淫羊藿苷介导MAPK信号通路促进间充质干细胞株C3H10T1/2成骨分化的体外研究

Analysis of the osteogenetic effects exerted on mesenchymal stem cell strain C3H10T1/2 by icariin via MAPK signaling pathway in vitro

目的:探索补肾中药淫羊藿有效成分淫羊藿苷对间充质干细胞株C3H10T1/2的促成骨分化作用,及该作用与丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)信号通路的相关性。方法:体外培养间充质干细胞株C3H10T1/2,给予淫羊藿苷(0、10-7、10-6、10-5和10-4mol/L)联合成骨诱导液干预26d后,碱性磷酸酶(alkaline phosphatase,ALP)染色观察成骨分化情况。10-5mol/L淫羊藿苷干预C3H10T1/2细胞2、8、24与48h后,实时荧光定量聚合酶链式反应法(polymerase chain reaction,PCR)检测p38、细胞外调节蛋白激酶(extracellular signal-regulated protein kinase,p42/44)mRNA的表达。10-5mol/L淫羊藿苷干预C3H10T1/2细胞10、30、60和120min后,蛋白质印迹法检测p38丝裂原活化蛋白激酶(p38 mitogen-activated protein kinase,p38)、p42和p44,以及翼式转录因子氨基末端激酶(c-Jun N-terminal kinase,JNK)及其磷酸化产物的蛋白表达情况。结果:10-5mol/L淫羊藿苷联合成骨诱导液的促进C3H10T1/2细胞成骨分化能力最强。PCR结果显示,淫羊藿苷作用2h后p38基因表达显著下调(P<0.01);p42和p44mRNA水平在淫羊藿苷干预2h后均显著下调(P<0.01),而在淫羊藿苷干预48h后表达均上调(P<0.05,P<0.01);其他时间点各通路基因表达情况均无显著变化(P>0.05)。10-5mol/L淫羊藿苷作用10min后,磷酸化p42/44蛋白表达减弱,磷酸化p38蛋白表达增强,并持续到30min,但对JNK蛋白的表达无明显影响。结论:淫羊藿苷促进间充质干细胞株C3H10T1/2向成骨细胞分化,其作用可能与激活p38和抑制ERK蛋白的表达有关。

OBJECTIVE. To investigate the effects of icariin, an effective extract from traditional Chinese medicine Epimedium pubescens with the function of tonifying kidney, in promoting osteogenesis of mesenchymal stem cell line C3H10T1/2, and to explore the underlying mechanism. METHODS： After culture with icariin （0, 10-7 , 10-6 , 10-5 , and 10-4 mol/L） and osteogenic supplement for 26 d in vitro, osteogenic differentiation of C3H10T1/2 cells was detected by alkaline phosphatase （ALP） assay. TheRNA was extracted from cells cultured with 10 5 mol/L icariin for 2, 8, 24 and 48 hours, and mRNA expressions of p38, p42 and p44 were measured using real-time reverse transcription-polymerase chain reaction （PCR） method. Three main proteins of MAPK signaling pathway （p38, and extracellular signal-regulated protein kinase （ERK）, also named p42/44） and c-Jun N-terminal kinase （JNK） and their phospho-products were examined using Western blotting after icariin treatments of 10, 30, 60 and 120 min. RESULTS.. Icariin at a dose of 10-5 mol/L, when combined with the osteogenic supplement, had the best ability to promote osteogenic differentiation on C3H10T1/2 cells. Based on real-time PCR, the authors found that after two-hour ICA treatment, the gene expression of p38 revealed a significant decline compared with the control group （P〈0.01）. The levels of p42 and p44 mRNAs were decreased greatly after two-hour ICA treatment, while increased after 48-hour ICA treatment （P〈0.05, P〈0.01）. There was no significant difference at other time points （P〈0.05）. Phospho-p42 was decreased after 10-minute icariin treatment, while phospho-p38 expression displayed an increase after 10- and 30-minute of treatment with icariin. There was no notable difference in phospho-JNK expression at these four time points. CONCLUSION. Icariin promotes differentiation of the mesenchymal stem cells C3H10T1/2 into osteoblasts, and its effect is related to the restraining of ERK expression and activation of p38 expression in the MAPK signaling pathway.