通窍散瘀方中有效成分鼻黏膜吸收及鼻—脑间接通路转运机制的研究

【作者】 张林；

【导师】 杜守颖；

【作者基本信息】 北京中医药大学 ， 中药制药学， 2016， 博士

【摘要】 经鼻入脑转运通路(即鼻-脑通路),是利用人体正常生理状态下鼻与脑解剖学特性之间的联系,为预防和治疗脑部疾病的药物提供的一条由鼻腔给药而在中枢神经系统中发挥作用的传递途径。利用鼻-脑通路输送药物的给药形式,具有有效提高药物生物利用度、使药物具有良好的脑部靶向性、快速发挥药物疗效、对机体无侵害性损伤、使用方便等特点,近年来受到广泛关注,而依据鼻-脑通路理论设计的鼻腔给药系统及制剂也已成为现代药剂学研究领域中的新热点。但是在使用鼻腔给药制剂后,常会出现脑内药物浓度测定值较低、靶向性不足的现象,这主要是由于鼻-脑通路涉及复杂的生理过程,并受药物自身诸多理化性质的影响所造成的。因此为了有效提高药物经鼻给药后的脑部生物利用度,对药物鼻-脑转运过程中,机体与药物间产生相互作用的机制进行研究,显得尤为重要。鼻-脑通路的药物转运形式包括绕过血脑屏障将药物由鼻腔直接运送至中枢神经系统的直接通路,和药物先经鼻腔内毛细血管吸收入血进入体循环后再透过血脑屏障抵达脑部的间接通路,并且二者往往同时发挥作用,但是,无论何种鼻-脑通路形式,药物都需要首先通过鼻黏膜屏障,才能进一步转运,抵达病灶,发挥疗效。目前对于鼻-脑通路药物递送的基础研究主要集中于对药物的脑部靶向性、入脑效率、血脑屏障透过能力进行评价和探讨,而对鼻黏膜渗透这一重要过程的研究相对较少,已有的相关研究也多采用大型哺乳动物离体鼻黏膜组织为模型,进行药物单一的渗透能力考察,限于离体组织活性较低,无法对药物与鼻黏膜间相互作用及影响机制进行深入挖掘。本课题组前期通过原代培养,构建人源鼻黏膜上皮细胞模型作为体外鼻黏膜细胞模型,对芳香开窍类药物的黏膜渗透增强作用进行相关研究,取得一定研究成果,但由于人源细胞来源有限,且取材多来自病变组织,故所构建细胞模型存在些许不足,并且对于鼻-脑通路的整体过程的探讨未能涉及,故认为有待于对研究进一步完善和补充。通窍散瘀方源自临床经验方,由葛根、白芍、薄荷等药物组成,具有散瘀活血、醒脑通窍等作用,用于治疗脑卒中等脑部疾病。本课题组选择实验常用动物大鼠作为鼻黏膜组织取材对象,利用细胞原代培养技术,作为原代鼻黏膜细胞模型,同时培养人肺腺癌细胞系Calu-3细胞,作为模拟体外鼻黏膜人源细胞模型,两者共同应用,全面地从原代和人源两种细胞模型角度对通窍散瘀方中主要药效成分葛根素、芍药苷、薄荷脑的鼻黏膜的吸收情况和药物配伍的黏膜渗透促进机制进行研究。并且借助细胞共培养技术,将Calu-3细胞与能够良好模拟血脑屏障的MDCK-MDRl细胞共同培养,用以构建完整地模拟鼻-脑间接通路的体外模型,对药物在整体的经鼻入脑转运过程中的传递情况进行详尽分析,期望对鼻-脑通路和鼻腔给药系统有更加深入的了解和认识。1鼠原代鼻黏膜上皮细胞体外模型、人Calu-3细胞模型的构建和鼻-脑间接通路共培养模型的构建本研究通过对大鼠鼻中隔上覆盖的鼻黏膜组织进行取材,采用蛋白酶消化法、细胞筛分选法分离、收集鼠鼻黏膜上皮细胞(rat nasal epithelial cell,RNEC),利用差速贴壁法去除成纤维细胞、内皮细胞等杂质细胞达到细胞纯化目的后,使用DMEM培养基(含10%胎牛血清、1%双抗)在饱和湿度、37℃、5%CO2条件下,培养24h,促使细胞贴壁生长,随后去除旧培养基,使用含有细胞生长添加因子的无血清BEGM培养基继续培养,可培养出形态良好、生长稳定的原代细胞。利用nti-pan Cytokeratin抗体(角蛋白PCK抗体)对细胞进行荧光标记,经免疫荧光化学反应鉴定,于激光共聚焦显微镜下观察,所培养的原代细胞PCK抗体染色呈阳性,证实培养出的细胞为鼻粘膜上皮细胞,且纯度较高。同时,在原代细胞培养过程中,考察不同种类的培养基(包括BEGM培养基、BEGM:DMEM/F12(1:1)培养基、DMEM培养基、含10%FBS的DMEM培养基、DMEM/F12培养基、含10%FBS的DMEM/F12培养基),对细胞生长的影响,发现使用无血清类培养基更适于原代细胞体外培养,其中使用BEGM培养基培养出的细胞,透明度高,折光性强,形态均一,呈铺路石状,可达到满意的培养效果。人肺腺癌细胞系(Calu-3细胞),作为体外模拟鼻黏膜的人源细胞模型,其培养过程属于常规细胞系培养操作,将细胞以含10%FBS、1%双抗、1NEAA的MEM培养基制成细胞悬液后,接种于培养瓶内,饱和湿度、37℃、5%CO2条件下的培养箱中,连续培养,隔天换液,5天左右之后细胞可生长至与鼻黏膜原代细胞形态相似、连接紧密、折光度高的融合状态。两种细胞模型的构建形式操作简便、重复性好,通过绘制细胞生长曲线,发现生长周期相对较短,均可稳定传代培养,通过冻存可有效保持细胞生理状态,复苏后细胞活力良好,并且在细胞成膜性和标准药物透过性能上,均表现出良好的模型特性,为后续药物在鼻黏膜吸收情况和作用机制的体外研究奠定坚实基础。为了能够将鼻-脑间接通路转运过程中,鼻黏膜屏障和血脑屏障这两道关键生理阻碍,紧密地联系在一起,真实反映体内生理情况,在体外构建合理的研究模型,本实验充分利用Transwell细胞培养板组件和细胞共培养技术,结合体内生理结构特性,选择模拟鼻黏膜屏障的Calu-3细胞和血脑屏障的MDCK-MDR1细胞进行间接共培养,首次构建出新颖的模拟鼻-脑间接通路的体外共培养细胞模型。通过显微镜下观察和电阻值测定,发现在共培养体系中,两种模拟不同组织结构的细胞均能够稳定生长,形成紧密单层膜结构,可用于药物在鼻-脑间接通路中连续转运过程中吸收、渗透情况的研究。2通窍散瘀方中主要有效成分的细胞毒性研究由于体外细胞模型的特殊性,在研究药物转运及配伍机制作用前,需要先对药物的细胞毒性进行考察,以确定各细胞模型中适宜的给药剂量,同时可以为后续鼻腔给药制剂的安全、合理的给药剂量提供依据。研究采用MTT比色法,对不同浓度药物和配伍在RNEC细胞和Calu-3细胞中的毒性进行考察。结果表明各个药物的细胞毒性均与给药剂量存在相关性,随着给药浓度升高细胞毒性作用也不断增强；RNEC细胞中,薄荷脑浓度在0～50μg/mL,芍药苷浓度在0-200μg/mL,葛根素浓度在0～300μg/mL,葛根素与芍药苷配伍(按组方比例1：0.4)浓度在0～100μg/mL(以芍药苷计),葛根素与薄荷脑配伍(按组方比例1：0.5)浓度在0～50μg/mL(以薄荷脑计),三者共同配伍(按组方比例1：0.4：0.5)浓度在0～50μg/mL范围内(以薄荷脑计),对细胞不产生毒性作用；Calu-3细胞中,薄荷脑浓度在0～60μg/ mL,芍药苷浓度在0～300μg/mL,葛根素浓度在0～350μgmL,葛根素与芍药苷配伍(按组方比例1：0.4)浓度在0～1OOμg/mL(以芍药苷计),葛根素与薄荷脑配伍(按组方比例1：0.5)浓度在0～60μg/mL(以薄荷脑计),三者共同配伍(按组方比例1：0.4：0.5)浓度在0～60μg/mL范围内(以薄荷脑计),对细胞不产生毒性作用；三种药物相比较,薄荷脑最大给药量最小,其细胞毒性最大,芍药苷次之,而葛根素的细胞毒性最小；与RNEC细胞毒性相比,Calu-3细胞中同种药物或配伍所产生毒性范围较大,可说明Calu-3细胞相应药物耐受强度较RNEC细胞稍强。3通窍散瘀方中主要有效成分黏膜细胞模型转运实验研究本研究分别将培养出的RNEC细胞和Calu-3细胞接种于Transwe 11细胞培养板的聚碳酸酯膜上,以气-液界面培养条件形成的细胞单层膜结构,模拟鼻腔黏膜,随后对通窍散瘀方中主要药效成分的转运进行研究,以评价药物的鼻黏膜渗透情况及组方配伍后对吸收的影响。