【作者】 李红艳；

【导师】 孙建宁；

【作者基本信息】 北京中医药大学 ， 中药药理， 2009， 博士

【摘要】 缺血性卒中因其高发病率、高死亡率、高致残率而极具危害性,缺血性卒中的最佳治疗时间窗是发病6小时以内,目前公认的有效方法是溶栓治疗,但受溶栓时间窗和治疗条件所限,仅2%-5%的患者能进行该项治疗;同时溶栓治疗虽可建立缺血区血流再灌注,但不能解决缺血引起的脑组织再灌注损伤。对于不能及时进行溶栓的大多数脑缺血患者来说,如何促进缺血损伤后的神经功能恢复,是脑缺血恢复早期治疗的重要内容。脑缺血可激活成年哺乳动物脑内的神经发生,诱导内源性神经前体细胞分裂、增殖并向损伤区域迁移,进而分化为成熟神经细胞,部分替代损伤后丢失的区域特异性神经元。神经发生与血液供应密切相关,尽快恢复缺血区的血供,挽救濒死的神经元、神经胶质细胞和血管内皮细胞,是治疗缺血性脑血管病的关键。除抗凝,溶栓等传统治疗手段外,刺激脑内的神经发生和血管新生可能成为新的临床治疗手段。血管内皮生长因子(vascular endothelial growth factor,VEGF)和血管生成素(angiopoietin,Ang)是重要的促血管生成因子,同时也有神经保护,促神经发生的作用,Ang1和Ang2依赖于VEGF而起作用。神经发生与血管新生的关系紧密,而VEGF很可能是连接二者的关键调控因子之一。Wnt/β-catenin是调控神经系统发育中,神经干细胞增殖和分化的经典通路,β-catenin能够促进神经干细胞的增殖,并影响着干细胞的分化方向,在缺血肢体的血管新生研究中也有重要作用。在脑缺血后VEGF,Ang1、Ang2与Wnt/β-catenin通路几个关键因子:β-catenin,GSK3β,c-myc水平的变化与神经发生和血管新生的关系是本课题探索的一个主要问题。甾体皂苷TSA是知母的有效成分之一,研究表明知母皂苷对急性脑缺血再灌注损伤具有确定的保护作用,其机制涉及调控缺血脑组织血管内皮功能,抗血栓形成和改善血液流变学等方面,而对于脑缺血损伤恢复早期神经发生和血管新生的研究作者尚未见报道,本课题采用大鼠大脑中动脉缺血(MCAO)再灌注以及永久性大脑中动脉缺血模型,观察TSA对脑梗死后恢复期神经功能的影响,着重探讨脑组织损伤及修复过程中TSA对缺血脑组织病理变化、神经发生以及血管新生的干预作用。为其脑缺血疾病的临床应用提供实验依据,同时也为脑梗死最佳治疗时间窗之外的中药干预时机的合理性进行探索。本课题主要分为三个部分:第一章甾体皂苷TSA对脑缺血再灌注大鼠神经功能的影响及其机制的研究第一节:TSA对脑缺血再灌注大鼠神经功能恢复及其血管新生的影响目的探讨TSA对脑缺血再灌注大鼠恢复早期神经功能及其血管新生的影响方法线栓法建立SD大鼠大脑中动脉缺血(MCAO)再灌注模型,随机分为模型组;TSA15mg/kg、30mg/kg、60mg/kg给药组;安宫牛黄丸400mg/kg阳性对照组,以及假手术组。于恢复早期3—14天灌胃给药,1次/天,缺血后3,7,10,14天进行横木行走实验,触觉刺激实验,抓握力量实验,观察缺血后神经功能的动态变化;缺血后14天,用HE染色及尼氏染色观察运动感觉皮层区神经元的病理改变,免疫组化方法检测脑梗死周围区VEGF/VEGFR2的表达以及CD34标记微血管的密度。结果与假手术组比较,脑缺血后模型组大鼠的神经功能评分均明显降低,(P＜0.01)与模型组比较,缺血14天,TAS 30mg/kg、60mg/kg给药组大鼠横木行走的能力明显增强,执行评分显著提高,(P＜0.05);触觉刺激敏感度恢复明显,揭除胶布的潜伏期明显缩短(P＜0.05,P＜0.01);大鼠抓握力量表现出增加的趋势,但是没有统计学意义。缺血14天,与假手术组比较,模型组大鼠皮层运动感觉区的神经细胞明显损伤,与模型组比较,TAS 30mg/kg、60mg/kg给药组皮层运动感觉区的完整神经细胞的数量增加(P＜0.01),皮层运动感觉区神经元尼氏体的表达增加(P＜0.05,P＜0.01);与假手术组比较,模型组梗死周围区VEGF的表达和微血管的密度明显增高,VEGFR2的表达降低;与模型组比较,TAS30mg/kg、60mg/kg给药组梗死周围区VEGF(P＜0.05,P＜0.01)和VEGFR2的表达(P＜0.05,P＜0.01)以及梗死周围区微血管的密度明显增加,(P＜0.05,P＜0.01)。结论TSA可加快大鼠脑缺血后神经功能的恢复,其可能的机制是通过上调VEGF和VEGFR2的表达促进了内源性的血管新生。第二节TSA对脑缺血再灌注大鼠内源性神经前体细胞增殖的影响目的探讨TSA脑缺血再灌注大鼠内源性神经前体细胞增殖的影响。方法线栓法建立SD大鼠大脑中动脉缺血(MCAO)再灌注模型,随机分为模型组;TSA15mg/kg、30mg/kg、60mg/kg给药组;安宫牛黄丸400mg/kg阳性对照组,以及假手术组。缺血后1至7天腹腔注射5’-溴脱氧尿核苷(BrdU),50mg/kg,1次/天,用于标记增殖的细胞。于恢复早期3—14天灌胃给药,1次/天,缺血后7天,14天,免疫荧光染色法检测BrdU标记的新生细胞,免疫组化法检测梗死周围区巢蛋白(Nestin)的表达,结果缺血7天、14天,与假手术组比较,模型组大鼠的SVZ区的Brdu阳性细胞数和梗死周围区Nestin的表达明显增加(P＜0.05,P＜0.01);与模型组比较,缺血7天、14天TSA 30mg/kg、60mg/kg给药组显著增加侧脑室下区的Brdu阳性细胞数(P＜0.05,P＜0.01)和梗死周围区Nestin蛋白的表达(P＜0.05,P＜0.01)。结论TSA可以促进脑缺血再灌注大鼠内源性的神经发生。TSA可能以此作用机制促进了大鼠脑缺血后神经功能的恢复。第二章TSA对永久性脑缺血大鼠恢复期神经功能的影响及其机制研究目的探讨TSA对永久性脑缺血大鼠恢复期神经功能的影响以及对脑内神经发生和血管新生的作用。方法线栓法建立SD大鼠永久性大脑中动脉缺血(pMCAO)模型,随机分为模型组;TSA 15mg/kg、30mg/kg、60mg/kg给药组;安宫牛黄丸400mg/kg对照组,必存7mg/kg对照组以及假手术组。术后1-7天腹腔注射BrdU,50mg/kg,1次/天,用于标记增殖的细胞。于恢复早期3—14天灌胃给药,1次/天,缺血后3,7,14,天进行横木行走实验,触觉刺激实验,抓握力量实验,观察神经功能的动态变化;缺血后14d,BrdU/Dcx免疫荧光双标记观察皮层和纹状体梗死周围区神经前体细胞的迁移情况;BrdU/FactorⅧRelatedAntigen免疫荧光双标记新生的微血管;缺血后28d,BrdU/NeuN以及BrdU/GFAP免疫荧光双标记鉴定新生细胞的存活和分化类型;结果与假手术组比较,缺血后3-14天,模型组各项神经功能检测指标均明显降低,(P＜0.05,P＜0.01),观察期内有自发恢复特点,但仍低于假手术水平;与模型组比较,缺血后14天,TAS30mg/kg、60mg/kg给药组能够促进脑缺血大鼠横木行走的能力,(P＜0.05,P＜0.01);缩短揭除胶布的潜伏期,(P＜0.05,P＜0.01);增加大鼠的抓握力,(P＜0.05,P＜0.01)。