淫羊藿甙促成骨细胞中Cbfa1表达的信号通路机制

目的 观察淫羊藿甙对成骨细胞中核心结合因子α1(cbfa1)蛋白和活性表达的调节,以及丝裂原活化蛋白激酶(MAPK)信号通路是否参与此过程.方法 用酶消化法分离24 h内新生SD大鼠颅盖骨成骨细胞,进行原代培养,经鉴定后用于实验.设立对照组、淫羊藿甙组(10 ng/ml)以及雌二醇组(10-8mol/L),分别用药物干预24 h,抽提核蛋白.利用转录因子活性ELISA法检测成骨细胞cbfa1与DNA结合的活性,Western印迹法检测成骨细胞中cbfa1蛋白的表达.将MAPK信号转导通路抑制剂U0126和SB203580分别与淫羊藿甙或雌二醇共同加入培养液中,培养24 h,同上法检测Cbfa1活性和Cbh1蛋白表达量的变化.结果 淫羊藿甙和雌二醇均可以促进成骨细胞中Cbfa1活性和Cbh1蛋白表达量的提高(P<0.05).加入细胞外信号调节激酶(ERK)途径的抑制剂UO126后,可以下调淫羊藿甙和雌二醇对成骨细胞中Cbfa1活性和Cbh1蛋白表达量的上调作用(P<0.05).加入p38MAPK途径的抑制剂SB203580后,也可以下调淫羊藿甙和雌二醇对成骨细胞中Cbfa1活性和Cbh1蛋白表达量的上调作用(P<0.05).结论 淫羊藿甙和雌激素均可上调体外培养大鼠成骨细胞转录因子Cbh1的蛋白表达和结合活性.MAPK信号转导通路抑制剂可以部分阻断淫羊藿甙和雌激素对成骨细胞转录因子Cbh1蛋白表达和活性的上调作用,说明MAPK可能是淫羊藿甙发挥抗骨质疏松作用的信号转导通路之一.

Signal pathway involved in regulation of Cbfa1 expression in osteoblasts by icariin

Objective To investigate the effects of icarrin on the activity and protein expression of core binding factor otl(Cbfa1) in rat osteoblasts cultured in vitro,and to explore whether mitogen-activated protein kinase (MAPK) pathway is involved in this process.Methods Calvarial osteoblasts were obtained from newborn (<24 h) SD rats by trypsin-coUagenase digestion method.The second generation osteoblasts were cultured in the medium containing icariin (10 ng/ml) or estradiol (10-8 mol/L) with or without extracellular-signal regulated kinase (ERK) inhibitor (UO126) or p38MAPK inhibitor (SB203580).Nuclear protein was extracted from osteoblasts.And then the activity of Cbfa1 was detected by ELISA.The amounts of Cbfa1 protein were detected by Western blot.Results Calvarial osteoblasts were obtained successfully and were used in this study after indentified by alkaline phosphatase and mineralized nodus staining.Cbfa1 expression and the activity in osteoblasts were up-regulated by both icariin and estradiol (P<0.05).The effects were partly inhibited by addition of U0126or SB203580 (P<0.05).Conclusions Either icarrin or estradiol can stimulate the proliferation and maturation of cultured osteoblasts in vitro via up-regulating the activity and expression of Cbfal.The MAPK signal pathway inhibitor seems to partly decrease Cbfa1 activity.It suggests that MAPK pathway may be involved in the transduction of icariin's impact on proliferation and mineralization of osteoblasts.