目的 探讨葛根素成骨细胞特异性转录因子（runt-related transcription factor 2,Runx2）对骨髓间充质干细胞（bone marrow mesenchymal stem/stromal cells, BMSCs）成骨分化影响的相关机制。方法 将BMSCs分为对照组和不同浓度葛根素处理组,采用CCK-8法检测细胞增殖,碱性磷酸酶（alkaline phosphatase,ALP）和茜素红染色测定评估成骨分化,Seahorse技术分析细胞糖酵解功能,q-PCR检测miR-141-3p、Runx2、神经生长因子（nerve growth factor,NGF）、脑源性神经营养因子（brain-derived neurotrophic factor,BDNF）、白细胞介素-10（interleukin-10,IL-10）、转化生长因子-β（transforming growth factor-β,TGF-β）、己糖激酶2（hexokinase 2,HK2）及乳酸脱氢酶A（lactate dehydrogenase A,LDHA）的mRNA表达,Western blot分析Runx2蛋白的表达。双荧光素酶报告基因实验测定微小RNA-141-3p（microRNA-141-3p,miR-141-3p）与Runx2靶向关系。结果 与对照组相比,葛根素浓度依赖性地促进BMSCs增殖（P<0.01）,增强细胞糖酵解功能（P<0.01）、提升ALP活性（P<0.01）和钙结节沉积（P<0.01）,显著下调miR-141-3p表达（P<0.01）,上调Runx2 mRNA和蛋白水平（P<0.01）和促进NGF、BDNF、IL-10和TGF-β的表达（P<0.01）,双荧光素酶报告基因实验证明miR-141-3p可直接靶向Runx2 3'UTR。结论 葛根素通过下调miR-141-3p解除对Runx2的抑制,促进成骨分化,同时通过激活神经营养因子表达和改善骨免疫微环境等多维度发挥促成骨作用。
Abstract
Objective To investigate the effects of puerarin on the osteogenic differentiation of bone marrow mesenchymal stem/stromal cells (BMSCs) and the mechanisms involved. Methods BMSCs were divided into a control group and treatment groups with different concentrations of puerarin. Cell proliferation was assessed using the CCK-8 assay. Alkaline phosphatase (ALP) and Alizarin Red staining were used to evaluate osteogenic differentiation. Seahorse technology was used to analyze cellular glycolytic function, while q-PCR detected the mRNA expression of miR-141-3p, Runx2, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), interleukin-10 (IL-10), transforming growth factor-β (TGF-β), hexokinase 2 (HK2), and lactate dehydrogenase A (LDHA). Western blot was employed to analyze the expression of Runx2 protein. The targeting relationship between miR-141-3p and Runx2 was determined through a dual-luciferase reporter gene experiment. Results Compared with the control group, puerarin concentration dependently promoted BMSCs proliferation (P<0.01), ALP activity (P<0.01) and calcium nodule deposition (P<0.01). Seahorse analysis showed that puerarin significantly enhanced the glycolytic function of BMSCs (P<0.01). Puerarin could also significantly down-regulated the expression of miR-141-3p (P<0.01), up-regulated the mRNA miR-141-3p (P<0.01) and the mRNA and protein levels of Runx2 (P<0.01). Meanwhile, puerarin promoted the expression of NGF, BDNF, IL-10 and TGF-β (P<0.01). Double luciferase reporter gene assay showed that miR-141-3p could directly target Runx2 3'UTR. Conclusion Puerarin alleviates the inhibition of Runx2 by downregulating miR-141-3p, promotes osteogenic differentiation, and exerts multidimensional bone promoting effects by activating neurotrophic factor signaling and improving the bone immune microenvironment.
关键词
葛根素 /
成骨细胞 /
微小RNA-141-3p /
神经营养因子
Key words
puerarin /
osteoblasts /
microRNA-141-3p /
neurotrophic factor
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