缺血后14天,与假手术组比较,模型组皮层和纹状体梗死周围区Brdu~+/Dcx~+细胞数,新生微血管的密度明显增加(P＜0.01);与模型组比较,TAS30mg/kg、60mg/kg给药组皮层梗死周围区Brdu~+/Dcx~+细胞数以及新生微血管的密度明显增加(P＜0.05,P＜0.01);对纹状体梗死周围区Brdu~+/Dcx~+细胞数以及新生微血管的密度没有影响。缺血后28天,与假手术组比较,模型组大脑梗死周围区Brdu~+/Neun~+细胞数,Brdu~+/GFAP~+细胞数目明显增加(P＜0.05);与模型组比较,TAS 30mg/kg、60mg/kg给药组皮层Brdu~+/Neun~+细胞数明显增加(P＜0.05),对纹状体和皮层Brdu~+/GFAP~+细胞数目没有影响。结论TSA可改善大鼠永久性脑缺血后神经功能的恢复,可能与其促进内源性的神经发生和血管新生从而实现神经血管网络结构和功能的修复有关。第三章TSA对永久性脑缺血大鼠恢复期神经发生及血管新生相关调节通路的影响第一节TSA对永久性脑缺血大鼠恢复期大脑皮层血管生成调节通路Ang1/Ang2,VEGF/VEGFR2水平的影响目的探讨TSA对大鼠永久性脑缺血损伤修复过程血管新生相关调节因子蛋白和基因水平的影响方法建立SD大鼠大脑中动脉(MCAO)永久性缺血模型,随机分为模型组;TSA 15mg/kg、30mg/kg、60mg/kg给药组;安宫牛黄丸400mg/kg照组,必存7mg/kg对照组以及假手术组。于恢复早期3—14天给药,缺血后3d,7d,14d酶联免疫吸附法(ELISA)检测缺血侧大脑皮层Ang1,Ang2,的水平;缺血后7d,14d Western-blot法检测缺血侧皮层VEGF的蛋白表达,Real-Time PCR法检测皮层VEGFmRNA,VEGFR2mRNA的水平。结果与假手术组比较,脑缺血后3-14天,模型组大鼠缺血侧皮层Ang1含量降低,其中缺血第7天降低明显且有统计学意义;缺血第7天,与模型组比较,TSA中,大剂量组以及安宫牛黄和必存组Ang1水平明显增高,(P＜0.05,P＜0.01);缺血第14天,各给药组与模型组Ang1水平无明显差异。脑缺血后3-14天,假手术组和各缺血组比较Ang2的表达均无明显差异。缺血后7d,TSA 60mg/kg明显增加大鼠大脑皮层VEGFR2 mRNA的水平,但对VEGF及VEGFmRNA水平无明显影响。结论TSA可改善大鼠脑缺血后神经功能的恢复,可能与其诱导脑缺血后皮层Ang1,VEGFR2 mRNA水平的上调,促进了梗死周围区血管新生的效应有关。第二节TSA对永久性脑缺血大鼠恢复期Wnt/β-catenin信号通路的影响目的探讨TSA对大鼠永久性脑缺血损伤恢复期Wnt/β-catenin信号通路β-catenin、c-myc,GSK3β水平的影响方法建立SD大鼠大脑中动脉(MCAO)永久性缺血模型,随机分为模型组;TSA 15mg/kg、30mg/kg、60mg/kg给药组;安宫牛黄丸400mg/kg照组,必存7mg/kg对照组以及假手术组。于恢复早期3—14天给药,缺血后3d,7d,14d酶联免疫吸附法(ELISA)检测缺血侧大脑皮层糖原合成酶3(GSK3β)的含量;缺血后7d,14d,Western-blot法检测缺血侧皮层:β连环素(β-catenin)以及c-myc的表达;Real-Time PCR法检测β-catenin mRNA、c-myc mRNA、的水平。结果与假手术组比较,模型组皮层GSK3β的含量在3-7d升高,14d下降,与模型组比较,缺血后3-14d,TSA对GSK3β的表达无统计学差异。缺血后7d,与假手术组比较,模型组c-myc、c-mycmRNA以及β-catenin、β-catenin mRNA含量明显增加。与模型组比较,TSA 60mg/kg大鼠大脑皮层c-myc蛋白含量明显增加(P＜0.01);TSA中剂量组c-mycmRNA表达量明显增加(P＜0.05);TSA中,大剂量组,β-catenin mRNA表达量显著增加(P＜0.05)。缺血后14d,与假手术组比较,模型组c-myc、c-mycmRNA以及β-catenin、β-catenin mRNA表达均降低。与模型组比较,TSA各剂量组β-cateninmRNA和c-myc mRNA表达量均明显增高,(P＜0.05);并且TSA促进β-cateninmRNA与c-myc mRNA表达的增加趋势一致;TSA大剂量组β-catenin蛋白和c-myc蛋白含量均明显增高(P＜0.05,P＜0.05),并且TSA促进β-catenin和c-myc蛋白表达的增加趋势一致。结论TSA通过上调β-catenin基因及蛋白的表达而激活了Wnt/β-catenin信号转导通路,TSA有可能通过这一途径促进了脑缺血后大鼠的内源性神经发生和血管新生的病理生理反应事件,增强了大鼠神经血管网络的重建和组织的自体修复,改善脑缺血后大鼠的神经功能障碍。课题创新点:1.本课题结合临床中多数脑缺血病人错过急性期治疗时间窗导致后遗症的现状,针对脑缺血神经功能的恢复早期进行药物TSA在动物模型中的干预,探讨TSA对这一阶段脑组织功能重建的影响,切入时间点具有新颖性,是对脑缺血急性期与恢复期之间中药干预时间窗可能性研究的一个积极补充。2.围绕脑缺血后神经功能缺损的行为学表现,结合促血管生成相关因子(VEGF/VEGFR2,Ang)和神经发生的Wnt/β-catenin经典信号调控通路中关键因子β-catenin、GSK3β,c-myc,水平的变化展开TSA对脑组织缺血后神经发生和血管新生作用的机理探讨。更多还原

【Abstract】 Ischemic stroke is extremely harmful because of its high morbidity,mortality and disability incidence.The effective treatments of ischemic stroke so far are thrombolysis with recombinant tissue plasminogen activator(rtPA) in 6h of onset.The restrictions on time and potential adverse effects have limited the use of rtPA to approximately 2%-5%of stroke patients in the United States.Meanwhile the ischemia-reperfuion lesion is the main issue of thrombolysis with rtPA. How to improve the neurological