石菖蒲活性成分醒脑开窍、透过血脑屏障作用机制研究
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摘要
血脑屏障(blood-brain bartier，BBB)是在脑和脊髓的毛细血管与神经组织之间存在的一个动态的调节界面，能够阻止绝大多数外源性物质透过，并选择性的将脑内有害物质或过剩物质泵出脑外，维持脑内微环境的稳定。但是，BBB对许多有价值的中枢神经系统(CNS)药物的通透性也很低，严重影响了药物的疗效。因此，关于BBB的研究越来越多的受到人们的关注。
中药中芳香类药物“醒脑开窍”的作用主要是指开窍启闭、宣发神气，调神定志，使心神复明，现代医学认为是增加BBB的通透性，促进其他CNS药物进入脑组织，发挥疗效。石菖蒲是传统的芳香开窍药之一，民间单用或与其他中药配伍使用广泛，主要用于脑病的治疗，有确切的疗效，提示石菖蒲对BBB有一定作用。本文主要研究石菖蒲醒脑开窍、透过血脑屏障的有效成分及其作用机制，为中药“芳香走窜、引药上行”提供理论基础和实验依据。
按照《中国药典》2005版一部附录挥发油提取法甲法，提取挥发油，提取率约为1.58％，去油水煎液浓缩至1g／ml。
采用气相色谱-质谱(GC／MS)联用仪检测挥发油样品中含有α—细辛醚、β—细辛醚、愈创木烯、菖蒲烯酮，相同条件下检测服用挥发油后大鼠的脑脊液，结果显示α—细辛醚，β—细辛醚可透过BBB进入脑脊液。
采用液相色谱-质谱(HPLC／MS)联用仪测定去油水煎液样品及灌服去油水煎液的大鼠脑脊液样品，检测到三个化合物，分子量分别为396，452，339，已知石菖蒲化合物中尚未包括。
透射电镜下观察给药前后大鼠大脑皮层BBB的超微结构，可发现石菖蒲各提取部分均可使BBB内皮细胞之间的紧密连接(tight junction，TJ)疏松，但BBB基本完整，尚未破坏。
以冰片为阳性药，初步探讨石菖蒲各提取部分对伊文思兰(EB)透过BBB的促进作用，即以EB在脑内的含量反映BBB的通透性，统计分析表明冰片在灌胃2.5h后脑内EB含量显著高于空白组(P＜0.01)，石菖蒲在11.7g／kg剂量下自给药0.5h至2.5h未见明显作用；延长石菖蒲给药时间，石菖蒲挥发油剂量为23.4g／kg和11.7g／kg时，5h后脑内EB含量显著高于空白组(P＜0.05)，且有剂量依赖性趋势；总药剂量为11.7g／kg时，5h后脑内EB含量显著高于空白组(P＜0.01)。
石菖蒲各提取部分给予小鼠灌胃一周后，腹腔注射苯妥英钠，采用HPLC方法检测苯妥英钠在脑内的浓度，结果显示：石菖蒲总组动物脑内苯妥英钠的含量较高于未给石菖蒲组动物脑内的苯妥英钠含量，4h后仍保持较高浓度(P＜0.01)。
利用HPLC-ECD检测脑组织内5-羟色胺(5-HT)的浓度，统计分析得：与空白组比较，挥发油组小鼠大脑中5-HT的含量增加(P＜0.05)，石菖蒲水煎液组及总药组中5-HT的含量显著增加(P＜0.01)。
应用Hela细胞系作为评价石菖蒲对BBB上P-糖蛋白活性的载体，0.5h时石菖蒲各部分(挥发油、水煎液、总药)可明显增强长春新碱(VCR)的细胞毒性(P＜0.01)；8h后挥发油部分仍可明显增强VCR的细胞毒性(P＜0.01)，挥发油的药效维持时间明显长于阳性药维拉帕米(VER)(P＜0.01)；24h后，阳性药及石菖蒲各部分对VCR的细胞毒性无明显影响(P＞0.05)。
完成上述工作的基础上，采用两次酶消化、梯度离心的方法分离得到纯度较高的脑微血管内皮细胞，原代培养大鼠脑微血管内皮细胞，尝试建立血脑屏障体外模型。
综上，石菖蒲能够影响大鼠BBB的超微结构，增强BBB对EB的通透性，促进CNS药苯妥英钠进入脑内的含量增加，具有开启BBB、引药入脑的药理作用。作用机制研究结果显示，石菖蒲调节5-HT的含量是其增加BBB通透性的作用机制之一，此外，石菖蒲有很强的抑制Hela细胞膜上P-糖蛋白的药物外排作用，这也是其增加BBB通透性的作用机制。初步建了BBB体外模型的方法。
Blood-brain barrier (BBB) is a dynamic adjustive interface which exists between capillary vessel on brain and spinal cord and nerve tissue, prevents most exogenous material from permeating into BBB, pumps the noxious or excess substance out of brain selectively in order to keep the microenvironment stable. However, the permeability of many central nervous system medicine is low, impacts the therapeutic effect of medicine seriously. Therefore, more and more attention is paid to the research of BBB.
