| Objective:1. Glycosides has extensive pharmacological activities, such as effective treatment of central nervous system disease, but the clinical application of it has been limited because its poor membrane permeability and low bioavailability. Gastrodin (GAS), paeoniflorin (PAE) and echinacoside (ECH) have been chose as model drugs in this article. The aim was to illustrate the transport mechanism of model drug across the BBB, and find out the reason of their poor membrane permeability by studying their bilateral apparent permeability coefficient through BBB and the changed bilateral apparent permeability coefficient of model drugs caused by protein inhibitors. The results will provide a theoretical basis for the next step to take effective measures to solve the poor membrane permeability.2. Document surveys showed that chuanxiong could increase the concentrations of glycosides in brain, and the combination of chuanxiong and other Tradition Chinese Medicine which contains glycoside is often used to treat brain diseases. And therefore, the promoting material basis will be clarified by the changed transport capacity of model drugs caused by chuanxiong. The results will solve the poor membrane permeability of glycosides by compatibility method, and provide a new approach for more effective using glycosides to treat CNS diseases.3. The mechanism of the interactions was analyzed by the effects of the active ingredients of chuanxiong on the expression of efflux proteins and tight junction proteins, as well as the changed expression of MDRl-mRNA. The results will provide a potential penetration enhancer and solve the poor membrane permeability of glycosides.Method:1. After intragastric administration of chuanxiong extract once or continuous for 7 days, the contents of EB in rat’s brain was detected by fluorometric method. Study the effects of chuanxiong on BBB permeability to determine it whether or not as message drug to promote other drugs through the BBB.2. The content determination methods for GAS, PAE and ECH were established by LC-MS/MS. The BBB model in vitro was establish by using MDCK and MDCK-MDR1 cells, and its integrity was verified by electron microscopy observation and sodium fluorescein leakage experiments. The bilateral apparent permeability coefficients Papp (A→B) and Papp (B→A) were measured after different concentrations of GAS, PAE and ECH across MDCK, MDCK-MDR1 cells. The changed bilateral apparent permeability coefficients of GAS, PAE and ECH which were caused by P-gp inhibitor verapamil, MRP1 inhibitor probenecid and tight junction regulator EDTA, were measured to explore possible ways to solve the poor membrane permeability of glycosides.3. The Papp(A-B) of GAS, PAE and ECH were measured after they across MDCK-MDR1 cells mixed with different concentration of ferulic acid (FA), tetramethylpyrazine (TMP), ligustilide (LIG), senkyunolide A (SENA) and senkyunolide I (SENI) respectively. When compared with control groups, the changed Papp would enable us to find the promoting ingredient of chuanxiong and the promoting dosage.4. The combination mode and the bonding strength between three glycosides, five active ingredients of chuanxiong, as well as P-gp inhibitor verapamil, MRP1 inhibitor probenecid, tight junction regulator EDTA between P-gp, MRP1, claudin and ZO-1 were analyzed by Molecular Docking method to clarify the mechanism from the point of competitive protein binding. The P-gp substrate rhodamine 123 (RH123) was selected as a fluorescent probe to study the effects of different concentrations of TMP, FA, LIG, SENA, SENI and verapamil on RH123 accumulation in MDCK-MDR1 cells, and elucidate the promoting mechanism from the point of the regulation of P-gp efflux function. The effects of five active ingredient of chuanxiong at different concentrations on the expression of P-gp, MRP1, claudin-5 and ZO-1 protein was studied by Western Blotting to clarify the mechanism from the protein molecular level.Results:1. The content of EB in rat’s brain increased with the increasing dosage of chuanxiong after intragastric administration once. Compared with the control group, there was no significant difference (p>0.05), and the content of EB was up to about 35%. The content of EB in brain after successive administration was more than single-dose administration group. The content of EB in rat’s brain increased with the increasing dosage of chuanxiong after intragastric successive administration. Chuanxiong extract at 1.26,2.52 and 3.78g·kg-1 dose have a significant effect (p<0.05) on the content of EB, which made its content increased more than 80%. Whether intragastric administration once or successively, when the dosage of chuanxiong was greater than 1.26 g·kg-1, the content of EB in brain was less affected. The results indicated that the adjustment ability of chuanxiong was limited. In addition, chuanxiong extract used in this article was extracted by water and mainly contained phenolic acids such as FA. Because the promoting effects of chuanxiong after intragastric administration successively were stronger than intragastric administration once, phenolic acids in chuanxiong extract have weak regulating ability on BBB, and the promoting ability was connected with the acting time.2. MDCK and MDCK-MDR1 cells were built as BBB model in vitro. Under the transmission electron microscope, cells showed paving stone shape, lined uptightly with visible tight junction; and meanwhile, the transendothelial electrical resistance (TEER) of cells was more than 500·Ωcm2 and sodium fluorescein had no leakage, which