function recovery after stroke is the important therapeutic items in early recovery stage for most patients which can not receive the therapy of thrombolysis in time.Cerebral ischemia can activate the neurogenesis in adult mammal brain,which induce neuroal progenitor cells to divide,proliferate and migrate to the lesion region and differentiate to be mature neurons that partly take place of the missing specific neuron after ischemia. Neurogenesis is close related to angiogenesis therefore the key therapeutic phase of ischemic cerebrovascular disease is to reconstruct the blood supply in ischemic area and to retrieve injured neurons,neuroglia cells and endothelia cells as soon as possible.Motivating endogenous neurogenesis and angiogenesis following cerebral ischemia could be an original clinical therapeutic tool.Vascular endothelial growth factor(VEGF) and angiopoietin(Ang) are valued angiogenesis factor which have the role to protect neurons and stimulate neurogenesis.The effect of Ang1 and Ang2 relies on the role of VEGF.VEGF is probably one of the key factors of regulation mechanism linking both neurogenesis and angiogenesis.Wnt/β-catenin is a classic signal pathway to regulate the proliferation and differentiation of neuroal stem cells on the nerves system development.β-catenin promote the proliferation of NSC and determine its differentiation fate.What the study mainly concerns is the level of Ang1,Ang2, VEGF/VEGFR2 and the expression of several key factorsβ-catenin,GSK3β,c-myc of Wnt/β-catenin signal pathway as well as the relationship between the cytokines and neurogenesis & angiogenesis following ischemic stroke.Steroidal Saponin TSA is the major active components of anemarrhenae.The activation of anemarrhenae has been reported in the neuroprotective manifestations on acute cerebral ischemia rats model.Regulating the blood vessel endothelium function of ischemic brain, anti-thrombopoiesis and improving blood supply are the effective mechanisms of TSA.While the researches to TSA on the neurogenesis and angiogenesis after cerebral ischemia haven’t been reported so far.Here,in the study we were interested in the effect of TSA on the recovery of neurological function after cerebral ischemia,using middle cerebral artery occlusion model during a relatively long term of 14 days.We investigated the effect of TSA on the pathogenicchange and neurogenesis and angiogenesis in the early reparative period post ischemia in brain.So that we might provide experimental evidence for TSA clinical application and explore the rationality of optimal therapeutic window with Chinese medicine intervention on cerebral infarction.1.Effects of TSA on the recovery of neurological function after cerebral ischemia in rats and its primary mechanisms1.1 Effects of TSA on the recovery of neurological function and angiogenesis after cerebral ischemia in ratsAim To investigate the effect of TSA on the recovery of neurological function after cerebral