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.

NADPH Oxidase Isoforms Are Involved in Glucocorticoid-Induced Preosteoblast Apoptosis.

Oxidative stress induced by long-term glucocorticoid (GC) use weakens the repair capacity of bone tissue. Nicotinamide adenine dinucleotide phosphate, reduced form (NADPH) oxidase (NOX) is a superoxide-generating enzyme that plays an important role in regulating bone metabolism. To clarify the role of nonphagocytic NOX isoforms in osteoblast reactive oxygen species (ROS) generation and apoptosis, dexamethasone was used to establish a high-dose GC environment in vitro. A dose-dependent increase in intracellular ROS generation was demonstrated, which was accompanied by increased osteoblastic MC3T3-E1 cell apoptosis. Addition of the ROS inhibitor NAC (N-acetyl-L-cysteine) or NOX inhibitor DPI (diphenyleneiodonium) reversed this effect, indicating that NOX-derived ROS can induce osteoblast apoptosis under high-dose dexamethasone stimulation. NOX1, NOX2, and NOX4 are NOX homologs recently identified in bone tissue. To clarify the NOX isoforms that play a role in osteoblast ROS generation, Nox1, Nox2, and Nox4 mRNA expression and NOX2 and NOX4 protein expression were analyzed. Nox1 and Nox4 mRNA expression was elevated in a dose-dependent manner after culture in 100 nM, 250 nM, 500 nM, or 1000 nM dexamethasone, and the increased expression of NOX1 mRNA was more significant compared with NOX4 mRNA. Small interfering RNAs (siRNAs) were used to confirm the role of NOX1 and NOX4 in ROS generation. To clarify the signaling pathway in ROS-induced osteoblast apoptosis, mitogen-activated protein kinase (MAPK) signaling molecules were analyzed. Phosphorylated ASK1 and p38 levels were significantly higher in the 1000 nM dexamethasone group, which NAC or DPI markedly attenuated. However, the total mRNA and protein levels of ASK1 and p38 between the dexamethasone group and control were not significantly different. This is related to ROS regulating the posttranslational modification of ASK1 and p38 in MC3T3-E1 cell apoptosis. Altogether, NOX1- and NOX4-derived ROS plays a pivotal role in high-dose dexamethasone-induced preosteoblast apoptosis by increasing phosphorylated ASK1 and p38 and may be an important mechanism in steroid-induced avascular necrosis of the femoral head (SANFH).

Unburied versus buried wires for fixation of pediatric lateral condyle distal humeral fractures: A meta-analysis.

Open reduction and internal fixation with Kirschner (K) wires has been reported as an efficient and convenient technique for pediatric lateral condyle distal humeral fractures. However, no single study has been large enough to definitively determine whether the K-wires should be buried or unburied. Therefore, we performed a meta-analysis pooling the results from several clinical trials to compare the outcome of using buried versus unburied K-wires. Potential academic articles were identified from the Cochrane Library, Medline (1966-2017.3), PubMed (1966-2017.3), Embase (1980-2017.3), ScienceDirect (1985-2017.3), and other databases. Gray studies were identified from the references of included literature reports. RevMan 5.1 was used to analyze the pooling of data. Nonrandomized controlled trials were included in this meta-analysis. There was a significant difference in the duration of wires in situ (MD = -13.28, 95% confidence interval: -16.42 to -10.14, P < .00001). No significant differences were found regarding infection, superficial infection, total complications, delayed union, or reoperation. Unburied K-wire fixation for treatment of lateral condyle distal humeral fractures in children does not increase the total infection rate, superficial infection, reoperation rate, or complications. However, unburied K-wire fixation is of benefit for early extraction and impartial cost savings.