摘要
目的:观察桑根酮C(SanC)对地塞米松(DEX)作用下小鼠MC3T3-E1成骨细胞增殖与分化的影响,并探讨其作用机制。方法:将SanC与同源建模所得的Runt-相关转录因子2(Runx2)蛋白结构进行分子对接。不同浓度SanC(8,16,32μmol·L^-1)和1μmol·L^-1DEX共同作用MC3T3-E1细胞,而后采用细胞增殖-毒性检测试剂盒(CCK-8)法检测SanC对MC3T3-E1成骨细胞增殖影响。试剂盒测定MC3T3-E1成骨细胞碱性磷酸酶(ALP)活性和茜素红染色检测骨矿化结节的形成。采用实时荧光定量聚合酶链反应(Real-time PCR)检测Runt-相关转录因子2(Runx2),ALP,和锌指结构转录因子(Osterix)mRNA的表达水平。蛋白免疫印迹法(Western blot)检测Runx2蛋白表达。结果:SanC与Runx2对接打分为-9.78。与正常组比较,DEX组显著降低细胞存活率(P<0.01),其中7 d存活率差异达到最大;与DEX组比较,SanC能显著促进MC3T3-E1的细胞增值(P<0.01),其中32μmol·L^-1SanC作用细胞7 d增殖率差异达到最大。与正常组比较,DEX组Runx2,ALP和Osterix mRNA的表达均有一定程度升高(P<0.05);与DEX组比较,不同浓度SanC组依赖性上调Runx2,ALP和Osterix mRNA的表达(P<0.01)。与正常组比较,DEX组Runx2蛋白表达明显下降(P<0.05);与DEX组比较,SanC干预下细胞Runx2蛋白表达显著升高(P<0.01)。结论:桑根酮C能促进MC3T3-E1成骨细胞增殖、分化和矿化,其机制可能与上调Runx2表达有关。
Objective:To observe the effect of sanggenone C(SanC)on the proliferation and differentiation of mouse MC3T3-E1 osteoblasts induced by dexamethasone(DEX),and to explore its mechanism.Method:Molecular docking was conducted between SanC and Runt-associated transcription factor 2(Runx2)protein structure obtained by homologous modeling.MC3T3-E1 cells were jointly treated by different concentrations of SanC(8,16,and 32μmol·L^-1)and 1μmol·L^-1 DEX,and then cell counting kit-8(CCK-8)method was used to detect the effect of SanC on the proliferation of MC3T3-E1 osteoblasts.The alkaline phosphatase(ALP)activity of MC3T3-E1 osteoblasts was determined by reagent kit and the formation of mineralized bone nodules were detected by alizarin red staining.Real-time fluorescent quantitative polymerase chain reaction(Real-time PCR)was used to detect the mRNA expression of Runx2,ALP and Osterix.The protein expression of Runx2 was detected by Western blot.Result:The docking score of SanC and Runx2 was-9.78.As compared with the normal group,DEX group significantly reduced the cell survival rate(P<0.01),and the greatest difference occurred on the seventh day.As compared with DEX group,SanC could significantly promote the cell proliferation of MC3T3-E1(P<0.01),in which 32μmol·L^-1 SanC had the largest difference in proliferation rate on seventh day.As compared with the normal group,the expression of Runx2,ALP and Osterix mRNA increased to a certain extent in DEX group(P<0.01).As compared with DEX group,the expression levels of Runx2,ALP and Osterix mRNA were up-regulated in different concentration groups of SanC in a dose-dependent manner(P<0.01).As compared with the normal group,the expression of Runx2 protein in DEX group decreased significantly(P<0.05),and as compared with DEX group,the expression of Runx2 protein in cells under the intervention of SanC increased significantly(P<0.01).Conclusion:SanC can promote the proliferation,differentiation and mineralization of MC3T3-E1 osteoblasts,and the mechanism may be related to the up-regulation of Runx2 expression.
作者
刘芬
党院霞
邢菊玲
冯萌
莫紫欣
罗瑞娇
周欣欣
LIU Fen;DANG Yuan-xia;XING Ju-ling;FENG Meng;MO Zi-xin;LUO Rui-jiao;ZHOU Xin-xin(School of Pharmaceutical Sciences,Guangzhou University of Chinese Medicine,Guangzhou 510006,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2020年第10期44-50,共7页
Chinese Journal of Experimental Traditional Medical Formulae
基金
国家自然科学基金项目(81473439)。
作者简介
第一作者:刘芬,硕士,从事中药内分泌药理学研究,E-mail:sixpoints0518@163.com;通信作者:周欣欣,教授,硕士生导师,从事中药新药研究与开发,E-mail:020465@gzucm.edu.cn。