ischemia in rats and whether it is involved in the procedure of angiogenesisMethods Cerebral ischemia was induced by middle cerebral artery occlusion in rats which were departed into 6 groups:sham-operated,vehicle-treated,TSA 15mg/kg、30mg/kg、60mg/kg, angongniuhuang 400mg/kg group,Ischemic medication Animals received intragastric administration once a day 3-14day post-ischemia.At the same time rats in sham operation and vehicle group were lavaged with 5%CMC which was used to suspend TSA.Neurological functionwas evaluated with beam-walking,adhesivetape-exposing and bar-grasping performance at 3,7,10,and 14day post ischemia.Animals were killed at 14day post ischemia. HE staining and nissl staining to observe the injury of neuron of sensorimotor region of cortex.Immunohistochemistry methods were used to evaluate the localization and expression of VEGF and VEGFR2 and C D34-labeled MVD of the ischemic boundary zone.Result Vehicle-treated rats showed severe neurological disfuction compared with sham-operated group.Rats receiving TSA at a dose of 30 mg/kg and 60mg/kg had a more rapid recovery of beam-walking and adhesivetape-exposing performance than vehicle-treated rats, and the improvement became significant at 14 days after ischemia(P＜0.05,P＜0.01).There is no significance between TSA treated and vehicle-treated rats on the bar-grasping evaluating. Treatment with TSA 30mg/kg、60mg/kg also significantly improved the expression of the VEGF and VEGFR2 and the micrvascular density(MVD) of the ischemic boundary zone 14days post ischemia.(P＜0.05,P＜0.01).Conclusion The results demonstrate that TSA can improve the recovery of sensorimotor function follwing focal cerebral ischemia in rats.The abitity of TSA might result from its promotion of angiogenesis mediated by VEGF and VEGFR2 after ischemia.1.2.Effects of TSA on the proliferation of neuronal progenitor cell after cerebral ischemia in ratsAim To investigate the effect of TSA on the proliferation of neural progenitor cell after cerebral ischemia in rats and whether it is involved in the procedure of n eurogenesis.Methods Cerebral ischemia was induced by middle cerebral artery occlusion in rats which were departed into 6 groups:sham-operated,vehicle-treated,TSA 15mg/kg、30mg/kg、60mg/kg and angongniuhuang 400mg/kg group,Ischemic medication Animals received intragastric administration once a day 3-14day post-ischemia at the same time rats in sham operation and vehicle group were lavaged with 5%CMC which was used to suspend TSA.BrdU i.p 50mg/kg.d,at 2-7day to label the proliferated cells post ischemia.Animals were killed on day7 and day14.Brains were made into brain sections.Imunofluorescence and immunohistochemistry methods were used to evaluate the proliferation of neuronal progenitor cell in SVZ by Brdu-labeled cells number and the expression of nestin protein of the ischemic boundary zone.Result:Vehicle-treated rats showed increase of Brdu positive cells in the SVZ and the expression