Abstract: BACKGROUND: Osteoarthritis is now considered a metabolic disease. Previous studies have shown that glycolysis plays an important role in the occurrence and development of osteoarthritis. Compound 3k, as a novel small molecule inhibitor of glycolysis, has anti-inflammatory and anti-tumor effects. Therefore, it can target glycolysis and is expected to provide new ideas for the treatment of osteoarthritis.
OBJECTIVE: To explore the role of Compound 3k in osteoarthritis caused by glycolytic overactivity based on the hypoxia-inducible factor 1 alpha (HIF-1α)/reactive oxygen species (ROS) pathway.
METHODS: ATDC5 chondroblasts at logarithmic growth phase were taken to induce osteoarthritis in an in vitro cellular model by the action of 10 ng/mL interleukin-1β for 24 hours. The cytotoxicity of Compound 3k at different concentrations (0.25, 0.5, 1, 2.5, 5, 10, 15 μmol/L) was detected by cell counting kit-8 assay, and the appropriate concentrations were selected for the subsequent experiments. The chondrocytes were randomly divided into control, model and treatment groups. The model group was induced with 10 ng/mL interleukin 1β, and the treatment group was pre-stimulated with Compound 3k for 2 hours and then co-cultured with interleukin 1β. The proliferation of the cells in each group was detected by the cell counting kit-8 assay; the inflammatory level of the cells in each group was detected by the ELISA kit; the ROS, extracellular lactate and glucose contents were detected using the kit; qRT-PCR and western blot were used to detect the levels of related inflammatory factors, interleukin-6 and tumor necrosis factor-α, glycolysis-related genes glucose transporter protein-1, glyceraldehyde 3-phosphate dehydrogenase, monocarboxylate transporter protein-1 and HIF-1α.
RESULTS AND CONCLUSION: Compared with the control group, the model group showed a decrease in cell proliferative activity, active glycolysis level, manifested by an increase in extracellular lactate content (P < 0.001) and a decrease in glucose content (P < 0.001), interleukin-6 (P < 0.000 1) and tumor necrosis factor-α (P < 0.001). The expression levels of glycolysis-related genes glucose transporter protein-1 (P < 0.001), glyceraldehyde 3-phosphate dehydrogenase (P < 0.001), monocarboxylic acid transporter protein-1 (P < 0.001) and HIF-1α (P < 0.001) in the model group were all up-regulated, accompanied by oxidative stress and overproduction of ROS. Compared with the model group, Compound 3k treatment effectively increased cell proliferation activity and inhibited the level of overactive glycolysis (P < 0.001), while suppressing the expression of genes related to inflammation (P < 0.001) and glycolysis in osteoarthritic chondrocytes, inhibiting oxidative stress, downregulating the expression level of HIF-1α (P < 0.000 1) and decreasing the content of ROS. To conclude, Compound 3k inhibits interleukin-1β induced chondrocyte inflammation, and its mechanism may be related to glycolysis and HIF-1α/ROS mediated oxidative stress.

Key words: osteoarthritis, glycolysis, Compound 3k, oxidative stress, HIF-1α/ROS, ATDC5 cells, glucose metabolism, interleukin-1β, small molecule drug