Role of N-acetylcysteine in preventing steroid-induced osteonecrosis of the femoral head by regulating NOX4/ROS pathway in rats
Objective To explore the impact and potential mechanism of N-acetylcysteine(NAC)on osteoblasts of the femoral head tissue and glucocorticoid-induced MC3T3-E1 cells in rats with steroid-induced osteonecrosis of the femoral head(SONFH).Methods Thirty-six male Wistar rats,weighing 250 to 280 g,were randomly divided into control group,experimental group and NAC group,with 12 rats each.The SONFH model was prepared by injecting methylprednisolone+lipopolysaccharide for 6 consecutive weeks in experimental group and NAC group.The rats in NAC group were injected with 120 mg/kg NAC into tail vein daily during modeling period.The rats in control group were injected with equal volume of normal saline into tail vein.All rats were sacrificed 6 weeks after modeling to collect the femoral head tissues.The morphology of the femoral head tissues was observed by HE staining,and the apoptosis of osteoblasts was observed by TUNEL staining.The MC3T3-E1 cells in logarithmic growth phase were divided into blank group(no treatment),dexamethasone group(treated with dexamethasone for 48 h)and NAC treatment group(treated with dexamethasone+NAC for 48 h).The cell proliferation rate of three groups was detected by CCK-8 method.The apoptosis was observed by TUNEL staining.The reactive oxygen species(ROS)level was detected by DCFH-DA staining.The relative expressions of caspase-3,caspase-9,B-cell lymphoma-2(Bcl-2),Bcl-2 associated X protein(Bax)and NOX4 proteins were detected by Western blot.Results The femoral head trabeculae exhibited integrity structure and regular distribution,with uniform and clearly visible bone cell distribution and occasional empty lacunae in control group;appeared irregular,thin,porous,fractured,and structurally disordered,with increased gaps between trabeculae,enlarged bone marrow cavities,and more empty lacunae in experimental group;was relatively intact,well-arranged,and showed a reduction in empty lacunae in NAC group compared with experimental group.A small number of apoptotic osteoblasts with yellow-stained nuclei were found in control group;a great number of apoptotic osteoblasts with yellow-stained nuclei were seen in experimental group.Normal osteoblasts with blue-stained nuclei were more numerous,and apoptotic osteoblasts with yellow-stained nuclei were fewer in NAC group compared with experimental group.The cell proliferation rates increased successively in dexamethasone group,NAC treatment group and blank group[(42.06±6.36)％,(72.82±3.68)％,(99.44±9.01)％](P＜0.05),and showed significant difference among three groups(F=18.300,P＜0.001).The apoptotic cells were less in blank group,were more in dexamethasone group,and were less in NAC treatment group than those in dexamethasone group.The ROS levels(598.60±22.68,387.20±20.34,170.40±43.65),and the relative expressions of caspase-3(0.86±0.01,0.78±0.01,0.62±0.01),caspase-9(0.70±0.01,0.60±0.01,0.11±0.01),Bax(1.03±0.01,0.79±0.01,0.64±0.01)and NOX4(0.31±0.01,0.20±0.01,0.12±0.01)decreased successively in dexamethasone group,NAC treatment group and blank group(P＜0.05),and showed significant differences among three groups(F=48.536,432.750,296.010,1 369.120,1 239.120;all P values＜0.05).The relative expression of Bcl-2 protein increased successively in dexamethasone group,NAC treatment group and blank group(0.29±0.01,0.50±0.01,0.76±0.01)(P＜0.05),and showed no significant difference among three groups(F=11 995.500,P＜0.05).Conclusion NAC may potentially relieve the bone damage of SONFH rats and glucocorticoid-induced osteoblasts injury by inhibiting the NOX4/ROS pathway to alleviate oxidative stress.
steroid-induced osteonecrosis of the femoral headN-acetylcysteineNOX4reactive oxygen speciesratsMC3T3-E1 cells
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