通塞脉微丸治疗缺血性脑中风的物质基础研究
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摘要
本论文由文献综述和实验研究两部分组成。
文献综述包括中药复方效应物质基础的研究思路与方法及脑微血管内皮细胞和缺血性脑损伤两篇。文献研究为本实验设计提供了思路与方法。
实验研究按照血清药物化学的研究方法，着眼于中药口服吸收后的血中移行成分，以药物能否透过血脑屏障和对靶细胞-脑微血管内皮细胞的保护作用两个关键环节为切入点来探讨通塞脉微丸治疗缺血性脑中风的效应物质基础。
第一部分对通塞脉微丸体内外化学成分进行了分析。首先采用HPLC-UV技术，对样品中有UV吸收的部分进行了较全面的定性、定量分析，建立了10批样品的指纹图谱，并对所有特征峰所归属的药材和化合物进行了推断。以HPLC双波长法对通塞脉微丸中含量较高的6个成分进行了定量分析。以此为化学基础，进一步开展通塞脉微丸的血清药物化学的研究，以大鼠为实验动物，灌胃给药后制备含药血清，采用HPLC-UV法比较通塞脉微丸原药液、大鼠灌胃给药后含药血清和空白血清，找出血中移行的色谱峰，并根据各峰的相对保留时间和在线UV光谱图，通过与原方的各单味药制剂含药血清相比较，判定血中移行成分来源的药材和归属的化合物。
第二部分开展通塞脉微丸有效成分透过大鼠血脑屏障的研究。首先研究通塞脉微丸有关成分透过正常血脑屏障的情况，采用体内法，选用大鼠作为模型动物，灌胃给药后将动物麻醉，于大鼠脑枕骨大孔处抽取脑脊液，适当处理后进行HPLC-UV和HPLC-MS分析。进一步研究了通塞脉微丸中相关成分透过缺血性损伤大鼠血脑屏障的情况。采用线栓法复制大鼠大脑中动脉栓塞(MACO)模型，造成脑缺血损伤而致的血脑屏障通透性改变，灌胃给药后，从心脏灌流冲洗至脑组织中无血液，然后取脑组织，适当处理后以HPLC-UV和HPLC-MS法检测分析透过血脑屏障的成分。
第三部分以通塞脉微丸复方为研究对象，采用线栓法复制大鼠大脑中动脉栓塞(MACO)模型，以脑组织TTC染色测量脑梗塞面积，神经功能检查法对脑缺血后动物进行行为测评，病理及超微组织观察作为药效评价指标，考察通塞脉微丸对缺血性脑损伤的保护作用。
第四部分研究通塞脉微丸及其有效成分对大鼠脑微血管内皮细胞缺血性损伤的干预作用。首先进行大鼠脑微血管内皮细胞(CMEC)的原代培养研究：采用出生5～10天的大鼠乳鼠，无菌获取脑组织，以胶原酶组织消化，分离脑微血管段，两次过筛提取脑微血管内皮细胞，并进行传代培养。经倒置显微镜下细胞形态学观察，Ⅷ因子免疫组化检测进行细胞鉴定。然后以化学性缺血的方法模拟建立大鼠CMEC体外缺血性损伤模型，以细胞形态学改变和MTT、LDH值作为细胞活性的评价指标。试验分别考察了通塞脉微丸及其有效成分对正常CMEC的干预作用和在CMEC缺血性损伤情况下药物的保护作用。
第五部分探讨了通塞脉微丸调控CMEC的黏附机制。采用双抗夹心法，选择性地检测了E-选择素、ICAM-1两个黏附蛋白的表达。
实验研究结果：
一．通塞脉微丸体内外化学成分分析结果
1．通塞脉微丸的HPLC-UV指纹图谱研究标定了以甘草苷为参照峰的18个共有指纹峰，获得了通塞脉微丸的HPLC指纹图谱的技术参数，采用《中药色谱指纹图谱相似度评价系统(2004A版)》，测定了10批样品的指纹图谱，整体相似度在0.968以上。对18个共有指纹峰的来源分析：有1个峰来源于当归药材，鉴定为阿魏酸；有5个峰来源于金银花，其中1个鉴定为绿原酸，其余4个从光谱图上推断可能为类绿原酸类化合物；有3个峰来源于玄参，2个分别鉴定为肉桂酸和哈巴俄苷，另1个未知；有8个峰来源于甘草，其中3个分别鉴定为甘草苷、甘草素和甘草酸铵，另4个峰具有甘草黄酮母核的吸收特征；还有1个未知归属的化合物。
2．以HPLC双波长法在一定色谱条件下一次进样同时检测通塞脉微丸中相对含量较高的绿原酸、阿魏酸、肉桂酸、哈巴俄苷、甘草苷、甘草素6个成分的含量，所测6个成分的色谱峰与相邻色谱峰均能得到良好的分离，方法学实验结果良好。针对丁基苯酞具有增加缺血区脑血流量、缩小脑梗塞面积、减轻脑水肿等与复方疗效密切相关的作用，特别建立了微丸中丁基苯酞的含量测定方法。为其可能和微丸中其它成分一起发挥抗缺血性脑损伤作用提供了化学基础。
3．通塞脉微丸血清药物化学研究结果：含药血清、空白血清、通塞脉微丸原液、已知7个混合对照品相比：有12个入血成分，其中有6个以原型吸收入血，经与对照品保留时间和在线UV光谱对照，发现绿原酸、阿魏酸、甘草素和肉桂酸四个已知成分以原型吸收入血，另各有一个来源于金银花和甘草的未知成分以原型吸收入血。有3个为代谢产物吸收入血，经与单味药制剂含药血清对照，探明有1个来源于当归的代谢产物，另2个来源于甘草的代谢产物。含药血清中尚有3个入血成分归属不明确。
二．通塞脉微丸有效成分透过大鼠血脑屏障的研究结果
1．对于正常大鼠，经HPLC-UV和HPLC-MS法检测，含药脑脊液与空白脑脊液相比，含药脑脊液中未检测到通塞脉微丸中相关成分。但在T_8=51.6min、56.2min、60.1min和61.9min处脑脊液中四个内源性物质色谱峰强度明显增强(从峰面积比值来看分别增加了为4.5倍、4倍、3.3倍和4.5倍)。
2．正常组与假手术组大鼠脑组织中可检测到有微量通塞脉微丸的成分，经HPLC-UV和HPLC-MS鉴定为阿魏酸、甘草素、肉桂酸；在线栓法复制大鼠MACO模型所致的脑缺血损伤情况下，阿魏酸、甘草素、肉桂酸进入脑组织的量显著增加，组间比较有显著性差异(p＜0.05)。该研究结果提示阿魏酸、甘草素、肉桂酸作为通塞脉微丸的部分药效学物质基础，在缺血损伤时可透过血脑屏障较大量进中枢神经系统，达到相应损伤区域，作用于中风病的关键部位上而发挥作用。
三．通塞脉微丸对MACO模型大鼠的保护作用研究
MACO术后所有试验动物均出现了不同程度的神经功能障碍。在24h后，通塞脉微丸各剂量组动物的行为学评分均呈下降趋势，且显著低于模型组(p＜0.05)，提示通塞脉微丸可以改善模型动物的行为学障碍。各给药组大鼠的脑梗塞面积和梗塞率均明显低于模型组(p＜0.05)，提示通塞脉微丸可明显缩小模型大鼠的脑梗塞面积，降低梗塞率。模型大鼠脑指数和含水量均明显高于假手术组，通塞脉微丸高剂量可降低模型大鼠的脑指数及脑含水量(p＜0.05)，提示通塞脉微丸可以减轻脑水肿，高中剂量组作用尤为显著。病理组织学观察，线栓法缺血性脑中风大鼠模型，表现为脑组织不同程度坏死，胶质细胞及间质水肿。通塞脉微丸对脑中风模型在不同程度上有减轻脑组织损伤作用。
