柚皮苷调控成骨细胞成骨分化的机制  

Mechanism by which naringin regulates osteogenic differentiation in osteoblasts

作　　者：吴晶晶 林海雄 孙伟鹏 李紫阁 姜自伟[4] Wu Jingjing;Lin Haixiong;Sun Weipeng;Li Zige;Jiang Ziwei(The First Clinical Medical College of Guangzhou University of Chinese Medicine,Guangzhou 510405,Guangdong Province,China;Ningxia Chinese Medicine Research Center,Yinchuan 750000,Ningxia Hui Autonomous Region,China;Institute of Tissue Engineering and Regenerative Medicine,The Chinese University of Hong Kong,Sha Tin 999077,Hong Kong Special Administrative Region,China;The First Affiliated Hospital of Guangzhou University of Chinese Medicine,Guangzhou 510405,Guangdong Province,China)

机构地区：[1]广州中医药大学第一临床医学院,广东省广州市510405 [2]宁夏回族自治区中医医院暨中医研究所,宁夏回族自治区银川市750000 [3]香港中文大学组织工程与再生医学研究所,中国香港特别行政区999077 [4]广州中医药大学第一附属医院,广东省广州市510405

出　　处：《中国组织工程研究》2023年第11期1722-1727,共6页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金(81774337),项目负责人:姜自伟。

摘　　要：背景:研究发现,柚皮苷主要通过Wnt、转化生长因子β、MAPK信号通路、破骨细胞信号通路等直接影响骨代谢,通过P13K-Akt、血管内皮生长因子信号通路等间接调节骨代谢,具有促进骨损伤修复、减缓骨质疏松的作用。目的:探讨柚皮苷通过下调miR-206表达靶向激活缝隙连接蛋白43/ERK1信号通路对成骨细胞增殖和成骨分化的影响。方法:将大鼠成骨细胞(ROB细胞)经不同质量浓度柚皮苷(1,10,100 mg/L及1,10 g/L)处理培养,并进行CCK-8实验检测细胞增殖情况,筛选出柚皮苷质量浓度100 mg/L及1,10 g/L用于后续实验,并以未给药培养做对照组。RT-qPCR检测ROB细胞中miR-206、缝隙连接蛋白43及ERK1 mRNA相对表达水平,验证其靶向关系;Western blot检测ROB细胞中ERK1/2、P-ERK蛋白的表达水平;碱性磷酸酶染色及茜素红染色观察ROB细胞内碱性磷酸酶活性及细胞钙化能力。结果与结论:①柚皮苷能显著促进ROB细胞的增殖活性(P<0.05),柚皮苷在一定质量浓度范围内,随着给药浓度升高,ROB细胞吸光度值和相对增殖率均相应升高,在质量浓度为1 g/L时细胞增殖活性最强;②柚皮苷能显著抑制ROB细胞内miR-206 mRNA的表达并靶向促进成骨分化指标缝隙连接蛋白43 mRNA及ERK1/2、P-ERK蛋白的表达(P<0.05);③碱性磷酸酶染色及茜素红染色结果提示柚皮苷能显著促进ROB细胞内碱性磷酸酶活性和表达以及细胞钙化能力水平(P<0.05),其中1 g/L质量浓度处理的效果最佳;④柚皮苷通过下调miR-206表达,靶向激活缝隙连接蛋白43/ERK1信号通路,进而促进ROB细胞增殖活性及成骨分化能力。BACKGROUND:Studies have found that naringin affects bone metabolism directly through Wnt,transforming growth factor β,MAPK signaling pathway,and osteoclast signaling pathway,as well as indirectly through P13K-Akt and vascular endothelial growth factor signaling pathway.Naringin can promote bone repair and retard osteoporosis.OBJECTIVE:To investigate the effect of naringin on the prolife ration and osteogenic differentiation of osteoblasts by to rgeting the activation of connexin 43/ERK1 signaling pathway through down-regulation of miR-206.METHODS:Rat osteoblasts(ROB cells) were treated and cultured with different concentrations of naringin(1,10,100 mg/L and 1,10 g/L),and the cell proliferation was detected by cell counting kit-8 assay.Naringin at 100 mg/L and 1,10 g/L were used for subsequent expe riments.Osteoblasts with nondrug culture were used as controls.RT-qPCR was used to detect the relative expression levels of miR-206,connexin 43 and ERK1 mRNAs in ROB cells to verify their to rgeting relationship.Western blot was used to detect the protein expression levels of ERK1/2 and P-ERK in ROB cells.Alkaline phosphatase activity and calcification ability in ROB cells were observed by alkaline phosphatase staining and alizarin red staining,respectively.RESULTS AND CONCLUSION:Naringin significantly promoted the proliferation activity of ROB cells(P <0.05).The absorbance value and relative proliferation rate of ROB cells increased as the administration concentration of naringin increased in a certain range.When the mass concentration was 1 g/L,the cell proliferation activity was the strongest.Naringin significantly inhibited the expression of miR-206 mRNA in ROB cells and promoted the expression of connexin43 mRNA and ERK1/2 and P-ERK protein(P <0.05).Naringin could significantly promote the activity and expression of alkaline phosphatase and the level of cellular calcification in ROB cells(P <0.05).Moreover,naringin at 1 g/L showed the best effect.To conclude,naringin can to rget and activate the connexin 43/ERK1 s

关 键 词：柚皮苷 miR-206 CX43 ERK1 成骨细胞 骨损伤 骨碎补 MIRNAS 

分 类 号：R446[医药卫生—诊断学] R496[医药卫生—临床医学] R318