Combining network Pharmacology, molecular Simulation, and animal experiments to explore the molecular mechanisms of Tongxie Yaofang in the treatment of Diarrhea-Characterized irritable bowel syndrome comorbid with type 2 diabetes mellitus.

To elucidate the mechanisms of Tongxie Yaofang (TXYF) for treating irritable bowel syndrome with diarrhea (IBS-D) comorbid with type 2 diabetes mellitus (T2DM) using integrated computational and experimental approaches, given its clinical efficacy but unclear pharmacological basis. Active ingredients of TXYF (fried Atractylodes Macrocephala Koidz., Paeoniae Radix Alba, Citrus Reticulata, Saposhnikoviae Radix) were screened (TCMSP: oral bioavailability ≥ 30%, drug-likeness ≥ 0.18). IBS-D and T2DM targets were sourced from GeneCards, TTD, and OMIM. Shared targets were identified. Networks (compound-target-disease, PPI) were constructed (Cytoscape, STRING). Core targets were analyzed via GO/KEGG enrichment. Molecular docking assessed binding affinities. Molecular dynamics simulations evaluated binding stability. Animal experiments (H&E staining) assessed TXYF effects on rat colon histopathology. The study identified 43 active ingredients, 477 TXYF targets, and 87 shared drug-disease targets. Core targets (JUN, TP53, AKT1) were enriched in xenobiotic response, oxidative stress, and inflammation. KEGG pathways included IBS-D/T2DM-related pathways, AGE-RAGE signaling, and lipid metabolism. Molecular docking confirmed strong binding (energy ≤ - 7.0 kcal/mol) between naringenin and JUN/TP53. Dynamics simulations revealed stable binding of AKT1, MAPK1, MAPK3 with naringenin. Animal experiments demonstrated TXYF significantly reduced inflammatory cell infiltration, preserved colon tissue architecture, and attenuated inflammation versus the model group. This integrated study revealed TXYF's potential mechanisms for IBS-D with T2DM, involving multi-target regulation of inflammation, stress response, and metabolism via components like naringenin, supported by computational and preliminary animal evidence. It provides a scientific basis for clinical application, though further cellular and biochemical validation is warranted.