四．通塞脉微丸及其有效成分对大鼠脑微血管内皮细胞的干预作用
1．本实验原代培养的大鼠CMEC呈长梭形或多角形，单层汇合呈铺路石征象；Ⅷ因子免疫细胞化学染色阳性。经鉴定符合脑微血管内皮细胞的特征。
2．以化学性缺氧的方法建立CMEC体外模拟缺血损伤模型，模型组细胞缺氧损伤后，从形态学观察细胞先肿胀，折光度减弱，然后皱缩。MTT法测定细胞OD值，造模后6～12h均处于明显下降阶段(P＜0.05)，最终确定终浓度为0.85mg·mL~(-1) Na_2S_2O_4溶液作用于正常细胞8h以造缺血损伤模型。
3．药物对正常脑微血管内皮细胞的干预作用：通塞脉微丸高剂量组与阴性对照组比较，有显著性差异(P＜0.05)，表现为抑制细胞活性，中剂量组表现为促进细胞活性(P＜0.05)，小剂量作用不显著。剂量和细胞活性的总趋势呈钟形，表现为大剂量抑制细胞活性，中剂量范围促进细胞活性。
通塞脉微丸大鼠灌胃给药后的血清，高剂量组与空白血清对照组相比，表现为促进细胞活性(P＜0.01)。通塞脉微丸中有效成分阿魏酸、甘草素在高剂量组与DMSO对照组相比，表现出促进细胞活性的作用(P＜0.05)。
微丸中其它相关成分：丁基苯酞、黄芪甲苷在高、中剂量组与DMSO对照组相比，表现为促进细胞活性(P＜0.05)。绿原酸在中剂量组与DMSO对照组相比，表现为促进细胞活性(P＜0.05)。高剂量绿原酸表现出抑制细胞活性(P＜0.05)。
4．药物对缺血性损伤脑微血管内皮细胞的干预作用：通塞脉微丸给药浓度1000、500μg·mL~(-1)高剂量组与模型组比较，有极显著性差异(P＜0.01)，表现为抑制细胞活性。小剂量组与模型组比较，有显著差异(P＜0.05)，表现为促进细胞活性。
通塞脉微丸大鼠灌胃给药后的血清，高、中剂量组与空白血清对照组相比，表现为促进细胞活性(P＜0.05)。微丸中有效成分：除肉桂酸、甘草素低剂量外，阿魏酸、甘草素、肉桂酸在各剂量组均表现出一定的促进细胞活性的作用，与DMSO对照组相比，有显著性差异(P＜0.05)。
微丸中其它相关成分：丁基苯酞、黄芪甲苷在高、中剂量组与DMSO对照组相比，均表现为促进细胞活性(P＜0.05)。绿原酸在中、低剂量组与DMSO对照组相比，表现为促进细胞活性(P＜0.05)。中剂量甘草苷、高剂量哈巴俄苷也表现出一定的细胞保护作用(P＜0.05)。
五．通塞脉微丸调控脑微血管内皮细胞的黏附机制
以双抗夹心法法检测E-选择素和ICAM-1的表达量，结果表明CMEC细胞缺血8h，E-选择素和ICAM-1均表达升高，与正常比较有显著性差异(P＜0.01)。通塞脉微丸作用于缺血CMEC后，能够下调E-选择素和ICAM-1，与缺血细胞比较，有显著差异(P＜0.05)，接近恢复到正常细胞的表达值(P＞0.05)。
结论：
通过以上实验，综合分析认为：
1．通塞脉微丸血清药物化学的初步研究结果，其制备的含药血清是微丸在体内真正发挥药效的“制剂”，探明的部分血中的移行成分绿原酸、阿魏酸、甘草素、肉桂酸是可能的部分效应物质基础。
2．阿魏酸、甘草素、肉桂酸，在脑缺血损伤时可透过血脑屏障较大量进中枢神经系统，作为通塞脉微丸的部分药效学物质基础，达到相应损伤区域，作用于中风病的关键靶点(神经元及神经胶质细胞)上而发挥作用。
3．通塞脉微丸及其相关成分、微丸含药血清能够促进正常CMEC增殖，提高缺血性损伤CMEC的活性。从药物作用的靶细胞-脑微血管内皮细胞的角度验证了阿魏酸、甘草素、肉桂酸是通塞脉微丸治疗缺血性脑中风的部分药效学物质基础。
4．CMEC缺血时启动了黏附机制，通塞脉微丸干预缺血CMEC，能够下调因缺血损伤而高表达的E-选择素和ICAM-1，提示通塞脉微丸可通过对E-选择素素和ICAM-1两条通道的影响，抑制了黏附反应的启动从而阻抑CMEC缺血损伤。
5．以现代细胞培养技术获得的脑微血管内皮细胞，可作为体外研究脑血管疾病的重要工具和载体。
This thesis is composed of two parts, literatures review and experimental research.
Literatures review contains two pieces, the thinking study and the method for the substancebasis of chinese traditional medicine compounds, and, brain capillary endothelial cells and hypox-ic-ischemic brain damage. Literatures research has offered clew and method for this experimentdesigning.
Experimental study is according to research methods of serum pharmacochemistry, with aview to the migration compositions in blood after chinese traditional medicine oral administratio--n, according to whether drugs can permeate the blood-brain barrier or have protection on targetcell-CMEC, the two key links as cut-in drop, investigating the substance basis of the effect oftreating ischemic apoplexy and paralysis by Tongsaimai.