对不同浓度的葛根素在RNEC细胞和Calu-3细胞单层膜模型中转运研究发现,药物的吸收、外排速率(dQ/dt)均随浓度升高而增大,不同浓度组间表观渗透系数(Papp)值无显著差异,药物外排率(ER)均在1左右,说明葛根素的黏膜转运在研究浓度范围内以被动扩散为主。葛根素配伍不同浓度的芍药苷时,其转运研究结果显示,与无配伍的葛根素转运相比较,葛根素渗透能力并未显著改变,提示组方过程中,芍药苷并不能促进葛根素的鼻黏膜吸收,而主要作为药效成分发挥治疗作用。当配伍中含有薄荷脑时,葛根素在两种细胞模型转运的表观渗透系数,与无配伍的葛根素转运相比较,均显著升高(P<0.05),表明借助薄荷脑渗透增强的作用,葛根素的鼻黏膜吸收可被显著增强,有利于提高葛根素鼻-脑通路转运过程中药物的生物利用度。4通窍散瘀方中主要有效成分鼻-脑间接通路共培养细胞模型中转运实验研究在构建Calu-3细胞与MDCK-MDRl细胞共培养模型的基础上,不同浓度的葛根素以及配伍芍药苷和薄荷脑,在鼻-脑间接通路转运过程中的情况进行研究,考察药物连续通过两侧细胞屏障过程中渗透能力,评价配伍药物对葛根转运能力和配伍渗透增强作用的影响。发现与单层细胞模型转运研究结果相似,葛根素在转运过程中以被动扩散为主,配伍薄荷脑达到一定浓度可有效促进其渗透,而配伍芍药苷未能促进。然而,在共培养模型中,由于多重因素影响,葛根素连续透过两层细胞屏障难度较大,在透过第一层后,难于再透过第二层被检查。研究中,仅在高浓度葛根素条件下,当配伍薄荷脑浓度达到60μg/mL时,葛根素在通过第二层细胞后可被检测到。为了详细对薄荷脑渗透促进能力进行阐述,在保证薄荷脑渗透作用的情况下,对不同浓度葛根素共培养转运情况进行深入研究,按照研究所采用的检测方法,证明了60gg/mL的薄荷脑在体外共培养模型中,可对100μg/ nL以上的葛根素起到显著地连续促进双层细胞屏障的渗透作用。由此可见,共培养模型转运研究结果一方面表明连续转运过程需要较高的药物浓度为保障,另一方面也体现了薄荷脑连续的渗透增强能力。5通窍散瘀方中主要有效成分的细胞转运作用机理研究药物在鼻黏膜上皮细胞的转运过程为多种形式的综合作用,因此本课题选择多种与细胞模型转运和体内相关生理指标对葛根素转运过程及组方配伍可能涉及到的影响因素进行分析。跨膜电阻值(TEER)常用于表征细胞单层膜结构中细胞间的疏密程度,本研究对葛根素、芍药苷、薄荷脑对细胞TEER值的影响进行考察。在使用电阻仪对给药后细胞的跨膜电阻值变化测定的研究中发现,两种细胞模型的TEER值均在配伍薄荷脑后显著下降,提示薄荷脑促进葛根素黏膜渗透的作用与其使单层细胞膜结构疏松、增强药物细胞旁路转运密切相关。鼻黏膜细胞的紧密连接(tight junction, TJ)结构是限制药物摄入的主要屏障,TJ结构主要由多种紧密连接蛋白(tight junction proteins)构成,因此本课题通过研究药物对两种细胞模型中的Occludin、Claudin-1、ZO-1、F-actin四种紧密连接蛋白的作用,、进一步从黏膜紧密程度影响的方面对药物的作用进行评价,采用免疫荧光染色方法,通过激光共聚焦显微镜观察及对荧光强度的分析,发现薄荷脑对此四种紧密连接蛋白均有一定程度的抑制作用,从而打开紧密连接结构,削弱紧密连接的屏障作用,促进药物的鼻粘膜渗透性。黏膜上皮细胞常表达的P糖蛋白,影响药物的透过,课题组采用流式细胞仪,对给药后细胞罗丹明123摄入所产生的荧光强度进行分析,与典型的P糖蛋白抑制剂维拉帕米对比,发现葛根素、芍药苷、薄荷脑均与P糖蛋白存在相互作用,在配伍应用的过程中,理论上存在竞争抑制作用,可促使葛根素透过增多。细胞膜流动性强弱同样同样影响着药物转运的程度,本课题采用荧光漂白恢复技术(FRAP),观测药物作用对细胞膜中荧光标记物迁移特性的影响,绘制荧光恢复曲线、计算荧光恢复率,发现薄荷脑达到一定浓度时,细胞荧光恢复率可显著升高,说明薄荷脑能够作用于细胞膜的磷脂双分子层,增加膜脂流动性,降低膜粘度,利于药物跨膜转运。此外,因为药物综合转运过程常与酶介导的代谢活动相关,故本研究通过考察药物对Na+-K+-ATP酶和Ca2+-ATP酶的影响,阐释药物单独使用及配伍使用在细胞模型转运过程中离子通道和能量代谢的相关情况,发现葛根素、芍药苷、薄荷脑三种药物成分,对于Na+-K+-ATP酶的能量代谢无显著影响,而对于Ca2+-ATP酶,薄荷脑可显著增强其活性,这不仅说明薄荷脑对于Ca2+-ATP酶的活性进行调控,同时由于紧密连接蛋白的表达抑制,同样与钙离子流动有关,进而阐释了薄荷脑对紧密连接蛋白调控的可能机制。更多还原

【Abstract】 The pathways of conveying from nose to brain (the nose-brain pathways) provide the transfer ways that target the central nervous system by nasal delivery for the drugs which is used for prevention or treatment of brain diseases, taking advantage of the normal physiological state of the connection of anatomical features between nose and brain. The administration method using the nose-brain pathways for drug delivery attracted much attention in recent years for it has many excellent characteristics, such as improving drug bioavailability effectively, better brain-targeting, rapid medicinal effect, no body invasive damage and easy to use, etc. nasal drug delivery system and preparation according to the theory of the nose-brain pathways also become the new hotspot in the field of modern pharmacy research. However, using nasal drug delivery preparation may bring about different results, such as the drug concentration measured in brain may be lower than the theoretical value, no enough targeting drug, because the nose-brain pathways involved in complex physiological process and influenced by many physical and chemical properties of drug