三七茎叶总皂苷促血管新生抗缺血性脑卒中的作用机制

Mechanism of total saponins in stems and leaves of Panax notoginseng on promoting angiogenesis and preventing ischemic stroke

目的探究三七茎叶总皂苷改善缺血性脑卒中、促进缺血性脑卒中损伤后血管新生作用及机制。方法采用网络药理学技术预测三七茎叶总皂苷促进缺血性脑卒中损伤后血管新生可能的机制,利用分子对接技术对筛选出的核心靶点与主要成分的结合进行初步验证。雄性SD大鼠随机分为假手术组、模型组及三七茎叶总皂苷低、中、高剂量(73、146、292mg/kg)组和3-正丁基苯酞(3-n-butylphthalide,NBP,60 mg/kg)组,每组20只。建立大鼠脑中动脉阻塞/再灌注(middle cerebral artery occlusion/reperfusion,MCAO/R)模型,给药7 d后取材。每隔1 d对大鼠进行神经功能评分;通过2,3,5-氯化三苯基四氮唑(2,3,5-triphenyltetrazolium chloride,TTC)染色、苏木素-伊红(hematoxylin-eosin,HE)染色观察大鼠脑梗死体积及脑神经细胞形态状况;通过激光多普勒血流仪观察大鼠梗死脑侧血流恢复情况;通过光学相干断层扫描血管成像观察大鼠脑微血管新生状况;通过ELISA和Western blotting测定与血管新生相关蛋白表达。结果网络药理学分析显示,共收集到60个三七茎叶总皂苷活性成分和297个关键靶点,与血管新生和缺血性脑卒中取交集后得到64个交集靶点,并且三七茎叶总皂苷主要成分与这些核心靶点均有较好结合力。三七茎叶总皂苷促进血管新生改善缺血性脑卒中的关键通路主要富集在磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinase,PI3K)/蛋白激酶B(protein kinase B,Akt)、缺氧诱导因子-1α(hypoxia inducible factor-1α,HIF-1α)等通路。动物实验结果显示,与模型组比较,三七茎叶总皂苷能够显著降低MCAO/R大鼠神经功能评分(P<0.01),改善大鼠神经功能,降低大脑梗死体积(P<0.01、0.001),减少脑组织损伤,恢复梗死脑侧脑血流(P<0.05、0.01、0.001),促进梗死脑侧皮层微血管密度(P<0.05、0.01、0.001),提高血管内皮生长因子(vascular endothelial growth factor,VEGF)、碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)水平和HIF-1α、PI3K、p-Akt/Akt蛋白表达(P<0.05、0.01、0.001)。结论三七茎叶总皂苷能够显著改善MCAO/R大鼠脑损伤,有效促进MCAO/R大鼠缺血脑区血管新生,促血管新生机制可能与提高MCAO/R大鼠大脑中VEGF、bFGF、HIF-1α表达和激活PI3K/Akt通路有关。

Objective To explore the effect and mechanism of total saponins in stems and leaves of Panax notoginseng(PNGL) on improving ischemic stroke and promoting angiogenesis after ischemic stroke injury.Methods Network pharmacology was used to predict the possible mechanism of PNGL on promoting angiogenesis after ischemic stroke injury,and molecular docking technology was used to verify the combination of the selected core targets and main components.Male SD rats were randomly divided into sham group,model group,PNGL low-,medium-,high-dose(73,146,292 mg/kg) groups and 3-n-butylphthalide(NBP,60 mg/kg) group,with 20 mice in each group.Middle cerebral artery occlusion/reperfusion(MCAO/R) model was established in rats,samples were taken after 7 d of administration.Neurological function scoring on rats was performed every two day;2,3,5-Triphenyltetrazolium chloride(TTC) staining and hematoxylin-eosin(HE) staining were used to observe the volume of cerebral infarction and the morphology of brain nerve cells in rats;The recovery of cerebral blood flow on the infarcted side of rats was observed using laser Doppler flow meter;Cerebral microvascular neovascularization in rats was observed through optical coherence tomography angiography;The expressions of proteins related to angiogenesis was determined by ELISA and Western blotting.Results The network pharmacology analysis showed that a total of 60 active ingredients and 297 key targets of PNGL were collected.After intersection with angiogenesis and ischemic stroke,64 intersection targets were obtained,and the main components of PNGL had good binding affinity with these core targets.The key pathway of PNGL on promoting angiogenesis and improving ischemic stroke was mainly enriched in phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt),hypoxia inducible factor-1α(HIF-1α) and other pathways.Animal experimental results showed that compared with model group,PNGL could significantly reduce the neurological function score of MCAO/R rats(P < 0.01),improve neurological function,reduce cerebral infarction volume(P < 0.01,0.001),reduce brain tissue damage,restore cerebral blood flow on the infarcted side(P < 0.05,0.01,0.001),promote microvascular density in the infarcted cerebral cortex(P < 0.05,0.01,0.001),and increase levels of vascular endothelial growth factor(VEGF),basic fibroblast growth factor(bFGF) and expressions of HIF-1α,PI3K and p-Akt/Akt proteins(P < 0.05,0.01,0.001).Conclusion PNGL can significantly improve brain injury in MCAO/R rats,effectively promote angiogenesis in the ischemic brain area of MCAO/R rats,and the mechanism of promoting angiogenesis may be related to the increasing of VEGF,bFGF and HIF-1α expressions in brain of MCAO/R rats and activation of PI3K/Akt pathway.