The first part of this thesis analyzes the chemical composition inside and outside body ofTongsaimai. First, adopting technique of HPLC-UV, and carry through quite overall qualitativeand quantitative analysis for the fractions having UV absorption, and build fingerprint of 10 bat--ch samples, and deduce the medicinal materials and compounds which are attributive to all cha-racteristic peaks. With double waves method of HPLC carry through quantitative analysis to the6 upper determination components in Tongsaimai pellets. As this as Chemical basis, further ca--rry on the preliminary study on serum pharmacochemistry of Tongsaimai pellets, as rats for ex--perimental animal, and prepare serum contained bushentiaochong after the rats are dosed by irri-gating stomach, applying the method of HPLC-UV compare the Tongsaimai pellets raw liquor,The serum contained medicine after the rats and blank serum, questing chromatographic peak ofmigration from blood, and according to the relative retention time and online UV spectrogramof each peak, deduce the medicinal materials where the the migration compositions in blood co--me from and the attributive compounds, by contrast to the serum contained medicine of eachsimple preparation.
The second part, carry on the study on effective component of Tongsaimai pellets enteringthe brain through the blood-brain barrier. First, research the attributive compounds of Tongsaimaipellets permeating the normal blood-brain barrier, adopting in vivo method, choice rats as anim-al pattern, anesthesia the animals after rats dosed by irrigating stomach, extracting cerebrospinal
fluid in the foramen magnum from the brain of rats, after some special process, analysis in HPLC-UV and HPLC-MS. Experiments do more study on correlation components of Tongsaimaipellets permeating the ischemia damage blood-brain barrier of rats. Rats cerebral MACO modelis replicated with the suture method, shaping ischemia damage which lead to the alter of perm-eability of blood brain barrier, after rats are dosed by irrigating stomach, chafe on from cardiacperfusion to there is no blood in brain tissue, then choose the brain tissue, after some specialprocess, analysis the component which permeating the blood brain barrier by HPLC-UV andHPLC-MS.
The third part, with the complex prescription of Tongsaimai pellets as research subjects, fro-m integer animal level, rats cerebral MACO model is replicated with the suture method, measu-re brain infarct size with TTC coloration, go along behavioral measurement and envalument forthe cerebral ischemia animal with neural function check method, and investigate the protectionof pharmic with the observation of pathological mechanism and ultramicro organizations.
The fourth part, we study on Tongsaimai pellets and their effective components' interventio--n effect on cerebral micrangium endothelial cells ischemic damage of rats. First, carry on thestudy on rats' cerebral micrangium endothelial cells primary culture: 5-10 days borned rats suckl-ing mouse are used as experiment animal, acquisition brain tissue in asepsis environment, digest-ion tissues by collagenase isolate microvessels, cribration twice for extracting cerebral micrangiu-m endothelial cells, and subculture. Identify cells by cellular morphology observation, factorⅧimmunohist-ochemical detection under the invertmicroscope. Then build rats in-vitro ischemicdamage modle by the method chemical ischemic, with the alterlation of cellular morphology andthe value of MTT and LDH as the evaluating indicator of cytoactive. Experiments investigatingintervention active of Tongsaimai pellets andtheir effective components on the normal CMECand the protection for the ischemic damage CMEC differently.
The fifth part, we discuss the adhesion mechanism of Tongsaimaipellets control CMEC. Ad--opting enzyme linked immune sandwich assay technique, we checkout the expression of two ad-hesion Protein selectivity, E-selection and ICAM-1.
Experimental findings:
Ⅰ. The analysis result of Tongsaimai pellets' chemical component inside and outside of bod--y
1. The study on HPLC-UV fingerprint of Tongsaimai pellets demarcates 18 common fingerp-rint peaks with glycyrrhiza uralensis glycoside as compared peak, get the technical parameter ofTongsaimai pellets' HPLC fingerprint, adopting＜similarity evaluation of chromatographic finge rprint fortcm (2004A Edition)＞, we measured10 batches samples chromatography fingerprint integ-er degree of similarity is above 0.968, and elicit the contrast fingerprint. The analysis of thesource of 18 common fingerprint peaks: one peak come from angelica sinensis materia medicwhich is identified as ferulic acid; five peaks come from flos lonicerae, one of them is identifie-d as chlorogenic acid, the others can be deduced as similar chlorogenic acid components fromthe spectrogram; there peaks come from ningpo figwort, the two of them are identified as cinna-mic acid and ahbar khozaschet glycoside, the other one is unknown; 8 peaks come from glycyr-rhiza uralensis, there of them are identified as glycyrrhiza uralensis glycoside, liquiriti genin, an-d ammonium glycyrrhizinate, the other four have absorbing character of licoflavone mother nucl-eus; and there are a compound we don't know the adscription.
2. We inject once and checkout the upper 6 components chlorogenic acid, ferulic acid, cinn--amic acid, haba khozaschet glycoside, glycyrrhiza uralensis glycoside, liquiriti genin in Tongsai--mai pellets at one time by the method HPLC double waves method, the chromatographic peakof the 6 components we measured can Separate from the adjacent ones finely, the experimentalresult of methodology is fine. In allusion to butyl phthalide having the function of increasing ofcerebral blood flow to ischemic tissue, shrink cerebral infarct size, and reduction of brain edem,which has intimate correlative with complex prescription curative effect, we build the contentmeasuring method of butyl phthalide especially. The result affords the experimental foundationfor the function of anti ischemia damage with other components in Yongsaimai pellets.
3.The preliminary findings of medicinal chemistry Tongsaimai pellets: The serum containedmedicine, blank serum, raw liquor of Tongsaimai pellets, and 7 known mixed reference substa-nce, they compare: there are 12 entered blood components, the 6 of them are absorbed intoblood by archetype, we find chlorogenic acid, ferulic acid, liquiriti genin, and cinnamic acid,the four known components are absorbed into blood by archetype, the other two are absorbedinto blood by archetype too, one is a unknown component of flos lonicerae, the other one is aunknown component of glycyrrhiza uralensis, by contrast to the retention time and UV actionspectrum of reference substance. The 3 of them are absorbed into blood by metabolic product,we prove one of them come from metabolic product of angelica sinensis, the other two comefrom metabolic product of glycyrrhiza uralensis, by contrast to the serum contained medicineof simple preparation. And the attribution of the 3 of components in-blood of the serum contai-ned medicine, are unknown, maybe are the metabolic product of drug interaction, and maybe are physiology stress substance brought by rats' body after dosed of Tongsaimai complex presc-ription.
Ⅱ. The findings of effective component of Tongsaimai pellets permeating the the blood-brai-n barrier of rats
1.For the normal rats, we compared the cerebrospinal fluid contained medicine to the blankcerebrospinal fluid, we didn't detect the correlation component of Tongsaimai pellets, by themethod of HPLC-UV and HPLC-MS. The peak intensity of four endogenous substance in cerebr-ospinal fluid chromatogram peak stronger obviously at retention time 51.6 min, 56.2 min, 60.1min, 61.9min (we can see they stronger 6.5, 4, 3.3,4.5 times from the specific value peak areadifferently).
