Multi-omics reveals the effects and mechanisms of Xinxue Granules against idiopathic pulmonary fibrosis through activating the cAMP/EPAC/CREB axis.

ETHNOPHARMACOLOGICAL RELEVANCE: Xinxue Granules (XXGR) is a traditional Chinese medicine formula derived from the classical prescription Zixue Powder, one of the three treasured formulas in traditional Chinese medicine. Based on the principle of heat-clearing and detoxification, it has long been used to treat respiratory diseases associated with heat-toxin accumulation. Owing to its multi-component nature, XXGR has been reported to modulate inflammatory responses and alleviate pulmonary injury. However, its protective effects and underlying mechanisms in pulmonary fibrosis-like injury remain unclear.

AIM OF THE STUDY: To investigate the protective effects and mechanisms of XXGR in pulmonary fibrosis. A bleomycin-induced pulmonary fibrosis-like injury mice model was established after random grouping. The therapeutic effects were assessed by histopathological and biochemical analyses. Network pharmacology, metabolomics, and transcriptomics were integrated to identify potential pathways. Additional in vitro experiments using TGF-β1-induced MLE-12 cells, including EPAC inhibition with ESI-09, were performed to further explore the underlying mechanism.

MATERIALS AND METHODS: A bleomycin (BLM)-induced pulmonary fibrosis (PF) mouse model was established to evaluate the antifibrotic effects of XXGR. Ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) was used to characterize the chemical constituents and serum-absorbed components of XXGR. Network pharmacology, transcriptomics, and non-targeted metabolomics were integrated to identify potential targets and pathways. In vivo experiments, including histopathology, biochemical assays, quantitative real-time PCR (qRT-PCR), and Western blotting (WB), were performed to validate key molecular mechanisms. In vitro experiments using TGF-β1-stimulated MLE-12 cells with EPAC inhibition (ESI-09) were performed to explore mechanisms. Molecular docking was conducted to evaluate compound-target interactions. All botanical names were verified according to the World Flora Online.

RESULTS: UPLC-Q-TOF/MS identified 20 blood-absorbed components of XXGR, and network pharmacology revealed 222 potential targets, highlighting the cyclic adenosine monophosphate (cAMP) signaling pathway. XXGR reduced lung injury, inflammatory cytokine levels, fibroblast activation, and extracellular matrix deposition in BLM-induced mice. Mechanistically, XXGR increased EPAC expression and CREB phosphorylation while decreasing GLI3 expression in vivo and in vitro, consistent with activation of cAMP signaling. EPAC inhibition with ESI-09 partially reversed the effects of XXGR on fibrosis- and EMT-related markers.

CONCLUSION: XXGR attenuated bleomycin-induced pulmonary fibrosis-like injury by suppressing inflammation and fibroblast activation. Its protective effects may be partially mediated via the cAMP/EPAC/CREB signaling pathway. These findings suggest that XXGR represents a promising multi-target therapeutic candidate for pulmonary fibrosis.