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

Abstract

Non-traumatic osteonecrosis of the femoral head (ONFH) often occurs after corticosteroid therapy in patients with inflammatory diseases. Recent studies suggest that toll-like receptor (TLR) signaling may contribute to the pathogenesis of inflammatory diseases, and that the reason for corticosteroid therapy for inflammatory diseases is related to the anti-inflammatory activities of corticosteroids through the reduction of NF-κB. We hypothesized that the administration of TLR ligands in combination with corticosteroid causes ONFH and that transcription factors may contribute to the pathogenesis of ONFH. The aim of the study was to evaluate (1) the incidence of ONFH in rats after the administration of TLR7 or TLR9 ligands together with methylprednisolone (MPSL) and (2) whether transcription factors contribute to the development of ONFH. Male Wistar rats (n=148) were divided into five groups as follows: Group 1: Saline+MPSL, Group 2: Imiquimod+Saline, Group 3: Imiquimod+MPSL, Group 4: CpG-C+MPSL, Group 5: Imiquimod+BAY11-7082+MPSL. As a result, ONFH was observed in 0 of 12 rats in Group 1, in 1 of 10 in Group 2, in 6 of 12 in Group 3, in 4 of 12 in Group 4, in 0 of 9 in Group 5. MPSL treatment did not significantly affect IRF7 activity, whereas NF-κB activity was significantly repressed in Group 2 and Group 3. Furthermore, the repression in interferon regulatory factor 7 (IRF7) activity by BAY11-7082 interfered with the development of ONFH simultaneously with the MPSL treatment-induced repression in NF-κB activity. In conclusion, in the present study, corticosteroid treatment after the administration of TLR7 or TLR9 ligands caused ONFH. Repression in NF-κB activity by corticosteroid treatment boosted the development of ONFH.

Figure 1

Histological appearance of the femoral head affected by osteonecrosis. Panels show hematoxylin and eosin-stained femurs. Typical images from the Saline+MPSL (a); Imiquimod+MPSL (b); and CpG-C+MPSL (c) groups are shown. The diffuse presence of empty lacunae and pyknotic osteocytic nuclei in the bone trabeculae accompanied by bone marrow cell necrosis was observed in the femoral head of Imiquuuuuimod+MPSL and CpG-C+MPSL groups. Scale bar: 100 m.

Figure 2

Histological appearance of the femoral head affected by osteonecrosis. Panels show hematoxylin and eosin-stained femurs. Typical images from the Imiquimod+Saline group with ONFH (a) and without ONFH (b) are shown. The diffuse presence of empty lacunae and pyknotic osteocytic nuclei in the bone trabeculae accompanied by bone marrow cell necrosis was also observed in the femoral head of rats with ONFH (a). Scale bar: 100 μm.

Figure 3

Activity of transcription factors. (a) EMSA for NF-κB and IRF7 in Imiquimod+Saline and Imiquimod+MPSL rats on Day 1. Lane 1 contains no extract. Lane 2 contains extract from untreated rat. Lane 3 contains extract from rat treated with Imiquimod+Saline. Lane 4 contains extract from rat treated with Imiquimod+MPSL. (b) Activity of NF-κB and IRF7. A repression in NF-κB activity was observed at Day 1 after corticosteroid treatment. Data represent the mean±s.e.m. **P<0.01 compared with Imiquimod+Saline rats.

Figure 4

Activity of transcription factors. EMSA for NF-κB and IRF7 in Imiquimod+MPSL and Imiquimod+BAY11+MPSL rats on Day 1. Lane 1 contains no extract. Lane 2 contains extract from untreated rat. Lane 3 contains extract from rat treated with Imiquimod+MPSL. Lane 4 contains extract from rat treated with Imiquimod+BAY11+MPSL. A repression in IRF7 activity was observed at Day 1 after co-administration of BAY11-7082 and corticosteroids. Data represent the mean±s.e.m. **P<0.01 compared with Imiquimod+MPSL rats.