白花蛇舌草-半枝莲药对乙酸乙酯组分对破骨细胞分化的抑制作用  

Inhibitory effects of ethyl acetate component of Hedyotis diffusa-Scutellaria barbata drug pair on osteoclast differentiation

作　　者：徐元 陈龙 章丹丹 XU Yuan;CHEN Long;ZHANG Dan-dan(Institute of Interdisciplinary Integrative Medicine Research,Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China;Analysis and Test Laboratory,Experiment Center for Science and Technology,Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China)

机构地区：[1]上海中医药大学交叉科学研究院,上海201203 [2]上海中医药大学科技实验中心分析测试室,上海201203

出　　处：《中成药》2019年第1期44-50,共7页Chinese Traditional Patent Medicine

基　　金：国家自然科学基金项目(81001666;81573673;81773946);上海教委创新项目(13YZ048);上海教委优青项目(SZY07029);上海卫计委青年项目(20144Y0143)

摘　　要：目的研究白花蛇舌草-半枝莲药对乙酸乙酯组分(YDW11)对破骨细胞分化的抑制作用。方法 UPLC-MS法鉴定YDW11中化学成分后,MTT法分析其对RAW264. 7细胞活力的影响。100 ng/mL核因子-κB受体活化因子配体(RANKL)诱导RAW264. 7细胞7 d以建立破骨细胞模型后,抗酒石酸酸性磷酸酶(TRAP)染色考察TRAP阳性多核细胞数,相应试剂盒测定TRAP酶活性。QRT-PCR检测TRAP、树突状细胞-特异性跨膜蛋白(DC-STAMP)、活化T细胞核因子cl (NFATc1)基因表达,Western blotting检测核因子κB受体活化因子(RANK)、肿瘤坏死因子受体相关因子6 (TRAF6)、NFATc1、组织蛋白酶K蛋白表达,Taqman MicroRNA RT-PCR检测miR-155表达。结果 YDW11中有16种成分,鉴定出对羟基苯乙酮、野黄芩苷、木犀草素、芹菜素。与模型组比较,YDW11组(25、50μg/mL)阳性多核细胞数显著减少(P<0. 01),并对细胞活力无明显影响(P> 0. 05);呈剂量依赖性地显著抑制酶活性,TRAP、DC-STAMP、NFATc1基因表达,RANK、TRAF6、NFATc1、组织蛋白酶K蛋白表达(P <0. 05),并显著提高miR-155表达(P<0. 05)。结论 YDW11可通过上调miR-155表达、下调相关基因和蛋白表达抑制RANKL诱导的破骨细胞分化。AIM To study the inhibitory effects of ethyl acetate component of Hedyotis diffusa Willd.-Scutellaria barbata D. Don drug pair ( YDW11) on osteoclast differentiation. METHODS After the identification of chemical constituents in YDW11 by UPLC-MS,their effect on RAW264.7 cell viability was analyzed by MTT. Osteoclast models were induced via RAW264. 7 cells by 7 d exposure to 100 ng /mL receptor activator for nuclear factor-κB ligand ( RANKL). And TRAP postive polykaryocyte count was investigated by tartrate-resistant acid phosphatase ( TRAP) staining,and the TRAP enzyme activity was determined by corresponding kit. Subsequently,the detection of TRAP gene expression,dendritic cells-specific transmembrane protein ( DC-STAMP ) and nuclear factor of activated T cells cl ( NFATc1) by QRT-PCR,the protein expression detection of receptor activator for nuclear factor-κB ( RANK),tumor necrosis factor receptor-associated factor 6 ( TRAF6),NFATc1 and cathepsin K by Western blotting,and the expression detection of miR-155 by Taqman MicroRNA RT-PCR were conducted. RESULTS Among the sixteen constituents in YDW11,p-hydroxyacetophenone,scutellarein,luteolin and apigenin were identified. Compared with the model group,YDW11 groups ( 25,50 μg /mL),leaving no obvious effect on cell viability ( P>0. 05),significantly reduced postive polykaryocyte count ( P<0. 01),enzyme activity, TRAP,DC-STAMP,NFATc1 gene expressions,RANK,TRAF6,NFATc1,cathepsin K protein expressions ( P< 0. 05),and increased dose-dependently miR-155 expression ( P<0. 05). CONCLUSION YDW11 can inhibit RANKL-induced osteoclast differentiation by up-regulating miR-155 expression and down-regulating related gene and protein expressions.

关 键 词：白花蛇舌草 半枝莲 药对 乙酸乙酯组分 破骨细胞 分化 

分 类 号：R285.5[医药卫生—中药学]