淫羊藿次苷Ⅰ及淫羊藿次苷Ⅱ通过BMP/Runx2/Osx信号通路促进大鼠骨髓间充质干细胞成骨分化的实验研究  

The study of Icariside Ⅰ and Icariside Ⅱ on promoting osteogenic differentiation by BMSCs through BMP/Runx2/Osx signaling pathway

作　　者：訾慧[1] 范颖[1] 蒋宁[1] 谷丽艳[1] 董秀[1] ZI Hui;FAN Ying;JIANG Ning;GU Liyan;DONG Xiu(The Key Laboratory of Ministry of Education for TCM Viscera-State Theory and Applications,Liaoning University of Traditional Chinese Medicine,Shenyang 110847,China)

机构地区：[1]辽宁中医药大学中医脏象理论及应用教育部重点实验室,辽宁沈阳110847

出　　处：《中国骨质疏松杂志》2019年第5期690-695,共6页Chinese Journal of Osteoporosis

基　　金：辽宁省自然科学基金指导计划项目(201602510);辽宁中医药大学中医脏象理论及应用国家教育部重点实验室开放基金项目(zyzx1507)

摘　　要：目的观察淫羊藿次苷Ⅰ及淫羊藿次苷Ⅱ对大鼠骨髓间充质干细胞(bone marrow stromal cells,BMSCs)成骨分化过程中BMP-2/Runx2/Osx信号通路的影响,探讨其可能的作用机制。方法贴壁筛选法体外培养BMSCs,随机分为空白对照组、阳性转化液组、抑制剂组、淫羊藿次苷Ⅰ组及淫羊藿次苷Ⅰ+抑制剂组、淫羊藿次苷Ⅱ组及淫羊藿次苷Ⅱ+抑制剂组。通过茜素红染色,对各组促进骨髓间充质干细胞向成骨细胞分化作用进行药效学评价; ELISA法检测各组ALP活性、BGP、COL-Ⅰ、BMP2蛋白分泌量;RTReal-TimePCR法检测各组ALP、BGP、Osterix和Runx-2 mRNA基因表达。结果茜素红染色结果表明,淫羊藿次苷Ⅰ组及淫羊藿次苷Ⅱ组均可明显观察到大量的矿化结节的形成;ELISA实验结果表明,与空白对照组比较,淫羊藿次苷Ⅰ及淫羊藿次苷Ⅱ均能明显促进ALP细胞活性及BGP、COL-Ⅰ、BMP-2蛋白分泌量,并且差异具有统计学意义(P<0.05);两组药物作用强度均已达到阳性转化液组的80%以上。同时,淫羊藿次苷Ⅰ对ALP活性、 BGP蛋白表达均明显强于淫羊藿次苷Ⅱ,并且差异具有统计学意义(P<0.05);对COL-Ⅰ、BMP-2的蛋白表达量,两者差异无统计学意义(P>0.05)。RT-PCR实验结果表明,与空白对照组相比,淫羊藿次苷Ⅰ及淫羊藿次苷Ⅱ均可显著上调成骨相关转录因子ALP、BGP、RunX2,并进一步调节其下游基因Osterix的转录表达,差异具有统计学意义(P<0.05);与阳性对照液组比较,淫羊藿次苷Ⅱ对ALP、Osterix的mRNA表达无明显差异(P>0.05);淫羊藿次苷Ⅰ与淫羊藿次苷Ⅱ组比较,淫羊藿次苷Ⅱ对Osterix mRNA的表达明显强于淫羊藿次苷Ⅰ,并且差异具有统计学意义(P<0.05)。对ALP、BGP、RunX2 mRNA的表达二者无显著性差异(P>0.05)。结论淫羊藿次苷Ⅰ和淫羊藿次苷Ⅱ均能促进BMSCs定向成骨转化,其作用机制可能与激活BMP/Runx2/Osx信号通路有关。淫羊藿次苷Ⅰ和淫羊藿Objective To study the effect of Icariside Ⅰ and Icariside Ⅱ on promoting osteogenic differentiation of bone marrow stromal cells(BMSCs) by activating the BMP-2/Runx2/OSX signaling pathway, and to explore its possible mechanism. MethodsBy using the adherence method, BMSCs were cultured and divided into blank control group, positive control group, inhibitor group, Icariside Ⅰ control and Icariside Ⅰ+ inhibitor group, Icariside Ⅱ and Icariside Ⅱ+ inhibitor group. Osteoblasts differentiation was evaluated using Alizarin red staining. The protein expression of ALP, BGP, COL-Ⅰ, and BMP-2 was detected with ELISA method. The expression of ALP, BGP, Osterix, and Runx-2 genes was detected with real-Time RT-PCR. Results The Result of Alizarin red staining showed that a large number of mineralized nodules were formed in Icariside I and Icariside II groups. The ELISA result showed that both Icariside I and Icariside II significantly promoted the activity of ALP and the secretion of BGP, COL-I, and BMP-2 proteins(P<0.05). The strength of action in both groups reached more than 80 % of that in the positive control group. Meanwhile, the effect of Icariside I on ALP activity and BGP protein was significantly stronger than that of Icariside II(P<0.05). The effect was not significantly different in the protein expression of COL-I and BMP-2(P>0.05). RT-PCR result showed that Icariside I and Icariside II significantly increased ALP, BGP, RunX2, and further regulated the transcription expression of the downstream gene Osterix, with significant differences compared with the blank control group. The mRNA expression of ALP and Osterix was not significantly different between Icariside II and positive contrast group(P>0.05). The expression of Osterix mRNA in Icariside II group was higher than that in Icariside I group, and there was significant difference(P<0.05). The mRNA expression of ALP, BGP, and RunX2 was not significantly different(P>0.05). Conclusion Both Icariside I and Icariside II promote osteogenesis by BMSCs. The

关 键 词：淫羊藿次苷Ⅰ 淫羊藿次苷Ⅱ 成骨分化 BMP-2/Runx2/OSX信号通路 骨髓间充质干细胞 大鼠 动物实验 

分 类 号：R332[医药卫生—人体生理学]