The action of aromatic Chinese traditional medicine is to restore consciousness, Acorus tatarinowii Schott is one of traditional "kaiqiao" drugs, used in folk alone or compatibility with other drugs, the effect is indeed, which cues us that acorus tatarinowii schott could increase permeability of BBB, so we research it to solve neck-problem in central nervous system drugs development, and set up new rational and experimental evidence for traditional application of aromatice Chinese medicine.
We extracted volatile oil according to Chinese pharmacopeia (2005 edition), extraction ratio was 1.58%, decoction was condensed to 1g/ml.
Volatile oil of Acorus tatarinowii Schott samples were analyzed by analytical technique. α-Asarone, β-asarone, guaiene, acorenone were detected in volatile oil with GC/MS and α-asarone, β-asarone was detected in rat cerebrospinal fluid barrier with GC/MS under the same condition after gastric infusion of volatile oil.
Decoction of acorus tatarinowii schott samples were analyzed with HPLC/MS, there're three compounds was detected.
The ultramicrostructure of BBB in rat cerebral cortex was observed under electron microscope, and acorus tatarinowii schott could relax the tight junction
(TJ), but BBB was still integrity.
Primarily study the promotion of evan's blue (EB) penetration into BBB administrated acorus tatarinowii schott and borneol which was control drug, namely the content of EB in brain reflected the permeability of BBB. Statistical analysis showed after administrating borneol 2.5h the content of EB in brain was high compared with blank group significantly(P<0.01), that is borneol could enhance permeability administrated 2.5h later, but acorus tatarinowii schott couldn't.
Prolong action time of acorus tatarinowii schott, observe effect to BBB with the high dose (23.4g/kg), middle dose (11.7g/kg), low dose (1.17g/kg) volatile oil. Statistical analysis indicated middle and high dose could promote the permeability of BBB compared with blank group significantly (P<0.05), and showed dose-dependent; at middle dose volatile oil and decoction could promote the permeability of BBB (P<0.05), the whole drag could promote the permeability of BBB compared with blank group significantly (P<0.01).
Administrated acorus tatarinowii schott a week, intraperitoneal injected phenytoin sodium, and the concentration of phenytoin sodium in brain was detected used by HPLC. At 4h after injected, the concentration of phenytoin sodium was increased significantly (P<0.01). In word, acorus tatarinowii schott appeared to be a prospecting assisting agent with auxo-action on the penetration of other substances across BBB.
Concentration of 5-HT was detected used by electrochemical detector, compared with blank group, the content of 5-HT in volatile oil group increased significantly (P<0.05), as well as decoction and whole drag (P<0.01), so we presumed one of molecular pathways which acorus tatarinowii schott influenced permeability of BBB was to regulate content of 5-HT in brain.
Cultivated Hela cells as career to evaluate the activity of P-glycoprotein, the result was that all parts of acorus tatarinowii could reinforce the cytotoxicity caused by VCR significantly(P<0.01) at 0.5h; after 1h, only volatile oil could
reinforce the cytotoxicity significantly (P<0.01) ; after 24h, neither control drug nor all parts of acorus tatarinowii schott could affect the cytotoxicity caused by VCR(P>0.05).
Cerebral microvessel fragments were obtained by two steps of enzyme digestions and gradient centrifugation with BSA and Percoll, and cultivated primary rat cerebral microvascular endothelial cells, we established a BBB model in vitro initially.
In short, the resuscitative effect of acorus tatarinowii schott is resulted from the comprehensive action of multiple components; It is of great importance to improve the blood concentration in brain and bioavailability of central nervous system drugs; Acorus tatarinowii Schott could open BBB by increasing the level of 5-HT in brain, or P-glycoprotein inhibitor can get drugs across the blood-brain barrier; relatively pure primary culture of RCMEC was successfully established using this method and the model system could be used to develop an in vitro model of the rat blood-brain barrier.
