【作者】 王虹；

【导师】 刘屏；

【作者基本信息】 中国人民解放军军医进修学院 ， 药理学， 2007， 硕士

【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。更多还原

【Abstract】 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 couldreinforce 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.更多还原