摘要
目的探讨黄芪和当归的主要活性成分黄芪甲苷、芒柄花素、毛蕊异黄酮、毛蕊异黄酮苷和阿魏酸配伍对衰老造血干细胞(HSCs)增殖的影响,并从细胞周期调控研究其作用机制。方法以三丁基过氧化氢(T-BHP)构建小鼠HSCs衰老模型,研究不同质量浓度5种活性成分对HSCs衰老和增殖的影响,寻找可促进细胞增殖的主要活性成分。在此基础上,采用正交试验研究5种活性成分配伍对HSCs衰老和增殖的影响,寻找5种活性成分促进细胞增殖的合理配伍。最后,采用HSCs衰老模型,制备黄芪-当归1∶1配伍含药血浆,设立对照组、模型组、空白血浆组、阿魏酸组、黄芪甲苷组、芒柄花素组、毛蕊异黄酮组、毛蕊异黄酮苷组、活性成分配伍组、黄芪-当归1∶1含药血浆组,药物作用于衰老细胞,以SA-β-半乳糖苷酶(SA-β-Gal)染色法测定HSCs衰老率,CCK-8法测定细胞增殖率,流式细胞术检测细胞周期分布,Western blotting法检测Cyclin D1、细胞周期蛋白依赖性激酶4(CDK4)蛋白表达。结果阿魏酸、黄芪甲苷、芒柄花素均可显著促进衰老HSCs增殖和降低细胞衰老率,而毛蕊异黄酮、毛蕊异黄酮苷单独应用对HSCs增殖和细胞衰老率无显著影响。正交试验发现,以阿魏酸、芒柄花素和黄芪甲苷为基础因子,毛蕊异黄酮和毛蕊异黄酮苷为次要因子进行配伍的5种活性成分组合促细胞增殖和降低细胞衰老率的作用最强。阿魏酸、黄芪甲苷、芒柄花素、活性成分配伍、含药血浆均可降低细胞衰老率,使G0/G1期细胞减少,G2/M+S期细胞增加,并使Cyclin D1和CDK4蛋白表达增强。以上效应以活性成分配伍组和含药血浆组最强。结论黄芪和当归的主要活性成分阿魏酸、黄芪甲苷和芒柄花素对衰老HSCs具有促增殖和改善衰老作用,而毛蕊异黄酮和毛蕊异黄酮苷单用时并无明显的作用。当5种活性成分配伍后对衰老HSCs的促增殖作用最强,并可改善HSCs衰老,促进衰老HSCs由静止期向增殖期转换。黄芪和当归的主要活性成分及其配伍促HSCs增殖和抗HSCs衰老的作用可能与调节细胞周期相关蛋白表达及促进细胞周期转换有关。
Objective To explore the effects of the combination of main active components of Astragalus membranaceus and Angelica sinensis such as astragaloside IV, formononetin, calycosin, campanulin, ferulic acid on aging hematopoietic stem cells(HSCs), and clarify its mechanism through cell cycle regulation. Methods The aging model of HSCs in mice was established with three butyl hydrogen peroxide(t-BHP) to research the effects of five active components of different concentrations on the senescence and the proliferation of HSCs, and seek the main active components which could promote cell proliferation. Finally, HSCs aging model was used to prepare the drug-containing plasm of A. membranaceus combined with A. sinensis at 1∶1 ratio. Furthermore, blank control group, model group, blank plasma group, ferulic acid group, astragaloside IV group, formononetin group, calycosin group, calycosin glycoside group, combination group of main active components, drug-containing plasma group of A. membranaceus combined with A. sinensis at 1∶1 ratio were acted on aging cells, HSCs senescence rate was tested by SA-β-galactosidase staining and cell proliferation rate was measured by CCK-8 method, cell cycle distribution was determined by flow cytometry, and the protein expression of Cyclin D1 and cyclin dependent kinase 4(CDK4) were detected by Western blotting. Results Ferulic acid, astragaloside IV, and formononetin significantly promoted the proliferation of aging HSCs and decreased the positive rate of senescent cells, but the effects of calycosin and calycosin glycoside on HSCs proliferation and the positive rate of senescent cells were not significant. The orthogonal experiment showed that the combination of five active components that ferulic acid, formononetin, astragaloside IV were taken as basic factors, and calycosin and calycosin glycoside were taken as secondary factors, had the strongest effect on promoting cell proliferation and decreasing the positive rate of senescent cells. Ferulic acid, astragaloside IV, formononetin, active component combination and drug-containing plasma decreased the positive rate of senescent cells, down-regulated G0/G1 phase cells while up-regulated G2/M + S phase cells, and increased the expression of Cyclin D1 and CDK4 proteins. The above effects in the active component combination group and the drug-containing plasma group were the best. Conclusion The main active components of A. membranaceus and A. sinensis such as ferulic acid, astragaloside IV, and formononetin can promote the proliferation and improve the senescent of aging HSCs, however, calycosin and calycosin glycoside have no obvious effect. The effect of promoting the proliferation is the strongest on aging HSCs when five active components are combined, and the combination can improve HSCs senescence, enhance the transformation of HSCs from static stage to proliferative stage. The main active components and the combination of A. membranaceus and A. sinensis can promote HSCs proliferation and antagonize HSCs senescent, which may be related to regulating the expression of cell cycle related proteins and promoting the transformation of cell cycle.
作者
朱嘉欢
黄小平
邓常清
ZHU Jia-huan;HUANG Xiao-ping;DENG Chang-qing(Molecular Pathology Laboratory,Hunan Key Laboratory of Prevention and Treatment of Integrated Traditional Chinese and Western Medicine on Cardio-cerebral Diseases,Hunan University of Chinese Medicine,Changsha 410208,China)
出处
《中草药》
CAS
CSCD
北大核心
2019年第1期111-119,共9页
Chinese Traditional and Herbal Drugs
基金
国家自然科学基金资助项目(81473581)
"中西医结合防治心脑血管疾病的相关基础研究"湖南省高校科技创新团队
"中医药防治心脑血管疾病基础研究"湖南省自然科学创新群体基金
关键词
造血干细胞
阿魏酸
黄芪甲苷
芒柄花素
毛蕊异黄酮
毛蕊异黄酮苷
黄芪
当归
细胞周期蛋白D1
细胞周期蛋白依赖性激酶
hematopoietic stem cells
ferulic acid
astragaloside Ⅳ
formononetin
calycosin
campanulin
Angelica membranaceus Bge. var. mongholicus(Bge.) Hsiao
Astragalus sinensis(Oliv.) Diels
cyclin D1
cyclin dependent kinase
