Rehmannin attenuates bleomycin-induced pulmonary fibrosis via THBS4-dependent modulation of fibroblast-dendritic cell interplay and matrix dynamics.

Abstract

OBJECTIVE: Rehmannia glutinosa, a renowned yin-enriching herb in traditional Chinese medicine (TCM), has been used to manage chronic pulmonary conditions marked by qi-yin deficiency and progressive fibrotic changes. Rehmannin, a representative iridoid glycoside derived from this herb, is regarded as one of its primary active components. However, the molecular basis underlying its antifibrotic activity remains insufficiently understood. This work aimed to assess the protective effects of Rehmannin against bleomycin-induced pulmonary fibrosis and to explore its mechanistic involvement in THBS4-associated stromal-immune regulation. METHODS: C57BL/6 mice were challenged with bleomycin via intratracheal instillation to establish a fibrosis model. Rehmannin was administered orally at doses of 20 and 40 mg/kg. Lung function parameters, tissue pathology, oxidative stress markers, apoptotic rates, and fibrosis-related proteins were comprehensively analyzed. Integrated transcriptomic and metabolomic profiling was employed to uncover key molecular targets. The role of THBS4 was investigated through surface plasmon resonance (SPR), immunofluorescence, quantitative PCR, and in vitro functional assays in NIH-3 T3 fibroblasts and bone marrow-derived dendritic cells (BMDCs), with additional gain- and loss-of-function approaches. RESULTS: Rehmannin treatment markedly reduced lung injury, improved pulmonary function, suppressed oxidative damage, and alleviated extracellular matrix deposition. Multi-omics analysis highlighted THBS4 as a central molecule responsive to Rehmannin intervention. Binding affinity between Rehmannin and THBS4 was confirmed by SPR assays. In vitro studies showed that Rehmannin diminished fibroblast migration and ECM-related protein expression, inhibited PI3K/AKT pathway activity, and facilitated M2 macrophage polarization in BMDCs. These regulatory effects were weakened upon THBS4 overexpression and strengthened following its knockdown. CONCLUSION: Our findings indicate that Rehmannin may exert its antifibrotic activity by modulating THBS4-mediated signaling between stromal and immune cells, thereby influencing matrix remodeling in pulmonary fibrosis. These mechanistic insights provide scientific support for the traditional application of Rehmannia glutinosa in respiratory disorders and suggest Rehmannin as a promising molecular candidate for further pharmacological exploration.