2.We can detect the components of Tongsaimai pellets in groups of normal and sham oper-ation, we prove them as ferulic acid, liquiriti genin, and cinnamic acid by HPLC-UV and HPLC-MS; In conditions of brain ischemia damage brought by the MACO model of replicated by ins-erting nylon thread, the capacity into brain tissue of ferulic acid, liquiriti genin, and cinnamicacid add notability (P＜0.05). The finding indicated ferulic acid, liquiriti genin, and cinnamic acid as the substance basis of part pharmacodynamics of Tongsaimai pellets, can permeate the blood-brain barrier when the brain is ischemia damage, can go to corresponding damage area, act on the key sites and bring into play effect.
Ⅲ.The study on the protection of Yongsaimai pellets for the MACO model of rats
All experimental animal appear differ degree nerve dysfunction. After 24h, the assign markof behavioristics of each dosage group animals appear downtrend, and lower than the model gro-w up obviously (P＜0.05), indicating Tongsaimai pellets can improve the nerve dysfunction ofmodel animals. The brain infarction area and infarct hasty of each dosed groups rats all lowerthan the model group obviously (P＜0.05), indicating Tongsaimai pellets can contract the braininfarction area, drop the infarct hasty. The cerebral exponential and moisture content of modelrats are upper than the sham operation group, the large dosage of Yongsaimai pellets can lowthe cerebral exponential and moisture content of model rats (P＜0.05), indicating Tongsaimai pel—lets can improve brain edema, the action of larger dosage is more obvious. From histopathology observation, the ischemic apoplexy rat model of replicated by inserting nylon thread, becomeas brain tissue differ degree necrosis, gliocyte and seroma edema. The Tongsaimai pellets havethe action lower the brain tissue damage on different degree on apoplexy model.
Ⅵ.Tongsaimai pellets and the effective components have intervention action on CMEC
1. The CMEC of rats primary cultured in this experiment are Long spindle and polygon,Signs were paving stones single convergence ;Ⅷfactor immune cytochemical staining are mascu-line. We proved it to be according to cha-racteristics of CMEC.
2. We build CMEC ischemia damage models in vitro by the method of chemical lack ano--xic, the model cells have been damage of anoxic, we can see from the morphological the cellswill strut first and the diopter will be bate, and than collapse. The method of MTT determinethe OD value of cells, after we building model 6-12h, the value is at a distinct decline phase(P＜0.05), we Ultimately determine to use the sugar Ealer's solution and the concentration of0.85 mg·mL~(-1) solution Na_2S_2O_4 role in normal cell 8h to create the model of ischemic injur--y.
3. For normal brain microvascular endothelial cells: We compare Tongsaimai pellets highdose group to negative control group, we can see a significant difference (P＜0.05), curb cell act-ivity, Performance for cell activity dose group (P＜0.05).Low dose group compared with the con-trol group, no significant difference. The general trend of dose and the cell activity is bell-shap-ed, the cells showed inhibition of high-dose, middle dose range Promote for the activity.
The serum after Tongsaimai pellets were administrated, High dose group compared with theblank group, Performance for cell activity (P＜0.01).The effective components in Yongsaimai pe--llets, ferulic acid, liquiriti genin in the high-dose group compared with DMSO control group,shown to promote cell activity (P＜0.05).
The other components in pellets: butylphthalide, astragalus A in the high and middle dosegroup compared with DMSO control group, performance for cell activity (P＜0.05). Chlorogenicacid in middle-dose group compared with the DMSO control group, the performance for cell act-ivity (P＜0.05). High doses of chlorogenic acid showed inhibition (P＜0.05)
4. For ischemic injury in brain microvascular endothelial cells: The dosage of Tongsaimaipellets at 1000,500μg·mL~(-1) of high-dose group compared with the model group, there was a sig--nificant difference (P＜0.01), showed inhibition activity. Low-dose group compared with the mod-el group, we can see a significant difference (P＜0.05), promote the performance of the activity.
The serum after Tongsaimai pellets were administrated, High and middle dose group compa-red with the blank group, performance for cell activity (P＜0.05). The active ingredients in Pell--ets: except cinnamic acid and low-dose licorice, ferulic acid, isoliquiritigenin, einnamie acid inall dose groups have shown activity in promoting the activity of cells, compare with the controlgroup DMSO, we can see a significant difference (P＜0.05).
The other components in pellets: butylphthalide, astragalus A in the high dose group comp--ared with the control group DMSO, have shown to promote cell activity (P＜0.05). Chlorogenicacid in the low-dose and middle-dose group compared with the control group DMSO, to pro--mote the performance of cells (P＜0.05). Glycyrrhiza uralensis glycoside of middle dose, harpag--oside of high dose, all have shown a protective effect (P＜0.05).
ⅤThe adhesion mechanism of Tongsaimai pellets control CMEC
Adopting enzyme linked immune sandwich assay technique, we checkout the expression oftwo adhesion Protein, E-selection and ICAM-1, we can see the results after CMEC 8h ischemiashowed that expression of both E-selection and ICAM-1 increased, a significant difference com-pared with normal (P＜0.01). After Tongsaimai pellets role in ischemia, reduced E-selection andICAM-1, compare with ischemic cells, we can see a significant difference (P＜0.05), which clos-e to the normal value of the expression (P＞0.05).
Conclusion:
By this experiment, a comprehensive analysis:
1.The preliminary findings of medicinal chemistry Tongsaimai pellets,pellets containing seru--m was prepared by its in vivo efficacy of the play "agents", among some of the blood compo--nents transitional chlorogenic acid, ferulic acid, liquiriti genin, cinnamic acid, it is possible toeffect a material foundation.
2.Ferulic acid, liquiriti genin, cinnamic acid can be much more into the central nervous sys-tem through the blood-brain barrier when ischemic injury, as part of Pharmacodynamics materia-1 foundation of Tongsaimai pellets, to achieve the corresponding injury area, act on the key tar-get of apoplexy (nerve cell and glial celt ) and bring into play effect.
3.Tongsaimai pellets and related components, and the serum Containing pellets, can promotenormal CMEC proliferation, enhance the activity of ischemic injury CMEC. We proved ferulicacid, liquiriti genin, cinnamic acid are the Pharmacodynamics material foundation of treatment ofischemic stroke, from the target cells-brain microvascular endothelial cells.
4.CMEC started the adhesion mechanism when ischemia, Tongsaimai pellets intervention is--chemic CMEC, can reduce the high expression of E-selection and ICAM-1 due to ischemic inj--ury, suggest Tongsaimai pellets can inhibit the adhesion inhibited CMEC ischemic injury, byimpacting E-selection and ICAM-1 in the two channels.
5.Brain microvascular endothelial cells drived from cell cultured by the modern technology,can be used as an important tool in vitro study of cerebrovascular disease and vector.
