Paeoniae decoction attenuates chronic colitis-associated intestinal fibrosis by suppressing TGF-β/Smad-mediated epithelial-mesenchymal transition and remodeling the metabolic microenvironment.

Paeoniae Decoction (PD) is a classic Chinese medicine formula for treating intestinal diseases. However, its direct intervention effect on intestinal fibrosis and the underlying molecular mechanisms have not been fully elucidated. To evaluate the effect of PD on inflammatory bowel disease (IBD) associated intestinal fibrosis and to systematically investigate its potential molecular mechanisms utilizing integrated multi-omics. The chemical constituents of PD were characterized and quantified utilizing ultra-high performance liquid chromatography-Quadrupole-Orbitrap mass spectrometry (LC-Q-Orbitrap-MS) and LC-MS/MS. A chronic experimental colitis-associated intestinal fibrosis mouse model lasting 56 days and involving four repeated cycles of dextran sulfate sodium (DSS) injury was established. The therapeutic efficacy of PD was evaluated through endoscopy, histopathological analysis, and collagen staining. Label-free quantitative proteomics and untargeted metabolomics and network pharmacology were employed to elucidate the target networks. In vitro mechanisms were further validated in TGF-β1-stimulated NCM460 cells using wound healing assays, immunofluorescence, and Western blotting to evaluate the epithelial-mesenchymal transition (EMT) process and the TGF-β/Smad signaling pathway. Finally, molecular docking was used to verify the direct binding of the main bioactive components of PD to the key pathway targets. Chemical profiling identified 16 common constituents across independent batches of PD. In vivo, PD administration significantly ameliorated intestinal fibrosis and reduced massive extracellular matrix (ECM) deposition in the mucosal and submucosal layers. Integrated omics analyses revealed that PD profoundly modulated the TGF-β signaling pathway, ECM-receptor interaction, and the Sphingolipid signaling pathway. Mechanistically, PD dose-dependently inhibited myofibroblast activation, downregulating the expression of α-SMA, Vimentin, Collagen I, and Collagen III. In vitro, PD serum suppressed TGF-β1-induced cell migration, preserved the epithelial marker E-cadherin, and inhibited the nuclear translocation of Snail by significantly decreasing the phosphorylation of Smad2 and Smad3. Furthermore, PD reversed pro-fibrotic sphingolipid metabolism and upregulated taurine and glutathione metabolism associated with antioxidant defense. PD effectively alleviates chronic colitis-associated intestinal fibrosis by inhibiting the TGF-β/Smad signaling pathway-mediated EMT process, reducing ECM deposition, and reprogramming the fibrotic metabolic microenvironment.