Integrated Dose‐Effect Weighted Method and Metabolomics Reveals Mechanism of Rhizoma Drynariae for the Treatment of Inflammatory Osteoporosis

ABSTRACT Rhizoma Drynariae (RD), derived from the desiccated rhizome of Drynaria fortunei (Kunze) J. Sm. , has been researched for its potential therapeutic efficacy in modulating inflammatory responses (IR) and treating osteoporosis (OP). Nevertheless, the molecular mechanisms through which RD attenuates OP by modulating the IR microenvironment have yet to be thoroughly elucidated. Consequently, this study aimed to assess the impact of RD on IR and OP through integrated zebrafish and cellular model assays, focusing on the quantification of neutrophil recruitment, bone mineralization area, and the levels of tumor necrosis factor‐α, nitric oxide, interleukin‐6, and alkaline phosphatase. The experimental data demonstrate that the < 3 kDa RD fraction (RDE‐2) effectively mitigates IR and OP models across both zebrafish and cellular models. Utilizing a novel dose‐effect weighted network pharmacology, we identified kaempferol‐3‐O‐rutinoside, procyanidin B2, and prunin as the primary bioactive constituents of RDE‐2. Combined with untargeted metabolomics analysis, potential targets for their action were identified. Subsequently, a network comprising active ingredients, core targets, metabolic targets, and metabolites was constructed to elucidate their functional interactions. RT‐qPCR analysis revealed a significant upregulation in the mRNA expression levels of AKT1, ESR1, ESR2, MMP9, PI3K, SRC, EP300, NCOA3, Runx2, and CREBBP. The findings suggest that RD has the potential to mitigate OP by modulating the IR microenvironment through the ER/PI3K‐EP300 signaling axis. Through the integration of dose‐effect weighted network pharmacology and metabolomic analysis, our study advances beyond existing descriptive research on RD and pioneers the elucidation of the ER/PI3K‐EP300 axis, thereby offering a novel mechanistic explanation.