通窍活血汤对脑缺血模型大鼠血脑屏障紧密连接等蛋白表达的影响及其脑内移行成分的分析

Blood-brain barrier（BBB）is a complex cell system which lies beween brain tissue and blood.It can regulate the transport of substances of two sides between brain tissue and blood.Whether the drug could penetrate the BBB is one of the key factors on the protection on the neurocytes.TongQiaoHuoXue Decoction(TQHXD) has affirmative effect on cerebral arterial thrombosis,on our previous study,we carred out preliminary resrarch on proctetive effect of TQHXD on nerve cells and analysis the chemical constituents from TQHXD.On basis of these research,the changes of expression of proteins on BBB such as tight junction proteins, transport protein which can affect drugs penetrating BBB will have been investigated.By the method of cerebrospinal fluid chemistry,the difference of constituents in cerebrospinal fluid in normal and cerebral ischemia rats will have been compared.Chemical constituents in the cerebrospinal fluid are oriented to trace its source by comparing the fingerprint of constituents in cerebrospinal fluid of cerebral ischemia rats undergoing intragastric administration single medicinal material,the fingerprint of chemical constituents from TQHXD and single medicinal materials.Thus we can analyse,identify,extract the effective constituents and confirmation its pharmacodynamics of protective effects on nerve cells and influence of expression of proteins on BBB models in vitro,confirm function of the constituents in cerebrospinal fluid in formulae, discuss compatible regularity of the formulae, uncover pharmacodynamic substance of protective effects of TQHXD on nerve cells damaged by glutamate,elucidate scientific connotation of "Blood-activating and orifice-opening",also provide train of thought for pharmacodynamics screening,the quality controlling of formulae on prevention and treatment of cerebral vascular disease.

血脑屏障（BBB）控制血脑两侧的物质转运。药物能否透过BBB成为研究中药神经保护作用的关键因素之一。通窍活血汤（TQHXD）治疗脑卒中疗效确切，在前期基础上，探讨TQHXD对脑缺血大鼠BBB上影响药物通过的蛋白如紧密连接蛋白、转运蛋白等表达变化的影响。采用脑脊液药物化学方法，比较大鼠正常及脑缺血状态下BBB对TQHXD中成分通透性的差别，以TQHXD在脑缺血大鼠脑内移行成分为指纹导向，将其分别和各单味药脑内移行成分、TQHXD及各单味药的指纹图谱进行比对，分析、鉴定脑内移行成分，追踪神经保护作用的药效学及其对体外血脑屏障模型相关蛋白表达的影响，明确移行成分在复方中的作用，探讨复方的配伍规律，揭示TQHXD神经保护作用的药效物质基础，阐明TQHXD"活血通窍"的科学内涵，也为防治脑血管疾病的中药复方的药效学筛选、质量控制等提供思路。

采用大脑中动脉阻塞法建立了局灶性脑缺血损伤大鼠模型；HE法观察通窍活血汤（TQHXD）对脑缺血损伤大鼠BBB组织病理变化；透射电镜观察TQHXD对脑缺血损伤大鼠海马神经元及半球额顶区皮质BBB的超微结构的影响；Western Blotting法检测TQHXD对脑缺血损伤大鼠BBB上紧密连接蛋白（ZO-1,Occludin,Claudin-5）、AQP-4和MMP-9的表达水平的影响。采用脑脊液药物化学方法，比较大鼠正常及脑缺血状态下BBB对TQHXD中成分通透性的差别，以TQHXD在脑缺血大鼠脑内移行成分为指纹导向，将其分别和各单味药脑内移行成分、TQHXD及各单味药的指纹图谱进行比对，分析、鉴定脑内移行成分，追踪移行成分神经保护作用的药效学及其对体外血脑屏障模型相关蛋白表达的影响，明确移行成分在复方中的作用，揭示TQHXD神经保护作用的药效物质基础，阐明TQHXD“活血通窍”的科学内涵。     .研究结果如下：HE染色及透射电镜观察结果显示，TQHXD各剂量可显著改善大鼠缺血侧大脑损伤的形态学结构，神经元的损伤情况较模型组大鼠有明显的改善作用，并可减少BBB紧密连接的开放。TQHXD可抑制脑缺血大鼠ZO-1，Occludin，Claudin-5表达的下降，抑制AQP-4蛋白、MMP-9表达量的增加。GC/MS分析显示在脑缺血大鼠脑脊液中检测到麝香酮、藁本内酯、羟基红花黄色素A，分别来自麝香、川芎、红花，在空白脑脊液中均未出现。麝香酮、藁本内酯、羟基红花黄色素A对谷氨酸损伤的PC12有显著的保护作用，改善损伤细胞的形态，提高活细胞比率。藁本内酯、麝香酮可减少谷氨酸所致的Ca2+内流, 抑制Caspase-3 活性,升高Bcl-2/Bax 比值来保护谷氨酸诱导的PC12 细胞凋亡。藁本内酯可降低缺糖缺氧损伤BBB的HIF-1α、VEGF表达的增加，抑制紧密连接蛋白ZO-1、Occludin表达的减少，抑制AQP-4表达的升高。 .综上所述，TQHXD对脑缺血模型大鼠海马区神经元具有一定保护作用，该作用与TQHXD上调ZO-1,Occludin,Claudin-5表达水平，下调AQP-4,MMP-9表达水平有关。麝香酮、藁本内酯、羟基红花黄色素A可以透过血脑屏障，且对损伤的神经细胞有保护作用，可作为通窍活血汤神经保护的生物活性成分。.