橘红素预处理对过氧化氢诱导成骨细胞损伤的保护作用及机制  

Protection and mechanism of tangeretin pretreatment against oxidative damage to osteoblasts induced by hydrogen peroxide

作　　者：李汁涵 陈艳莉 田欣 路鸿瑜 殷航 邬钰 黄宏靓 陈珺 LI Zhi-han;CHEN Yan-li;TIAN Xin;LU Hong-yu;YIN Hang;WU Yu;HUANG Hong-liang;CHEN Jun(School of Biosciences and Biopharmaceutics,2.Center for Bioresources&Drug Discovery,Guangdong Pharmaceutical University,Guangzhou 510006,China)

机构地区：[1]广东药科大学生命科学与生物制药学院,广东广州510006 [2]生物资源与创新药物研究中心,广东广州510006

出　　处：《中国药理学与毒理学杂志》2022年第4期267-273,共7页Chinese Journal of Pharmacology and Toxicology

基　　金：2020年广东大学生科技创新培育专项资金“(攀登计划)”(pdjh2020b0305)。

摘　　要：目的研究橘红素预处理对过氧化氢(H_(2)O_(2))诱导的成骨细胞氧化损伤的保护作用和机制。方法①酶消化法分离培养大鼠颅骨来源的原代成骨细胞,分别培养7和21 d后,采用碱性磷酸酶(AKP)染色和茜素红染色鉴定细胞。②橘红素5~40μmol·L^(-1)孵育成骨细胞48 h后,加H_(2)O_(2)300μmol·L^(-1)1 h,采用CCK-8法测定细胞存活率,AKP试剂盒测定AKP活性。③培养的成骨细胞分为细胞对照组、模型组(H_(2)O_(2)300μmol·L^(-1),孵育1 h)、模型+橘红素组(橘红素10μmol·L^(-1)预处理48 h后加H_(2)O_(2)300μmol·L^(-1),1 h)。茜素红染色检测钙化结节数量,流式细胞术检测成骨细胞内活性氧(ROS)水平,超氧化物歧化酶(SOD)活性检测试剂盒检测SOD活性,Western印迹法检测成骨细胞转录因子(OSX)和骨保护素(OPG)蛋白表达水平。结果①AKP染色成骨细胞细胞胞质呈蓝色,茜素红染色可见钙化结节,结节呈深红色。②与模型组相比,橘红素(10~30μmol·L^(-1))预处理组成骨细胞存活率显著升高(P<0.01),橘红素(10~20μmol·L^(-1))预处理组成骨细胞AKP活性显著增加(P<0.05)。③与细胞对照组相比,模型组成骨细胞矿化结节数量显著降低(P<0.05),ROS水平明显升高(P<0.01),SOD活性明显降低(P<0.01),OSX和OPG蛋白表达水平显著降低(P<0.05);与模型组相比,模型+橘红素10μmol·L^(-1)预处理组矿化结节数量明显增加(P<0.05),ROS水平显著降低(P<0.01),SOD活性显著升高(P<0.01),OSX和OPG的蛋白表达水平显著升高(P<0.05)。结论橘红素可通过激活SOD、减少ROS累积而降低氧化应激,对H_(2)O_(2)诱导的成骨细胞氧化损伤具有保护作用。OBJECTIVE To study the protective effects and mechanism of tangeretin pretreatment against oxidative damage to osteoblasts induced by hydrogen peroxide(H_(2)O_(2)).METHODS①Osteo⁃blasts were isolated from the skull of rats by enzymes digestion and identified by alkaline phosphatase staining and Alizalin red staining after 7 and 21 days of culture,respectively.②Osteoblasts were pretreated with tangeretin 5-40μmol·L^(-1) for 48 h before being exposed to H_(2)O_(2)300μmol·L^(-1) for 1 h,cell viability was evaluated by CCK-8 assay kit and AKP activity was detected by AKP assay kit.③Osteoblasts were divided into cell control group,model group(H_(2)O_(2)300μmol·L^(-1),1 h)and model+tangeretin group(pretreated with tangeretin for 48 h,and then induced by H_(2)O_(2)300μmol·L^(-1),1 h).The number of calci⁃fied nodules was detected by Alizalin red staining,reactive oxygen species(ROS)by flow cytometry,super⁃oxide dismutase(SOD)activity by superoxide dismutase assay kit,and the protein expressions of osterix(OSX)and osteoprotegerin(OPG)were determined by Western blotting.RESULTS①Cyto⁃plasm of osteoblasts was stained blue after AKP staining.Calcified nodules were formed after 21 consecutive days of culture of osteoblasts,and were stained dark red by alizalin red staining.②Compared with model group,cell viability was significantly increased in the model+10-30μmol·L^(-1) tangeretin group(P<0.01),so was AKP activity in the model+tangeretin 10-20μmol·L^(-1) group(P<0.05).③Compared with cell control group,the number of calcified nodules was significantly decreased in the model group(P<0.05),the ROS level was significantly increased(P<0.01),the SOD activity significantly reduced(P<0.01),and the expressions of OSX and OPG decreased significantly(P<0.05).Compared with model group,the number of calcified nodules was significantly increased in model+tangeretin 10μmol·L^(-1) group(P<0.05),the ROS level was reduced significantly(P<0.01),the SOD activity was increased signifi⁃cantly(P<0.01),and the expr

关 键 词：橘红素 氧化损伤 成骨细胞 成骨功能 过氧化氢 

分 类 号：R285[医药卫生—中药学]