银杏叶中异鼠李素对RAW264.7细胞向破骨细胞分化的影响及其分子机制  

Effect and molecular mechanism of isorhamnetin extracted from Ginkgo biloba on the differentiation of RAW264.7 cells into osteoclasts

作　　者：李婧[1] 程梁[1] 郭吕华[2] 李彤[1] 杨沫扬 王俊妹[1] 吴哲[1,2] LI Jing;XHENG Liang;GUO Lühua;LI Tong;YANG Moyang;WANG Junmei;WU Zhe(Department of Prothodontics, School and Hospital of Stomatology, Jilin University, ChangchunDepartment of Prothodontics, School and Hospital of Stomatology, Jilin University, Changchun;Department of Prothodontics, Stomatology School of Guangzhou Medical University, Guangzhou510500, China)

机构地区：[1]吉林大学口腔医学院修复科,吉林长春130021 [2]广州医科大学口腔医学院修复科,广东广州510500

出　　处：《口腔疾病防治》2018年第3期158-165,共8页Journal of Prevention and Treatment for Stomatological Diseases

基　　金：吉林省教育厅“十二五”科学技术研究项目(吉教科合字[2015]第530号);广东省科技计划国际合作项目(2017A050501041)

摘　　要：目的探讨银杏叶中异鼠李素(isorhamnetin,ISO)对RAW264.7细胞向破骨细胞分化的影响及其可能的相关分子机制。方法在核因子κB受体活化因子配基(receptor activator of NF-κB ligand,RANKL)诱导RAW264.7细胞分化为破骨细胞的同时,加入不同浓度的ISO(1~10μM),研究其对RAW264.7细胞向破骨细胞分化的影响,通过CCK8法检测及评估各作用浓度ISO的细胞毒性,通过抗酒石酸酸性磷酸酶(tartrate-resis-tant acid phosphatase staining,TRAP)染色及骨片吸收效果判定其对RAW264.7细胞向破骨细胞分化的影响。实时定量PCR检测破骨细胞分化过程的标志性基因:TRAP、组织蛋白酶K(cathepsin K,Ctsk)、金属基质蛋白酶9(matrix metallo protein,MMP-9);分化相关转录因子:原癌基因c-Fos(proto-oncogene protein,c-Fos)、核转录因子激活的T细胞1(nuclear factor of activated T-cells cytoplasmic 1,NFATc1)的表达及破骨细胞分化下游因子NF-κB p65信号通路的磷酸化水平。结果不同浓度的ISO(1~10μM)对RAW264.7细胞无细胞毒性,并且能抑制TRAP活性,减少骨吸收陷窝数量,在浓度为10μM时最为显著;ISO能显著降低TRAP、Ctsk、MMP-9、c-Fos、NFATc1及NF-κB p65的m RNA表达。结论 ISO对RAW264.7细胞向破骨细胞的分化具有抑制作用,其机制可能与经典NF-κB通路的抑制有关。Objective To investigate the effect and potential molecular mechanisms of isorhamnetin(ISO)extracted from Ginkgo biloba on the differentiation of osteoclasts.Methods Osteoclast precursor RAW264.7cells were induced with RANKL to differentiate into mature osteoclasts.Different concentrations of ISO were added to RAW264.7cells to determine its effect on osteoclast differentiation.CCK8was used to evaluate the effect of ISO on cytotoxicity.The impact of ISO on the osteoclast differentiation process was investigated by analyzing tartrate resistance and bone resorption lacuna.Real-time PCR was performed to analyze the levels of differentiation marker genes,including tartrate resistant acid phosphatase(Trap),cathepsin K(Ctsk),and matrix metalloproteinase9(MMP-9);differentiation-related transcription factors,including the proto-oncogene protein c-Fos,nuclear factor of activated T-cells cytoplasmic1(NFATc1);and the levels of downstream NF-κB p65signaling pathway phosphorylation.Using the above-described method,we verified that ISO exerted an inhibitory effect on osteoclast differentiation and explored related molecular mechanisms.Results Different concentrations of ISO(1-10μM)had no cytotoxic effects on RAW264.7cells,inhibited TRAP activity and decreased the number of bone resorption lacuna during osteoclast differentiation.When applied at a concentration of10μM,its inhibitory effect was significant.In addition,ISO significantly reduced the expression levels of Trap,Ctsk,MMP-9,c-Fos,NFATc1and NF-κB p65mRNA.Conclusion ISO extracted from Ginkgo biloba extract exerted aninhibitory effect on osteoclast differentiation,and the mechanism underlying its activity may involve the inhibition of the classical NF-κB pathway.

关 键 词：银杏叶提取物 黄酮类化合物 异鼠李素 破骨细胞 破骨分化 核因子κB受体活化因 

分 类 号：R783.4[医药卫生—口腔医学]