黄芪当归主要活性成分抗血管平滑肌细胞增殖和迁移的作用

Effects of main active components of Astragalus membranaceus and Angelica sinensis combination on VSMCs proliferation and migration

目的:研究黄芪和当归6种主要活性成分芒柄花素、黄芪皂苷Ⅰ、黄芪甲苷、毛蕊异黄酮、阿魏酸、毛蕊异黄酮苷单用及其配伍对大鼠血管平滑肌细胞(VSMCs)增殖与迁移的影响。方法:以血小板衍生生长因子BB(PDGF-BB)诱导VSMCs增殖,随机分为对照组、模型组、各成分单用组、各成分剔除组、六药配伍组。CCK-8检测VSMCs增殖,流式细胞术检测细胞周期,划痕实验检测细胞迁移能力,免疫荧光法检测增殖细胞核抗原(PCNA)、平滑肌α肌动蛋白(α-SMA)、骨桥蛋白(OPN)的表达。结果:各成分单用组(除芒柄花素单用组外)、各成分剔除组、六药配伍组均可显著抑制VSMCs增殖、细胞周期转化、PCNA的表达(P<0.05,P<0.01)。与六药配伍组比较,黄芪皂苷Ⅰ剔除组、黄芪甲苷剔除组、毛蕊异黄酮剔除组细胞增殖抑制率显著降低,增殖指数(PI)、PCNA蛋白表达显著升高(P<0.05,P<0.01);单用黄芪皂苷Ⅰ组、单用毛蕊异黄酮组、单用阿魏酸组、各成分剔除组、六药配伍组均可使细胞迁移降低,α-SMA表达上调,OPN蛋白表达下调(P<0.01),单用毛蕊异黄酮苷组可上调α-SMA表达(P<0.05)。与六药配伍组比较,黄芪皂苷Ⅰ剔除组、黄芪甲苷剔除组、毛蕊异黄酮剔除组均可使细胞迁移率增加,α-SMA表达降低,OPN表达增强(P<0.01);阿魏酸剔除组可使细胞迁移率和OPN表达增加(P<0.01)。结论:黄芪当归主要活性成分配伍对VSMCs表型转化、增殖和迁移的抑制作用强于药物单用,其中黄芪皂苷Ⅰ、黄芪甲苷、毛蕊异黄酮主要起抑制VSMCs增殖和迁移的作用,阿魏酸起抑制VSMCs迁移的作用,毛蕊异黄酮苷可抑制VSMCs增殖。黄芪和当归主要活性成分配伍的作用可能是通过抑制VSMCs表型转化和细胞周期进程而发挥的。

Objective: To study the effects of the compatibility of the six main active components of Astragalus membranaceus and Angelica sinensis, such as formononetin, astragaloside-Ⅰ, astragaloside Ⅳ, flavonoids, ferulic acid and flavonoid glycosides on the proliferation and migration of vascular smooth muscle cells(VSMCs) in rats. Methods: The proliferation of VSMCs was induced by platelet-derived growth factor(PDGF-BB). The cells were randomly divided into control group, model group, drug single-use groups, drug elimination groups, and six components combination group. The proliferation rate of VSMCs was detected by CCK-8 method, cell cycle was determined by flow cytometry, cell migration ability was detected by cell scratch test, the expression of proliferating cell nuclear antigen(PCNA), alpha-smooth muscle actin(α-SMA)and osteopontin(OPN) were detected by immunofluorescence assay. Results: Drug single-use groups(except for formononetin single-use group), drug elimination groups and six components combination group could significantly inhibit the proliferation of VSMCs, the transformation of cell cycle and the expression of PCNA(P<0.05, P<0.01). Compared with the six components combination group, the inhibition rate of cell proliferation in astragaloside Ⅰ elimination group, astragaloside Ⅳ elimination group and flavonoids elimination group decreased significantly, while the proliferation index(PI) and PCNA protein expression increased significantly(P<0.05, P<0.01). Astragaloside Ⅰ single-use group, flavonids single-use group, ferulic acid single-use group, drug elimination groups, six components combination group could all significantly inhibit cell migration, up-regulate the expression of α-SMA and down-regulate the expression of OPN protein(P<0.01). Flavonoids glycosides single-use group could up-regulate the expression of α-SMA(P<0.05). Compared with the six components combination group, astragaloside Ⅰelimination group, astragaloside Ⅳ elimination group and flavonoids elimination group could increase the cell mobility, decrease the expression of α-SMA and increase the expression of OPN(P<0.01). Ferulic acid elimination group could increase the cell mobility and the expression of OPN(P<0.01). Conclusion: The compatibility of the main active components combination of Astragalus membranaceus and Angelica sinensis was stronger than that of single-use. Among them, astragaloside Ⅰ, astragaloside Ⅳ and flavonids mainly acted as inhibitors of cell proliferation. The ferulic acid could inhibit the migration of VSMCs, flavonoids glycosides could inhibit the proliferation of VSMCs. The compatibility of the main active components combination of Astragalus membranaceus and Angelica sinensis may played a role by the phenotypic transformation of VSMCs and the inhibition of cell cycle progression.