Integrated serum pharmacochemistry, network pharmacology and transcriptomics reveals the mechanism of Periploca forrestii on rheumatoid arthritis bone erosion.

ETHNOPHARMACOLOGICAL RELEVANCE: Periploca forrestii Schltr. (PF), classified among the "Ten Major Miao Medicinal Herbs" of Guizhou, China, has been traditionally used in Miao medicine to treat fractures and rheumatoid arthritis (RA), in accordance with the principle of "using vines to alleviate obstruction-related pain." However, the mechanism underlying its bone-protective effects, particularly regarding bone metabolism regulation in RA, remains incompletely elucidated.

AIM OF THE STUDY: To investigate the potential pharmacodynamic material basis and mechanisms of PF against RA-associated bone erosion by integrating serum pharmacochemistry, network pharmacology, and transcriptomics, with in vivo experimental validation, thereby providing scientific evidence for its traditional application.

MATERIALS AND METHODS: Chemical constituents of PF and its plasma-absorbed components were characterized using UPLC-Q-TOF-MS and GNPS molecular networking, while potential targets and pathways were predicted through network pharmacology. The anti-arthritic and bone-protective effects were assessed in a collagen-induced arthritis (CIA) rat model via Micro-CT, histopathology, and immunohistochemistry. Key targets within the RANKL-RANK-OPG axis and BMP2-SMAD signaling pathway were further validated by transcriptomics, Western blot, immunohistochemistry and molecular docking.

RESULTS: Fifty-three compounds were identified from PF. In the serum of PF-treated CIA rats, 57 compounds were detected in the serum of CIA rats treated with PF, mainly caffeoylquinic acids and steroids, comprising 8 prototype components and 49 metabolites. Integrated network pharmacology and transcriptomics analysis indicated that PF alleviates bone destruction in RA by inhibiting osteoclast differentiation and promoting osteoblast differentiation. In CIA rats, PF treatment markedly reduced joint swelling and lowered serum levels of pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α. Micro-CT, Safranin O staining, and TRAP staining, together with upregulated ALP mRNA and OCN protein expression, collectively confirmed that PF mitigates bone erosion. Moreover, IHC, RT-qPCR, and Western blot analyses revealed that PF effectively decreased the RANKL/OPG ratio in ankle tissues while significantly suppressing RANK and its key downstream transcription factor NFATc1. PF also restored the diminished levels of BMP-2, SMAD1, p-SMAD1, and SMAD4 in CIA rats and normalized the abnormally elevated RUNX2 protein. These findings demonstrate that PF ameliorates bone erosion in CIA rats via a multi-targeted and bidirectional regulatory mechanism, simultaneously inhibiting osteoclast differentiation and enhancing osteoblast activity.

CONCLUSION: PF alleviates bone erosion in CIA rats by inhibiting osteoclast differentiation and promoting osteogenic differentiation through modulation of the RANKL-RANK-OPG and BMP2-SMAD signaling pathways. This finding validates its traditional use for bone protection.