This study aimed to investigate the therapeutic mechanisms of Wangbi(WB) in osteoarthritis (OA), specifically focusing on its modulatory effects on the AIM2 inflammasome-mediated pyroptosis pathway. We sought to confirm these effects through a comprehensive approach encompassing network pharmacology predictions, in vitro cellular assays, in vivo animal models, and a preliminary clinical assessment, thereby providing a scientific basis for WB's clinical application in OA.

Network Pharmacology: We identified potential active compounds in WB and their corresponding targets. These targets were then integrated with known OA- and pyroptosis-related genes, particularly those associated with AIM2, to predict WB's multi-targeted mechanisms and enriched pathways (e.g., NF-κB, MAPK, IL-17 signaling).

In Vitro Cell Experiments: SW1353 chondrocytes were used to establish an AIM2 pyroptosis model by Poly(dA:dT) transfection. The cells were then treated with WB-containing medium. Pyroptosis was assessed by measuring LDH release, and the expression levels of key pyroptotic proteins (AIM2, Cleaved Caspase-1, GSDMD-N) and inflammatory cytokines (IL-1β, TNF-α) were evaluated using Western Blot and ELISA.

In Vivo Animal Experiments: A rat model of OA was induced by intra-articular injection of monoiodoacetate (MIA). Rats were subsequently treated with different doses of WB via gavage. Joint pathology was evaluated through gross observation, joint swelling measurements, Micro-CT, histological analysis (HE, Safranin-O/Fast Green staining), and OARSI scoring. The expression of AIM2 pyroptosis pathway molecules (AIM2, Cleaved Caspase-1, GSDMD-N) and inflammatory factors (IL-1β) in joint tissues was determined by WB, RT-qPCR, and ELISA.

Clinical Study: This preliminary study involved healthy volunteers and OA patients. OA patients were divided into diacerein-treated and diacerein-plus-WB-treated groups. Peripheral blood mononuclear cells (PBMCs) were collected to measure the mRNA expression (by RT-qPCR) and protein expression (by Western Blot) of pyroptosis-related molecules (AIM2, Caspase-1, GSDMD, IL-1β). Serum levels of IL-1β, TNF-α, and cartilage degradation markers were also assessed by ELISA. Clinical symptoms were evaluated using WOMAC, VAS, and Lequesne scores, and correlations between molecular markers and clinical outcomes were analyzed.

Network Pharmacology: Analysis revealed 65 core common targets for WB, OA, and AIM2 pyroptosis. GO and KEGG enrichment indicated these targets are critically involved in regulating inflammatory responses, stress responses to bacterial molecules, and inflammasome activation, suggesting WBF's multi-component synergistic action on inflammation and pyroptosis pathways.

In Vitro Findings: Poly(dA:dT) transfection successfully induced AIM2-mediated pyroptosis in SW1353 chondrocytes, characterized by increased LDH release and upregulation of AIM2, Cleaved Caspase-1, GSDMD-N, and IL-1β. Importantly, WB-containing medium significantly inhibited these pyroptotic events and inflammatory cytokine release, confirming its suppressive effect on AIM2 pyroptosis.

In Vivo Findings: MIA-induced OA rats exhibited typical pathological changes, including joint swelling, cartilage damage, and elevated OARSI scores. Concomitantly, AIM2, Cleaved Caspase-1, GSDMD-N, and IL-1β levels were significantly increased in joint tissues. WBF treatment markedly improved joint pathology, reduced OARSI scores, and suppressed the activation of the AIM2 pyroptosis pathway and expression of associated inflammatory mediators.

Clinical Observations: In OA patients, combined treatment with WB and diacerein led to significantly greater improvements in clinical symptom scores (WOMAC, VAS, Lequesne, phGA) and a reduction in ESR levels compared to diacerein monotherapy. Furthermore, this combined treatment significantly downregulated the mRNA and protein expression of key pyroptosis components (AIM2, Caspase-1, GSDMD, IL-1β) in PBMCs and reduced serum IL-1β levels, suggesting WB’s additive clinical benefits by modulating the pyroptosis pathway.

This study provides compelling evidence, spanning from network pharmacology predictions to in vitro, in vivo, and preliminary clinical data, that Wangbi  exerts significant therapeutic effects in osteoarthritis. Its mechanism involves the critical inhibition of the AIM2 inflammasome-mediated pyroptosis pathway, thereby mitigating cartilage damage and inflammatory responses. These findings highlight WB's potential as a promising multi-targeted therapeutic agent for OA, warranting further clinical investigation.