OBJECTIVE To investigate the mechanism by which Xibining Ⅱ alleviates chondrocyte injury in knee osteoarthritis (KOA) through the PI3K/Akt/NF-κB signaling pathway.

METHODS Network pharmacology was used to screen the active components of Xibining Ⅱ and KOA-related targets, followed by construction of a protein-protein interaction network, GO and KEGG enrichment analyses, and molecular docking to predict the binding ability of representative key active components to core target proteins. An inflammatory injury model was established in primary mouse chondrocytes induced by IL-1β. According to the CCK-8 results, 5% and 10% Xibining Ⅱ medicated serum was selected as low- and high-concentration groups for subsequent experiments, and the PI3K agonist 740Y-P was used for rescue verification. ELISA was used to detect the levels of IL-6, TNF-α, and IL-4. TUNEL staining was used to assess apoptosis. Western blot and immunofluorescence were performed to detect the expression of cartilage matrix metabolism-related proteins and PI3K/Akt/NF-κB pathway-related proteins.

RESULTS A total of 135 overlapping targets between Xibining Ⅱ and KOA were identified by network pharmacology. The core targets included AKT1, TNF-α, IL-6, IL-1β, and NF-κB1. Enrichment analysis indicated that the PI3K-Akt signaling pathway, apoptosis, and the MAPK signaling pathway might be major pathways involved in the effects of Xibining Ⅱ. Molecular docking showed that representative key active components had good binding activities with core target proteins such as AKT1, NFKB1, TNF, IL-6, IL-1β, and CASPASE3. CCK-8 results showed that different concentrations of blank serum and Xibining Ⅱ medicated serum had no significant cytotoxicity on primary chondrocytes. Based on the overall trend of cell viability changes, 5% and 10% medicated serum were selected as the subsequent intervention concentrations. In vitro experiments showed that, compared with the model group, Xibining Ⅱ medicated serum reduced the levels of IL-6 and TNF-α, increased the level of IL-4, decreased nuclear TUNEL fluorescence intensity, upregulated the expression of COL2A1, ACAN, and SOX9, downregulated the expression of MMP13, and decreased the ratios of p-PI3K/PI3K, p-Akt/Akt, and p-NF-κB/NF-κB, with better effects in the high-concentration group（P<0.05）. After addition of the PI3K agonist 740Y-P to the high-concentration group, the above protective effects were partially reversed（P<0.05）.

CONCLUSION Xibining Ⅱ may exert protective effects on KOA chondrocytes by inhibiting abnormal activation of the PI3K/Akt/NF-κB signaling pathway, thereby reducing inflammation and apoptosis and improving cartilage matrix metabolic imbalance.