阿魏酸对过氧化氢诱导成骨细胞损伤的影响

Effects of ferulic acid on osteoblasts induced by hydrogen peroxide

目的研究阿魏酸(ferulic acid,FA)对过氧化氢诱导氧化损伤的成骨细胞功能的影响。方法酶消化法分离提取大鼠颅骨成骨细胞并进行体外传代培养,以碱性磷酸酶(alkaline phosphatase,ALP)染色和茜素红(alizarin red S,ARS)染色进行细胞鉴定;通过Cell Counting Kit-8(CCK-8)法测定细胞存活率及ALP检测试剂盒检测ALP分泌,并筛选出适合的过氧化氢损伤浓度和FA作用浓度。将细胞分为对照组(C组)、氧化损伤模型组(M组)和FA给药组(M+FA组),流式细胞术检测各组成骨细胞内活性氧簇(reactive oxygen species,ROS)含量,ARS染色检测各组钙化结节形成大小,并以实时荧光定量PCR(realtime fluorescence quantitative PCR,RTFQ-PCR)检测成骨功能相关基因的表达情况。结果(1)确定过氧化氢致损浓度为200μmol/L、作用时间1 h;FA给药浓度为640μmol/L,作用时间为72 h。(2)与C组相比,M组成骨细胞的存活率降低了60%(P<0.0001),细胞培养上清液中ALP含量显著降低(P<0.0001),细胞ROS含量上升约为C组3倍,细胞着色变浅、未能生成明显的钙化结节,成骨特异性转录因子(runt-related transcription factor 2,RUNX-2)、骨保护蛋白(osteoprotegerin,OPG)、成骨相关转录因子(osterix,OSX)等成骨功能相关基因的表达亦有所降低;与M组相比,M+FA组成骨细胞的存活率显著提高(P<0.01),细胞上清液中ALP含量显著提高(P<0.01),ROS含量降低了一半,钙化结节数量增加、细胞着色加深,成骨功能相关基因OPG、OSX的表达均显著升高(P<0.05),但FA对RUNX-2表达无明显影响。结论FA干预能明显改善过氧化氢所致氧化损伤成骨细胞的存活能力,增加细胞ALP分泌水平和细胞体外矿化能力,亦明显上调成骨功能相关基因的表达。

Objective To study the effects of ferulic acid on the function of osteoblasts induced by hydrogen peroxide.Methods Osteoblasts isolated from the skull of rat by enzymes digestion were identified by alkaline phosphatase(ALP)staining and alizarin red S(ARS)staining;Cell viability and alkaline phosphatase content were evaluated by Cell Counting Kit-8(CCK-8)assay kit and ALP assay kit respectively,and according to which the suitable concentration of hydrogen peroxide and ferulic acid were screened out.Osteoblasts were divided into control group(C),oxidative-damage model group(M),and ferulic acid group(M+FA).Reactive oxygen species(ROS)in each group were detected by flow cytometry.The size of calcified nodules in each group was detected by ARS staining,and the expression of osteogenesis related genes were detected by realtime fluorescence quantitative PCR(RTFQ-PCR).Results(1)Cells were treated with 200μmol/L hydrogen peroxide for 1 hour to establish the hydrogen peroxide-impaired model and the dosage regimens of ferulic acid were 640μmol/L for 72 hours incubation.(2)Compared with group C,the cell viability of M group were decreased by 60%(P<0.0001),and ALP content in cell culture supernatant of group M was reduced significantly(P<0.0001),the ROS level was increased by 3 times.And the calcified nodules were small and cells stained lightly in group M.Moreover,the expression of Runt-related transcription factor 2(RUNX-2),osteoprotegerin(OPG)and osterix(OSX)were decreased in group M.Compared with group M,the viability of cells in M+FA group was significantly increased(P<0.01),and ALP content in supernatant was increased significantly(P<0.01).The ROS level of group M+FA was only half of group M.The calcium nodules increased and the staining was darker in M+FA group.Furthermore,the expression of OPG and OSX was increased significantly(P<0.05),while the decrease of RUNX-2 expression was no significant difference in M+FA group.Conclusion Ferulic acid not only stimulates the cell viability and the secretion of alkaline phosphatase in osteoblasts induced by hydrogen peroxide,but also increases the mineralization function and enhances the expression of osteogenesis related genes,indicating that ferulic acid can repair the injury of osteoblasts induced by hydrogen peroxide.