itself. Therefore, studies on the mechanism of interaction between body and drugs in the process of the intranasal administration are particularly important.There are two kinds of nasal-brain pathways for drug delivery, one is the direct path, which bypass the blood brain barrier to deliver drugs directly by the nasal cavity to the central nervous system; the other is the indirect pathway, in which the drugs enter the systemic circulation by the nasal cavity capillary absorption and then pass into the brain through the blood brain barrier. They often play a role at the same time, and both of the ways need to make the drugs get through nasal mucosa barrier first, after that, the drugs can make further transfer to arrive in lesions and show effect. Nowadays, the drug delivery theory research of the nose-brain pathways mainly focused on the brain-targeting, the efficiency of brain uptakes, and the ability of getting through blood brain barrier. The study of primary process, the nasal mucous membrane permeability, more often than not is ignored. Existing related research always put an eye on single drug penetration test using model which based on the in vitro nasal mucosa tissue of large mammals, without lucubrating the mechanism of interaction between the nasal mucosa and drugs. Our research group has developed model of human original nasal mucosa epithelial cells as the nasal mucosa in vitro cell model by primary culture of nasal mucosa tissues from patients with nasosinusitis or nasal polyps in earlier stage. The research also focused on the function of aromatic orifice-opening medicinal which enhance the penetration on drugs through mucous membrane, and try to elucidate the mechanisms of drug absorption and infiltration promotion on account of compatibility of medicines. But as a result of the limit of sources and health status of human original cells, the cell model was constructed with some deficiencies and failed to involve the overall process of nose-brain pathways. The further research and supplement is wanted.Tongqiaosanyu is the prescription which promotes blood flow and removes stasis, shaped up through long clinical practice. It is composed of radix puerariae, radix paeoniae alba and mint. Possessing the effects of scattering stasis and waking up the brain, the prescription is frequently used for some brain disease like cerebral apoplexy. Our research group gets the nasal mucosa tissues from rats, the commonly used experimental animals, as the primary nasal mucosa cell model by primary culture. At the same time, we culture the Calu-3 cells, the human lung adenocarcinoma cell line, as the model of the in vitro human original nasal mucosa cell. By utilizing both of the models above, we roundly studied the nasal mucosa absorption of main efficacy components, puerarin, paeoniflorin and menthol, and the mechanism of compatibility. What is more, we co-culture Calu-3 cells with the MDCK-MDR1 cells, which simulate the BBB well. Relying on this method, we are capable to build the complete in vitro model of the nose-brain pathway, in order to detailed analyze the process of drug conveying in the nose-brain pathway. We hope that the research can enable us to know more about the nose-brain pathway and the nasal drug delivery system. 