当归芍药散活血利水不同配伍对缺血性脑卒中小鼠血管新生的影响

Danggui Shaoyao San regulates angiogenesis in ischemic stroke mice through’blood activating’and/or’body fluidtransportation and transformation’actions

目的观察当归芍药散活血利水不同配伍对缺血性脑卒中雄性小鼠血管新生的影响,并探讨其对血管新生相关因子的影响。方法采用成年C57雄性小鼠制备大脑中动脉远端梗阻(dMCAO)模型,随机分为7组,假手术组、模型组、原方组(当归、川芎、白芍、泽泻、茯苓、白术生药剂量分别为68.3、68.3、363.8、181.9、91.0、91.0 mg/kg)、在原方基础上,将活血的3味药加倍的活血倍量(当归、川芎、白芍、泽泻、茯苓、白术生药剂量分别为136.6、136.6、727.6、181.9、91.0、91.0 mg/kg)组、将利水的3味药加倍的利水倍量(当归、川芎、白芍、泽泻、茯苓、白术生药剂量分别为68.3、68.3、363.8、363.8、180.2、180.2 mg/kg)组、仅有活血的3味药且加倍的活血(当归、川芎、白芍生药剂量分别为136.6、136.6、727.6 mg/kg)组,以及仅有利水的3味药且加倍的利水(泽泻、茯苓、白术生药剂量分别为363.8、180.2、180.2 mg/kg)组。采用转棒实验对小鼠进行神经功能评分;通过免疫荧光染色检测血管新生;采用Pearson相关性分析进行神经功能与血管数量相关性分析;采用通过蛋白芯片及Western blotting检测对血管新生相关因子的影响。结果与模型组及原方组比较,活血倍量组小鼠在转棒上的停留时间显著增加,差异有统计学意义(P<0.05、0.01);CD31+细胞数及CD31+/BrdU+细胞数显著增加(P<0.05、0.01),且活血倍量组改善神经功能与血管数量具有相关性;进一步分子机制研究发现,活血倍量组显著增加了碱性成纤维细胞生长因子(FGF-2)、基质细胞衍生因子-1α(SDF-1α)、血管内皮生长因子(VEGF)的表达(P<0.05、0.01、0.001)。结论当归芍药散的活血倍量配伍对缺血性脑损伤小鼠血管新生的贡献度最大。

Objective To observe the effects of different compatibility of Danggui Shaoyaosan(DSS)for activating blood circulation and diuresis on the angiogenesis of male mice with ischemic stroke,and explore its effect on angiogenesis-related factors.Methods Adult C57 male mice were made with a model of distal middle cerebral artery obstruction(dMCAO model).They were randomly divided into seven groups,the sham group,the model group and the original prescription group(the crude drug dosage of Angelicae Sinensis Radix,Chuanxiong Rhizoma,Angelicae Dahuricae Radix,Alismatis Rhizoma,Poria,Atractylodis Macrocephalae Rhizoma were 68.3,68.3,363.8,181.9,91.0,91.0 mg/kg respectively).On the basis of the original formula,the doubling blood activating volume group(the crude drug doses of Angelicae Sinensis Radix,Chuanxiong Rhizoma,Angelicae Dahuricae Radix,Alismatis Rhizoma,Poria,Atractylodis Macrocephalae Rhizoma were 136.6,136.6,727.6,181.9,91.0,91.0 mg/kg respectively),the doubling body fluidtransportation and transformation volume group(the crude drug doses of Angelicae Sinensis Radix,Chuanxiong Rhizoma,Angelicae Dahuricae Radix,Alismatis Rhizoma,Poria,Atractylodis Macrocephalae Rhizoma were 68.3,68.3,363.8,363.8,180.2,and 180.2 mg/kg respectively),blood activating group(the crude drug dosage of Angelicae Sinensis Radix,Chuanxiong Rhizoma,Angelicae Dahuricae Radix were 68.3,68.3,363.8 mg/kg),body fluidtransportation and transformation(the crude drug dosage of Alismatis Rhizoma,Poria,Atractylodis Macrocephalae Rhizoma were 363.8,180.2,180.2 mg/kg).Mice were scored for neurological function.The angiogenesis was detected by immunohistochemistry.The angiogenesis-related factors were detected by protein array and Western blotting.Results Compared with the model group and the original prescription group,the residence time of mice in the doubling blood activating volume group was significantly increased(P<0.05,0.01);the number of CD31+cells and the number of CD31+/BrdU+cells were significantly increased(P<0.05,0.01),and the improvement of nerve function was correlated with the number of blood vessels in the doubling blood activating volume group;further molecular mechanism study found that the number of CD31+cells and CD31+/BrdU+cells in the doubling blood activating volume group was significantly increased(P<0.05,0.01)The expression of basic fibroblast growth factor(FGF-2),stromal cell-derived factor-1α(SDF-1α)and vascular endothelial growth factor(VEGF)were increased(P<0.05,0.01,0.001).Conclusion The DSS’s blood activating compatibility is the most significant contribution to nerve regeneration in mice with ischemic brain injury.