Multi-omics analysis of the active components and mechanisms of Baojin Chenfei formula in silica-induced murine silicosis.

BACKGROUND

Silicosis is a severe fibrotic lung disease resulting from exposure to silica, with limited therapeutic options despite recent advances. The Baojin Chenfei Formula (BCF), a traditional Chinese medicine, has shown promise in alleviating silicosis, yet its precise mechanisms of action remain unclear.

PURPOSE

This study aimed to elucidate the antifibrotic mechanisms of BCF, focusing particularly on its regulation of peroxisome proliferator-activated receptor gamma (PPARγ) and lipid metabolism in silica-induced pulmonary fibrosis.

METHODS

Silicosis was induced in mice via intratracheal instillation of crystalline silica (CS), followed by oral administration of BCF. Lung function, histopathology (H&E and Masson's staining), fibrosis markers, collagen deposition, and PPARγ signaling pathway activity were assessed. Active fractions of BCF were separated using macroporous resin adsorption technology, and the cellular activity of these fractions was evaluated. The active fraction BCF5 was analyzed by mass spectrometry to identify its components. RNA-Seq analysis was conducted to uncover molecular pathways by which BCF inhibits fibroblast activation. Co-immunoprecipitation (Co-IP) and GFP reporter assays assessed BCF's effects on PPARγ-RXRα complex formation and PPARγ transcriptional activity. Molecular docking and biolayer interferometry (BLI) evaluated binding affinities between BCF5 components and PPARγ. Expression of PPARγ target genes was quantified by PCR, and lipid metabolomics identified lipid metabolites modulated by BCF5. The role of PPARγ-mediated lipid synthesis in BCF's therapeutic effects was further investigated using siPPARγ, PPARγ inhibitors, siFASN and FASN-EGFP-Reporter plasmids.

RESULTS

BCF treatment improved lung function, reduced fibrosis markers, and attenuated histopathological damage in silicosis mice. The active fraction BCF5 significantly inhibited TGF-β-induced fibroblast activation and contained 188 identified active constituents. RNA-Seq analysis revealed that BCF5 regulates the PPAR signaling pathway.Co-IP showed that BCF5 promoted PPARγ-RXRα complex formation, and PPRE-EGFP-Reporter assays confirmed enhanced PPARγ transcriptional activity. PCR analysis revealed increased expression of PPARγ target genes following BCF5 treatment. The antifibrotic effect of BCF5 was notably diminished by siPPARγ and PPARγ inhibitors. Molecular docking, cellular assays, and BLI identified Magnoloside A as a potent PPARγ agonist capable of inhibiting fibroblast activation. Lipid metabolomics demonstrated that BCF5 markedly increased total fatty acid levels, with lauric acid exhibiting significant inhibitory effects on fibroblast activation. Analysis via the JASPAR database suggested fatty acid synthase (FASN) as a PPARγ target gene, and FASN silencing significantly reversed BCF's inhibitory effect on fibroblast activation. FASN-EGFP reporter gene analysis showed that BCF-driven FASN transcription depends on the transcriptional activity of PPARγ. Importantly, the therapeutic efficacy of BCF in silicosis was substantially reduced upon administration of a PPARγ inhibitor.

CONCLUSION

BCF exerts antifibrotic effects by activating the PPARγ signaling pathway, upregulating FASN expression, and promoting lauric acid biosynthesis, a novel mechanistic insight into silicosis treatment.