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目的 探讨高浓度地塞米松(dexamethasone,DEX)激活还原型烟酰胺腺嘌呤二核苷酸(nicotinamide adenine dinucleotide phosphate-reduced,NADPH)氧化酶-4(NADPH oxidase 4,Nox4)途径产生氧自由基对成骨细胞凋亡的影响和作用机制.方法 设正常对照组,DEX组,DEX+N-乙酰半胱氨酸(n-acetylcysteine,NAC)组,NAC组,DEX+二亚苯基碘(diphenyleneiodonium chloride,DPI)组,DPI组.干预培养24h后,倒置显微镜下观察成骨细胞形态;MTT法检测细胞活性;荧光探针DCFH-DA检测细胞内氧自由基水平;Hoechst染色检测成骨细胞凋亡;荧光定量PCR及Western-Blot检测Nox4/NADPH氧化酶基因和蛋白的表达;Nox4 siRNA沉默细胞内Nox4/NADPH氧化酶表达后,通过荧光探针检测氧自由基水平.结果 采用1 000 nmol/L地塞米松处理成骨细胞,按照实验分组干预培养24 h,倒置相差显微镜下观察和MTT结果提示,与正常对照组相比,DEX组成骨细胞出现明显的皱缩、变形,细胞存活率显著降低,加入NAC或DPI后细胞形态良好,生长旺盛.荧光探针检测结果显示,DEX组成骨细胞内氧自由基生成(45.14％±1.49％)与正常对照组(5.86％±0.28％)相比,差异有统计学意义(P=0.000);Hoechst染色结果显示,DEX组成骨细胞凋亡(29.60％±1.52％)与正常对照组(4.12％±0.67％)相比,差异有统计学意义(P=0.000).DEX组成骨细胞内氧自由基生成与DEX+NAC组(28.06％±1.61％)和DEX+DPI组(23.70％±1.28％)相比,差异均有统计学意义(均P=0.000),提示NAC或DPI显著降低了成骨细胞内氧自由基的产生.DEX组成骨细胞凋亡与DEX+NAC组(8.94％±1.47％)和DEX+DPI组(12.96％±2.03％)相比,差异均有统计学意义(均P=0.000),提示NAC或DPI显著降低了成骨细胞凋亡.Nox4的荧光定量PCR显示DEX组Nox4的基因表达是正常对照组的2.67倍(t=-10.301,P=0.009),Western Blot检测显示DEX组Nox4的蛋白表达是正常对照组的2.37倍(t=-15.542,P=0.004),差异有统计学意义.进一步通过Nox4 siRNA靶向沉默Nox4的表达,DEX+Nox4 siRNA组成骨细胞内氧自由基的生成(14.53％±1.00％)比DEX组(31.45％±0.72％)降低了16.92％,两者比较差异有统计学意义(P=0.000).结论 Nox4介导的氧自由基生成在高浓度地塞米松诱导成骨细胞凋亡过程中起重要作用,Nox4可能是糖皮质激素性股骨头坏死治疗过程中的重要靶点.“,”Objective To investigate the role and mechanism of nico-tinamide adenine dinucleotide phosphate oxidase 4 (NAPHD oxidase 4,Nox4)-mediated reactive oxygen species (ROS) generation on high-dose dexamethasone (DEX) induced apoptosis in osteoblasts.Methods According to culture conditions,3rd passage of murine osteoblastic MC3T3-E 1 cells were divided into control group,Dexamethasone group,Dexamethasone+NAC (N-acetyl-L-cysteine) group,NAC group,Dexamethasone+DPI (Diphenyleneiodonium) group and DPI group.24 hours after culture,the morphology of osteoblasts was observed by inverted phase contrast microscope.Cell viability was determined by MTT assay.The generation of ROS in osteoblasts was measured using a fluorescent probe DCFH-DA.The apoptosis of each group was observed through Hoechst staining.The mRNA level and protein expression of Nox4 were detected by real-time quantitative PCR and Western Blot.In addition,after silence of Nox4 with small interfering RNA (siRNA),the ROS generation was further detected by a fluorescent probe DCFH-DA.Results After treatment with 1000 nmol DEX for 24 hours,compared to control group,the results of inverted phase contrast microscope and MTT showed that osteoblasts in DEX group exhibited more obvious signs of shrinkage and deformation with decreased cell viability.After intervene with NAC and DPI,morphology of osteoblasts was good with increased viability of osteoblasts.Compared to control group (5.86％± 0.28％),the production of ROS in DEX group (45.14％±1.49％) was significantly increased (P=0.000).The apoptotic rates in DEX group (29.60％± 1.52％) was significantly increased compared with control group (4.12％±0.67％) (P=0.000).Compared to DEX group,the production of ROS generation in DEX+NAC group (28.06％±1.61％) and DEX+DPI group (23.70％±1.28％) was significantly decreased (P=0.000).It presented that NAC or DPI significantly decreased the formation of ROS.Compared to DEX group,the apoptotic rate in DEX+NAC group (8.94％± 1.47％) and DEX+DPI group (12.96％±2.03％) was significantly decreased (P=0.000).It presented that NAC or DPI significantly decreased osteoblast apoptosis.In addition,the Nox4 mRNA level in DEX group was 2.67-fold compared with control group (t=-10.301,P=0.009).The difference had statistically significance.The protein expression of Nox4 in DEX group was 2.37-fold compared with control group (t=-15.542,P=0.004).The difference has statistically significance.After silence of Nox4 by siRNA,the generation of ROS in DEX+Nox4 siRNA group (14.53％± 1.00％) was decreased by 16.92％ compared with DEX group 31.45％±0.72％ (P=0.000).The difference had statistically significance.Conclusion Nox4-mediated ROS generation plays an important role in osteoblasts apoptosis induced by high-dose dexamethasone.It provided us the new target in the management of Nox4 to provide possible therapy for steroid-induced avascular necrosis of the femoral head (SANFH).