Network pharmacology and in-depth blood proteomics reveal the mechanism of Buqi Tongluo capsules in treating bone destruction in osteoarthritis

Abstract

Background

Osteoarthritis (OA) is the most prevalent form of arthritis, characterized by a rapidly increasing global incidence. Once OA develops, it initiates an irreversible process of bone destruction, which can lead to joint dysfunction or disability, and currently, no cure exists. Traditional Chinese medicine formulations, such as Buqi Tongluo Capsule (BQTL), are known for their ability to promote blood circulation, relieve pain, and unblock meridians, and are widely used to treat pain-related conditions. However, the effects of BQTL on OA-related bone destruction remain unclear.

Purpose

This study aims to investigate the safety and efficacy of BQTL in treating OA bone destruction using a multi-method approach, including bioinformatics analysis, network pharmacology, in-depth blood proteomics, and an ACLT-induced OA rat model. The study also seeks to identify active components of BQTL using UHPLCHRMS, LC-MS/MS and SPR techniques and to explore its potential mechanisms of action in BMMs-induced osteoclasts.

Methods

Network pharmacology analysis was used to predict the main active ingredients, key pathways, and targets of BQTL for OA treatment. An ACLT-induced OA rat model was employed to evaluate the in vivo efficacy of BQTL. Articular bone destruction and serum marker expression were assessed using Micro CT and ELISA. Hepatotoxicity and nephrotoxicity were evaluated via ELISA and HE staining. Key protein expression related to cartilage, osteoclastogenesis, and inflammation was detected using TRAP staining, Safranin O-Fast Green staining, and immunohistochemistry. UHPLCHRMS and LC-MS/MS was used to identify potential active components and compound concentrations of BQTL in vivo. Surface plasmon resonance (SPR), molecular docking, and dynamics simulations were employed to validate interactions between active ingredients and key targets. In-depth blood proteomics was used to explore the mechanism of action of BQTL in OA treatment. Cytotoxicity of Buqi Tongluo capsule-containing serum (BQTLS) was assessed using the CCK-8 assay. The inhibitory effects of BQTLS on osteoclastogenesis were evaluated in an osteoclast model induced by BMMs, with TRAP staining used to analyze osteoclast number and average area. F-actin staining and immunofluorescence were used to assess osteoclast morphology, function, and ROS production. IF, WB, and RT-PCR were employed to detect protein expression related to key signaling pathways, osteoclastogenesis, and ROS. Lentiviral transfection with overexpressed RAGE was used for rescue verification of the molecular mechanism of upstream and downstream pathways.

Results

In vivo findings demonstrated that BQTL reduced proteins associated with osteoclast activity and inflammation in subchondral bone, decreased osteoclastogenesis, and mitigated articular cartilage degradation. Oral administration of BQTL did not induce significant hepatorenal toxicity and reduced the expression of inflammatory factors and bone turnover markers in rat serum. In-depth blood proteomics identified 180 differentially expressed proteins, with GO enrichment analysis indicating correlations with cellular responses to redox states and KEGG pathway analysis highlighting regulation of the actin cytoskeleton and the AGE-RAGE signaling pathway. Ingredient identification results confirmed that Formononetin may be the predominant active ingredient of BQTL for the treatment of OA in vivo. Target validation confirmed that Formononetin directly binds to and targets RAGE receptor expression on critical pathways. In vitro experiments showed that BQTLS inhibits osteoclastogenesis by targeting the RAGE-RAS pathway, regulating the MAPK/NF-κB pathway, reducing ROS production, and decreasing NFATc1 nuclear transcription. Overexpression of RAGE reversed the inhibitory effects of BQTLS and Formononetin on osteoclastogenesis and rescued the decreased phosphorylation expression of the MAPK/NF-κB pathway.

Conclusion

Formononetin, the principal active compound in BQTL, suppresses OA bone destruction progression by inhibiting abnormal osteoclastogenesis in subchondral bone through targeting the RAGE-RAS signaling pathway and regulating the MAPK/NF-κB pathway. These findings provide an experimental foundation for expanding the clinical application of BQTL and developing alternative therapies for OA treatment.