文献综述包括中药复方效应物质基础的研究思路与方法及脑微血管内皮细胞和缺血性脑损伤两篇。文献研究为本实验设计提供了思路与方法。
实验研究按照血清药物化学的研究方法，着眼于中药口服吸收后的血中移行成分，以药物能否透过血脑屏障和对靶细胞-脑微血管内皮细胞的保护作用两个关键环节为切入点来探讨通塞脉微丸治疗缺血性脑中风的效应物质基础。
第一部分对通塞脉微丸体内外化学成分进行了分析。首先采用HPLC-UV技术，对样品中有UV吸收的部分进行了较全面的定性、定量分析，建立了10批样品的指纹图谱，并对所有特征峰所归属的药材和化合物进行了推断。以HPLC双波长法对通塞脉微丸中含量较高的6个成分进行了定量分析。以此为化学基础，进一步开展通塞脉微丸的血清药物化学的研究，以大鼠为实验动物，灌胃给药后制备含药血清，采用HPLC-UV法比较通塞脉微丸原药液、大鼠灌胃给药后含药血清和空白血清，找出血中移行的色谱峰，并根据各峰的相对保留时间和在线UV光谱图，通过与原方的各单味药制剂含药血清相比较，判定血中移行成分来源的药材和归属的化合物。
第二部分开展通塞脉微丸有效成分透过大鼠血脑屏障的研究。首先研究通塞脉微丸有关成分透过正常血脑屏障的情况，采用体内法，选用大鼠作为模型动物，灌胃给药后将动物麻醉，于大鼠脑枕骨大孔处抽取脑脊液，适当处理后进行HPLC-UV和HPLC-MS分析。进一步研究了通塞脉微丸中相关成分透过缺血性损伤大鼠血脑屏障的情况。采用线栓法复制大鼠大脑中动脉栓塞(MACO)模型，造成脑缺血损伤而致的血脑屏障通透性改变，灌胃给药后，从心脏灌流冲洗至脑组织中无血液，然后取脑组织，适当处理后以HPLC-UV和HPLC-MS法检测分析透过血脑屏障的成分。
第三部分以通塞脉微丸复方为研究对象，采用线栓法复制大鼠大脑中动脉栓塞(MACO)模型，以脑组织TTC染色测量脑梗塞面积，神经功能检查法对脑缺血后动物进行行为测评，病理及超微组织观察作为药效评价指标，考察通塞脉微丸对缺血性脑损伤的保护作用。
第四部分研究通塞脉微丸及其有效成分对大鼠脑微血管内皮细胞缺血性损伤的干预作用。首先进行大鼠脑微血管内皮细胞(CMEC)的原代培养研究：采用出生5～10天的大鼠乳鼠，无菌获取脑组织，以胶原酶组织消化，分离脑微血管段，两次过筛提取脑微血管内皮细胞，并进行传代培养。经倒置显微镜下细胞形态学观察，Ⅷ因子免疫组化检测进行细胞鉴定。然后以化学性缺血的方法模拟建立大鼠CMEC体外缺血性损伤模型，以细胞形态学改变和MTT、LDH值作为细胞活性的评价指标。试验分别考察了通塞脉微丸及其有效成分对正常CMEC的干预作用和在CMEC缺血性损伤情况下药物的保护作用。
第五部分探讨了通塞脉微丸调控CMEC的黏附机制。采用双抗夹心法，选择性地检测了E-选择素、ICAM-1两个黏附蛋白的表达。
实验研究结果：
一．通塞脉微丸体内外化学成分分析结果
1．通塞脉微丸的HPLC-UV指纹图谱研究标定了以甘草苷为参照峰的18个共有指纹峰，获得了通塞脉微丸的HPLC指纹图谱的技术参数，采用《中药色谱指纹图谱相似度评价系统(2004A版)》，测定了10批样品的指纹图谱，整体相似度在0.968以上。对18个共有指纹峰的来源分析：有1个峰来源于当归药材，鉴定为阿魏酸；有5个峰来源于金银花，其中1个鉴定为绿原酸，其余4个从光谱图上推断可能为类绿原酸类化合物；有3个峰来源于玄参，2个分别鉴定为肉桂酸和哈巴俄苷，另1个未知；有8个峰来源于甘草，其中3个分别鉴定为甘草苷、甘草素和甘草酸铵，另4个峰具有甘草黄酮母核的吸收特征；还有1个未知归属的化合物。
2．以HPLC双波长法在一定色谱条件下一次进样同时检测通塞脉微丸中相对含量较高的绿原酸、阿魏酸、肉桂酸、哈巴俄苷、甘草苷、甘草素6个成分的含量，所测6个成分的色谱峰与相邻色谱峰均能得到良好的分离，方法学实验结果良好。针对丁基苯酞具有增加缺血区脑血流量、缩小脑梗塞面积、减轻脑水肿等与复方疗效密切相关的作用，特别建立了微丸中丁基苯酞的含量测定方法。为其可能和微丸中其它成分一起发挥抗缺血性脑损伤作用提供了化学基础。
3．通塞脉微丸血清药物化学研究结果：含药血清、空白血清、通塞脉微丸原液、已知7个混合对照品相比：有12个入血成分，其中有6个以原型吸收入血，经与对照品保留时间和在线UV光谱对照，发现绿原酸、阿魏酸、甘草素和肉桂酸四个已知成分以原型吸收入血，另各有一个来源于金银花和甘草的未知成分以原型吸收入血。有3个为代谢产物吸收入血，经与单味药制剂含药血清对照，探明有1个来源于当归的代谢产物，另2个来源于甘草的代谢产物。含药血清中尚有3个入血成分归属不明确。
二．通塞脉微丸有效成分透过大鼠血脑屏障的研究结果
1．对于正常大鼠，经HPLC-UV和HPLC-MS法检测，含药脑脊液与空白脑脊液相比，含药脑脊液中未检测到通塞脉微丸中相关成分。但在T_8=51.6min、56.2min、60.1min和61.9min处脑脊液中四个内源性物质色谱峰强度明显增强(从峰面积比值来看分别增加了为4.5倍、4倍、3.3倍和4.5倍)。
2．正常组与假手术组大鼠脑组织中可检测到有微量通塞脉微丸的成分，经HPLC-UV和HPLC-MS鉴定为阿魏酸、甘草素、肉桂酸；在线栓法复制大鼠MACO模型所致的脑缺血损伤情况下，阿魏酸、甘草素、肉桂酸进入脑组织的量显著增加，组间比较有显著性差异(p＜0.05)。该研究结果提示阿魏酸、甘草素、肉桂酸作为通塞脉微丸的部分药效学物质基础，在缺血损伤时可透过血脑屏障较大量进中枢神经系统，达到相应损伤区域，作用于中风病的关键部位上而发挥作用。
三．通塞脉微丸对MACO模型大鼠的保护作用研究
MACO术后所有试验动物均出现了不同程度的神经功能障碍。在24h后，通塞脉微丸各剂量组动物的行为学评分均呈下降趋势，且显著低于模型组(p＜0.05)，提示通塞脉微丸可以改善模型动物的行为学障碍。各给药组大鼠的脑梗塞面积和梗塞率均明显低于模型组(p＜0.05)，提示通塞脉微丸可明显缩小模型大鼠的脑梗塞面积，降低梗塞率。模型大鼠脑指数和含水量均明显高于假手术组，通塞脉微丸高剂量可降低模型大鼠的脑指数及脑含水量(p＜0.05)，提示通塞脉微丸可以减轻脑水肿，高中剂量组作用尤为显著。病理组织学观察，线栓法缺血性脑中风大鼠模型，表现为脑组织不同程度坏死，胶质细胞及间质水肿。通塞脉微丸对脑中风模型在不同程度上有减轻脑组织损伤作用。