of nestin protein compared with sham-operated group.TSA 30mg/kg、60mg/kg increased the number of Brdu positive cells in the SVZ and the expression of nestin protein at day7 and day14 post ischemia(P＜0.05,P＜0.01).Conclusion The results demonstrate that TSA can improve the proliferation of neural progenitor cell follwing focal cerebral ischemia in rats.The ability of TSA might result from its promotion of neurogenesis by upgrading VEGF and VEGFR2 after ischemia.And the promotion of neurological function induced by TSA might come from its effect of neurogenesis promotion.2 Effects of TSA on the recovery of neurological function after permanent cerebral ischemia in rats and its primary mechanismsAim To investigate the effect of TSA o n the recovery of neurological function after permanent cerebral ischemia in rats and whether it is involved in the procedure of angiogenesis and neurogenesis.Methods Permanent Cerebral ischemia was induced by permanent middle cerebral artery occlusion(pMCAO) in rats which were departed into7groups:sham-operated,vehicle-treated, TSA 15mg/kg、30mg/kg、60mg/kg and angongniuhuang 400mg/kg and bicun 7mg/kg group, Ischemic medication Animals received intragastric administration except bicun i.p.once a day 3-14day post-ischemia at the same time rats in sham operated and vehicle group were lavaged with 5%CMC which was used to suspend TSA.BrdU i.p 50mg/kg.d,at 1-7day to label the proliferated cells post ischemia.Neurological functionwas evaluated with beam-walking, adhesivetape-exposing and bar-grasping performance at 3,7 and 14day post ischemia.Animals were partly killed 14days post ischemia.Brains were made into ice brain sections. Imunofluorescence methods were used to evaluate the proliferation,migration and differentiation of neuronal progenitor cell in cortex and stritum of ischemic boundary zone by Brdu/Dcx, Brdu/Neun,Brdu/GFAP labeled cells.Angiogenesis was valued with microvascular density (MVD) labeled by Brdu/factorⅧin cortex and stritum of ischemic boundary zone.Result:Vehicle-treated rats showed severe neurological disfuction compared with sham-operated group.Rats receiving TSA at a dose of 30 mg/kg and 60mg/kg had a more rapid recovery of beam-walking,adhesivetape-exposing and bar-grasping performance than vehicle -treated rats,and the improvement became significant at 14 days after ischemia(P＜0.05,P＜0.01).Vehicle-treated rats showed increase number of Brdu/Dcx,Brdu/Neun,Brdu/GFAP positive cells and MVD labeled by Brdu/factorⅧin the ischemic boundary zone compared with sham-operated group.TSA 30mg/kg、60mg/kg increased the number of Brdu/Dcx positive cells and MVD in ischemic boundary zone of cortex 14days(P＜0.05,P＜0.01) and the number of Brdu/Neun cell in ischemic boundary zone of cortex 28days after ischemia (P＜0.05).There is no significance on Brdu/GFAP labeled cells in cortex and stritum compared with vehicle treated. Conclusion The results demonstrate that TSA can improve the neurological