1 The development of in vitro primary rat nasal mucosa epithelial cells model, Calu-3 cells model and co-culture cells modelIn this study, we isolate the nasal mucosa tissue of rat nasal upper cover, separate and collect rat nasal epithelial cells (RNEC) by using the methods of protease digestion and cell screening, remove impurities such as fibroblasts, endothelial cells using the method of differential sticking wall. After the purification of RNEC cells, using DMEM medium (containing 10% fetal bovine serum,1% double resistance), in saturated humidity, under the condition of 37℃ and 5% CO2, cultivate for 24 hours to make the cells grown with adherence, then remove the old medium, add the serum-free medium BEGM with cell growth factor for further culture. Then, we gain the primary cells with good shape and stable growth. Using the Anti-pan Cytokeratin antibody (keratin antibody of PCK) to make fluorescent tags for cell, after the identification by the chemical reaction of immunofluorescence, the staining reaction of PCK antibody in primary cells was positive under the laser confocal microscope, confirmed that the cells are nasal mucosal epithelial cells in high purity. At the same time, in the process of primary culture, study the influence of different kinds of culture medium on cell growth, including BEGM medium, BEGM: DMEM/F12 (1:1) medium, DMEM medium DMEM medium containing 10% FBS, DMEM/F12 medium, DMEM/F12 medium containing 10% FBS. It finds that serum-free medium is more suitable for in vitro primary culture, and the cells grown in BEGM medium achieve satisfactory result with high transparency, strong refraction, uniform size and paving stone shaped.The human lung adenocarcinoma cell line (Calu-3 cells), as the in vitro simulation model of human original cells of the nasal mucosa, the cultivation process is normal operation of cell lines. Add the MEM medium with 10% FBS,1% of the double resistance and 1% NEAA to the cells to gain the suspension, remove it to the culture bottle, saturated humidity, under the condition of 37℃ and 5% CO2 incubator, continuous culturing, Refresh the medium every other day, the cells will show the fusion state in the similar shape with primary cell, compact connection and high diopter in about five days.The developments of the two kinds of cell models are in simple operation and have good repeatability. It found that the growth cycles are relatively short by drawing the cell growth curve, and they are stable to passage and culture. It is effective to keep the cell in good physiological condition by cryopreservation, showing good vitality after the recovery. It provides powerful guarantees for the in vitro research of the mechanism of nasal mucosa absorption of drugs.Inorder to connect both barrier of nasal mocusa and blood brain, we created a co-culture cells model stimulated the indirect pathway from nose to brain, to reflect the body physiology. The reaearch took advantage of the technology of transwell and co-culture and chose Calu-3 cells and MCK-MDRl cells to stimulate nasal mocusa and BBB. The