【作者】 刘杨； 薛强； 杨盛； 何然； 李许； 徐晓玉；

【Author】 Yang Liu;Qiang Xue;Sheng Yang;Ran He;Xu Li;Xiaoyu Xu;College of Pharmaceutical Sciences & College of TCM and Pharmacology,Southwest University;Institute of Chinese Medicine,Southwest University;

【机构】 西南大学药学院&中医药学院； 西南大学中药研究所；

【摘要】 实验前期研究报道梓葛冻干粉针能够促进梗死灶周围的血管新生减少脑梗死体积,从而显著改善永久性卒中大鼠损伤的神经功能,但其促缺血半暗带内血管新生的机制尚未阐明。试验方法:该研究主要从在体和体外进行观察。在体实验:主要采用免疫荧光法观察梓葛对永久性大脑中动脉栓塞大鼠(PMCAO)血管新生以及HIF-1a、EIF-4E以及p4E-BP表达情况;体外实验:在低氧、低营养液培养下的原代脑微血管内皮细胞上,采用MTT、BrdU标记以及Matrigel培养内皮细胞观察梓葛对细胞存活、增殖以及血管形成的作用。并采用荧光结合WB的方法检测药物对HIF-1a的表达及核转移,以及对EIF-4E、p4E-BP表达的影响。实验结果:脑组织切片免疫荧光显示梓葛能够促进PMCAO大鼠缺血侧脑皮层血管新生以及EIF-4E、p4E-BP/4E-BP蛋白表达:MTT和BrdU实验结果显示,梓葛能增加OGD损伤8h后低氧、低营养液培养下的细胞活性以及增殖;在iatrigel上进行低氧、低营养液培养6h后,梓葛显著增加小管形成的数量;免疫荧光以及WB结果提示,梓葛能够促进低氧、低营养液培养下的内皮细胞HIF-1a的表达和核转移。同时,梓葛也能上调HIF-1a上游蛋白EIF-4E、p4E-BP。结论:梓葛能通过促进内皮细胞在卒中后低血流灌注下的存活、增殖以及小管形成从而促进卒中后的血管新生。而这可能与其上调p4E-BP/EIF-4E/HIF-1a有关。更多还原

【Abstract】 The previous study has reported a potential new drug,Lyophilized Powder of Catapol and Puerarin,that ameliorates the stroke-induced neurological deficiency by several pharmacological effects,as anti-oxidative stress,anti-apoptosis,and enhanced angiogenesis as well.So,this research focuses on the prominent promotion of angiogenesis after brain stroke by the chemical compounds of the drug,Catapol and Puerarin(C-P).Method:The angiogenesis and its related proteins after administration of C-P were observed in vivo and in vitro.Experiments in vivo:the brain slices were used to observe the angiogenesis and related protein expression(HIF-1a,EIF-4E and p4E-BP) by immunofluorescent after mediating with C-P on rats those suffered from Permanent Middle Cerebral Artery Oclusion(PMCAO);Experiments in vitro:all primary endothelial cells from brain cortex were cultured in hypoxia and diluted culture media that aimed to mimic the situation of ischemic penumbra.MTT and BrdU marking were used to determinate the cellular viability and proliferation.And the cellular capability of tube formation under hypoxia was observed on Matrigel.WB and immunofluorescent technique were performed to observe the expression of HIF-la,EIF-4E and p4E-BP.Results:The immunofluorescent observation on brain slices suggests that C-P prominently enhances the angiogenesis on the ischemia side of brain cortex,and increases expression of HIF-1a,EIF-4E and p4E-BP as well.The MTT and BrdU show that these mixture compounds increases the cellular viability and proliferation while the OGD-damaged endothelia cells were cultured in low oxygen and diluted culture media.Also,the endothelial capability of tube formation under deficient of oxygen and nutrition is enhanced by treating C-P for 6 h.Simultaneously,an increased expression of HIF-1a,EIF-4E and p4E-BP was observed in these endothelial cells.Conclusion:Depending on the research,C-P promotes the angiogenesis via surviving and proliferating endothelial cells,and also increasing the tube formation.And most parts are potentially depending on emphasized p4E-BP / EIF-4E / HIF-1a.更多还原