Abstract: BACKGROUND: Yougui Pill is derived from Jingyue Quanshu. Studies have confirmed that Yougui Pill is highly effective in treating patients with knee osteoarthritis, but its mechanism of action remains unclear.
OBJECTIVE: To explore the potential molecular mechanism of Yougui Pill in improving knee osteoarthritis in rats. 
METHODS: Forty-eight SPF-grade Sprague-Dawley rats were randomly divided into four groups: blank control group, model group, Yougui Pill group and celecoxib group. Modified Hulth modeling method was used to establish the left knee joint model in the latter three groups. After wound healing, the rats were urged to move for 8 weeks. After modeling, the celecoxib group was given celecoxib suspension by gavage, the Yougui Pill group was given Yougui Pill decoction by gavage, and the blank control group and the model group were given the same volume of normal saline by gavage, once a day for 4 continuous weeks. The pathological changes of rat cartilage tissue were observed by hematoxylin-eosin staining, toluidine blue staining, and safranin O-fast green staining, and the ultrastructure of rat chondrocytes was observed by transmission electron microscope. ELISA was used to detect the levels of interleukin-18, interleukin-1β, and tumor necrosis factor-α in serum. The protein expressions of PI3K, AKT, NF-κB, p-PI3K, p-AKT, p-P65, NLRP3, GSDMD, GSDMD-N, Caspase1, Cleaved-Caspase1, interleukin-18, and interleukin-1β in cartilage tissues of rats were detected by western blot. 
RESULTS AND CONCLUSION: (1) Compared with the blank control group, the rats in the model group had severe cartilage edge destruction, cartilage tissue defect, thinning and disordered arrangement of cartilage layer cells, subchondral bone hyperplasia, disordered tide line, severe structural damage of chondrocytes and formation of pyroptosis. The levels of tumor necrosis factor-α, interleukin-18 and interleukin-1β in serum were significantly increased (P < 0.05). The protein expressions of p-PI3K, p-Akt, p-p65, NLRP3, GSDMD-N, Cleaved Caspase1, interleukin-18, and interleukin-1β in cartilage tissue were significantly increased (P < 0.01). (2) Compared with the model group, the Yougui Pill group and celecoxib group had normal cartilage structure, darker cartilage color, thicker cartilage, more complete chondrocyte membrane, and significantly reduced serum levels of tumor necrosis factor-α, interleukin-18 and interleukin-1β (P < 0.05). The protein expressions of p-PI3K, p-Akt, p-p65, NLRP3, GSDMD-N, Cleaved Caspase1, interleukin-18, and interleukin-1β in cartilage tissue were significantly decreased (P < 0.01). (3) Compared with the celecoxib group, the chondrocytes in the Yougui Pill group were arranged more regular, the articular cartilage surface was relatively smooth, the cartilage layer was thickened, the tide line was relatively complete, and the cell membrane of chondrocytes was relatively complete. The levels of tumor necrosis factor-α, interleukin-18 and interleukin-1β in serum were significantly decreased (P < 0.05). The protein expressions of p-PI3K, p-Akt, p-p65, NLRP3, GSDMD-N, Cleaved Caspase1, interleukin-18, and interleukin-1β in cartilage tissue were significantly decreased (P < 0.01). In conclusion, Yougui Pill can improve the inflammatory response of chondrocytes in rats with knee osteoarthritis, and its mechanism may be related to the inhibition of PI3K/AKT/NF-kB pathway activation, thereby regulating pyroptosis mediated via the NLRP3/Caspase-1/GSDMD pathway.

Key words: Yougui Pill, knee osteoarthritis, traditional Chinese medicine, cartilage, inflammation, pyroptosis, signaling pathway