function follwing focal permanent cerebral ischemia in rats.Which might related to the promotion of neurogenesis and angiogenesis and the reestablishing of function of neuron-vascular system improved by TSA.3 Effects of TSA on the signaling pathway of neurogenesis and angiogenesis after permanent cerebral ischemia in rats3.1 Effect of TSA on the regulating pathway of Ang1/Ang2 and VEGF/VEGFR2 in angiogenesis after permanent cerebral ischemia in ratsAim To investigate the effect of TSA on the expression of Ang1/Ang2 and VEGF/VEGFR2 in angiogenesis after permanent cerebral ischemia in ratsMethods Permanent Cerebral ischemia was induced by permanent middle cerebral artery occlusion(pMCAO) in rats which were departed into7groups:sham-operated,vehicle-treated, TSA 15mg/kg、30mg/kg、60mg/kg and angongniuhuang 400mg/kg and bicun 7mg/kg group, Ischemic medication Animals received intragastric administration except bicun by i.p.once a day 3-14day post-ischemia at the same time rats in sham operated and vehicle group were lavaged with 5%CMC which was used to suspend TSA.Animals were killed at 3,7 and 14 days post ischemia.ELISA methods were used to evaluate the expression of Ang 1 and/Ang2 of ischemic cortex.3,7 and 14 days post ischemia.Western blot methods were used to evaluate the expression of VEGF and real time PCR methods were used to evaluate the expression of VEGF mRNA and VEGFR2 mRNA of ischemic cortex 7 and 14 days post ischemia.Result:the expression of Ang1 of Vehicle-treated rats decreased compared with sham-operated group 3,7 and 14 days post ischemia.Rats receiving TSA at a dose of 30 mg/kg and 60mg/kg and angongniuhuang group showed a significant increase of expression of Ang1 at 7days post ischemia compared with vehicle-treated rats(P＜0.05,P＜0.01),and the expression of Ang2 was not significant difference within all groups at 3,7 and 14 days post ischemia.TSA at a dose of 60mg/kg significantly increased the expression of VEGFR2mRNA but had no effect on the expression of VEGF and VEGFmRNA. Conclusion The results demonstrate that TSA can improve the neurological function follwing focal permanent cerebral ischemia in rats.Which might related to the upregulating of the expression of Ang1 and VEGFR2mRNA and the angiogenesis induced by Ang1.3.2 Effect of TSA on the Wnt/β-catenin signaling pathway after permanent cerebral ischemia in ratsAim To investigate the effect of TSA on the expression ofβ-catenin、c-myc,GSK3βin the Wnt/β-catenin signaling pathway after permanent cerebral ischemia in rats.Methods Permanent Cerebral ischemia was induced by permanent middle cerebral artery occlusion(pMCAO) in rats which were departed into7groups:sham-operated,vehicle-treated, TSA 15mg/kg、30mg/kg、60mg/kg and angongniuhuang 400mg/kg and bicun 7mg/kg group, Ischemic medication Animals received intragastric administration except bicun by i.p.once a day 3-14day post-ischemia at the same time rats in sham operated and vehicle group were lavaged