An integrative strategy combining network pharmacology, metabolomics, and gut microbiota analyses to elucidate the bioactive ingredients and underlying mechanisms of Xin-Shu-Ning tablets against myocardial ischemia.

Xin-Shu-Ning tablets (XSNT) are clinically used for the treatment of angina pectoris and coronary insufficiency; however, their bioactive constituents and cardioprotective mechanisms remain insufficiently clarified. In this study, an integrated strategy combining network pharmacology, metabolomics, and 16S rDNA amplicon sequencing was employed to systematically elucidate the mechanisms of XSNT and identify its potential active components. The blood-absorbed constituents of XSNT were first characterized using an HPLC-MS/MS method. Network pharmacology analysis was then conducted to predict potential therapeutic targets and pathways, which were further validated by serum metabolomics and Western blot analysis. In parallel, fecal metabolomics was applied to evaluate metabolic alterations following XSNT administration. Differential metabolites were analyzed using the MetOrigin platform to predict associated gut microbiota and metabolic pathways, and these predictions were further validated by 16S rDNA sequencing. A total of 76 compounds were identified in XSNT, including 40 blood-absorbed constituents. Integrated network pharmacology and serum metabolomics analyses indicated that XSNT exerts cardioprotective effects mainly through modulation of the tyrosine metabolism pathway, with tyrosine hydroxylase and catechol-O-methyltransferase identified as potential key cardiac targets. Additionally, XSNT significantly regulated gut microbiota composition and amino acid metabolism, including arginine, phenylalanine, and lysine pathways. Notably, phenylalanine, as a precursor of tyrosine, may function as a metabolic bridge linking gut microbial regulation to cardiac tyrosine metabolism. Overall, XSNT exerts cardioprotective effects through coordinated direct cardiac actions and systemic regulation mediated by the heart-gut axis, providing a systems-level understanding of its multi-component and multi-target therapeutic mechanisms.