褪黑素对H_(2)O_(2)诱导的成骨细胞氧化应激损伤的保护作用  

Study on the protective mechanism of melatonin on the oxidative stress damage of osteoblasts induced by hydrogen peroxide

作　　者：刘合栋 任茂贤 李杨 蒋文凯 周志 杨民 LIU Hedong;REN Maoxian;LI Yang;JIANG Wenkai;ZHOU Zhi;YANG Min(Department of Trauma Orthopedics,The First Affiliated Hospital of Wannan Medical College,Yijishan Hospital,Wuhu 241001,China)

机构地区：[1]皖南医学院第一附属医院/弋矶山医院创伤骨科,安徽芜湖241001

出　　处：《中国骨质疏松杂志》2022年第8期1093-1098,共6页Chinese Journal of Osteoporosis

基　　金：国家自然科学基金(81341054,81171732);皖南医学院人才引进基金(YR201917);弋矶山医院攀峰计划(PF2019005)。

摘　　要：目的探讨褪黑素对H_(2)O_(2)诱导的成骨细胞氧化应激损伤的保护作用机制。方法取24 h内新生SD大鼠10只,采用组织块贴壁法提取原代成骨细胞并行差速贴壁提纯细胞。细胞传至第2代时行碱性磷酸酶及茜素红染色进行鉴定。将鉴定后的成骨细胞用不同浓度的H_(2)O_(2)作用不同时间并行CCK8试验检测细胞增殖情况。选择合适的浓度和时间建立氧化应激损伤模型,并用褪黑素及EX527进行干预。实验分为对照组、H_(2)O_(2)组、褪黑素组及EX527组。分别对四组细胞行碱性磷酸酶染色、茜素红染色,检测四组细胞活性氧、丙二醛、超氧化物歧化酶含量,同时用流式细胞仪检测四组细胞凋亡率,免疫印迹法检测凋亡相关蛋白Bax、Bcl2和成骨相关蛋白BMP2、RUNX2以及SIRT1和p66SHC的表达量。结果经鉴定,成功提取原代成骨细胞。CCK8结果显示,400μmol/L H_(2)O_(2)作用4 h成骨细胞活性降至52%,适宜用于建立氧化应激损伤模型。H_(2)O_(2)作用后成骨细胞活性及矿化能力降低,ROS含量、MDA含量升高,SOD活性降低,细胞凋亡率升高,伴随有SIRT1、BMP2、RUNX2、Bcl2表达降低,p66SHC和Bax表达升高。褪黑素预处理后缓解了H_(2)O_(2)对成骨细胞的氧化应激损伤作用,部分恢复了成骨细胞的活性及矿化能力,SIRT1抑制剂EX527能够逆转褪黑素的上述作用。结论褪黑素通过调节SIRT1/p66SHC通路来抑制H_(2)O_(2)诱导的成骨细胞的氧化应激损伤并促进成骨。Objective To explore the protective mechanism of melatonin on the oxidative stress damage of osteoblasts induced by hydrogen peroxide.Methods Primary osteoblasts were extracted by tissue explants adherent method from ten newborn SD rats within 24 hours and purified by differential adhesion method.Cells were identified by alkaline phosphatase and Alizarin Red S staining when passaged to the second generation.The identified primary osteoblasts were treated with different concentrations of H_(2)O_(2) for different time and CCK8 assay was used to detect cell proliferation.Choose an appropriate concentration and time to establish an oxidative stress damage model and intervene with melatonin and EX527.The experiment was divided into control group,H_(2)O_(2) group,melatonin group and EX527 group.Alkaline phosphatase and Alizarin Red S staining were performed on the four groups,the content of reactive oxygen species,malondialdehyde and superoxide dismutase were detected,the apoptosis rate was detected by flow cytometry and the western blotting method was used to detect the expression of Bax,Bcl2,BMP2,RUNX2,SIRT1 and p66SHC.Results The primary osteoblasts were successfully extracted after identification.After treated with 400μmol/L H_(2)O_(2) for 4 hours,the cell viability decreased to 52%which was suitable for establishing oxidative stress damage model,alkaline phosphatase activity and mineralization ability decreased,ROS content,MDA content increased and SOD activity decreased,the apoptosis rate increased accompanied by SIRT1,BMP2,RUNX2,Bcl2 expression decreased and p66SHC,Bax expression increased.Melatonin pretreatment alleviated the oxidative damage of H_(2)O_(2) on osteoblasts and partially restored the activity and mineralization ability of osteoblasts.The SIRT1 inhibitor EX527 can reverse the above effects of melatonin.Conclusion Melatonin can inhibit the H_(2)O_(2)-induced oxidative damage of osteoblasts and promote osteogenesis via SIRT1/p66SHC pathway.

关 键 词：褪黑素 成骨细胞 氧化应激 骨质疏松 SIRT1/p66SHC通路 

分 类 号：R336[医药卫生—人体生理学]