| 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.
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