indicated that the cell model was successfully built. Both Papp (A→B) of GAS, PAE and ECH was less than 1x10-6 cm·s1 in MDCK and MDCK-MDR1 cells, which showed that they were low permeability. ECH has the worst permeability of all. There was a linear relationship between the concentration and transport rate, so the three model drugs were not saturated and mainly passive diffusion in the concentration of 100-80μg·mL-1. The ER of GAS in MDCK-MDR1 cell was different from it in MDCK cell. ER of GAS in MDCK-MDR1 cells was between 1 and 1.5, and the P-gp inhibitor verapamil could significantly increase the transport of GAS, which indicated that GAS was a substrate of P-gp and its transport was affected by P-gp. ER of PAE in MDCK-MDR1 cells was more than 2, both of the P-gp inhibitor verapamil and MRP1 inhibitor probenecid significantly increased the transport of PAE, which indicated that PAE was a substrate of P-gp and MRP1. ER of ECH in MDCK-MDR1 cells was more than 2, and both of verapamil and probenecid significantly increased the transport of ECH, which indicated that ECH was a substrate of P-gp and MRP1. EDTA significantly increased the Papp (A→B) of GAS, PAE and ECH (p<0.01), which showed that GAS, PAE, ECH were partly paracellular transport.3. LIG, SENA and SENI significantly increased the Papp (A→B) of GAS, PAE and ECH to play a penetration enhancement in a concentration-dependent manner. LIG had strongest promoting effects on the transport amounts of GAS. 30μg·mL-1LIG (LIG:GAS=1:20, g/g) significantly increased the Papp (A→B)of GAS (p<0.01). SENA and SENI had stronger promoting effects on the transport amounts of PAE than LIG. Both 20μgmL-SENA and 20μg·mL-1 SENI (SENA:PAE=120, SENI:PAE=1:20, g/g) significantly increased the Papp (A→B)of PAE (p＜0.05). LIG and SENI had stronger promoting effects on the transport amounts of ECH than SENA. Both 20μg·mL-1 LIG and 20μg·mL-1 SENA(LIG:ECH=1:20, SENI:ECH=1:20, g/g) significantly increased the Papp (A→B)of ECH (p<0.05). Different concentrations of FA and TMP had no significant influences on the transports of three model drugs (p＞.05).4. Molecular docking results showed that GAS, PAE and ECH could be combined with P-gp protein through intramolecular hydrogen bonding. LibDock score of ECH was higher than positive drug, and meanwhile LibDock scores of GAS and PAE were close to positive drug, which indicated that GAS, PAE and ECH were P-gp substrates. PAE and ECH also could be combined with MRP1 protein through intramolecular hydrogen bonding, and therefore they were substances of MRP1. Because ECH contained two glycosyl with many phenolic hydroxyl groups, the LibDock score was highest. The combining abilities between five bioactive ingredients of chuanxiong and related proteins were weak and LibDock Socre were less than three glycosides, so the ingredients of chuanxiong were not through competitively binding with proteins to play the role of permeation enhancement. LIG, SENA and SENI significantly increased the accumulation of P-gp substrate rhodamine 123 in cells and significantly reduced the expression of P-gp protein, which indicated that the three components of chuanxiong promoted GAS, PAE and ECH across BBB by inhibiting the expression of P-gp protein. High concentration of SENI also could inhibit the expression of MRP1 to promote PAE and ECH across BBB. LIG, SENA and SENI also inhibited the expression of claudin-5 and ZO-1, and promoted GAS, PAE and ECH across BBB by opening the tight junctions. Although FA and TMP were P-gp substrates and could inhibit the expressions of P-gp and MRP1, in this article, they didn’t increase the transport of GAS, PAE and ECH, the reason maybe that the inhibiting abilities of GAS, PAE and ECH were stronger than FA and TMP. FA and TMP also inhibited the expressions of claudin-5 and ZO-1, but TEER did not significantly change after permeation, it may be explained that no significant enhancement of FA and TMP was due to the different acting time between transport experiment and western blotting experiment. The time of western blotting experiment was 10h, and the transport experiment was 2.5h, so the shorter time in transport experiment made FA and TMP difficultly play the role of opening the tight junction to increase the transport of GAS, PAE and ECH.The anisotropies of fluorescent probe were significantly reduced after the compatibility, which indicated that the three components of chuanxiong promoted GAS, PAE and ECH across BBB by enhance the membrane fluidity, and meanwhile, the promoting effects was positive correlated with concentrations.Conclusion:GAS, PAE and ECH were difficult to penetrate the blood-brain barrier, and ECH has the worst permeability of all. All of them were mainly passive diffusion and partly paracellular transport. The transport of GAS was affected by the efflux of P-gp, and the transport of PAE and ECH were affected by the efflux of P-gp and MRP1. FA and TMP did not increase the transport of GAS, PAE and ECH. LIG, SENA and SENI in the volatile oil of chuanxiong could promote GAS, PAE and ECH across BBB. LIG, SENA and SENI inhibited the expression of P-gp, tight junction protein claudin-5 and ZO-1 to enhance the transport of GAS. LIG, SENA and SENI also inhibited the expression of P-gp, claudin-5 and ZO-1 to enhance the transport of PAE and ECH, and SENI has a synergistic effect on the expression of MRP 1. LIG, SENA and SENI increased the membrane fluidity of MDCK-MDR1 to enhance the transport of GAS, PAE and ECH to act as penetration enhancers.
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