四．通塞脉微丸及其有效成分对大鼠脑微血管内皮细胞的干预作用
1．本实验原代培养的大鼠CMEC呈长梭形或多角形，单层汇合呈铺路石征象；Ⅷ因子免疫细胞化学染色阳性。经鉴定符合脑微血管内皮细胞的特征。
2．以化学性缺氧的方法建立CMEC体外模拟缺血损伤模型，模型组细胞缺氧损伤后，从形态学观察细胞先肿胀，折光度减弱，然后皱缩。MTT法测定细胞OD值，造模后6～12h均处于明显下降阶段(P＜0.05)，最终确定终浓度为0.85mg·mL~(-1) Na_2S_2O_4溶液作用于正常细胞8h以造缺血损伤模型。
3．药物对正常脑微血管内皮细胞的干预作用：通塞脉微丸高剂量组与阴性对照组比较，有显著性差异(P＜0.05)，表现为抑制细胞活性，中剂量组表现为促进细胞活性(P＜0.05)，小剂量作用不显著。剂量和细胞活性的总趋势呈钟形，表现为大剂量抑制细胞活性，中剂量范围促进细胞活性。
通塞脉微丸大鼠灌胃给药后的血清，高剂量组与空白血清对照组相比，表现为促进细胞活性(P＜0.01)。通塞脉微丸中有效成分阿魏酸、甘草素在高剂量组与DMSO对照组相比，表现出促进细胞活性的作用(P＜0.05)。
微丸中其它相关成分：丁基苯酞、黄芪甲苷在高、中剂量组与DMSO对照组相比，表现为促进细胞活性(P＜0.05)。绿原酸在中剂量组与DMSO对照组相比，表现为促进细胞活性(P＜0.05)。高剂量绿原酸表现出抑制细胞活性(P＜0.05)。
4．药物对缺血性损伤脑微血管内皮细胞的干预作用：通塞脉微丸给药浓度1000、500μg·mL~(-1)高剂量组与模型组比较，有极显著性差异(P＜0.01)，表现为抑制细胞活性。小剂量组与模型组比较，有显著差异(P＜0.05)，表现为促进细胞活性。
通塞脉微丸大鼠灌胃给药后的血清，高、中剂量组与空白血清对照组相比，表现为促进细胞活性(P＜0.05)。微丸中有效成分：除肉桂酸、甘草素低剂量外，阿魏酸、甘草素、肉桂酸在各剂量组均表现出一定的促进细胞活性的作用，与DMSO对照组相比，有显著性差异(P＜0.05)。
微丸中其它相关成分：丁基苯酞、黄芪甲苷在高、中剂量组与DMSO对照组相比，均表现为促进细胞活性(P＜0.05)。绿原酸在中、低剂量组与DMSO对照组相比，表现为促进细胞活性(P＜0.05)。中剂量甘草苷、高剂量哈巴俄苷也表现出一定的细胞保护作用(P＜0.05)。
五．通塞脉微丸调控脑微血管内皮细胞的黏附机制
以双抗夹心法法检测E-选择素和ICAM-1的表达量，结果表明CMEC细胞缺血8h，E-选择素和ICAM-1均表达升高，与正常比较有显著性差异(P＜0.01)。通塞脉微丸作用于缺血CMEC后，能够下调E-选择素和ICAM-1，与缺血细胞比较，有显著差异(P＜0.05)，接近恢复到正常细胞的表达值(P＞0.05)。
结论：
通过以上实验，综合分析认为：
1．通塞脉微丸血清药物化学的初步研究结果，其制备的含药血清是微丸在体内真正发挥药效的“制剂”，探明的部分血中的移行成分绿原酸、阿魏酸、甘草素、肉桂酸是可能的部分效应物质基础。
2．阿魏酸、甘草素、肉桂酸，在脑缺血损伤时可透过血脑屏障较大量进中枢神经系统，作为通塞脉微丸的部分药效学物质基础，达到相应损伤区域，作用于中风病的关键靶点(神经元及神经胶质细胞)上而发挥作用。
3．通塞脉微丸及其相关成分、微丸含药血清能够促进正常CMEC增殖，提高缺血性损伤CMEC的活性。从药物作用的靶细胞-脑微血管内皮细胞的角度验证了阿魏酸、甘草素、肉桂酸是通塞脉微丸治疗缺血性脑中风的部分药效学物质基础。
4．CMEC缺血时启动了黏附机制，通塞脉微丸干预缺血CMEC，能够下调因缺血损伤而高表达的E-选择素和ICAM-1，提示通塞脉微丸可通过对E-选择素素和ICAM-1两条通道的影响，抑制了黏附反应的启动从而阻抑CMEC缺血损伤。
5．以现代细胞培养技术获得的脑微血管内皮细胞，可作为体外研究脑血管疾病的重要工具和载体。
This thesis is composed of two parts, literatures review and experimental research.
Literatures review contains two pieces, the thinking study and the method for the substancebasis of chinese traditional medicine compounds, and, brain capillary endothelial cells and hypox-ic-ischemic brain damage. Literatures research has offered clew and method for this experimentdesigning.
Experimental study is according to research methods of serum pharmacochemistry, with aview to the migration compositions in blood after chinese traditional medicine oral administratio--n, according to whether drugs can permeate the blood-brain barrier or have protection on targetcell-CMEC, the two key links as cut-in drop, investigating the substance basis of the effect oftreating ischemic apoplexy and paralysis by Tongsaimai.
