Study On The Transport Mechanism Of Hyaluronic Acid With Different Molecular Weights In The Caco-2 Cell Monolayer Model

As a kind of natural polysaccharide,Hyaluronic Acid comes from a wide range of sources and has good biological activities.It has been widely used in medicine and daily life.At present,the Commission of Health and Development has approved sodium hyaluronate as a food additive material,but the biological activity of HA is often different due to the large difference in molecular weight.Therefore,elucidate the oral transport mechanism of HA with different molecular weights will provide theoretical guidance for better play of its biological activities.On this basis of our laboratory successfully established a 5-day Caco-2 cell monolayer model in the early stage.,FITC-HA of 10 kD,40～100 kD and 100～200 kD were synthesized by fluorescein isothiocyanate(FITC)labeling method to study the oral transport mechanism and differences of HA with different molecular weights.To provide experimental basis for the molecular weight selection of HA oral absorption.The main achievements of this subject are as follows:1.Fluorescence labeling and results verification of HA with different molecular weightsIn this study,10 kD,40～100 kD and 100～200 kD HA were conjugated with FITC by tyramine method,and then the products were separated and purified by dialysis and AKTAPure,and the fluorescence absorption and sugar content of the purified products were verified by agarose-gel electrophoresis,multifunctional enzyme labeling instrument and phenol-sulfuric acid method.Three kinds of HA with different molecular weight have been successfully labeled.The average value of FITC-HA labeling per mole of 10 kD,40～100 kD and 100～200 kD are 25.19,11.24 and 6.22,respectively.2.Optimization of 5-day Caco-2 cells monolayer modelThe 5-day model was established by adding 50 μg/mL fucoidan to the 7-day Caco-2 cells monolayer model.It was confirmed that there was no difference between the 5-day model and the 7-day model in the transmembrane resistance values,alkaline phosphatase activity,permeability markers fluorescence yellow,FITC,transferrin and glucan absorption rates.Then,the in vitro transport experiments of large molecule drugs(FITC-HA synthesized above)and small molecule drug Doxubicin were conducted for the first time on the 5-day model,and the experimental results were compared with those in the 7-day model.In view of the differences in the transport results of HA,a reasonable use method was proposed to improve the 5-day model.The Apparent permeability coefficient(permeability coefficient)of 10 kD and 100～200 kD FITC-HA in this model was significantly improved.Papp)were 3.016×10-6 cm/s and 2.332×10-6 cm/s,respectively.The transport results of dextran,transferrin and small molecule drug DOX in the improved model were also compared with that in the 7-day model,which indicated that the improved method was in line with the standards for the use of Caco-2 cells monolayer model,and also provided a more efficient model selection for the in vitro transport experiments of other drugs.At the same time,due to the difference in molecular weight,the transport effect of 10 kD HA is better than 100～200 kD HA,and the Papp value is also higher.3.Study on transport mechanism of HA with different molecular weight in Caco-2 monolayer cell modelIn the modified model,the transport mechanism of 10 kD and 100～200 kD HA across intestinal epithelial cells was studied.The results showed that the intake of 10 kD and 100～200 kD HA by Caco-2 cells increased with time,and the transport process required energy consumption,but with the extension of time,there would be effection.The transshipment volume does not increase continuously,which indicates that the number of transporters on the cell is limited and there will be saturation.With the addition of inhibitors Chlorpromazine and Dynasore,Papp values of 10 kD and 100～200 kD FITC-HA decreased by 84.61%,28.15%,41.41%and 60.06%,but MβCD has little effect on 10 kD and 100～200 kD FITC-HA,suggesting that the transport of 10 kD and 100～200 kD FITC-HA may be affected by clathrin-mediated endocytosis pathway,but the influence of fossa(lipid raft)protein is relatively small.For giant pinocytosis,10 kD and 100一 200 kD FITC-HA showed significant differences after administration of TRPP3channel inhibitor(EIPA),with transport inhibition rates of 6.18%and 89.25%,respectively.These results suggest that 100-200 kD HA may enter cells through the giant pinocytosis pathway much more than 10 kD HA.In the intracellular transport process,the transport inhibition rates were 89.99%and 60.47%after the addition of Monension,respectively,which indicated that the transport of 10 kD and 100～200 kD HA in the cell may be related to the early endosome acidification to lysosome maturation.After addition of Nocodazole,the transport rate of 100～200 kD HA decreased by 82.26%,but the effect on 10 kD HA was not significant,which indicated that part of 100～200 kD HA was dependent on tubulin for intracellular transport.In conclusion,FITC was used to fluorescently label HA of 10 kD,40～100 kD and 100～200 kD,and the absorption and transport mechanism and differences of FITC-HA of 10 kD and 100～200 kD were studied in an improved 5-day Caco-2 cell model.The results show that the transport of 10 kD and 100～200 kD HA is time-dependent and energy-dependent.In the endocytosis pathway,both 10 kD and 100～200 kD HA exhibit clathrin-dependent endocytosis and transport mechanisms,but 100～200 kD HA also depends on giant pinocytosis.In the intracellular transport process of Caco-2,both 10 kD and 100～200 kD HA are affected by endosomal acidification,and part of 100～200 kD HA depends on tubulin mediated transport.This master’s project comprehensively elaborated the oral absorption and transport mechanism of HA with different molecular weight and its differences.