中药中芳香类药物“醒脑开窍”的作用主要是指开窍启闭、宣发神气，调神定志，使心神复明，现代医学认为是增加BBB的通透性，促进其他CNS药物进入脑组织，发挥疗效。石菖蒲是传统的芳香开窍药之一，民间单用或与其他中药配伍使用广泛，主要用于脑病的治疗，有确切的疗效，提示石菖蒲对BBB有一定作用。本文主要研究石菖蒲醒脑开窍、透过血脑屏障的有效成分及其作用机制，为中药“芳香走窜、引药上行”提供理论基础和实验依据。
按照《中国药典》2005版一部附录挥发油提取法甲法，提取挥发油，提取率约为1.58％，去油水煎液浓缩至1g／ml。
采用气相色谱-质谱(GC／MS)联用仪检测挥发油样品中含有α—细辛醚、β—细辛醚、愈创木烯、菖蒲烯酮，相同条件下检测服用挥发油后大鼠的脑脊液，结果显示α—细辛醚，β—细辛醚可透过BBB进入脑脊液。
采用液相色谱-质谱(HPLC／MS)联用仪测定去油水煎液样品及灌服去油水煎液的大鼠脑脊液样品，检测到三个化合物，分子量分别为396，452，339，已知石菖蒲化合物中尚未包括。
透射电镜下观察给药前后大鼠大脑皮层BBB的超微结构，可发现石菖蒲各提取部分均可使BBB内皮细胞之间的紧密连接(tight junction，TJ)疏松，但BBB基本完整，尚未破坏。
以冰片为阳性药，初步探讨石菖蒲各提取部分对伊文思兰(EB)透过BBB的促进作用，即以EB在脑内的含量反映BBB的通透性，统计分析表明冰片在灌胃2.5h后脑内EB含量显著高于空白组(P＜0.01)，石菖蒲在11.7g／kg剂量下自给药0.5h至2.5h未见明显作用；延长石菖蒲给药时间，石菖蒲挥发油剂量为23.4g／kg和11.7g／kg时，5h后脑内EB含量显著高于空白组(P＜0.05)，且有剂量依赖性趋势；总药剂量为11.7g／kg时，5h后脑内EB含量显著高于空白组(P＜0.01)。
石菖蒲各提取部分给予小鼠灌胃一周后，腹腔注射苯妥英钠，采用HPLC方法检测苯妥英钠在脑内的浓度，结果显示：石菖蒲总组动物脑内苯妥英钠的含量较高于未给石菖蒲组动物脑内的苯妥英钠含量，4h后仍保持较高浓度(P＜0.01)。
利用HPLC-ECD检测脑组织内5-羟色胺(5-HT)的浓度，统计分析得：与空白组比较，挥发油组小鼠大脑中5-HT的含量增加(P＜0.05)，石菖蒲水煎液组及总药组中5-HT的含量显著增加(P＜0.01)。
应用Hela细胞系作为评价石菖蒲对BBB上P-糖蛋白活性的载体，0.5h时石菖蒲各部分(挥发油、水煎液、总药)可明显增强长春新碱(VCR)的细胞毒性(P＜0.01)；8h后挥发油部分仍可明显增强VCR的细胞毒性(P＜0.01)，挥发油的药效维持时间明显长于阳性药维拉帕米(VER)(P＜0.01)；24h后，阳性药及石菖蒲各部分对VCR的细胞毒性无明显影响(P＞0.05)。
完成上述工作的基础上，采用两次酶消化、梯度离心的方法分离得到纯度较高的脑微血管内皮细胞，原代培养大鼠脑微血管内皮细胞，尝试建立血脑屏障体外模型。
综上，石菖蒲能够影响大鼠BBB的超微结构，增强BBB对EB的通透性，促进CNS药苯妥英钠进入脑内的含量增加，具有开启BBB、引药入脑的药理作用。作用机制研究结果显示，石菖蒲调节5-HT的含量是其增加BBB通透性的作用机制之一，此外，石菖蒲有很强的抑制Hela细胞膜上P-糖蛋白的药物外排作用，这也是其增加BBB通透性的作用机制。初步建了BBB体外模型的方法。
Blood-brain barrier (BBB) is a dynamic adjustive interface which exists between capillary vessel on brain and spinal cord and nerve tissue, prevents most exogenous material from permeating into BBB, pumps the noxious or excess substance out of brain selectively in order to keep the microenvironment stable. However, the permeability of many central nervous system medicine is low, impacts the therapeutic effect of medicine seriously. Therefore, more and more attention is paid to the research of BBB.