The first part of this thesis analyzes the chemical composition inside and outside body ofTongsaimai. First, adopting technique of HPLC-UV, and carry through quite overall qualitativeand quantitative analysis for the fractions having UV absorption, and build fingerprint of 10 bat--ch samples, and deduce the medicinal materials and compounds which are attributive to all cha-racteristic peaks. With double waves method of HPLC carry through quantitative analysis to the6 upper determination components in Tongsaimai pellets. As this as Chemical basis, further ca--rry on the preliminary study on serum pharmacochemistry of Tongsaimai pellets, as rats for ex--perimental animal, and prepare serum contained bushentiaochong after the rats are dosed by irri-gating stomach, applying the method of HPLC-UV compare the Tongsaimai pellets raw liquor,The serum contained medicine after the rats and blank serum, questing chromatographic peak ofmigration from blood, and according to the relative retention time and online UV spectrogramof each peak, deduce the medicinal materials where the the migration compositions in blood co--me from and the attributive compounds, by contrast to the serum contained medicine of eachsimple preparation.
The second part, carry on the study on effective component of Tongsaimai pellets enteringthe brain through the blood-brain barrier. First, research the attributive compounds of Tongsaimaipellets permeating the normal blood-brain barrier, adopting in vivo method, choice rats as anim-al pattern, anesthesia the animals after rats dosed by irrigating stomach, extracting cerebrospinal
fluid in the foramen magnum from the brain of rats, after some special process, analysis in HPLC-UV and HPLC-MS. Experiments do more study on correlation components of Tongsaimaipellets permeating the ischemia damage blood-brain barrier of rats. Rats cerebral MACO modelis replicated with the suture method, shaping ischemia damage which lead to the alter of perm-eability of blood brain barrier, after rats are dosed by irrigating stomach, chafe on from cardiacperfusion to there is no blood in brain tissue, then choose the brain tissue, after some specialprocess, analysis the component which permeating the blood brain barrier by HPLC-UV andHPLC-MS.
The third part, with the complex prescription of Tongsaimai pellets as research subjects, fro-m integer animal level, rats cerebral MACO model is replicated with the suture method, measu-re brain infarct size with TTC coloration, go along behavioral measurement and envalument forthe cerebral ischemia animal with neural function check method, and investigate the protectionof pharmic with the observation of pathological mechanism and ultramicro organizations.
The fourth part, we study on Tongsaimai pellets and their effective components' interventio--n effect on cerebral micrangium endothelial cells ischemic damage of rats. First, carry on thestudy on rats' cerebral micrangium endothelial cells primary culture: 5-10 days borned rats suckl-ing mouse are used as experiment animal, acquisition brain tissue in asepsis environment, digest-ion tissues by collagenase isolate microvessels, cribration twice for extracting cerebral micrangiu-m endothelial cells, and subculture. Identify cells by cellular morphology observation, factorⅧimmunohist-ochemical detection under the invertmicroscope. Then build rats in-vitro ischemicdamage modle by the method chemical ischemic, with the alterlation of cellular morphology andthe value of MTT and LDH as the evaluating indicator of cytoactive. Experiments investigatingintervention active of Tongsaimai pellets andtheir effective components on the normal CMECand the protection for the ischemic damage CMEC differently.
The fifth part, we discuss the adhesion mechanism of Tongsaimaipellets control CMEC. Ad--opting enzyme linked immune sandwich assay technique, we checkout the expression of two ad-hesion Protein selectivity, E-selection and ICAM-1.
Experimental findings:
Ⅰ. The analysis result of Tongsaimai pellets' chemical component inside and outside of bod--y
1. The study on HPLC-UV fingerprint of Tongsaimai pellets demarcates 18 common fingerp-rint peaks with glycyrrhiza uralensis glycoside as compared peak, get the technical parameter ofTongsaimai pellets' HPLC fingerprint, adopting＜similarity evaluation of chromatographic finge rprint fortcm (2004A Edition)＞, we measured10 batches samples chromatography fingerprint integ-er degree of similarity is above 0.968, and elicit the contrast fingerprint. The analysis of thesource of 18 common fingerprint peaks: one peak come from angelica sinensis materia medicwhich is identified as ferulic acid; five peaks come from flos lonicerae, one of them is identifie-d as chlorogenic acid, the others can be deduced as similar chlorogenic acid components fromthe spectrogram; there peaks come from ningpo figwort, the two of them are identified as cinna-mic acid and ahbar khozaschet glycoside, the other one is unknown; 8 peaks come from glycyr-rhiza uralensis, there of them are identified as glycyrrhiza uralensis glycoside, liquiriti genin, an-d ammonium glycyrrhizinate, the other four have absorbing character of licoflavone mother nucl-eus; and there are a compound we don't know the adscription.
2. We inject once and checkout the upper 6 components chlorogenic acid, ferulic acid, cinn--amic acid, haba khozaschet glycoside, glycyrrhiza uralensis glycoside, liquiriti genin in Tongsai--mai pellets at one time by the method HPLC double waves method, the chromatographic peakof the 6 components we measured can Separate from the adjacent ones finely, the experimentalresult of methodology is fine. In allusion to butyl phthalide having the function of increasing ofcerebral blood flow to ischemic tissue, shrink cerebral infarct size, and reduction of brain edem,which has intimate correlative with complex prescription curative effect, we build the contentmeasuring method of butyl phthalide especially. The result affords the experimental foundationfor the function of anti ischemia damage with other components in Yongsaimai pellets.
3.The preliminary findings of medicinal chemistry Tongsaimai pellets: The serum containedmedicine, blank serum, raw liquor of Tongsaimai pellets, and 7 known mixed reference substa-nce, they compare: there are 12 entered blood components, the 6 of them are absorbed intoblood by archetype, we find chlorogenic acid, ferulic acid, liquiriti genin, and cinnamic acid,the four known components are absorbed into blood by archetype, the other two are absorbedinto blood by archetype too, one is a unknown component of flos lonicerae, the other one is aunknown component of glycyrrhiza uralensis, by contrast to the retention time and UV actionspectrum of reference substance. The 3 of them are absorbed into blood by metabolic product,we prove one of them come from metabolic product of angelica sinensis, the other two comefrom metabolic product of glycyrrhiza uralensis, by contrast to the serum contained medicineof simple preparation. And the attribution of the 3 of components in-blood of the serum contai-ned medicine, are unknown, maybe are the metabolic product of drug interaction, and maybe are physiology stress substance brought by rats' body after dosed of Tongsaimai complex presc-ription.
Ⅱ. The findings of effective component of Tongsaimai pellets permeating the the blood-brai-n barrier of rats
1.For the normal rats, we compared the cerebrospinal fluid contained medicine to the blankcerebrospinal fluid, we didn't detect the correlation component of Tongsaimai pellets, by themethod of HPLC-UV and HPLC-MS. The peak intensity of four endogenous substance in cerebr-ospinal fluid chromatogram peak stronger obviously at retention time 51.6 min, 56.2 min, 60.1min, 61.9min (we can see they stronger 6.5, 4, 3.3,4.5 times from the specific value peak areadifferently).
