还原型烟酰胺腺嘌呤二核苷酸氧化酶-4在成骨细胞凋亡中的作用  

Effect of Nox4/NADPH oxidase on the process of osteoblast apoptosis in vitro

作　　者：张华峰[1] 白树财 徐倩[3] 李晖[1] 李东[1] 李呈凯 宋秀钢 秦亚飞 马信龙[4] Zhang Huafeng;Bai Shucai;Xu Qian;Li Hui;Li Dong;Li Chengkai;Song Xiugang;Qin Yafei;Ma Xinlong(Department of Orthopaedics, the General Hospital of Tianjin Medical University, Tianjin 300052, Chin;Graduate School of Tianjin Medical University, Tianjin 300070, Chin;School of Integrative Medicine, the Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China;Tianjin Hospital, Tianjin 300211, Chin)

机构地区：[1]天津医科大学总医院骨科,300052 [2]天津医科大学研究生院,300070 [3]天津中医药大学基础实验教学部,300193 [4]天津市天津医院,300211

出　　处：《中华骨科杂志》2018年第12期742-751,共10页Chinese Journal of Orthopaedics

基　　金：国家自然科学基金（81501915）;天津中医药大学优秀教师培养计划

摘　　要：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-aeetylcysteine，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1000nmol／L地塞米松处理成骨细胞，按照实验分组干预培养24h，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南基的产生。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�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 DP] 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 apopt

关 键 词：地塞米松 细胞凋亡 活性氧 NADPH氧化酶 

分 类 号：R681.8[医药卫生—骨科学]