Absorption Mechanism Of Paeonol Nanoemulsion In Intestinal Epithelial Cell Model

Paeonol is one of the main active components of the dried root or the whole plant of Cynanchum paniculatum,which has a wide range of pharmacological activities but poor water solubility and low bioavailability.In order to improve the bioavailability of paeonol,pre-project group incorporated paeonol into the nanoemulsion.The results of pharmacokinetics showed that the prepared nanoemulsion can indeed improve the bioavailability and absorption of paeonol,however,the mechanism of paeonol absorption improvement is not clear.As we know the absorption site for oral drugs are mainly located in small intestine,and that the human colon adenocarcinoma Caco-2 cells are similar to small intestinal epithelial cells in morphology,marker enzyme expression,transportation and permeability characteristics under certain condition of culture.Therefore,Caco-2 cells are often used to study the absorption mechanism of drugs through normal intestinal epithelial cells.In addition to a large number of normal intestinal epithelial cells,M cells existing in lymphatic follicle of small intestine can form channels on intestinal mucosa,which is also the main way to absorb nanoparticles through the small intestine.The co-culture of Caco-2 cells and Raji B cells can form a monolayer cell model of mucosal follicular epithelial cells(FAE),FAE cells monolayer model has also become an important cell model for studying M cells uptake mechanism of nanoparticle drug delivery systems.Based on the previous work,we established Caco-2 cells model and FAE cells monolayer model to explore the uptake mechanism and transport characteristics of paeonol nanoemulsion through intestinal epithelial cells.The mechanism of absorption promotion of oral Pae nanoemulsion will provide basis for the performulation of paeonol oral nano preparation,as well as new research methods and thoughts for the application of nanoemulsion drug delivery system in the improvement of bioavailability for some active components of insoluble Chinese Medicine phenolic.OBJECTIVEOBJECTIVE Explore the absorption mechanism of paeonol nanoemulsion through investigating the uptake and transport characteristics of paeonol nanoemulsion based on Caco-2 cells model and FAE cells model.METHODS Screened the cell safety concentration range of paeonol nanoemulsion by CCK-8and LDH method;The concentration of paeonol in Caco-2 cells was determined by HPLC;Established an in vitro FAE cells model and M cells were identified by transepithelial electrical resistance and expression of Galectin-9;The concentration of paeonol in FAE cells model was determined by HPLC;Established the model of Caco-2cells transport;The compactification and polarization of the monolayer was determined by transepithelial electrical resistance and the alkaline phosphatase activity;The distribution of ZO-1 was detected by laser scanning confocal microscopy;Investigated separately the effects of efflux protein inhibitors on the transportation of paeonol nanoemulsion though Caco-2 cell monolayer.The expression of Occludin,Claudin-1,ZO-1,P-gp,BCRP and MRP2 was detected by Western blot.RESULTS1.Uptake characteristics of paeonol nanoemulsion on Caco-2 cells and FAE cells monolayer model The results of the relevant indicators in the Caco-2 cell model indicated that the cell model was successfully established;The Caco-2 cell uptake amounts of paeonol nanoemulsion gradually increased with time and reached the maximum at 2 h;Within a certain concentration range,the cell uptake amounts was proportional to the concentration of paeonol;The uptake amounts of paeonol nanoemulsion did not differ significantly at 4 °C and 37 °C;The results of FAE monolayer cell model related indicators showed that the cell model was established successfully;the FAE cell uptake amounts of paeonol nanoemulsion in FAE cell monolayer model increased significantly with time,concentration and temperature,and the uptake amounts is greater than Caco-2 cell's.2.The transport characteristics of paeonol nanoemulsion on Caco-2 cells monolayer.Compared to the paeonol group,the paeonol nanoemulsion group had a significantly increased Papp from the apical side to the basolateral side in the Caco-2cell monolayer,while the basal side to the apical side Papp decreased significantly and the efflux rate decreased significantly;The tight junction protein ZO-1 of paeonol nanoemulsion group showed a discontinuous shape,and the expression of ZO-1decreased significantly.The P-gp inhibitor Ver and the MRP2 inhibitor MK-571 could reduce the efflux rate of paeonol group.However,the efflux rate of paeonol nanoemulsion was not affected by efflux inhibitors.Compared with paeonol,Pae nanoemulsion could significantly reduce the expression of efflux protein P-gp,MRP2 and BCRP.CONCLUSION1.Results of uptake experiments of nanoemulsion on Caco-2 cells and FAE cells monolayer models show that nanoemulsion can increase the normal intestinal epithelial cells' passive uptake amounts of paeonol,M cells' endocytosis amounts,and the uptake ability of nanoemulsion by M cells is stronger than that of normal intestinal epithelial cells.2.Results of transportation experiments of nanoemulsion on Caco-2 cells monolayer model show that paeonol is susceptible to be effluxed by P-gp and MRP2,resulting in poor absorption of paeonol through the intestinal epithelium;Nanoemulsion opens the tight junction between cells to promote the transport of paeonol from paracellular pathway and reduces the expression of efflux protein to decrease efflux rate of paeonol,which may be the reason why nanoemulsion increase intestinal epithelial absorption of paeonol.