The “Tianyu” Formulation Alleviates Rheumatoid Arthritis by Modulating the <scp>NLRP3</scp> /Caspase‐1/ <scp>GSDMD</scp> ‐Mediated Pyroptosis Pathway

This research focuses on exploring the modulation of pyroptosis and its underlying mechanisms via the "Tianyu" formulation (TY), a traditional Chinese medicine, in human rheumatoid arthritis fibroblast-like synovial cells (RA-HFLS) and a collagen-induced arthritis (CIA) rat model. In vitro, RA-HFLS cells were induced with tumor necrosis factor α (TNF-α). Cell viability, proliferation, migration, and invasion were assessed using CCK-8, EdU, and Transwell assays, respectively. Pyroptosis was evaluated by flow cytometry, acridine orange/ethidium bromide (AO/EB) staining, and by measuring the release of lactate dehydrogenase (LDH) and inflammatory factors (interleukin [IL]-1β, IL-18, TNF-α) in the cell culture supernatants. The expression of pyroptosis-related mRNAs and proteins was examined by RT-qPCR, immunofluorescence, and Western blotting. For in vivo analysis, a CIA rat model was established. Joint swelling was evaluated using arthritis scores and paw volume measurements, while histopathological changes in the joint tissues were examined by hematoxylin and eosin (HE) staining and Safranin O-Fast Green staining. Serum levels of autoantibodies (anti-CCP, RF), LDH, and inflammatory cytokines (IL-1β, IL-18, TNF-α) were quantified concurrently. Furthermore, the expression of pyroptosis-related molecules in joint tissues was determined using RT-qPCR, immunohistochemistry, and Western blot analysis. The major components of TY were identified by UHPLC-Q Exactive HFX. Subsequently, molecular docking simulations were performed to evaluate the binding affinities of the main bioactive compounds to the NLRP3, Caspase-1, and GSDMD proteins. In vitro experiments showed that TY significantly reduced the viability, proliferation, migration, and invasion abilities, as well as the LDH levels of TNF-α-induced RA-HFLS cells, and decreased the levels of IL-1β, IL-18, and TNF-α in the supernatant. Additionally, TY downregulated the expression of key proteins involved in pyroptosis mediated by the NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome. In the CIA rat model, TY treatment alleviated arthritis symptoms, reduced paw swelling and bone erosion, improved joint pathology, decreased serum levels of IL-1β, IL-18, and TNF-α, and suppressed the expression of key proteins involved in the NLRP3 inflammasome-mediated pyroptosis pathway in synovial tissue. A total of 95 compounds belonging to 16 classes were identified from TY in both positive and negative ion scanning modes. These included 42 phenylpropanoid and polyphenolic compounds, as well as 12 organic oxides. Molecular docking showed that all five major compounds (Apigenin, Isorhamnetin, Kaempferol, Quercetin, and Salidroside) bound well to NLRP3, Caspase-1, and GSDMD, with all binding energies below -5 kcal/mol. Among them, Quercetin exhibited the highest affinity for GSDMD (-9.1 kcal/mol). By regulating the NLRP3/Caspase-1/GSDMD pathway, TY alleviates joint inflammation in CIA rats and reduces pyroptosis in RA-HFLS cells.