BMP⁃2/Smad信号通路探讨金匮肾气丸对BMSCs成骨分化的影响  

Exploring the BMP⁃2/Smad signaling pathway and the effect of Jin Kui Shen Qi Wan on the osteogenic differentiation of BMSCs

作　　者：孟菲菲 高志礼 李娜[1,2] 王花欣 WANG Jiayun[1,2] MENG Feifei;GAO Zhili;WANG Jiayun;LI Na;WANG Huaxin(Shandong University of Traditional Chinese Medicine,Jinan 250355,China;Shandong Province Collaborative Innovation Center of Traditional Chinese Medicine Classics,Jinan 250355,China)

机构地区：[1]山东中医药大学,山东济南250355 [2]山东省中医经典名方协同创新中心,山东济南250355

出　　处：《中国骨质疏松杂志》2024年第3期379-384,共6页Chinese Journal of Osteoporosis

基　　金：国家自然科学基金委员会青年科学基金项目(82004087);山东中医药大学青年创新团队项目(2020⁃54⁃19)。

摘　　要：目的观察金匮肾气丸含药血清对大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)成骨分化的影响并探讨其发生的作用机制。方法制备金匮肾气丸含药血清,CCK⁃8法筛选出最佳浓度的金匮肾气丸含药血清对BMSCs向成骨分化的影响。将BMSCs分为空白组(control组)、成骨诱导组(OIS)、空白血清组(ROIS组)及含药血清组(JKSQ⁃OIS),分别对各组BMSCs进行成骨诱导。连续干预14 d及21 d后,分别进行碱性磷酸酶(ALP)染色和茜素红染色镜下观察各组碱性磷酸酶活性和成骨矿化水平;连续干预14 d后,实时荧光定量PCR(q⁃PCR)和蛋白免疫印迹法(Western blot)分别检测各组BMSCs中骨形态发生蛋白2(BMP⁃2)、Smad1、ALP、Runt相关转录因子2(runt⁃related transcription factor 2,Runx2)及成骨细胞特异性转录因子(Osterix,OSX)mRNA和蛋白的相对表达量。结果与control组相比,OIS组与ROIS组出现点状矿化结节,ALP染色轻微变深,ALP和Runx2 mRNA及蛋白的表达上升(P<0.05,P<0.01),OSX的表达上升但不明显(P>0.05);与OIS组相比,JKSQ⁃OIS组矿化结节成片分布,ALP染色明显加深,BMP⁃2、Smad1、ALP和Runx2 mRNA及蛋白的表达上升(P<0.05,P<0.01),OSX mRNA及蛋白表达有上升趋势(P>0.05)。结论金匮肾气丸含药血清可能通过调控BMP⁃2/Smad信号通路,上调ALP、Runx2和OSX的表达,促进BMSCs向成骨分化,改善OP。Objective To observe the effect of serum containing Jinguishenqi pill on the osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs)in rats and to explore the mechanism of its occurrence.Methods The serum containing Jinguishenqi pill was prepared.CCK⁃8 method was used to screen out the effect of the optimal concentration of the serum containing Jinguishenqi pill on the osteogenic differentiation of BMSCs.BMSCs were divided into blank group(control group),osteogenic induction group(OIS),blank serum group(ROIS group),and drug⁃containing serum group(JKSQ⁃OIS).The osteogenic induction was carried out for BMSCs in each group respectively.After 14 or 21 consecutive days of intervention,alkaline phosphatase staining and Alizarin red staining were performed to observe the alkaline phosphatase activity and osteogenic mineralization level in each group under microscopic observation,respectively.After 14 consecutive days of intervention,real⁃time fluorescence quantitative PCR(q⁃PCR)and protein immunoblotting(Western blotting)were used to detect the mRNA and protein relative expressions of BMP⁃2,Smad1,ALP,Runx2,and OSX.Results Compared to the control group,the OIS and ROIS groups showed punctuate mineralized nodules,slightly darkened ALP staining,increased expressions of ALP and Runx2 mRNA and protein(P<0.05,P<0.01),and increased but not significant expression of OSX(P>0.05).Compared to that in the OIS group,the mineralized nodules in the JKSQ⁃OIS group were in patches.ALP staining was obviously deepened,the expressions of BMP⁃2,Smad1,ALP,and Runx2 mRNA and protein increased(P<0.05,P<0.01),and the expressions of OSX mRNA and protein had a rising trend(P>0.05).Conclusion The drug⁃containing serum of Jinguishenqi pill may promote the differentiation of BMSCs towards osteogenesis and relieve OP by regulating the BMP⁃2/Smad signaling pathway and up⁃regulating the expressions of ALP,Runx2,and OSX.

关 键 词：金匮肾气丸 骨髓间充质干细胞 成骨 BMP⁃2/Smad信号通路 

分 类 号：R285.5[医药卫生—中药学]