novelty co-culture cells model was builted for the first time. By observed under the microscope and measured TEER, in the system of co-culture cells model, two kinds of cell which stimulated the different tissues could stably grew and formed cell monolayer, which could be used in the research of the absorption and the permeability o drugs in the indirect pathway from nose to brain.2 Cytotoxicity studies of the main efficacy components in TongqiaosanyuDue to the particularity of in vitro cell model, evaluating the cytotoxicity of drug comes before mechanism research of drug transport and compatibility to determine appropriate dose for the cell models and provide the basis for subsequent study on safe and rational drug doses of nasal drug delivery preparation.The study intends to explore the toxicity of different concentrations of drugs and compatibility in RNEC cells and Calu-3 cells by MTT colorimetric method. The results show that the cell toxicity of various drugs exist correlation with drug dose, for cytotoxic effect increased as the drug concentration increased. For the RNEC cells, when the concentration of menthol is in 0～50 μg/mL, or paeoniflorin in 0～200 μg/mL, or puerarin in 0～300 μg/mL, or the compatibility of puerarin and paeoniflorin (according to the formula ratio of 1:0.4 to paeoniflorin) in 0～100 μg/mL, or puerarin and menthol compatibility (according to the formula ratio of 1:0.5 to menthol) in 0～50μg/mL, or the three compatibility (according to the formula ratio of 1:0.4:0.5 to menthol) in 0～50 μg/mL, the cells show no toxic effects.～As to the Calu-3 cells, when the concentration of menthol is in 0～60 μg/mL, or paeoniflorin in 0～300μg/mL, or puerarin in 0～350 μg/mL, or the compatibility of puerarin and paeoniflorin (according to the formula ratio of 1:0.4 to paeoniflorin) in 0～100 μg/mL, or puerarin and menthol compatibility (according to the formula ratio of 1:0.5 to menthol) concentration in 0～60 μg/mL, or the three compatibility (according to the formula ratio of 1:0.4:0.5 to menthol) concentration within 0～60μg/mL, the cells do not show toxic effects. Compared the three drugs, the menthol with the minimum maximum dosage has the largest cytotoxicity, while the puerarin with minimum cytotoxicity. Compared with RNEC cells, the range of toxicity in Calu-3 cells caused by the compatibility or the single drug is larger, which confirm that the drug tolerance of Calu-3 cells is a little better than RNEC cells’.3 Cellular transport studies of the main efficacy components in TongqiaosanyuThe study develop RNEC cells and Calu-3 cells on the polycarbonate membrane of the Transwell cell culture plate respectively, with the culture conditions of gas-liquid interface, the cell formed monolayer membrane in order to simulate the nasal mucosa. Then study the transport of the main efficacy components in Tongqiaosanyu to evaluate the nasal mucous membrane permeability and the influence on absorption of single drugs and the prescription.Then the transport study with different concentrations of puerarin in RNEC cells and Calu-3 cells monolayer models found that the absorption of drug, efflux rate (dQ/dt) increase with the increase