基于网络药理学与实验验证探讨鸡血藤总黄酮抑制破骨细胞分化的作用机制  

Mechanism of Total Flavonoids from SPATHOLOBI CAULIS in Inhibiting Osteoclast Differentiation Based on Network Pharmacology and Experimental Validation

作　　者：赖克道 胡筱希 陆国寿 黄建猷 黄周锋 袁健童 LAI Kedao;HU Xiaoxi;LU Guoshou;HUANG Jianyou;HUANG Zhoufeng;YUAN Jiantong(Guangxi Institute of Traditional Medical and Pharmaceutical Science,Nanning 530022;Guangxi Key Laboratory of Traditional Chinese Medicine Quality Standards,Nanning 530022)

机构地区：[1]广西壮族自治区中医药研究院,南宁530022 [2]广西中药质量标准研究重点实验室,南宁530022

出　　处：《中药药理与临床》2023年第5期62-69,共8页Pharmacology and Clinics of Chinese Materia Medica

基　　金：广西中医药适宜技术开发与推广项目(编号:GZSY22-07);中央补助广西中药资源质量评价与标准化公共服务检测平台建设项目(桂中医药发(2021)8号);中央补助广西道地药材生态种植及质量保障项目(桂中医药发(2021)15号)。

摘　　要：目的:对鸡血藤总黄酮提取物中的黄酮类成分进行辨识和鉴定,并对其治疗骨质疏松的靶点进行预测,观察其对核因子κB受体活化因子配体(sRANKL)诱导RAW264.7向破骨细胞分化的影响,并对破骨细胞分化产生影响的作用成分及作用机制进行探讨。方法:采用液相色谱-离子阱-静电轨道阱杂交质谱(HPLC-LTQ/Orbitrap MS)法采集数据后,对各色谱峰的质谱图高分辨一级和二级质谱数据进行解析,与文献数据库进行对比,对各色谱峰进行结构推测和确认。运用Swiss Target Prediction、GeneCards、String平台预测鸡血藤总黄酮18个成分与骨质疏松相关的潜在靶点,在DAVID 6.8平台进行基因功能富集和KEGG通路富集分析,Cytoscape软件构建“成分-靶点”网络。体外培养RAW264.7细胞,sRANKL诱导破骨细胞分化,鸡血藤总黄酮干预后,通过抗酒石酸酸性磷酸酶(TRAP)活性检测盒TRAP染色法评价破骨细胞形成和分化能力,Western blot法检测雌激素受体(Estrogen receptor, ESR)相关蛋白的表达。结果:从鸡血藤总黄酮提取物中共鉴定了18个黄酮类化合物(芒柄花素、染料木素及美迪紫檀素等),网络药理学预测共得到鸡血藤黄酮化合物治疗骨质疏松的潜在共同作用靶点33个,筛选出16条与骨质疏松相关的信号通路。GO分析和KEGG分析显示关键靶点涉及催乳素(Prolactin)、内分泌(Endocrine)、雌激素(Estrogen)、缝隙连接(Gap junction)、酪氨酸激酶受体(ErbB)、等信号通路,生物过程主要涉及以DNA为模板的转录调控、蛋白质自磷酸化、血管收缩的正向调节、等;分子功能涉及蛋白酪氨酸激酶活性、跨膜受体蛋白酪氨酸激酶活性及类固醇结合等;细胞组分涉及细胞膜上的膜筏、原生质膜、神经元末端、等。实验验证结果显示:与正常对照组相比,sRANKL 50 ng/mL组耐酒石酸盐酸性磷酸酶(TRAP)活性明显升高,ESR1、ESR2蛋白表达明显下调(P<0.05或P<0.01),与sRAObjective:To identify the total flavonoids from SPATHOLOBI CAULIS,predict the potential target for the treatment of osteoporosis,ob-serve the effect of the total flavonoids from SPATHOLOBI CAULIS on the differentiation of RAW264.7 cells into osteoclasts induced by receptor activator of nuclear factor-κB ligand(sRANKL),and explore the components and mechanism of osteoclast differentiation.Methods:The high-performance liquid chromatography-linear ion trap quadrupole/orbitrap mass spectrometer(HPLC-LTQ/Orbitrap MS)was used to collect data,and high-resolution MS and MS2 spectra of mass spectrogram of chromatographic peaks were analyzed.Compared with the literature database,the structure of each chromatographic peak was calculated and confirmed.The Swiss Target Prediction,GeneCards,and String platform were used to predict 18 potential targets of total flavonoids from SPATHOLOBI CAULIS.Gene Ontology(GO)functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis were performed on the DAVID 6.8 platform,and the“component-target”network map was constructed by Cytoscape.RAW 264.7 cells were cultured in vitro and osteoclast-like cells differentiation were induced by sRANKL.After treatment,the osteoclast formation and differentiation were identified by tartrate resistant acid phosphatasw(TRAP)staining and TRAP enzyme activity.The expression of estrogen receptor(ESR)protein was determined by Western blot.Results:Eighteen flavonoids from SPATHOLOBI CAULIS(formononetin,genistein,medicarpin,etc.)were identified.Thirty-three potentail targets of flavonoids in the treatment of osteoporpsis were predicted by network pharmacology,and 16 signaling pathways related to osteoporosis were screened out.GO analysis and KEGG analysis showed that key targets involved prolactin,endocrine,estrogen,gap junction,and tyrosine kinase receptor(ErbB)signaling pathway.Biological process mainly involved DNA-templated positive regulation of transcription,protein autophosphorylation,and positive regul

关 键 词：鸡血藤 黄酮 液相色谱-离子阱-静电轨道阱杂交质谱 雌性激素 破骨细胞 

分 类 号：R285.5[医药卫生—中药学]