MAPK信号通路在淫羊藿甙促进成骨细胞Cbfa1蛋白表达中的作用

目的 观察淫羊藿甙对体外培养大鼠成骨细胞丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)信号通路的影响以及MAPK信号通路在淫羊藿甙促成骨细胞核心结合因子α1(core binding factor-1,Cbfa1)蛋白表达中的作用,以探讨淫羊藿甙对成骨细胞作用的信号传导机制.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.在培养液中加入淫羊藿甙(10 ng/mL),作用于成骨细胞5 min、10 min、30 min、60 min,抽提总蛋白,用Western blot法检测细胞中ERK、p-ERK、P38和p-P38蛋白的表达.用雌二醇(10-8 mol/L)作用于成骨细胞5 min、10 min、30 min,同上法检测细胞中ERK、p-ERK、P38和p-P38蛋白的表达.用淫羊藿甙(10 ng/mL)、雌二醇(10-8 mol/L)和u0126或SB203580单独或共同干预成骨细胞24 h,抽提核蛋白,用Western blot法检测Cbfa1蛋白的表达.结果 ①淫羊藿甙作用于成骨细胞,30 min时可促进ERK蛋白的磷酸化,并可持续至60 min,和空白组比较,差异有显著性(P<0.05);淫羊藿甙作用于成骨细胞,5 min时即可促进P38蛋白的磷酸化,在30 min时达高峰,并可持续至60 min,和空白组比较,差异有显著性(P<0.05).②雌二醇作用于成骨细胞,在30 min时可促进ERK蛋白的磷酸化,和空白组比较,差异有显著性(P<0.05);雌二醇作用于成骨细胞,在10 min时可促进P38蛋白的磷酸化,并可持续至30 min,和空白组比较,差异有显著性(P<0.05).③淫羊藿甙和雌二醇均能促进成骨细胞中Cbfa1蛋白的表达,和空白组相比,差异有显著性(P<0.05).u0126和SB203580可以抑制淫羊藿甙和雌二醇促进Cbfa1蛋白表达的作用.结论 ①淫羊藿甙和雌二醇均可以激活成骨细胞中ERK/MAPK和P38/MAPK信号通路;②淫羊藿甙和雌二醇均能促进成骨细胞中核转录因子Cbfa1的表达,并且ERK/MAPK和P38/MAPK信号通路参与了此过程.

Role of MAPK signal pathyway in the regulation of Cbfa1 protein expression in osteoblast by Icariin

Objective To investigate the effect of icariin (ICA) on mitogen-activated protein kinase (MAPK) signal pathway in rat osteoblasts cultured in vitro and the role of MAPK signal pathway in the icariin-promoting expression of core binding factor-1 (Cbfa1) in osteoblasts, and to elucidate the signal mechanism of icariin on osteoblasts. Methods Calvarial osteoblasts were obtained from newborn (<24 h) SD rats by trypsin-collagenase digestion method. After 5 min, 10 min, 30 min, 60 min of treatment with icariin (10 ng/mL) or estrodial (E2) (10-8 mol/L), total protein was isolated from osteoblasts and proteins of ERK, p-ERK, P38 and p-P38 were detected by western-blot analysis. Then calvarial osteoblasts were cultured in the medium containing icariin (10 ng/mL), estrodial (10-8 mol/L) with or without u0126, SB203580 for 24 h respectively, nucleus protein was isolated from osteoblasts and protein of Cbfa1 was detected by western-blot analysis. Results 1.The protein of p-ERK in calvarial osteoblasts increased at 30 min and lasted for 60 min (P<0.05, contrast to the blank group) when treating osteoblasts with icariin(10 ng/mL); the protein of p-P38 in calvarial osteoblasts increased at 5 min and was at the peak at 30 min, and lasted for 60 min (P<0.05, contrast to the blank group) when treating osteoblasts with icariin(10 ng/mL). 2.The protein of p-ERK in calvarial osteoblasts increased at 30 min (P<0.05, contrast to the blank group) when treating osteoblasts with estrodial (10-8 mol/L); the protein of p-P38 in calvarial osteoblasts increased at 10 min and continuously increased to 30 min(P<0.05, contrast to the blank group) when treating osteoblasts with estrodial (10-8 mol/L). 3. Both icariin and estrodial could promote the expression of Cbfa1 protein (P<0.05, contrast to the blank group) and this effect could be weakened by SB203580 or u0126. Conclusion Icariin and estrodial can activate ERK/MAPK and P38/MAPK signal pathways and promote the expression of Cbfa1 protein in osteoblasts respectively. ERK/MAPK and P38/MAPK signal pathway are involved in the processes of icariin-promoting and estrodial-promoting Cbfa1 expression.