To explore the pathological role of high mobility group box chromosomal protein 1 (HMGB1) in osteoarthritis (OA) by comparing the difference of HMGB1 in the synoviocytes between OA and normal knees.
Synoviocyte lines from OA and normal knees were collected and cultured. Immunohistochemistry and Western blot were applied to identify the difference of HMGB1 between the OA and normal synoviocyte lines. The eukaryotic expression vector containing human Pgenesil-1/HMGB1 small interfering RNA (siRNA) were constructed and identified. The synoviocyte lines were transfected with the eukaryotic expression vector of Pgenesil-1/HMGB1 siRNA (Pgenesil-1/HMGB1 siRNA group) and with Pgenesil-1 plasmid (Pgenesil-1 group) and were not transfected as a control (untransfected group). Western blot was applied to identify the difference of HMGB1 among groups, and the levels of interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α) protein synthesis in the supernatants were measured by ELISA.
Primary knee synoviocytes cultured in vitro were fibroblast-like cells with long- spindle shape. The immunohistochemistry and immunofluorescence results showed positive staining for HMGB1 in cytoplasm and weak positive staining in the nucleus in the OA synoviocyte line, but positive staining for HMGB1 in the nucleus and weak positive staining in the cytoplasm in the synoviocyte line of normal knee. The level of HMGB1 in the OA synoviocytes (0.687 ± 0.025) was significantly higher than that of normal synoviocytes (0.172 ± 0.030) (t = 32.159, P = 0.000) by Western blot. The recombinant plasmid Pgenesil-1/HMGB1 siRNA was successfully constructed. The expression of HMGB1 protein in Pgenesil-1/HMGB1 siRNA group (0.134 ± 0.048) was significantly lower than that of Pgenesil-1 group (0.581± 0.032) and untransfected group (0.514 ± 0.069) (P < 0.05). ELISA results showed that IL-1β and TNF-α in supernatants of Pgenesil-1/HMGB1 siRNA group were significantly lower than those of Pgenesil-1 group and untransfected group (P < 0.05).
The up-regulated expression and expressed location (from nucleus to cytoplasm) of HMGB1 in the synoviocyte are closely related to CA. The siRNA targeting inhibition of HMGB1 gene expression can obviously inhibit II-lp and TNF-a in supernatants of the CA synoviocyte line and delayed the inflammation of CA.