 in vivo metabolites were screened for network pharmacology analysis, with metabolites C16 and C19 showing higher binding affinities to EGFR and TNF. Results:: RC treatment modulated fecal metabolites, primarily affecting the amino acid metabolism, urea cycle, oxidative stress, and glycolysis pathways. It restored gut microbiota balance by enriching beneficial genera (Muribaculaceae, Akkermansia, Alloprevotella, and Alistipes), which was associated with increased production of short-chain fatty acids and reduced low-density lipoprotein cholesterol (LDL-C). Conversely, the abundance of harmful genera (Dubosiella, Erysipelotrichaceae, Streptococcus, Faecalibaculum) decreased. Network pharmacology identified three RC isoflavonoids and 31 in vivo metabolites as the key active constituents against HLP. Metabolites C16 and C19 showed strong binding affinities to EGFR and TNF, key targets in HLP regulation. Discussion:: RC prototype compounds altered gut microbiota composition by enriching beneficial bacteria, including Muribaculaceae and Akkermansia, and suppressing harmful ones such as Faecalibaculum. These beneficial genera correlated with reduced LDL-C and increased SCFAs. Increased SCFA production improved gut barrier function and altered amino acid metabolism, a key pathway identified in 1H NMR-based fecal metabolomics analysis. In addition, RC's effects on urea cycle modulation, oxidative stress reduction, and glycolysis regulation contributed to improved lipid homeostasis. Secondly, the RC in vivo metabolites, particularly C16 and C19, directly targeted TNF and EGFR, inhibiting inflammation and oxidative stress, as supported by the fecal metabolomics analysis. The anti-inflammatory effects of RC metabolites create a more favorable gut environment for beneficial gut bacteria. Conclusion:: RC exerts anti-hyperlipidemic effects through a dual mechanism involving modulation of the gut microbiota and direct targeting of TNF and EGFR by its in vivo metabolites. This synergistic interplay underscores the potential of RC as a holistic therapeutic agent for HLP, targeting both microbial and molecular pathways.