Study On The Transport Mechanisms Of Gastrodin Across The Intestine Barrier And The Blood-brain Barrier Based On The Glucose Transporters

Research background and purpose:Gastrodia elata is a kind of valuable traditional Chinese medicine.The content of gastrodin is the highest among the pharmacologically active components of gastrodia,therefore,gastrodin is recognized as the main effective component.Gastrodin has a variety of pharmacological effects such as sedation,analgesia,anticonvulsant with low toxicity and few adverse reactions.It has been widely used in the treatment of dizziness,insomnia,and neuralgia clinically.Gastrodin acts on the benzodiazepine receptors in the brain to enhance the function of theγ-aminobutyric acid and benzodiazepine receptor complex so that it can exert a central nervous system inhibitory effect.Because gastrodin is a water-soluble drug with strong polarity,it is generally considered that gastrodin possesses weak transmembrane ability and poor oral bioavailability so it needs to be hydrolyzed into aglycon to be absorbed.However,oral gastrodin is absorbed rapidly(Tmax=0.8 h)with high bioavailability(about 86%)in fact.Based on this background,our team conducted research on the intestinal absorption mechanism of gastrodin.Due to high water-solubility of gastrodin,it is speculated that it transports through membrane not mediated by passive diffusion but by carrier mediation.Since the structure of gastrodin contains a molecule of glucose structure,it is predicted that it is likely to complete the transport and absorption through the glucose transport pathway.The results showed that glucose can competitively inhibit the absorption rate of gastrodin,and the addition of SGLT1 inhibitor phlorizin can significantly inhibit the absorption of gastrodin.In addition,some research have confirmed that gastrodin can enter the brain through the blood-brain barrier(BBB),but so far it is not clear about the mechanism of gastrodin across the BBB.Because the target organ of gastrodin is the brain,the study of transport mechanism across the BBB is of great importance.According to the results of previous studies,we speculate that gastrodin across the BBB may be mediated by the glucose transport pathway,just like its intestinal absorption mechanism.Based on the above research,this project focuses on the transport mechanisms of gastrodin across the intestine barrier and the BBB based on the glucose transporters.Research methods and content:1.To investigate the effects of glucose and phlorizin on the absorption of gastrodin into the blood,we used Sprague-Dawley rats to conduct pharmacokinetic experiments.q-PCR experiment was used to explore relative mRNA expression of glucose transporters Sgltl and Glut2 in each intestinal segment.Western blot experiment was used to explore relative protein expression of glucose transporters SGLT1 and GLUT2 in each intestinal segment.2.The factors affecting the expression of glucose transporter were investigated by cell experiment,and then the transport mechanism of gastrodin across intestinal barrier was studied.Firstly,the effects of different concentrations of glucose and SGLT1 inhibitor phlorizin on gastrodin uptake were investigated.Then the transport models of Caco-2 and MDCK were constructed to predict the intestinal transport capacity of gastrodin.Finally,the mechanism of gastrodin transport across the intestinal barrier was explored by adding different glucose transporter inhibitors.The results of Caco-2 and MDCK transport experiments were compared to analyze the correlation and the applicability of two cell models.3.In order to determine whether gastrodin can enter the brain tissue and the effect of GLUT1 inhibitor phloretin on gastrodin entering the brain,gastrodin was injected into the tail vein of mice,and the content in the brain tissue of mice was measured by UPLC method.BBB cell model was constructed by co-culture of HBMEC and HA cells to explore the transport mechanism of gastrodin across BBB.4.To dock gastrodin with glucose transporters SGLT1 and GLUT1,we simulated the binding sites and binding capacity of gastrodin with both glucose transporters mentioned above.Then we contrated the prediction results with glucose,and further compared gastrodin with glucose about the degree of superimposition of the binding mode with the corresponding glucose transporter to determine whether gastrodin is transported via above two glucose transporters.Results and conclusions:1.The results of pharmacokinetics in rats showed that glucose could compete with gastrodin for absorption,and the competitive effect enhanced with the increase of glucose concentration.Phlorizin could inhibit the absorption of gastrodin,and the inhibition increased with the increase of phlorizin concentration.Phlorizin inhibits gastrodin intestinal absorption by down-regulating the intestinal expression of SGLT1,which further indicates that gastrodin intestinal absorption is mediated by SGLT1.2.The results of Caco-2 uptake experiment were consistent with those of pharmacokinetics experiment.The addition of glucose or phlorizin could reduce the uptake of gastrodin,and phlorizin could decrease the expression of SGLT1 on Caco-2 cell.In the Caco-2 cell transport model,the absorption of gastrodin showed a certain concentration dependence.Adding the inhibitor phlorizin can significantly inhibit the transport of gastrodin.In the MDCK cell transport model,the transport capacity of gastrodin was not dose-dependent.Adding the inhibitor phlorizin can also significantly inhibit the transport of gastrodin.All the above experiments indicated that gastrodin was mainly mediated by SGLT1 to transport across the intestinal barrier.3.The Papp values of gastrodin in Caco-2 and MDCK transport models were more than 10-6 cm/s,which indicated that oral absorption of gastrodin was pretty good,and the results were consistent with its high bioavailability.However,the absorption results of gastrodin in the Caco-2 and MDCK cell models were slightly different due to the inconsistent expression levels of glucose transporters in different cells.The expression level of glucose transporters in MDCK cells is lower than that in Caco-2 cells,the corresponding glucose transporter was saturated when gastrodin at low concentration so there was no dose-dependent.4.The results of injecting gastrodin via tail vein showed that gastrodin can cross the BBB.After adding the GLUT1 inhibitor phloretin,the content of gastrodin into the brain was significantly reduced.The Papp value of gastrodin on the BBB cell model was 10-8 cm/s,which indicated the poor ability of gastrodin to cross the BBB.The transport ability of gastrodin weakened significantly after adding GLUT1 inhibitor phloretin,which proved that gastrodin was mainly mediated by GLUT1 to transport across the BBB.5.The binding sites and modes of gastrodin with SGLT1 and GLUT1 were very similar to those of glucose,so it can be considered that transport process of gastrodin is similar to that of glucose.Gastrodin can extremely be transported by SGLT1 in the intestine and transported by GLUT1 in the brain.In summary,this research has conducted in vivo pharmacokinetic experiments,in vitro cell transport models,molecular biology experiments and computer simulation molecular docking technology to jointly prove that gastrodin can extremely be transported by SGLT1 in the intestine and transported by GLUT1 in the brain.