Metabolomics-guided analysis combined with network pharmacology and molecular simulations reveals the mechanism of JHT in treating septic intestinal dysfunction.

Ethnopharmacological Relevance: Jinhong Tang (JHT), recorded in Surgery of Traditional Chinese Medicine, is a famous modern prescription for acute abdomen. Our previous study demonstrated that JHT can improved the immune function and organ function of patients with sepsis, achieving favorable clinical outcomes. However, the basis of these effects and the underlying mechanisms remain unclear. We aimed to explore the efficacy and mechanism of JHT in reducing septic intestinal dysfunction.

Methods: Active components of JHT, identified in prior HPLC-IT/MS analyses, were used for network pharmacology, molecular docking, and molecular dynamics simulations to predict key therapeutic targets and pathways. To functionally validate these predictions, untargeted metabolomics of colonic tissues was performed followed by pathway enrichment analysis to assess metabolic alterations. A cecal ligation and puncture (CLP)-induced septic mouse model was used to evaluate the effects of JHT on intestinal dysfunction in vivo. Histological changes were assessed via hematoxylin-eosin (HE) staining, and serum inflammatory cytokines were quantified using ELISA. Gene expression of key metabolic enzymes was measured by real-time quantitative PCR (qPCR), and protein levels of signaling molecules were analyzed by Western blotting.

Results: Network pharmacology suggested that JHT may exert therapeutic effects through the AMPK/SIRT1/NF-κB signaling pathway. Metabolomics revealed that JHT reversed sepsis-induced disruptions in glycolysis, tricarboxylic acid (TCA) cycle, and tryptophan metabolism, while regulating mitochondrial-associated energy metabolism. In vivo, JHT significantly reduced serum TNF-α, IL-6, and IL-1β levels compared to the model group. HE staining showed reduced mucosal edema, attenuated inflammatory infiltration, and preserved colonic architecture. Western blot analysis demonstrated upregulation of AMPK, phosphorylated AMPK (p-AMPK), and SIRT1, along with downregulation of p-NF-κB and pro-inflammatory cytokines. Glycolysis-related enzymes PFKFB3 and LDHA were also significantly reduced in JHT-treated animals.

Conclusion: JHT alleviated septic intestinal dysfunction by modulating the AMPK/SIRT1/NF-κB signaling axis, suppressing inflammatory responses, and reprogramming abnormal glycolytic metabolism. These findings supported the multi-target, multi-pathway mechanism of JHT and provided a pharmacological basis for its application in the treatment of sepsis-associated intestinal injury.