of concentration, the values of apparent permeability coefficient (Papp) between the different concentration groups shows no significant difference, drug efflux rate (ER) are around 1, illustrating that the mucous membrane transport of puerarin in this concentration range is given priority to passive diffusion. When puerarin compatibility of different concentrations of paeoniflorin, the transfer results show no significantly change on penetration ability compared with single puerarin part. It prompts that paeoniflorin cannot promote the nasal mucosa absorption of puerarin and play an important role of efficacy components in the prescription. When puerarin compatibility of different concentrations of menthol, the transfer results show significantly increased (P<0.05) on penetration ability compared with single puerarin part, which indicates that the menthol can enhance penetration and nasal mucosa absorption of puerarin, improving the bioavailability during the transfer process of puerarin through the nose-brain pathways.4 Transfer researches of main efficacy components in Tongqiaosanyu in the co-culture model of nose-brain pathwayBased on the co-culture cells model of Calu-3 cells and MDCK-MDR1 cells, the transports of different concentrations of puerarin and plus paeoniflorin and menthol were researched. The continuous permeability of puerarin and the enhancing penetration of compatibility were evaluated. The results were similar to the one monolayer model:the way of puerarin transport in co-cultured cells model was passive diffusion, and the permeability of puerarin could be enhanced by menthol but not by paeoniflorin.However, in the co-cultured cells model, due to many infactors, it was difficult that puerarin could tansport across two monolayers. Puerarin could only be detected by through Calu-3 cell monolayer, but could not be detected by through MDCK-MDR1 cell monolayer. In our research, only when puerarin was high concentration and menthol was 60μg/mL, puerarin could be detected by through MDCK-MDR1 cell monolayer. In order to detailed describe the function of menthol enhancing penetration, when keeping the menthol playing a role, puerarin which was over 100μg/mL could be detected by through two cell monolayers. Therefore, it could be suspectd that in one hand high concentration was the necessary condition of passing across two cell monolayers, and in the other hand it confirmed the ability of continous enhancing penetration of menthol in the co-cultured cells model.5 Research on the mechanism of cell transport of main efficacy components in TongqiaosanyuDrug transport in the nasal mucosa epithelial cells contains both the active form of transportation and passive diffusion. So the research analyzes the puerarin transfer process and influencing factors involved in the prescription by various indicators.The research of the mechanism of drug transport starts from the passive diffusion. Transepithelial electrical resistance (TEER) generally used to characterize the structures of monolayer cell membrane, our studies centre around the influence that puerarin, paeoniflorin and menthol make on cells’ TEER values. The results of