补肾活血方促进骨髓间充质干细胞的成骨及其防治大鼠骨质疏松症机制研究  

Study on Mechanism of Bushen Huoxue Recipe(补肾活血方)Promoting Osteogenesis of BMSCs and Preventing and Treating Osteoporosis in Rats

作　　者：许旻鸣 楼红侃 高麟超 金甬 方剑利 XU Minming;LOU Hongkan;GAo Linchao;JIn Yong;FANG Jiani(Ningbo Hospital of Traditional Chinese Medicine,Ningbo 315000,Zhejiang,China)

机构地区：[1]宁波市中医院,浙江宁波315000

出　　处：《中华中医药学刊》2024年第9期144-150,I0007,共8页Chinese Archives of Traditional Chinese Medicine

基　　金：国家中医药管理局全国名老中医药专家传承工作室项目(国中医药人教发[2016]42号);浙江省中医药科学研究基金项目(2022ZB312)。

摘　　要：目的 研究表明微核糖核酸(microRNA,MiRNA)和Kruppel样因子10(Kruppel-like factor10, KLF10)调控骨质疏松症的进程。研究旨在探讨miR-26b/KLF10信号调控骨髓间充质干细胞(bone marrow-derived mesenchymal stem cells, BMSCs)分化的机制。方法 流失鉴定分离的大鼠BMSCs,不同剂量的补肾活血方处理BMSCs,检测其^([1-2])增殖能力,碱性磷酸酶活性和茜素红染色检测BMSCs的成骨分化潜能。qPCR检测不同剂量的补肾活血方处理BMSCs后,miR-26b和KLF10的表达水平。mirtarBase数据库检测KLF10 mRNA的3’非翻译区(untranslated regions, 3'UTR)上是否有miR-26b的结合位点,双荧光素酶报告实验检测miR-26b对KLF10启动子活性的影响。qPCR检测miR-26b/KLF10信号对BMSCs增殖和成骨分化标志物的表达的影响。Western blot检查Wnt/β-catenin信号是否被激活。结果 流式分析显示分离的原代细胞大部分是BMSCs。补肾活血方显著促进骨髓间充质干细胞的增殖,补肾活血方显著提高了BMSCs中碱性磷酸酶的水平和成骨分化的潜能。补肾活血方促进BMSCs中miR-26b和KLF10的表达。在KLF10 mRNA的3’UTR上有miR-26b的结合位点。在BMSCs中,miR-26b抑制KLF10的3’UTR荧光素酶活性。此外,miR-26b也抑制了KLF10的表达水平。CCK8(Cell Counting Kit-8)显示KLF10促进BMSCs增殖,qPCR显示miR-26b显著抑制成骨分化标志物的表达。而KLF10能够逆转miR-26b的抑制效应,提示miR-26b/KLF10信号介导了补肾活血方在BMSCs增殖和成骨分化中的效应,且补肾活血方通过激活Wnt/β-Catenin信号通路促进成骨。结论 补肾活血方促进BMSCs的成骨由miR-26b/KLF10/Wnt/β-Catenin信号轴调控。Objective Studies have shown that microRNA(miRNA)and Kruppel-like factor 10(KLF10)regulate the pro-gression of osteoporosis.The purpose of this study was to explore the mechanism of Bushen Huoxue Recipe(补肾活血方)/miR-26b/KLF10 signal regulating bone marrow mesenchymal stem cells(BMSCs)differentiation.Methods The BMSCs of rats were identified and separated.Different doses of Bushen Huoxue Recipe were used to treat BMSCs,and the proliferation effect was detected.Alkaline phosphatase activity and alizarin red staining were used to detect the osteogenic differentiation potential of BMSCs.The qPCR was used to detect the expressions of miR-26b and KLF10 in BMSCs treated with different doses of Bushen Huoxue Recipe.MirtarBase database was used to detect the binding site of miR-26b on the 3'UTR(untranslated region)of KLF10mRNA,and double luciferase report test was used to detect the effect of miR-26b on the activity of KLF10 promoter.The qPCR was used to detect the effect of miR-26b/KLF10 signal on the expressions of BMSCs proliferation and osteogenic differen-tiation markers.Western blot was used to examine whether the Wnt/β-catenin signal was activated.Results Fluorescence-ac-tivated cell sorting analysis showed that most of the isolated primary cells were BMSCs.Bushen Huoxue Recipe significantly pro-moted the proliferation of BMSCs,significantly increased the level of alkaline phosphatase in BMSCs and the potential of osteogen-ic differentiation.Bushen Huoxue Recipe promoted the expressions of miR-26b and KLF10 in BMSCs.There was an miR-26b binding site on the 3'UTR(untranslated region)of KLF10 mRNA.In BMSCs,miR-26b inhibited the activity of KLF103'UTR luciferase.In addition,miR-26b also inhibited the expression of KLF10.CCK8 showed that KLF10 promoted the proliferation of BMSCs,while qPCR showed that miR-26b significantly inhibited the expressions of osteogenic differentiation markers.KLF10 can reverse the inhibitory effect of miR-26b,suggesting that miR-26b/KLF10 signal mediates the effect of Bushen Huoxue R

关 键 词：补肾活血方 骨质疏松症 骨髓间充质干细胞 增殖 成骨 

分 类 号：R289.5[医药卫生—方剂学]