The action of aromatic Chinese traditional medicine is to restore consciousness, Acorus tatarinowii Schott is one of traditional "kaiqiao" drugs, used in folk alone or compatibility with other drugs, the effect is indeed, which cues us that acorus tatarinowii schott could increase permeability of BBB, so we research it to solve neck-problem in central nervous system drugs development, and set up new rational and experimental evidence for traditional application of aromatice Chinese medicine.
We extracted volatile oil according to Chinese pharmacopeia (2005 edition), extraction ratio was 1.58%, decoction was condensed to 1g/ml.
Volatile oil of Acorus tatarinowii Schott samples were analyzed by analytical technique. α-Asarone, β-asarone, guaiene, acorenone were detected in volatile oil with GC/MS and α-asarone, β-asarone was detected in rat cerebrospinal fluid barrier with GC/MS under the same condition after gastric infusion of volatile oil.
Decoction of acorus tatarinowii schott samples were analyzed with HPLC/MS, there're three compounds was detected.
The ultramicrostructure of BBB in rat cerebral cortex was observed under electron microscope, and acorus tatarinowii schott could relax the tight junction
(TJ), but BBB was still integrity.
Primarily study the promotion of evan's blue (EB) penetration into BBB administrated acorus tatarinowii schott and borneol which was control drug, namely the content of EB in brain reflected the permeability of BBB. Statistical analysis showed after administrating borneol 2.5h the content of EB in brain was high compared with blank group significantly(P<0.01), that is borneol could enhance permeability administrated 2.5h later, but acorus tatarinowii schott couldn't.
Prolong action time of acorus tatarinowii schott, observe effect to BBB with the high dose (23.4g/kg), middle dose (11.7g/kg), low dose (1.17g/kg) volatile oil. Statistical analysis indicated middle and high dose could promote the permeability of BBB compared with blank group significantly (P<0.05), and showed dose-dependent; at middle dose volatile oil and decoction could promote the permeability of BBB (P<0.05), the whole drag could promote the permeability of BBB compared with blank group significantly (P<0.01).
Administrated acorus tatarinowii schott a week, intraperitoneal injected phenytoin sodium, and the concentration of phenytoin sodium in brain was detected used by HPLC. At 4h after injected, the concentration of phenytoin sodium was increased significantly (P<0.01). In word, acorus tatarinowii schott appeared to be a prospecting assisting agent with auxo-action on the penetration of other substances across BBB.
Concentration of 5-HT was detected used by electrochemical detector, compared with blank group, the content of 5-HT in volatile oil group increased significantly (P<0.05), as well as decoction and whole drag (P<0.01), so we presumed one of molecular pathways which acorus tatarinowii schott influenced permeability of BBB was to regulate content of 5-HT in brain.
Cultivated Hela cells as career to evaluate the activity of P-glycoprotein, the result was that all parts of acorus tatarinowii could reinforce the cytotoxicity caused by VCR significantly(P<0.01) at 0.5h; after 1h, only volatile oil could
reinforce the cytotoxicity significantly (P<0.01) ; after 24h, neither control drug nor all parts of acorus tatarinowii schott could affect the cytotoxicity caused by VCR(P>0.05).
Cerebral microvessel fragments were obtained by two steps of enzyme digestions and gradient centrifugation with BSA and Percoll, and cultivated primary rat cerebral microvascular endothelial cells, we established a BBB model in vitro initially.
In short, the resuscitative effect of acorus tatarinowii schott is resulted from the comprehensive action of multiple components; It is of great importance to improve the blood concentration in brain and bioavailability of central nervous system drugs; Acorus tatarinowii Schott could open BBB by increasing the level of 5-HT in brain, or P-glycoprotein inhibitor can get drugs across the blood-brain barrier; relatively pure primary culture of RCMEC was successfully established using this method and the model system could be used to develop an in vitro model of the rat blood-brain barrier.
引文
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[3] Olesen SP.A calcium—dependent reversible permeability increase in microvessels in frog brain incluced by serotonin [J]. J Physiol, 1985, 36:103—113
[4] Winkler T, Sharma H. S, Stalberg E, et al. Impairment of blood-brain barrier function by serotonin induces desynchronization of spontaneous cerebral cortical activity: experimental observations in the anaesthetized rat [J]. Neuroscience, 1995, 68(4): 1097-1104.
[5] Tohrta T, Suta, Mikihiko, et al. Function involement of p-glycol protein in the blood-brain barrier. The Journal Biological Chemistry, 1992, 267: 20383.
