Oxidative Stress Induced Osteocyte Apoptosis in Steroid-Induced Femoral Head Necrosis

Objective To investigate the effect and mechanism of Glucocorticoids (GCs) induced oxidative stress and apoptosis on necrosis of the femoral head in patients and rats. Methods Eight patients with steroid-induced avascular necrosis of the femoral head (SINFH) and eight patients with developmental dysplasia of the hips (DDH) were enrolled in our study. In animal model, twenty male Sprague-Dawley rats were randomly divided into two groups (SINFH group and NS group). The SINFH model group received the methylprednisolone (MPS) injection, while control group was injected with normal saline (NS). MRI was used to confirm SINFH rat model was established successfully. Then, the rats were sacrificed 4 weeks later and femoral head samples were harvested. Histopathological staining was preformed to evaluate osteonecrosis. TUNEL staining was performed with 8-OHdG and DAPI immunofluorescence staining to evaluate oxidative injury and osteocyte apoptosis. Immunohistochemistry staining was used to detect Nox1, Nox2, and Nox4 protein expression. Results MRI showed signs of typical osteonecrosis of femoral head in SIHFH patients. Histopathological staining showed that the rate of empty lacunae in SINFH patients was significantly higher (56.88% +/- 9.72% vs 19.92% +/- 4.18%, T = -11.04, P < 0.001) than that in DDH patients. The immunofluorescence staining indicated that the TUNEL-positive cell and 8-OHdG-positve cell in SINFH patients were significantly higher (49.32% +/- 12.95% vs 8.00% +/- 2.11%, T = -7.04, P = 0.002, 54.6% +/- 23.8% vs 9.75% +/- 3.31%, T = -4.17, P = 0.003) compared to the DDH patients. The immunohistochemistry staining showed that the protein expression of NOX1, NOX2 and NOX4 in SINFH patients were significantly increased (64.50% +/- 7.57% vs 37.58% +/- 9.23%, T = -3.88, P = 0.018, 90.84% +/- 2.93% vs 49.56% +/- 16.47%, T = -5.46, P = 0.001, 85.46% +/- 9.3% vs 40.69% +/- 6.77%, T = -8.03, P = 0.001) compared to the DDH patients. In animal model, MRI showed signs of edema of femoral head in MPS group, which represents SINFH rat model was established successfully. Histological evaluation showed the rate of empty lacunae in MPS group was significantly higher (25.85% +/- 4.68% vs 9.35% +/- 1.99%, T = -7.96, P < 0.001) than that in NS group. The immunofluorescence staining indicated that the TUNEL-positive cell and 8-OHdG-positve cell (in MPS group were significantly increased (31.93% +/- 1.01% vs 11.73% +/- 1.16%, T = -32.26, P < 0.001, 47.59% +/- 1.39% vs 22.07% +/- 2.45%, T = -22.18, P < 0.001) compared to the NS group. The immunohistochemistry staining showed that the expression of NOX2 in MPS group was significantly increased (76.77% +/- 8.34% vs 50.32% +/- 10.84%, T = -4.74, P = 0.001) compare with NS group. Conclusion Our findings indicated that GC-induced NOXs expression may be an important source of oxidative stress, which could lead to osteocyte apoptosis in the process of SINFH