机构地区:[1]天津医科大学朱宪彝纪念医院、天津市内分泌研究所、卫健委激素与发育重点实验室、天津市代谢性疾病重点实验室,天津300134 [2]锦州医科大学医疗学院
出 处:《中国慢性病预防与控制》2020年第10期748-752,共5页Chinese Journal of Prevention and Control of Chronic Diseases
基 金:国家自然科学基金青年基金项目(81603461);天津医科大学朱宪彝纪念医院基金项目(2019ZXY06)。
摘 要:目的研究依达拉奉(EDA)对过氧化氢(H2O2)诱导的成骨细胞氧化应激损伤的影响,为骨质疏松的治疗提供依据。方法取24 h内新生SD大鼠头盖骨进行成骨细胞原代培养,传代至第3代进行成骨细胞鉴定。将鉴定后的成骨细胞分别用H2O2或(和)EDA以不同浓度作用2、4、6 h进行MTT法检测细胞增殖情况;选择合适的作用浓度和作用时间进行H2O2诱导成骨细胞氧化损伤模型的建立,测定超氧化物歧化酶(SOD)、丙二醛(MDA)、碱性磷酸酶(ALP)等指标,分析EDA对H2O2诱导成骨细胞氧化应激损伤的影响。同时通过免疫印迹法测定药物处理分组后bax,bcl-2凋亡相关蛋白的变化。用SPSS 23.0软件进行方差分析。结果400μmol/L H2O2作用4 h是建立成骨细胞氧化应激模型的适宜浓度和作用时间。MTT显示,不同浓度(10、20、40、80μmol/L)EDA作用2、4、6 h成骨细胞成活率均高于空白组,差异均有统计学意义(P<0.05)。EDA(80μmol/L)+H2O2(400μmol/L)组较H2O2(400μmol/L)组成骨细胞成活率高,差异有统计学意义(P<0.05)。细胞氧化结果显示,MDA浓度随EDA浓度增加而逐渐下降,SOD活性则逐渐升高,均有统计学意义(P<0.05)。EDA(80μmol/L)+H2O2(400μmol/L)组ALP水平高于H2O2组,差异有统计学意义(P<0.01)。western blot显示,EDA(10、20、40、80μmol/L)+H2O2(400μmol/L)组bcl-2表达增强,bax表达递减,差异有统计学意义(P<0.05)。结论EDA对H2O2诱导的成骨细胞氧化应激损伤具有保护作用,可以减少损伤后成骨细胞的氧化和凋亡。Objective To study the inhibition effects of Edaravone(EDA)on the osteoblast oxidative stress damage induced by hydrogen peroxide(H2O2),and to provide the evidence for the treatment of osteoporosis.Methods Osteoblasts were obtained directly from neonatal Sprague-Dawley rats,and primary cells were cultured for three generations then identified.The identified osteoblasts were treated with H2O2 or H2O2 plus the EDA at the different concentrations for 2,4,6 h,the cell proliferation was detected by MTT assay;the oxidative stress damage model was established at the suitable concentration and time point,the superoxide dismutase(SOD),malondialdehyde(MDA)and alkaline phosphatase(ALP)were measured to analyze the inhibition effects of EDA on the osteoblast oxidative stress damage induced by H2O2.The western blot assay was used to detect the change of bax and bcl-2 related to the apoptosis.The ANOVA was used to analyze the data,the used software was SPSS 23.0.Results The400μmol/L and 4 h exposed to H2O2 were the suitable concentration and time point for estalishing the osteoblast oxidative stress damage model.The MTT assay results showed that,as compared with the control group,osteoblast viability of EDA group(10,20,40 and 80μmol/L)at 2,4 and 6 h significantly enhanced,P<0.05.The osteoblast viability of EDA(80μmol/L)+H2O2(400μmol/L)group was significantly higher than that of H2O2(400μmol/L)group(P<0.05).The oxidative stress damage results showed that MDA concentration decreased with EDA,SOD activity increased with EDA(P<0.05).The ALP level of EDA(80μmol/L)+H2O2(400μmol/L)group was significantly higher than that of H2O2(400μmol/L)group(P<0.05).The western blot assay results showed that the protein expression levels of bcl-2 in EDA(10、20、40、80μmol/L)+H2O2(400μmol/L)groups increased,but the protein expression levels of bax decreased(P<0.05).Conclusion EDA has the protective effects on the osteoblasts oxidative stress damage induced by H2O2,and can reduce the oxidation and apoptosis of damaged osteoblasts.
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