[6] 李伟荣，姚丽梅，宓穗卿，等。冰片对大鼠下丘脑组胺、5-羟色胺含量的影响。中药材，2004，27(12)：937-939
[7] Sarker M H, et al. The role of guanylyl cyclases in the permeability response to inflammatory mediators in pial venular capillaries in the ratl J Physiol, 2002,540(Pt 1): 209
[8] Sharma H S, et al. p-Chlorophenylalanine, an inhibitor of serotonin synthesis reduces blood-brain barrier permeability, cerebral blood flow, edema formation and cell injury following trauma to the rat brainl Acta Neurochir Suppl, 2000,769:1
[9] Peter Brust, Anne Friedrich, Istvan A. Krizbai, et al. Functional Expression of the Serotonin Transporter in Immortalized Rat Brain Microvessel Endothelial Cells[J]. Journal of Neurochemistry, 2000, 74(3): 1241-1248.
[10] 陈艳明，王宁生．冰片对P-糖蛋白的影响．中国新药与临床药理，2003，14(2)：96-99
[11] 黄哲玮，孙皎，孟爱英．两种体外细胞毒性检测方法的比较研究．上海生物医学工程，2005，26(4)：205—207
[12] 金雪红，包仕尧．P—糖蛋白与血脑屏障．国际脑血管病杂志，2006，14(4)：282—285
[13] Germann UA.P-glycoprotein a mediator of multidrug resistance in tumor cells [J]. Eur J cancer, 1996, 17(2): 433-444
[14] 吴云娟，方晓玲．P—糖蛋白在药物脑部转运中作用的研究进展．中国临床药学杂志，2004，13：382-385
[1] Masaru Honda, Shinsuke Nakagawa, Kentaro Hayashi, et al. Adrenomedullin Improves the Blood-Brain Barrier Function Through the Expression of Claudin-5. Cellular and Molecular Neurobiology, 2006, 26 (2): 109-118
[2] 许熊飞，李润平，李泉，等．大鼠脑微血管内皮细胞的分离与原代培养。细胞生物学杂志，2005，27：84-88
[3] Joo F. Endothelial cells of the brain and other organ systems: some similarities and difference. Progr Neurobiol,1996,48:255-73
[4] 鲍欢，张志琳，包仕尧，等．实验性血脑屏障细胞模型及其评价．中国临床神经科学，2004，12(1)：73—76
[5] 胡利民，范祥，张艳军，等．大鼠脑微血管内皮细胞与星形胶质细胞共培养血脑屏障体外模型的建立．天津中医药，2005，22(2)：149—151
[6] Andreas Reichel, David J. Begley, N. Joan Abbott. An Overview of in vitro techniques for blood-brain barrier studies. Biology and Research Protocols Biology, 307-324
[1] Schlosshauer B. The blood-brain barrier morphology, molecules, and neurothelin [J]. Bioassays, 1993, 1: 341-346
[2] 包金风．血脑屏障上的P-糖蛋白。国外医学药学分册，2000，27(3)：154-158
[3] 高瑞峰．血脑屏障的通透性对药物的选择作用．中华实用中西医杂志，2004，4(17)：62—64
[4] 唐忠．血脑屏障基础与临床的某些新进展．国外医学神经病学神经外科学分册，2004，31(2)：193-196
[5] McAllister MS, Krizanac-Bengez L, Macchia F, et al. Mechanisms of glucose transport at the blood-brain barrier: an in vitro study. Brain Res. 2001, 904 (1): 20-30
[6] 林凤云，朱照静．血脑屏障转运机制的研究进展．中国药房，2006，17(10)：783-785
[7] Yasuto K, Ikumi T, Akira T, et al. Functional Clarification of MCT1-mediated Transport of Monocarboxylic Acids at the Blood-brain Barrier Using in vitro Cultured Cells and in vivo BUI Studies. Pharm Research, 2000, 1:55
[8] 左明新，刘晓东．血脑屏障上的P-糖蛋白与药物转运功能．中国现代应用药学杂志，1999，16(2)：1-3
[9] O'Kane RL, Vina JR, Simpson I, et al. Na~+ -dependent neutral amino acid transporters A, ASC, and N of the blood-brain barrier: mechanisms for neutral amino acid removal. Am J Physiol Endocrinol Metab. 2004; 287(4): E622-9
[10] Zhang Y, Bachmeier C, Miller DW, In vitro and in vivo models for assessing drug effiux transporter activity [J]. Adv Drug Delivery Rev, 2003, 55 (1): 31.
