基于均匀试验设计的黄芪-当归主要化学成分剂量配伍对血管平滑肌细胞增殖的影响

Effect of Dose Compatibility of Main Chemical Components from Astragali Radix-Angelicae Sinensis Radix on Proliferation of VSMCs Based on Uniform Design

目的:采用均匀试验设计方法,构建黄芪-当归中6种主要成分配伍抗血管平滑肌细胞(VSMCs)增殖的函数模型,分析这6种成分配伍与抑制VSMCs增殖的相关性,探讨黄芪-当归主要化学成分配伍对VSMCs增殖的影响、均匀设计试验在中药多成分配伍研究中的可行性。方法:采用细胞增殖及活性检测试剂盒(CCK-8)法测定黄芪-当归中6种成分配伍对血小板衍生生长因子-BB(PDGF-BB)诱导的大鼠VSMCs增殖的抑制作用,求得各成分半数抑制浓度(IC_(50))后,以芒柄花素、黄芪皂苷Ⅰ、黄芪甲苷、毛蕊异黄酮、阿魏酸、毛蕊异黄酮苷6种成分分别为自变量X_(1)、X_(2)、X_(3)、X_(4)、X_(5)、X_(6),细胞增殖抑制率为因变量Y,选用U_(24)^(*)(24^(9))均匀设计表和线性、二次多项式2种数学模型,以逐步回归方法筛选变量,得出理论上拟合较好的方程式。通过设定不同剂量的6种成分配伍进行验证试验,筛选出实际较优的关系方程式,并根据实验目的对筛选出的回归方程进行数据中心化处理,比较方程的各回归系数,以分析药物与药效的相关性。结果:黄芪-当归中6种成分均能抑制VSMCs增殖,且呈浓度依赖性。芒柄花素、黄芪皂苷Ⅰ、黄芪甲苷、毛蕊异黄酮、阿魏酸、毛蕊异黄酮苷的IC_(50)分别为123.453、113.184、81.655、141.159、101.187、151.016 mg·L^(-1)。经验证试验筛选出2个较优的数学模型,方程1和方程2分别为Y_(1)=73.137897-0.207888X_(1)+0.000508X_(2)^(2)+0.000347X_(1)X_(6)、Y_(2)=70.038433-0.236665X_(1)+0.000577X_(2)^(2)+0.000260X_(1)X_(4)+0.000415X_(1)X_(6)+0.000370X_(3)X_(5)。方程2经数据中心化处理后为Y=-1.480260-0.146281X_(1)+0.000673X_(2)^(2)+0.000314X_(1)X_(4)+0.000456X_(1)X_(6)+0.000737X_(3)X_(5),回归系数表明黄芪皂苷Ⅰ对细胞增殖抑制率起正向作用,芒柄花素与毛蕊异黄酮、芒柄花素与毛蕊异黄酮苷、黄芪甲苷与阿魏酸是以交互作用形式对细胞增殖发挥抑制效应,且黄芪甲苷与阿魏酸的协同作用高于其他交互项。结论:黄芪-当归中6种主要成分配伍后对VSMCs异常增殖有抑制作用,证明均匀试验设计可用于预测中药化学成分配伍与药效的相关性,可为合理运用均匀设计与回归分析构建数学模型研究中药配伍关系提供实验依据。

Objective:The functional model of six major components of Astragali Radix-Angelicae Sinensis Radix combination against the proliferation of vascular smooth muscle cells(VSMCs)was constructed by uniform design,the relationship between the compatibility of these six main components and the inhibition of VSMCs proliferation was analyzed,and the effect of the compatibility of these main components of Astragali Radix-Angelicae Sinensis Radix on the proliferation of VSMCs as well as the feasibility of uniform design test in the study of multi-component compatibility of Chinese medicines were discussed.Method:Cell proliferation and toxicity assay kit(CCK-8)method was used to determine the inhibitory effect of the six components of Astragali Radix-Angelicae Sinensis Radix on platelet derived growth factor-BB(PDGF-BB)-induced VSMCs proliferation in rats and the half inhibitory concentration(IC_(50))of each component were obtained.Six chemical components of Astragali Radix-Angelicae Sinensis Radix(formononetin,astragaloside,astragalosideⅣ,calycosin,ferulic acid and calycosin-7-O-β-D-glucoside)were taken as the independent variables X_(1),X_(2),X_(3),X_(4),X_(5),X_(6),respectively,and the cell proliferation inhibition rate as the dependent variable Y.U_(24)^(*)(24^(9))uniform design table and mathematical models(including linear and quadratic polynomial)were selected,the stepwise regression method was used to screen the variables,and the better equations were obtained in theory.The actually better relationship equations were screened by setting the different dose compatibility of these six components for verification tests.According to the purpose of the experiment,the selected regression equation was processed by data centralization,so as to compare the regression coefficients of the data centralization equation and analyze the correlation between components and efficacy.Result:The six components of Astragali Radix-Angelicae Sinensis Radix could inhibit abnormal proliferation of VSMCs in a dose-dependent manner.IC50values of formononetin,astragalosideⅠ,astragalosideⅣ,calycosin,ferulic acid and calycosin-7-O-β-D-glucoside were 123.453,113.184,81.655,141.159,101.187,151.016 mg·L^(-1),respectively.Two superior models were selected by experimental verification,including equation 1 of Y_(1)=73.137897-0.207888X_(1)+0.000508X_(2)^(2)+0.000347X_(1)X_(6)and equation 2 of Y_(2)=70.038433-0.236665X_(1)+0.000577X_(2)^(2)+0.000260X_(1)X_(4)+0.000415X_(1)X_(6)+0.000370X_(3)X_(5).After data centralization,equation 2 was adjusted to Y=-1.480260-0.146281X_(1)+0.000673X_(2)^(2)+0.000314X_(1)X_(4)+0.000456X_(1)X_(6)+0.000737X_(3)X_(5),the regression coefficients showed that the astragalosideⅠhad a positive effect on the inhibition rate of cell proliferation,formononetin-calycosin,formononetin-calycosin-7-O-β-D-glucoside and astragalosideⅣ-ferulic acid had an interaction effect on the inhibition rate of cell proliferation,and the synergistic effect of astragalosideⅣand ferulic acid was higher than other interaction items.Conclusion:The combination of six main components of Astragali Radix-Angelicae Sinensis Radix can inhibit the abnormal proliferation of VSMCs,and it is demonstrated that the uniform test design can be used to predict the correlation between the chemical composition of Chinese medicines with their pharmacological effects,which can provide an experimental basis for the rational use of uniform design and regression analysis to construct mathematical models to study the compatibility relationship of Chinese medicines.