2.We can detect the components of Tongsaimai pellets in groups of normal and sham oper-ation, we prove them as ferulic acid, liquiriti genin, and cinnamic acid by HPLC-UV and HPLC-MS; In conditions of brain ischemia damage brought by the MACO model of replicated by ins-erting nylon thread, the capacity into brain tissue of ferulic acid, liquiriti genin, and cinnamicacid add notability (P＜0.05). The finding indicated ferulic acid, liquiriti genin, and cinnamic acid as the substance basis of part pharmacodynamics of Tongsaimai pellets, can permeate the blood-brain barrier when the brain is ischemia damage, can go to corresponding damage area, act on the key sites and bring into play effect.
Ⅲ.The study on the protection of Yongsaimai pellets for the MACO model of rats
All experimental animal appear differ degree nerve dysfunction. After 24h, the assign markof behavioristics of each dosage group animals appear downtrend, and lower than the model gro-w up obviously (P＜0.05), indicating Tongsaimai pellets can improve the nerve dysfunction ofmodel animals. The brain infarction area and infarct hasty of each dosed groups rats all lowerthan the model group obviously (P＜0.05), indicating Tongsaimai pellets can contract the braininfarction area, drop the infarct hasty. The cerebral exponential and moisture content of modelrats are upper than the sham operation group, the large dosage of Yongsaimai pellets can lowthe cerebral exponential and moisture content of model rats (P＜0.05), indicating Tongsaimai pel—lets can improve brain edema, the action of larger dosage is more obvious. From histopathology observation, the ischemic apoplexy rat model of replicated by inserting nylon thread, becomeas brain tissue differ degree necrosis, gliocyte and seroma edema. The Tongsaimai pellets havethe action lower the brain tissue damage on different degree on apoplexy model.
Ⅵ.Tongsaimai pellets and the effective components have intervention action on CMEC
1. The CMEC of rats primary cultured in this experiment are Long spindle and polygon,Signs were paving stones single convergence ;Ⅷfactor immune cytochemical staining are mascu-line. We proved it to be according to cha-racteristics of CMEC.
2. We build CMEC ischemia damage models in vitro by the method of chemical lack ano--xic, the model cells have been damage of anoxic, we can see from the morphological the cellswill strut first and the diopter will be bate, and than collapse. The method of MTT determinethe OD value of cells, after we building model 6-12h, the value is at a distinct decline phase(P＜0.05), we Ultimately determine to use the sugar Ealer's solution and the concentration of0.85 mg·mL~(-1) solution Na_2S_2O_4 role in normal cell 8h to create the model of ischemic injur--y.
3. For normal brain microvascular endothelial cells: We compare Tongsaimai pellets highdose group to negative control group, we can see a significant difference (P＜0.05), curb cell act-ivity, Performance for cell activity dose group (P＜0.05).Low dose group compared with the con-trol group, no significant difference. The general trend of dose and the cell activity is bell-shap-ed, the cells showed inhibition of high-dose, middle dose range Promote for the activity.
The serum after Tongsaimai pellets were administrated, High dose group compared with theblank group, Performance for cell activity (P＜0.01).The effective components in Yongsaimai pe--llets, ferulic acid, liquiriti genin in the high-dose group compared with DMSO control group,shown to promote cell activity (P＜0.05).
The other components in pellets: butylphthalide, astragalus A in the high and middle dosegroup compared with DMSO control group, performance for cell activity (P＜0.05). Chlorogenicacid in middle-dose group compared with the DMSO control group, the performance for cell act-ivity (P＜0.05). High doses of chlorogenic acid showed inhibition (P＜0.05)
4. For ischemic injury in brain microvascular endothelial cells: The dosage of Tongsaimaipellets at 1000,500μg·mL~(-1) of high-dose group compared with the model group, there was a sig--nificant difference (P＜0.01), showed inhibition activity. Low-dose group compared with the mod-el group, we can see a significant difference (P＜0.05), promote the performance of the activity.
The serum after Tongsaimai pellets were administrated, High and middle dose group compa-red with the blank group, performance for cell activity (P＜0.05). The active ingredients in Pell--ets: except cinnamic acid and low-dose licorice, ferulic acid, isoliquiritigenin, einnamie acid inall dose groups have shown activity in promoting the activity of cells, compare with the controlgroup DMSO, we can see a significant difference (P＜0.05).
The other components in pellets: butylphthalide, astragalus A in the high dose group comp--ared with the control group DMSO, have shown to promote cell activity (P＜0.05). Chlorogenicacid in the low-dose and middle-dose group compared with the control group DMSO, to pro--mote the performance of cells (P＜0.05). Glycyrrhiza uralensis glycoside of middle dose, harpag--oside of high dose, all have shown a protective effect (P＜0.05).
ⅤThe adhesion mechanism of Tongsaimai pellets control CMEC
Adopting enzyme linked immune sandwich assay technique, we checkout the expression oftwo adhesion Protein, E-selection and ICAM-1, we can see the results after CMEC 8h ischemiashowed that expression of both E-selection and ICAM-1 increased, a significant difference com-pared with normal (P＜0.01). After Tongsaimai pellets role in ischemia, reduced E-selection andICAM-1, compare with ischemic cells, we can see a significant difference (P＜0.05), which clos-e to the normal value of the expression (P＞0.05).
Conclusion:
By this experiment, a comprehensive analysis:
1.The preliminary findings of medicinal chemistry Tongsaimai pellets,pellets containing seru--m was prepared by its in vivo efficacy of the play "agents", among some of the blood compo--nents transitional chlorogenic acid, ferulic acid, liquiriti genin, cinnamic acid, it is possible toeffect a material foundation.
2.Ferulic acid, liquiriti genin, cinnamic acid can be much more into the central nervous sys-tem through the blood-brain barrier when ischemic injury, as part of Pharmacodynamics materia-1 foundation of Tongsaimai pellets, to achieve the corresponding injury area, act on the key tar-get of apoplexy (nerve cell and glial celt ) and bring into play effect.
3.Tongsaimai pellets and related components, and the serum Containing pellets, can promotenormal CMEC proliferation, enhance the activity of ischemic injury CMEC. We proved ferulicacid, liquiriti genin, cinnamic acid are the Pharmacodynamics material foundation of treatment ofischemic stroke, from the target cells-brain microvascular endothelial cells.
4.CMEC started the adhesion mechanism when ischemia, Tongsaimai pellets intervention is--chemic CMEC, can reduce the high expression of E-selection and ICAM-1 due to ischemic inj--ury, suggest Tongsaimai pellets can inhibit the adhesion inhibited CMEC ischemic injury, byimpacting E-selection and ICAM-1 in the two channels.
5.Brain microvascular endothelial cells drived from cell cultured by the modern technology,can be used as an important tool in vitro study of cerebrovascular disease and vector.
引文
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