[11] Wakayama K, Ohtsuki S, Takanaga H, et al. Localization of norepinephrine and serotonin transporter in mouse brain capillary endothelial cells [J]. Neurosci Res, 2002, 44(2): 173-180
[12] O'Kane RL, Martinez-Lopez I, DeJoseph MR, et al. Na~+-dependent glutamate transporters ( EAAT1 , EAAT2 , and EAAT3) of the blood-brain barrier. A mechanism for glutamate removal [J]. J Biol Chem, 1999, 274 (45): 31891-31895
[13] Kanner BI. Sodium-coupled neurotransmitter transport: structure function and regulation [J]. J Exp Biol, 1994, 196: 237-249
[14] Zhang Y, Liu GQ. Sodium and chloride-dependent high and low-affinity uptakes of GABA by brain capillary endothelial cells [J]. Brain Res, 1998, 808: 1-7
[15] Kakee A, Takanaga H, Terasaki T, et al. Efflux of a suppressive neurotrans — mitter, GABA, across the blood-brain barrier [J]. J Neurochem, 2001, 79(1): 110-118
[16] Liu QR, Lopez-Corcuera B, Mandiyan S, et al. Molecular characterization of four pharmacologically distinct gamma-aminobutyric acid transporters in mouse brain [J]. J Biol Chem , 1993 , 268(3):2106-2112.
[17] Zhang Y, Liu GQ. A novel method to determine the location of high and low affinity GABA transporters to the luminal and antiluminal membranes of brain capillary endothelial cells [J]. Brain Res Protocols, 1999,4:288-294.
[18] Neuwelt EA, Maravilla KR, Frenkel EP, et al. Use of enhanced computerized tomography to evaluate osmotic blood-brain barrier disruption. Neurosurgery, 1980, 6 (1): 49—56
[19] Neuwelt EA, Barnett PA, Glasberg M, Ea et al. Pharmacology and neuro— toxicity of cis-diamminedichloroplatinum, bleomycin, 5-fluorouracil, and cyclophosphamide administration following osmotic blood-brain barrier modification. Cancer Res, 1983, 43 (11): 5278—5285
[20] Song BW, Vinters HV, Wu D, et al. Enhanced neuroprotective effects of basic fibroblast growth factor in regional brain ischemia after conjugation to a blood-brain barrier delivery vector. J pharamacol Exp Ther, 2002, 301 (2): 605—610
[21] Alyaudtin RN, Reichel A, Lobenberg R, et al. Interaction of poly(butylcyanoacrylate)nanoparticles with the blood-brain barrier in vivo and in vitro. J Drug Targe, 2001, 9(3): 209—221
[2] 魏刚，方永奇，刘东辉，et al．GC—MS建立石菖蒲挥发油特征指纹图谱方法学研究．2004，29(8)：764—768
[3] 唐洪梅，李锐．石菖蒲透过大鼠血脑屏障的化学成分分析．中医药研究，2002，18(1)：40-41
[4] 吴启端，方永奇，魏刚，et al．石菖蒲去油水煎液的高效液相色谱分析．时珍国医国药，2002，13(7)：385—387
[5] 刘春海，刘西京，杨华生．石菖蒲挥发油的GC-MS分析．中医药学刊，2006，24(7)：1280-1281
[6] 吕刚，李婷婷．石菖蒲的研究进展．时珍国医国药，2006，17(7)：1291-1292
[7] 唐洪梅，席萍，薛秀清．石菖蒲不同提取物化学成分的GC-MS分析．广东药学，2001，11(6)：33—35
[8] 金维岳．石菖蒲注射液治疗肺性脑病279例临床观察小结[J]．中成药研究，1982，(10)：22
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[1] 韦献良，叶峻，周燕．海洛因成瘾大鼠脑血脑屏障超微结构的变化。广西医科大学学报，2004，21(4)：495-497
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[3] 朱明启．血脑屏障的研究进展．锦州医学院学报．2005，26(1)：53—56
[4] 吴启端，方永奇，李翎，等．石菖蒲醒脑开窍的有效部位筛选。时珍国医国药，2002，13(5)：260—261
[5] 梁美蓉，叶少梅，张银卿，等．冰片对兔、大鼠脑组织伊文氏蓝染色作用的观察[J]．广州中医学院学报，1993，10(4)：211—213
[6] Carrera RM, Pacheco AM Jr, Mastroti RA. Qualitative evaluation of the blood-brain barrier after the use of hypertonic saline solution in young rats[J]. Eur Surg Res,2001,33(5～6):311.
