基于cAMP-PKA信号通路的淫羊藿苷促进骨形成研究

Icariin Promote Bone Formation Dependent on c AMP-PKA Signaling Pathway

目的研究淫羊藿苷是否通过c AMP-PKA信号通路来促进成骨细胞(osteoblast,OB)成熟矿化。方法以1×10?5 mol·L?1淫羊藿苷分别处理体外培养的大鼠颅骨OB细胞和人类乳腺癌细胞(MCF-7)不同时间后,免疫荧光染色法检测2种细胞内雌激素受体(ERα)的核转位情况。待P1代OB细胞铺满80%皿底后,采用1×10?5 mol·L?1的2’,3’-双脱氧腺苷(2’,3’-dideoxyadenosine,DDA)抑制胞内腺苷酸环化酶(adenylate cyclase,AC),同时以1×10?5 mol·L?1淫羊藿苷分别作用于正常组和信号阻断组不同时间后,ELISA检测细胞内c AMP的含量。以终浓度为1×10?6 mol·L?1蛋白激酶A(protein kinase A,PKA)抑制剂KT5720处理细胞,同时以1×10?5 mol·L?1淫羊藿苷分别作用于信号阻断组和正常组,3 d和6 d后检测胞内碱性磷酸酶(ALP)活性;药物处理48 h后,Real-time PCR检测I型胶原(collagen I,COL I)、Runx-2、ALP m RNA的表达量;Western blot检测COL I、Runx-2蛋白表达量。结果淫羊藿苷作用于细胞4 h后,MCF-7胞内ERα发生明显的核转位,而OB细胞在所有时间点都未检测到明显核转位现象,结合本课题组前期的淫羊藿苷雌激素比较试验说明,淫羊藿苷促进成骨细胞成骨性分化并不主要依赖于其雌激素活性。淫羊藿苷促OB细胞后,胞内c AMP迅速升高,处理1 h后与对照组相比具有显著性差异。加入DDA阻断AC后,淫羊藿苷促胞内c AMP升高的作用消失。1×10?5 mol·L?1淫羊藿苷能够显著地促进胞内ALP的活性,成骨性分化相关的因子COL I、Runx-2和ALP的基因表达也相应增高,同时COL I和Runx-2蛋白表达量也显著增加。当采用KT5720抑制PKA的活性之后,ALP活性下降,成骨性分化的指标也随之降低。结论淫羊藿苷促进OB细胞成骨性分化并不主要依赖于其雌激素活性,而是通过迅速提高成骨细胞内c AMP的含量,激活胞内c AMP-PKA信号通路,进而促进成骨性相关因子的表达,来促进OB细胞成熟矿化。

OBJECTIVE To study the role of cAMP-PKA signaling pathway in the osteogenic differentiation process of rat calvarial osteoblasts（OB） induced by icariin. METHODS Osteoblasts were obtained by enzyme digestion from the segregated neonatal SD rat skull. Serial subcultivation was proceeded when cells covered with 80% culture dish. OB and MCF-7 cells were treated by 1×10^-5 mol·L^-1 icariin separately, nuclear translocation of estrogen receptor a （ERa） in cells was detected by immunofluorescence. Icariin and 2＇,3＇-dideoxyadenosine （DDA） were supplemented into the culture separately at 1×10^-5 mol·L^-1 After further incubation for different time, cAMP level in the cultured cells was determined using a cAMP enzyme immune assay system in accordance with the manufacturer＇s instructions. Cells were treated with icariin （1×10^-5 mol·L^-1） and KT5720 （1×10^-5 mol·L^-1） separately. The alkaline phosphatase ALP activity was determined at the 3rd and 6th day. Total RNA was isolated and the gene expression of ALP, collagen I and Runx-2 were investigated by real-time PCR. Finally total protein was also isolated and the secretion of collagen I and Runx-2 was examined by Western blot. RESULTS ERa nuclear translocation were found in MCF-7 treated by icariin after 4 h, but this phenomenon coundn＇t be detected in OB cells. Elevation of intracellular cAMP concentration was consistently noted in all cultured ceils following icariin treatment. The increasing of cAMP was blocked after the treatment of DDA. icariin significantly improved ALP activity, the level of factors related to the osteogenic differentiation included collagen I, Runx-2 and ALP also enhanced. The secretion of collagen and Runx-2 also promotedsignificantly. But all of these effects could be inhibited by KT5720 which could specifically inhibit the activity of protein kinase A（PKA）. CONCLUSION Icariin can elevate intracellular cAMP concentration and promote bone formation dependent on cAMP-PKA signaling pathway.