基于体外细胞模型的痹祺胶囊药效物质基础研究  

Study on pharmacodynamic material basis of Biqi Capsules based on in vitro cell model

作　　者：宋紫腾 卜睿臻 韩彦琪[1,2,3] 王磊 姚鹏飞 许浚[1,2,3] 张铁军[1,2,3] 刘昌孝[1,2,3] SONG Zi-teng;BU Rui-zhen;HAN Yan-qi;WANG Lei;YAO Peng-fei;XU Jun;ZHANG Tie-jun;LIU Chang-xiao(Tianjin Key Laboratory of Quality Marker of Traditional Chinese Medicine,Tianjin Institute of Pharmaceutical Research,Tianjin 300462,China;National&Local Jointed Engineering Laboratory of Modern Preparation and Quality Control Technology of Traditional Chinese Medicine,Tianjin Institute of Pharmaceutical Research,Tianjin 300462,China;National Key Laboratory of Druggability Evaluation and Systematic Translational Medicine,Tianjin Institute of Pharmaceutical Research,Tianjin 300462,China;Tianjin Darentang Jingwanhong Pharmaceutical Co.,Ltd.,Tianjin 300112,China;Tianjin Pharmaceutical Darentang Group Co.,Ltd.,Tianjin 300193,China;Tianjin University of Traditional Chinese Medicine,Tianjin 301617,China)

机构地区：[1]天津药物研究院,天津市中药质量标志物重点实验室,天津300462 [2]天津药物研究院,中药现代制剂与质量控制技术国家地方联合工程实验室,天津300462 [3]天津药物研究院,药物成药性评价与系统转化全国重点实验室,天津300462 [4]天津达仁堂京万红药业有限公司,天津300112 [5]津药达仁堂集团股份有限公司,天津300193 [6]天津中医药大学,天津301617

出　　处：《中草药》2023年第23期7673-7686,共14页Chinese Traditional and Herbal Drugs

基　　金：国家自然科学基金重点项目(U21A20406)。

摘　　要：目的 探究痹祺胶囊祛风除湿、活血止痛功能的药效物质基础。方法 通过建立脂多糖(lipopolysaccharide,LPS)诱导的单核巨噬细胞白血病细胞RAW264.7体外炎症模型、细胞核因子-κB受体活化因子配体(receptor activator of nuclear factorκB ligand,RANKL)和巨噬细胞集落刺激因子(macrophage-stimulating factor,M-CSF)与RAW264.7细胞共培养建立破骨细胞分化模型、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)诱导的人类风湿关节炎成纤维样滑膜细胞RA-HFLS增殖模型,分别探讨痹祺胶囊发挥抗炎活性、抑制破骨细胞形成、抑制RA-HFLS细胞增殖的作用机制及药效物质基础。MTS法检测细胞增殖活性,ELISA法检测细胞上清中一氧化氮(nitric oxide,NO)、TNF-α和白细胞介素-6(interleukin-6,IL-6)的含量,TRAP染色法检测破骨细胞分化数量,流式细胞术检测细胞凋亡情况,RT-qPCR法检测细胞中组织蛋白酶K(cathepsin K,CTSK)、抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphatase,TRAP)、基质金属蛋白酶-3(matrix metalloproteinase-3,MMP-3)、RANKL mRNA相对表达情况。结果 痹祺胶囊及其19个单体成分均能显著抑制LPS诱导的RAW264.7细胞内NO、TNF-α和IL-6的释放,推测19个化合物是痹祺胶囊发挥抗炎作用的药效物质基础。痹祺胶囊及其13个单体成分均能显著减少RANKL和M-CSF诱导RAW264.7分化为破骨细胞的数量,并能明显降低破骨细胞标志基因CTSK和TRAP的相对表达量,推测马钱子碱、士的宁、党参炔苷、茯苓酸、丹参素、丹酚酸B、迷迭香酸、三七皂苷R_(1)、人参皂苷Rg_(1)、人参皂苷Rb_(1)、藁本内酯、甘草次酸和甘草苷为痹祺胶囊发挥治疗骨破坏、缓解关节疼痛作用的主要物质基础。痹祺胶囊及其9个单体成分能明显抑制TNF-α诱导的RA-HFLS细胞增殖,并且能显著促进RA-HFLS细胞的凋亡以及明显降低MMP-3和RANKL基因的相对表达量,推测马钱子碱、士的宁、丹参素、丹Objective To explore the pharmacological material basis of effects of dispelling wind and eliminating dampness,promoting blood circulation and relieving pain of Biqi Capsules(痹祺胶囊).Methods The mechanism and material basis of the antiinflammatory activity,inhibition of osteoclast formation and inhibition of RA-HFLS cell proliferation of Biqi Capsules were respectively investigated based on the established lipopolysaccharide(LPS)-induced RAW264.7 inflammation model in vitro,receptor activator of nuclear factor kappa-B ligand(RANKL)and macrophage-stimulating factor(M-CSF)co-cultured with RAW264.7 cells to establish osteoclast differentiation model and tumor necrosis factor-α(TNF-α)induced proliferation model of human fibroblast-like synoviocyte of rheumatoid arthritis(RA-HFLS).Cell proliferation was detected by MTS assay.The levels of nitric oxide(NO),TNF-αand interleukin-6(IL-6)of cells in the supernatant were detected by ELISA.TRAP staining was used to detect the number of osteoclast differentiation,and flow cytometry was used to detect cell apoptosis.RT-qPCR was used to detect the mRNA relative expression of cathepsin K(CTSK),tartrate-resistant acid phosphatase(TRAP),matrix metalloproteinase-3(MMP-3),and RANKL.Results Biqi Capsules and its 19 monomer components could significantly inhibit the release of NO,TNF-αand IL-6 in RAW264.7 induced by LPS,suggesting that the 19 compounds were the material basis for the anti-inflammatory effect of Biqi Capsules.Biqi Capsules and 13 monomer compounds could significantly reduce the number of RAW264.7 differentiated into osteoclasts induced by RANKL and M-CSF,and remarkably reduce the relative expression of osteoclast marker genes CTSK and TRAP.It was speculated that brucine,strychnine,lobetyolin,pachymic acid,tanshinol,salvianolic acid B,rosmarinic acid,notoginsenoside R_(1),ginsenoside Rg_(1),ginsenoside Rb_(1),ligustilide,glycyrrhetinic acid and liquiritin were the main material basis of Biqi Capsules for playing a role in treating bone destruction and relievin

关 键 词：痹祺胶囊 类风湿关节炎 药效物质基础 祛风除湿 活血止痛 马钱子碱 士的宁 丹参素 丹参酮Ⅱ_(A) 阿魏酸 藁本内酯 蜕皮甾酮 次黄嘌呤 甘草次酸 

分 类 号：R285[医药卫生—中药学]