Development of non-traumatic osteonecrosis of the femoral head requires toll-like receptor 7 and 9 stimulations and is boosted by repression on nuclear factor kappa B in rats

Okazaki, Shunichiro; Nagoya, Satoshi; Matsumoto, Hiroshi; Mizuo, Keisuke; Sasaki, Mikito; Watanabe, Satoshi; Yamashita, Toshihiko; Inoue, Hiromasa

The bone samples were decalcified with Kalkitox (Wako Pure Chemical Industries, Osaka, Japan) and then neutralized with a 5% sodium sulfate buffer. The tissues were then processed for routine hematoxylin and eosin staining to assess the general architecture and ONFH. Osteonecrosis was defined, in the present study, as the diffuse presence of empty lacunae or pyknotic nuclei in osteocytes within the bone trabeculae, accompanied by surrounding bone marrow cell necrosis. 3, 22, 23 Electrophoretic Mobility Shift Assay NF-kB and IRF7 activity were assessed by electrophoretic mobility shift assay as described previously. 24 In brief, equal amounts of liver nuclear extract (2.0 mg of protein) were incubated for 1 h at room temperature with 32 P-labelled NF-kB or IRF7 consensus oligonucleotide probes (5 0 -AGTTGA GGGGACTTTCCCAGGC-3 0 or 5 0 -ACTGATCGGAACCGA ACGATCTATG-3 0 , respectively) in binding buffer (10 mM HEPES (pH 7.9), 50 mM KCl, 0.2 mM ethylenediaminetetraacetic acid, 2.5 mM dithiothreitol, 10% glycerol, and 0.05% NP-40). The DNA protein complexes were separated on 7% non-denaturing polyacrylamide gels at a constant voltage of 100 V at room temperature. The gels were then exposed to an Image Plate (Fuji Film, Tokyo, Japan) at room temperature. The radioactivity of the DNA-binding complexes in the gel was analyzed using an FLA3000 Image Analyzer (Fuji Film) and ImageQuant Software (Molecular Dynamics, Sunnyvale, CA, USA).