Abstract: BACKGROUND: Warm acupuncture has a significant therapeutic effect on knee osteoarthritis. It can improve articular cartilage degeneration by inhibiting periarticular inflammation. However, whether this effect is related to pyroptosis mediated by the Toll-like receptor 4/nuclear factor-κB (TLR4/NF-κB) signaling pathway remains unclear.
OBJECTIVE: To investigate the mechanism of warm acupuncture in the treatment of knee osteoarthritis from the perspective of pyroptosis.
METHODS: Forty New Zealand rabbits were randomly divided into blank, model, western medicine, and warm acupuncture groups using a random number table, with 10 rabbits in each group. The blank group received no intervention, while knee osteoarthritis models were established in the other three groups using the plaster cast fixation method. Four weeks after modeling, the model group was immobilized in a fixator for 15 minutes once daily for 2 consecutive weeks. The warm acupuncture group received warm acupuncture at “Heding” (EX-LE2), “Neixiyan” (EX-LE4), and “Waixiyan” (ST35) for 15 minutes once daily for 2 weeks. The western medicine group received celecoxib by gavage once daily for 2 weeks. Samples were collected on the second day after treatment. Interleukin-1β and interleukin-18 levels in synovial fluid were detected by ELISA. Morphological changes in knee cartilage were observed by hematoxylin-eosin staining. The expression of TLR4, NF-κB p65, Caspase-1, and gasdermin D in knee cartilage was detected by qPCR, immunohistochemistry, and western blot.
RESULTS AND CONCLUSION: (1) Interleukin-1β and interleukin-18 levels were significantly higher in the model group than in the blank group (P < 0.05), and significantly lower in the warm acupuncture and western medicine groups than in the model group (P < 0.05). (2) Hematoxylin-eosin staining results showed that the cartilage surface in the model group was rough and significantly damaged, with irregular and disorganized chondrocyte distribution. In the warm acupuncture and western medicine groups, the cartilage surface was relatively smooth, and chondrocytes were more uniformly and regularly distributed. (3) Results from qPCR, immunohistochemistry, and western blot showed that compared with the blank group, the mRNA and protein expression levels of TLR4, NF-κB p65, Caspase-1, and gasdermin D were significantly increased in the model group (P < 0.05). Compared with the model group, the mRNA and protein expression levels of TLR4, NF-κB p65, Caspase-1, and gasdermin D were significantly decreased in the warm acupuncture and western medicine groups (P < 0.05). These findings indicate that warm acupuncture can alleviate articular cartilage degeneration in rabbits with knee osteoarthritis by regulating the TLR4/NF-κB signaling pathway to downregulate the expression of pyroptosis-related proteins Caspase-1 and gasdermin D, as well as downstream inflammatory factors interleukin-1β and interleukin-18, thereby inhibiting pyroptosis.

Key words: warm acupuncture, knee osteoarthritis, pyroptosis, Toll-like receptor 4/nuclear factor-κB signaling pathway, Caspase-1, gasdermin D