with 5%CMC which was used to suspend TSA.Animals were killed at 3,7 and 14 days post ischemia.ELISA methods were used to evaluate the expression of GSK3βof ischemic cortex.3,7 and 14 days post ischemia.Western blot methods were used to evaluate the expression ofβ-catenin and c-myc and real time PCR methods were used to evaluate the expression ofβ-catenin mRNA and c-myc mRNA of ischemic cortex 7 and 14 days post ischemia.Result:The expression of GSK3βof Vehicle-treated rats increased in 3-7day and decreased in 14days post ischemia compared with sham-operated group.Rats receiving TSA group showed no significant of expression of GSK3βat 3-14days post ischemia compared with vehicle-treated rats.The expression of c-myc of vehicle-treated rats significantly increased 7days post ischemia compared with sham-operated group(p＜0.01).TSA 60mg/kg significantly increased the expression of c-myc(p＜0.01) and TSA 30mg/kg significantly increased the expression of c-mycmRNA7days post ischemia compared with vehicle-treated.TSA30mg/kg,60mg/kg significantly increased the expression ofβ-cateninmRNA (p＜0.05) 7days post ischemia compared with vehicle-treated.The expression of c-myc, c-mycmRNA andβ-catenin,β-catenin mRNA of vehicle-treated decreased compared with sham-operated group 14days post ischemia.TSA at all dose group significantly increased the expression ofβ-catenin mRNA and c-mycmRNA compared with vehicle-treated(P＜0.05) 14days post ischemia and the increased trend is same betweenβ-catenin mRNA and c-mycmRNA.Besides TSA 60mg/kg group significantly increased the expression ofβ-catenin and c-myc compared with vehicle-treated(P＜0.05) and the increased trend is same betweenβ-catenin and c-myc 14days post ischemia.Conclusion The results demonstrate that TSA can stimulatethe the Wnt/β-catenin signaling pathway after permanent cerebral ischemia in rats by upregulating the the expression ofβ-cateninmRNA andβ-catenin protein.Which might related to the promotion of angiogenesis and neurogenesis procedure and neurological function recovery follwing focal permanent cerebral ischemia in rats.The innovation of the research1.Thinking of the situation that most stroke patients miss the best theropetic window and leave Sequelae,in the study,TSA was adopted to intervene in the early recovery stage of neurological function in animal models after cerebral ischemia to explore the effect of TSA on the reconstruction of brain function.The timepoint of the intervention is a novelty and also is a positive complement of drug intervention on the subacute and early stage of recovery of cerebral ischemia.2.The researcher observed the behavioral performance of neurological deficit and detect the level of VEGF,VEGFR2 and Angiopoietin in angiogenesis-regulated path way andβ-catenin,GSK3-β,c-myc,in neurogenesis-regulated classic Wnt/β-catenin signal pathway to explore the effect and mechanism of TSA on neurogenesis and angiogenesis after cerebral ischemia in rats.更多还原