Intestinal Absorption Characteristics And Mechanism Of Chitooligosaccharides With Single Degree Of Polymerization

Chitooligosaccharides.as an important functional oligosaccharide,have important physiological activities such as improving immunity,liver protection and tumor suppression.They are important new food raw materials.However,the absorption mechanism and transmembrane transport process of chitooligosaccharides in the intestinal are not clear,which limits the product development and further research of their physiological activities.We intend to study the vitro absorption and bioavailability of chitooligosaccharides with single degree of polymerization by setting up everted-gut model and human intestinal Caco-2 monolayer cell absorption model.In this study,intestinal absorption characteristics and transmembrane transport mechanisms of chitooligosaccharides with different degrees of polymerization were elucidated and intracellular accumulation sites of chitooligosaccharides were determined to provide theoretical support for further understanding their absorption characteristics and metabolic processes.Firstly,everted-gut model was established,and the feasibility of the model was verified by glucose transport.Lactate dehydrogenase method and paraffin section method were used to determine the dosing time and concentration of chitooligosaccharides.The intestinal absorption of glucose and chitooligosaccharides was determined by high performance liquid chromatography-ELSD.Results showed that the junction of small intestine is tight and the villus structure is relatively complete within 100 min.LDH enzyme activities were increased to 1.5 times of the control group only at 120 min under the three experimental concentrations.The difference was significant.In summary,the time range of subsequent chitooligosaccharides transport experiments was selected to be within 100 min^.and the experimental concentrations were selected to be 2 mg/mL,4 mg/mL,and 8 mg/mL,respectively.Secondly,the everted-gut model was used to determine the effects of concentration,time,SGLTs and GLUT2 active transporters on the absorption of chitooligosaccharides with different degrees of polymerization.The results showed that the transport capacity of chitooligosaccharides in everted-gut sac was not correlated with the concentration of 2 mg/mL and 4 mg/mL.Linear correlation was found between transport volume and concentration at 8 mg/mL.It indicated that at low and medium concentrations(≤4 mg/mL),chitooligosaccharides were mainly transported by carrier proteins.At higher concentrations(8 mg/mL),the membrane transport mechanism of chitooligosaccharides was passively diffused.After the GLUT2 transporter inhibitor was added,the apparent permeability coefficient of chitobiose decreased from(13.99 ± 0.40)×10-6 cm/s to(11.30 ± 0.12)×10-6 cm/s,the inhibition rate was 19%;chitotriose decreased from(13.99 ± 0.40)×10-6 cm/s to(11.30 ±0.12)×10-6 cm/s,the inhibition rate was 19%.The inhibition rate of chitotriose is 18%.Chitotetraose and chitopentaose decreased from(12.43 ± 0.17)×10-6 cm/s and(6.62 ±0.18)×10-6 cm/s to(9.72 ± 0.17)×10-6 cm/s and(5.92 ± 0.01)×10-6 cm/s,respectively.The inhibition rates were 22%and 26%.When the SGLTs transporter inhibitor was added,only the cumulative penetration of chitotetraose and chitopentaose were significantly reduced,with inhibition rates of 9%and 11%.respectively.The inhibitory effect of GLUT2 transporter inhibitors was better than that of SGLTs transporter inhibitors,indicating that chitooligosaccharides were transported mainly through GLUT2 proteinThirdly,the Caco-2 cell model was used to verify the absorption mechanism of chitooligosaccharidcs.MTT assay was used to determine the effect of chitobiose to chitopentaose on the cell viability of Caco-2 cells.The results showed that incubation of chitobiose to chitopentaose below 2 mM for 4 h had no significant effect on the cell viability of Caco-2 cells.0.5,1,2 mM was selected as the dosing concentration in Caco-2 cells.At low and medium concentrations(0.5,1 mM),the transports of chitooligosaccharides were independent of concentration,and at high concentrations(2 mM),the transport amount increased significantly.At the same time,the PDR values of chitobiose to chitopentaose were more than 1.5.indicating that active transport existed in the absorption process.The results of cell model were consistent with the results in the everted-gut model.Thus,the results showed that chitooligosaccharides in the small intestine were absorbed by multiple ways-coexist of passive diffusion and carrier protein transport.The active transporter inhibition assay also showed that the GLUT2 transporter is involved in the transport of chitobiose to chitopentaose.while the SGLTs transporter is involved in the transport of chitotetraose chitopentaose.Chitobiose to chitopentaose were absorbed through the GLUT2 protein preferentially.Finally,fluorescence microscopy was used to investigate the localization of chitobiose and chitopentaose into living cells.The results showed that the rate of chitobiose and chitopentaose entering the cells was the concentration-dependent manner.Incubation of chitobiose and chitopentaose with low-concentration(10,100 μM)in Caco-2 cells,chitooligosaccharides were concentrated on the surface of the nuclear membrane or cell membrane.At high concentration(500 μM).chitobiose and chitopentaose were dispersed in the cells.Chitobiose entered the cell faster than chitopentaose and could be detected at 10 min,and the fluorescence signal of chitopentaose could be detected only after incubation for 30 min.The chitobiose and chitopentaose were not degraded by intracellular lysosomes within 240 minutes after in vitro absorption,and both of them could aggregate in the endoplasmic reticulum and mitochondria.The chitobiose is preferentially localized to the endoplasmic reticulum,and the chitopentaose is preferentially localized to the mitochondria.It is revealed that chitooligosaccharide may exert certain physiological activities by regulating endoplasmic reticulum and mitochondria-related signaling pathways.