[7] 徐伟，王宗锐．薄荷醇及冰片对磺胺嘧啶和伊文思兰在脑中分布的影响．中药药理与临床，1995，6：31—33
[8] 马爱梅，赵永波，陈英辉．苯妥英钠在大鼠血液和脑细胞外液药动学研究．中国临床神经科学，2005，13(2)：118-122
[9] 马爱梅，赵永波，陈英辉．P-糖蛋白对卡马西平和苯妥英钠通过大鼠血脑屏障影响的研究．中国临床神经科学，2005，38(7)：438-441
[10] Steffen Baltes, Alexandra M. Gastens, et al. Differences in the transport of the antiepileptic drugs phenytoin, levetiracetam and carbamazepine by human and mouse P-glycoprotein. Neuropharmacology, 52 (2): 333-346
[1] 朱明启．血脑屏障的研究进展．锦州医学院学报，2005，26(1)：53—56
[2] Preston E, Foster DO. Evidence for pore-like opening of blood-brain barrier following forebrain ischemia in rats[J]. Brain Res, 1997, 761: 4-10
[3] Olesen SP.A calcium—dependent reversible permeability increase in microvessels in frog brain incluced by serotonin [J]. J Physiol, 1985, 36:103—113
[4] Winkler T, Sharma H. S, Stalberg E, et al. Impairment of blood-brain barrier function by serotonin induces desynchronization of spontaneous cerebral cortical activity: experimental observations in the anaesthetized rat [J]. Neuroscience, 1995, 68(4): 1097-1104.
[5] Tohrta T, Suta, Mikihiko, et al. Function involement of p-glycol protein in the blood-brain barrier. The Journal Biological Chemistry, 1992, 267: 20383.
[6] 李伟荣，姚丽梅，宓穗卿，等。冰片对大鼠下丘脑组胺、5-羟色胺含量的影响。中药材，2004，27(12)：937-939
[7] Sarker M H, et al. The role of guanylyl cyclases in the permeability response to inflammatory mediators in pial venular capillaries in the ratl J Physiol, 2002,540(Pt 1): 209
[8] Sharma H S, et al. p-Chlorophenylalanine, an inhibitor of serotonin synthesis reduces blood-brain barrier permeability, cerebral blood flow, edema formation and cell injury following trauma to the rat brainl Acta Neurochir Suppl, 2000,769:1
[9] Peter Brust, Anne Friedrich, Istvan A. Krizbai, et al. Functional Expression of the Serotonin Transporter in Immortalized Rat Brain Microvessel Endothelial Cells[J]. Journal of Neurochemistry, 2000, 74(3): 1241-1248.
[10] 陈艳明，王宁生．冰片对P-糖蛋白的影响．中国新药与临床药理，2003，14(2)：96-99
[11] 黄哲玮，孙皎，孟爱英．两种体外细胞毒性检测方法的比较研究．上海生物医学工程，2005，26(4)：205—207
[12] 金雪红，包仕尧．P—糖蛋白与血脑屏障．国际脑血管病杂志，2006，14(4)：282—285
[13] Germann UA.P-glycoprotein a mediator of multidrug resistance in tumor cells [J]. Eur J cancer, 1996, 17(2): 433-444
[14] 吴云娟，方晓玲．P—糖蛋白在药物脑部转运中作用的研究进展．中国临床药学杂志，2004，13：382-385
[1] Masaru Honda, Shinsuke Nakagawa, Kentaro Hayashi, et al. Adrenomedullin Improves the Blood-Brain Barrier Function Through the Expression of Claudin-5. Cellular and Molecular Neurobiology, 2006, 26 (2): 109-118
[2] 许熊飞，李润平，李泉，等．大鼠脑微血管内皮细胞的分离与原代培养。细胞生物学杂志，2005，27：84-88
[3] Joo F. Endothelial cells of the brain and other organ systems: some similarities and difference. Progr Neurobiol,1996,48:255-73
[4] 鲍欢，张志琳，包仕尧，等．实验性血脑屏障细胞模型及其评价．中国临床神经科学，2004，12(1)：73—76
[5] 胡利民，范祥，张艳军，等．大鼠脑微血管内皮细胞与星形胶质细胞共培养血脑屏障体外模型的建立．天津中医药，2005，22(2)：149—151
[6] Andreas Reichel, David J. Begley, N. Joan Abbott. An Overview of in vitro techniques for blood-brain barrier studies. Biology and Research Protocols Biology, 307-324
[1] Schlosshauer B. The blood-brain barrier morphology, molecules, and neurothelin [J]. Bioassays, 1993, 1: 341-346
[2] 包金风．血脑屏障上的P-糖蛋白。国外医学药学分册，2000，27(3)：154-158
[3] 高瑞峰．血脑屏障的通透性对药物的选择作用．中华实用中西医杂志，2004，4(17)：62—64
