Computational Screening and Experimental Validation of Natural Compounds that Enhance Butyrate Production in Gut Bacteria and Promote Muscle Cell Mass

Butyrate produced by gut microbiota influences host metabolism and the gut-muscle axis, but systematic approaches to identify natural compounds (NCs) that enhance butyrate production are limited. To identify NCs that increase butyrate production in <i>Faecalibacterium prausnitzii</i> and <i>Anaerostipes hadrus</i> and evaluate their effects on muscle cell growth. Methods: Molecular docking screened 25,000 NCs against three butyrate biosynthesis enzymes: butyryl-CoA dehydrogenase (BCD), β-hydroxybutyryl-CoA dehydrogenase (BHBD), and butyryl-CoA:acetate CoA-transferase (BCoAT). Selected NCs were cultured with bacteria in monoculture and coculture systems for 0-48 h. Bacterial growth (OD600), butyrate production (gas chromatography), gene expression (qRT-PCR), and signaling pathway (immune blot) were assessed. C2C12 myocytes were cultured with NC-treated bacterial supernatants to evaluate muscle cell effects. Molecular docking identified 109 NCs with high binding affinity (≤-10 kcal/mol). Network analysis revealed interactions between 19 NCs, 19 genes, and 5 pathways. Coculture systems produced more butyrate (0.31-0.58 mM) than monocultures. Key NCs and their butyrate production were: hypericin (0.58 mM), piperitoside (0.54 mM), luteolin 7-glucoside (0.39 mM), and khelmarin D (0.41 mM). Hypericin showed the highest gene upregulation: 2.5-fold for BCD, 1.8-fold for BCoAT, and 1.6-fold for BHBD (<i>P</i> < 0.001). C2C12 myocytes treated with NC-bacterial supernatants demonstrated enhanced viability (1.6-2.5-fold increase), upregulated myogenic genes (<i>MYOD1</i>: 1.55-1.75-fold; <i>myogenin</i>: 1.76-2.15-fold), improved insulin sensitivity related genes (<i>PPARA</i>: 1.75-1.97-fold; <i>PPARG</i>: 1.51-1.73-fold), reduced lipid accumulation (to 0.2 μmol/mg protein), suppressed inflammatory markers (PTGS2:0.53-0.72-fold; NF-κB: 0.61-0.79-fold; IL-2:0.57-0.76-fold), and reduced phosphorylation of STAT3 (by 14-19%) and NF-κB (by 43-44%). In conclusion, this integrated approach identified NCs that enhance butyrate production through enzyme targeting and bacterial synergy. Hypericin, piperitoside, and khelmarin D show promise for muscle cell growth through the gut-muscle axis with potential applications in managing metabolic and inflammatory diseases.