measurements of TEER value of Cells after treated show that the TEER values of the two cell models are declined significantly after compatibility menthol, suggesting that menthol may enhance mucous membrane permeability by loosing single-layer structure of cytomembrane and enhancing transmembrane transport of drugs via the paracellular route. The tight junction(TJ) of nasal mucosa cells is the main barrier of limiting drug intake.TJ is mainly composed of a variety of tight junction proteins, and our research will evaluate the effect of drugs by the density of the human nasal mucosa, to be more clear, we focus on the drugs" influence on the four kinds of tight junction proteins, Occludin, Claudin 1, ZO-1, F-actin, using immunofluorescence staining method, with a laser confocal microscope to observe and analysis the fluorescence intensity. All the four kinds of tight junction proteins found a certain degree of inhibition by menthol, for menthol can open close connection structure and weaken the close connection of barrier function to promote the nasal mucous membrane permeability of drugs.As for the research in active transport, research group starts study with the drugs affects on the expression of P-lycoprotein of mucosa epithelial cells. After using flow cytometry instrument to analyze the fluorescence intensityon, which comes from the intake rhodamine 123 in the cell after the treatment, compared with the typical P glycoprotein inhibitor, verapamil. It found that the puerarin, paeoniflorin and menthol all interact with P-glycoprotein existence, so the coordinating makes competitive inhibition in theory, which may enhance the competitive of puerarin. The fluidity of cytomembrane also affects the extent of the drug transport across the membrane. The studies adopt fluorescent bleaching recovery technique (FRAP) to observe the drug effect on migration characteristics of fluorescent markers on cell membrane, draw the fluorescence recovery curve, calculate fluorescence recovery rate. It found that when menthol reaches a certain concentration, the cell fluorescence recovery rate can be increased significantly, which means menthol can act on the phospholipid bilayer of cytomembrane, increasing the membrane lipid fluidity, reducing the viscosity of membrane, to facilitate the drug transport across the membrane. In addition, the active transport is often associated with enzyme mediated energy metabolism, therefore, this study examines the influence of drugs on Na+-K+ atpase and Ca2+ atpase, which makes interpretation of the process of energy metabolism when drug across the plasma membrane alone or compatibility. It found that all the three drug ingredients have no obvious effect on the energy metabolism of Na+-K+ atpase. As for the Ca2+ atpase, menthol can significantly enhance its activity, which shows that menthol can adjust the activity of ATP enzyme, promoting the active transport associated with calcium ion pump. At the same time, it explains the possible mechanism of the adjustment of the menthol to the tight junction proteins, for the expression suppression of the tight junction protein is also associated with calcium ion flow.更多还原