基于均匀设计分析黄芪汤活性组分抗二甲基亚硝胺大鼠肝纤维化的配伍作用

Uniform Designed Research on the Active Ingredients Assembling of Huangqi Decoction for Inhibition of DMN-induced Liver Fibrosis

目的筛选古典方剂黄芪汤抗肝纤维化的有效组分及配伍作用。方法 (1)4周内给大鼠腹腔注射二甲基亚硝胺(dimethylnitrosamine,DMN)12次制备肝纤维化模型,于造模第3周首日开始给药干预至4周末。采用均匀设计对黄芪汤中4种组分(分别为黄芪总皂苷、黄芪总黄酮、甘草酸、甘草总黄酮)以4因素8水平安排给药;以肝组织羟脯氨酸(Hyp)含量和丙氨酸氨基转移酶(ALT)活性为观测指标,采用逐步回归法分析其抗肝纤维化效应的最优组方。(2)组分复方的再验证:模型制备及给药方法同上,采用上述实验获得的组分复方,与原方对照,观测大鼠肝组织胶原染色、Hyp含量及血清肝功能等变化。结果通过均匀设计研究,确定最优组方为组分复方(黄芪总皂苷、甘草酸的配伍比例为164:48),并得到有效验证。与模型组比较,组分复方及黄芪汤均可显著改善肝纤维化病理组织学变化,显著降低肝组织Hyp含量、血清ALT、天冬氨酸氨基转移酶(AST)以及碱性磷酸酶(ALP)活性(P<0.05);组分复方在降低肝组织Hyp含量及降低血清ALT活性方面均优于其分别单用组(P<0.05),降低血清ALT活性的作用优于黄芪汤原方(P<0.05)。结论黄芪总皂苷和甘草酸是黄芪汤发挥抗DMN大鼠肝纤维化作用的有效组分;两个组分配伍在降低肝组织胶原沉积及降低血清ALT活性方面具有明显的协同效应。

Objective To screen out effective ingredients of Huangqi Decoction（HQD） on dimethylnitrosamine（DMN） induced liver fibrosis and its assembling actions.Methods（1） DMN solution（0.5%） was peritoneally injected to rats to prepare the liver fibrosis model for 12 times,starting from the 1st day of modeling to the end of the 4th week.Uniform design method with 4-factor 8-level table was used to optimize the proportion of four ingredients from HQD,including astragalosides（AS）,astragalus flavonoids（AF）,glycyrrhizae acid（GA）,and glycyrrhizae flavonoids（GF）.Moreover,the changes of hydroxyproline（Hyp） content in the liver issue and the level of alanine aminotransferase（ALT） in serum were observed as screen indices,and the method of regression analysis was used to find out an optimal combination.（2） A further study for comparing and verifying the efficacy of the obtained optimized prescription was conducted by observing the changes of fibrosis pathology,the content of Hyp in the liver tissue and serum enzyme activity after medication.Results The optimal proportion of AS and GA was 164：48.Compared with the model group,the content of Hyp in the liver tissue and the levels of ALT,aspartate aminotransferase（AST）,and alkaline phosphatase（ALP） in serum decreased significantly,indicating the inhibiting effect of HQD and the AS/GA combination group on hepatic fibrosis formation（P0.05）.The AS/GA combination group was better than AS/GA used alone group in reducing the content of Hyp in the liver tissue and the level of ALT in serum.Furthermore,the AS/GA combination group was better than the HQD group in reducing the level of ALT in serum.Conclusions AS and GA were effective ingredients of HQD,and the combination of AS and GA had obvious synergistic effect in reducing liver collagen deposition and decreasing serum ALT activity in DMN-induced liver fibrosis.