基于miR-146a-5p/Notch1信号通路探讨补肾壮筋汤对骨质疏松小鼠骨密度及成骨分化的影响  

Exploring effect of Bushen Zhuangjin Decoction on bone density and osteogenic differentiation in osteoporosis mice based on miR-146a-5p/Notch1 signaling pathway

作　　者：杨彬 程韶 杨顺 许俊杰 陶广义 王上增 YANG Bin;CHENG Shao;YANG Shun;XU Junjie;TAO Guangyi;WANG Shangzeng(Department of Pain,Henan Hospital of Traditional Chinese Medicine(the Second Affiliated Hospital of Henan University of Chinese Medicine),Zhengzhou 450053,China;Department of Arthropathy,Henan Hospital of Traditional Chinese Medicine(the Second Affiliated Hospital of Henan University of Chinese Medicine),Zhengzhou 450053,China;College of Bone Injury,Henan University of Chinese Medicine,Zhengzhou 450046,China)

机构地区：[1]河南省中医院(河南中医药大学第二附属医院)疼痛科,河南郑州450053 [2]河南省中医院(河南中医药大学第二附属医院)关节病科,河南郑州450053 [3]河南中医药大学骨伤学院,河南郑州450046

出　　处：《药物评价研究》2025年第1期73-84,共12页Drug Evaluation Research

基　　金：河南省自然科学基金青年项目(232300420269);2023年度河南省中医药科学研究专项课题重大专项(2023ZYZD06);河南省高等学校重点科研项目(24A360002)。

摘　　要：目的基于miR-146a-5p/Notch1信号通路探究补肾壮筋汤(BZD)对骨质疏松小鼠骨密度及成骨分化的影响。方法采用地塞米松诱导的方法构建小鼠骨质疏松症模型,造模成功60只小鼠,将其随机分为模型组,BZD低、高剂量(5.4、10.8 g·kg^(-1))组,BZD+agomir NC(10.8 g·kg^(-1)+8 mg·kg^(-1))组,BZD+miR-146a-5p agomir(10.8 g·kg^(-1)+8 mg·kg^(-1))组,每组12只。另取12只小鼠正常饲养,记为对照组。造模成功后开始ig BZD,每天1次,连续8周;agomir NC、miR-146a-5p agomir采用尾iv给药,隔天给药,共8周。给药结束后测定小鼠骨密度和骨结构参数:骨矿物质含量(BMC)、骨矿物质密度(BMD)、总体积(TV)、骨体积(BV)、骨体积分数(BV/TV)、骨小梁数量(Tb.N)、骨小梁厚度(Tb.Th)和骨小梁分离度(Tb.Sp);酶联免疫吸附试验(ELISA)检查小鼠血清骨代谢指标:骨钙素(OCN)、骨源性碱性磷酸酶(BALP)、Ⅰ型前胶原羧基端前肽(PINP)、抗酒石酸酸性磷酸酶(Trap);HE染色观察小鼠骨组织的结构变化;实时荧光定量PCR(qRTPCR)法测定小鼠股骨组织miR-146a-5p、Notch1 m RNA的表达水平。Western blotting检查小鼠股骨组织Notch1蛋白表达水平。从Balb/c小鼠中分离骨髓间充质干细胞(BMSCs)并诱导成骨分化,分为对照组,模型组(1×10^(-5) mol·L^(−1)地塞米松处理48 h),BZD低、高浓度组(造模后用100、200μg·mL^(−1) BZD处理48 h),BZD+agomir NC组(造模后用200μg·mL^(−1) BZD和转染agomir NC处理48 h),BZD+miR-146a-5p agomir组(造模后用200μg·mL^(−1) BZD和转染miR-146a-5p agomir处理48 h)。CCK-8实验检测细胞增殖,ALP染色和茜素红染色分析细胞成骨分化,qRT-PCR测定细胞miR-146a-5p及Notch1m RNA表达,Western blotting检查细胞成骨标志蛋白及Notch1蛋白表达,双荧光素酶实验检测miR-146a-5p及Notch1的相互作用。结果与对照组比较,模型组小鼠BMC、BMD、BV/TV、Tb.N、Tb.Th水平,血清BALP、PINP水平,股骨组织Notch1 mRNA和蛋白表达水平显著降Objective To investigate the effect of Bushen Zhuangjin decoction(BZD)on bone density and osteogenic differentiation in osteoporosis mice based on the miR-146a-5p/Notch1 signaling pathway.Methods A mouse model of osteoporosis was constructed using dexamethasone induction,and 60 mice were successfully modeled.They were randomly divided into model groups,BZD low and high dose(5.4,10.8 g·kg^(-1))groups,BZD+agomir NC(10.8 g·kg^(-1)+8 mg·kg^(-1))group,and BZD+miR-146a-5p agomir(10.8 g·kg^(-1)+8 mg·kg^(-1))group,with 12 mice in each group.Another 12 mice were taken from normal feeding and designated as the control group.After successful modeling,start ig BZD once a day for 8 consecutive weeks;agomir NC and miR-146a-5p agomir were administered via tail vein injection every other day for a total of eight weeks.After administration,the bone density and structural parameters of the mice were measured,including bone mineral content(BMC),bone mineral density(BMD),total volume(TV),bone volume(BV),bone volume fraction(BV/TV),trabecular number(Tb.N),trabecular thickness(Tb.Th),and trabecular separation/spacing(Tb.SP).Enzyme-linked immunosorbent assay(ELISA)was applied to examine serum bone metabolism indicators in mice,including osteocalcin(OCN),bone alkaline phosphatase(BALP),N-terminal propeptide of type I procollagen(PINP),and tartrate acid-resistant phosphatase(Trap).HE staining was applied to observe structural changes in bone tissue.Real-time fluorescence quantitative PCR(RT-qPCR)method was applied to determine the expression levels of miR-146a-5p and Notch1 mRNA in femoral tissue.Western Blot was applied to examine the expression level of Notch1 protein in mouse femoral tissue.Bone marrow mesenchymal stem cells(BMSCs)were isolated from Balb/c mice,induced osteogenic differentiation,and then divided into control group,model group(treated with 1×10^(-5) mol·L^(−1) dexamethasone for 48 h),BZD low and high concentration groups(treated with 100 and 200μg·mL^(−1) BZD after modeling for 48 h),BZD+agomir NC group(tr

关 键 词：补肾壮筋汤 骨质疏松 miR-146a-5p/Notch1信号通路 骨密度 成骨分化 

分 类 号：R285.5[医药卫生—中药学]