[4] 唐忠．血脑屏障基础与临床的某些新进展．国外医学神经病学神经外科学分册，2004，31(2)：193-196
[5] McAllister MS, Krizanac-Bengez L, Macchia F, et al. Mechanisms of glucose transport at the blood-brain barrier: an in vitro study. Brain Res. 2001, 904 (1): 20-30
[6] 林凤云，朱照静．血脑屏障转运机制的研究进展．中国药房，2006，17(10)：783-785
[7] Yasuto K, Ikumi T, Akira T, et al. Functional Clarification of MCT1-mediated Transport of Monocarboxylic Acids at the Blood-brain Barrier Using in vitro Cultured Cells and in vivo BUI Studies. Pharm Research, 2000, 1:55
[8] 左明新，刘晓东．血脑屏障上的P-糖蛋白与药物转运功能．中国现代应用药学杂志，1999，16(2)：1-3
[9] O'Kane RL, Vina JR, Simpson I, et al. Na~+ -dependent neutral amino acid transporters A, ASC, and N of the blood-brain barrier: mechanisms for neutral amino acid removal. Am J Physiol Endocrinol Metab. 2004; 287(4): E622-9
[10] Zhang Y, Bachmeier C, Miller DW, In vitro and in vivo models for assessing drug effiux transporter activity [J]. Adv Drug Delivery Rev, 2003, 55 (1): 31.
[11] Wakayama K, Ohtsuki S, Takanaga H, et al. Localization of norepinephrine and serotonin transporter in mouse brain capillary endothelial cells [J]. Neurosci Res, 2002, 44(2): 173-180
[12] O'Kane RL, Martinez-Lopez I, DeJoseph MR, et al. Na~+-dependent glutamate transporters ( EAAT1 , EAAT2 , and EAAT3) of the blood-brain barrier. A mechanism for glutamate removal [J]. J Biol Chem, 1999, 274 (45): 31891-31895
[13] Kanner BI. Sodium-coupled neurotransmitter transport: structure function and regulation [J]. J Exp Biol, 1994, 196: 237-249
[14] Zhang Y, Liu GQ. Sodium and chloride-dependent high and low-affinity uptakes of GABA by brain capillary endothelial cells [J]. Brain Res, 1998, 808: 1-7
[15] Kakee A, Takanaga H, Terasaki T, et al. Efflux of a suppressive neurotrans — mitter, GABA, across the blood-brain barrier [J]. J Neurochem, 2001, 79(1): 110-118
[16] Liu QR, Lopez-Corcuera B, Mandiyan S, et al. Molecular characterization of four pharmacologically distinct gamma-aminobutyric acid transporters in mouse brain [J]. J Biol Chem , 1993 , 268(3):2106-2112.
[17] Zhang Y, Liu GQ. A novel method to determine the location of high and low affinity GABA transporters to the luminal and antiluminal membranes of brain capillary endothelial cells [J]. Brain Res Protocols, 1999,4:288-294.
[18] Neuwelt EA, Maravilla KR, Frenkel EP, et al. Use of enhanced computerized tomography to evaluate osmotic blood-brain barrier disruption. Neurosurgery, 1980, 6 (1): 49—56
[19] Neuwelt EA, Barnett PA, Glasberg M, Ea et al. Pharmacology and neuro— toxicity of cis-diamminedichloroplatinum, bleomycin, 5-fluorouracil, and cyclophosphamide administration following osmotic blood-brain barrier modification. Cancer Res, 1983, 43 (11): 5278—5285
[20] Song BW, Vinters HV, Wu D, et al. Enhanced neuroprotective effects of basic fibroblast growth factor in regional brain ischemia after conjugation to a blood-brain barrier delivery vector. J pharamacol Exp Ther, 2002, 301 (2): 605—610
[21] Alyaudtin RN, Reichel A, Lobenberg R, et al. Interaction of poly(butylcyanoacrylate)nanoparticles with the blood-brain barrier in vivo and in vitro. J Drug Targe, 2001, 9(3): 209—221