Hypericin attenuates pathological scarring through concurrent modulation of transforming growth factor-β1 and bone morphogenetic protein 2 signaling in vitro and in vivo.

BACKGROUND: Pathological scarring is driven by persistent crosstalk between profibrotic cytokine signaling and extracellular matrix remodeling. Transforming growth factor-β1 (TGF-β1)/Smad2/3 signaling broadly activates fibroblasts, whereas bone morphogenetic protein 2 (BMP2)/Smad1/5/9 signaling reinforces matrix-producing fibroblast states. Concurrent modulation of these pathways may therefore provide a more comprehensive therapeutic strategy.

PURPOSE: To determine whether hypericin (HYP), prioritized through receptor-based virtual screening, attenuates pathological scarring while concurrently modulating TGF-β1 and BMP2 signaling.

METHODS: HYP was prioritized through receptor-based virtual screening involving transforming growth factor beta receptor 1 and bone morphogenetic protein receptor type 1A and evaluated in human skin fibroblasts using low-dose viability assays, single-ligand and TGF-β1/BMP2 co-stimulation models, reference inhibitors, cellular thermal shift assays, quantitative polymerase chain reaction, immunoblotting, immunofluorescence, proliferation and migration assays, and integrated transcriptomic analysis. HYP-loaded micelles were characterized and assessed in a rabbit ear hypertrophic scar model, whereas non-micellar HYP was evaluated in a human keloid patient-derived xenograft model.

RESULTS: Virtual screening prioritized HYP as a candidate compound with predicted interactions with transforming growth factor beta receptor 1 and bone morphogenetic protein receptor type 1A. HYP at 1 and 2 μM did not significantly affect fibroblast viability, whereas viability remained above 90% at 4 μM. Cellular thermal shift assays demonstrated enhanced thermal stabilization of both receptor proteins in HYP-treated cells, supporting cellular target engagement. HYP suppressed TGF-β1/Smad2/3- and BMP2/Smad1/5/9-associated fibrotic responses. Under simultaneous TGF-β1 and BMP2 stimulation, HYP attenuated both signaling branches, whereas SB-431,542 and LDN-193,189 predominantly inhibited their respective pathways. Integrated transcriptomic analysis identified 96 shared HYP-suppressed genes and convergent downregulation of extracellular matrix, collagen, focal adhesion, integrin, ossification, and chondrogenesis programs. HYP-loaded micelles exhibited a mean particle size of 148.1 ± 15.1 nm, a polydispersity index of 0.219 ± 0.015, an encapsulation efficiency of 72.18 ± 4.05%, and a drug loading of 11.65 ± 1.28%. Local HYP treatment reduced scar elevation, collagen deposition, xenograft volume, and pathway activation in vivo.

CONCLUSION: HYP coordinately suppresses TGF-β1/Smad2/3 and BMP2/Smad1/5/9 signaling and exerts consistent antifibrotic effects across cellular, rabbit ear, and human keloid xenograft models. These findings provide a preclinical rationale for further investigation of HYP as a locally delivered antifibrotic therapy with concurrent TGF-β1- and BMP2-pathway modulatory activity.