Study On The Regulation Of Intestinal Epithelial Tight Junction

Intestinal mucosal barrier, which could effectively block the invasion of intestinal microorganisms and toxins, was an important protective barrier. Cell junction is an important component of the intestinal mucosal barrier, influencing the absorption efficiency of hydrophilic macromolecules such as protein drugs and vaccines through the paracellular route. Recent studies had found that some pathogens could secret toxins to disrupt intestinal mucosal barrier after colonization, including the damages to intestine epithelial cells and the destruction of cell junction proteins as well as cytoskeletal proteins. The whole study was divided into two major parts. First, through the Caco-2 cells (human colon carcinoma cell) monolayer model and the mice experiment to study the modulation of absorption enhancers to cell junction (tight junction and adherent junction) and the paracellular route, as well as detection the promotion effects of absorption enhancers to the fluorescent molecules and collagenase. Second, through the Caco-2 cells monolayer model and the rats experiment to study the effects of lactobacilli on the expression and distribution of cell junction proteins and observe the inhibition ability of lactobacilli to the adhesion of pathogenic bacteria, in order to reveal the mechanism of lactobacilli's antagonistic ability against pathogenic bacteria. The details were divided into four experiments as follows:Experiment one:The impact of absorption enhancers on cell junction of Caco-2 cells Caco-2 cells, cultured in vitro, could differentiate into intestinal epithelial cells. Its structure and biochemical functions were very similar to the human intestinal epithelial cells, possessing microvilli and tight junction and so on. The Caco-2 cells monolayer was an ideal model to study the function of intestinal epithelial cells, such as drug absorption, intestinal barriers and cell differentiation. In this study, six absorption enhancers were selected, including:EDTA, SDS, sodium cholate, glucose, ethanol, and menthol. We examined the effects of absorption enhancers on the viability of Caco-2 cells, the expression and distribution of cell junction proteins and the paracellular route. The results of MTT cytotoxicity test demonstrated that compared with the control group, EDTA (2.5mmol/L), SDS (0.2mmol/L), sodium cholate (10mmol/L), glucose (750mmol/L), ethanol (10%), and menthol (1%) showed no toxicity to Caco-2 cells under 2h. The results of transport experiments showed that the six absorption enhancers all had a positive role in promoting the absorption of fluorescent molecule, but the degrees of promotion were various. Compared with the control group, EDTA (2.5mmol/L) significantly enhanced the absorption of FITC in 2h (P＜0.01); SDS (0.2mmol/L) and menthol (1%) significantly increased the absorption of FD20S in 2h (P<0.01); glucose (750mmol/L) and menthol (1%) significantly increased the transport amount of FD2000S in 2h (P<0.01). We simultaneously found that in control group, cell junction proteins ZO-1, claudin-1, occludin, and E-cadherin all distributed along the cell membrane and showed clear boundaries, outlining the shape of epithelial cells. When the absorption enhancers were added to the Caco-2 cells, the expression of Zo-1, claudin-1, occludin, and E-cadherin were decreased. Some proteins scattered in the cytoplasm and the fluorescence intensity decreased, suggesting that the tight junction and adherent junction were disrupted at different degrees. This experiment proved that glucose (750mmol/L) and menthol (1%), which had less effect on the activity of Caco-2 cells and cell junction proteins, significantly increased the absorption of the large molecular weight marker FD2000S, showing a promising application prospect.Experiment two:The effects of absorption enhancers on the absorption of collagenase Collagenase, which can dissolve fibrin directly and activate plasminogen indirectly, is an ideal clinical thrombolytic drug to improve microcirculation. Through the Caco-2 cells monolayer model (in vitro) and mice administered (in vivo) tests, we studied the promotion effects of absorption enhancers on FITC (fluorescein isothiocyanate, FITC) labeled collagenase across intestinal epithelium. The results of transport assay on Caco-2 cells demonstrated that compared with the control group, the absorption enhancers all significantly increased the absorption of collagenase (P<0.05), although the promotion degrees were various. The sequence of enhanced efficiencies in 2h on Caco-2 cells was as follows:glucose (12.87±1.0%)> EDTA (12.71±0.74%)> menthol (11.71±0.73%)>sodium cholate (11.27±1.21%)> SDS (9.17±1.74%)> ethanol (8.37±0.74%)> control (6.60±1.70%). The results gotten from mice assay further demonstrated that absorption enhancers significantly promoted transport efficiency of collagenase (P<0.05). In 2h, The sequence of enhanced efficiencies in vivo was as follows:glucose (29.25±3.43%)> EDTA (26.25±2.60%)> sodium cholate (22.50±2.60%)> menthol (17.24±1.33%)> SDS (14.25±1.50%)> ethanol (11.25±1.30%)> control (10.5±1.98%). HE staining showed that the morphology of intestinal mucosa were in good condition, without inflammatory. This experiment proved that absorption enhancers could not only increase the transport amount of collagenase across intestinal epithelium, resolving the problem of oral administration of collagenase with low bioavailability; but also simultaneously did not cause damage to the intestinal mucosa, showing good security.Experiment three:The effects of lactobacilli on the cell junction of intestinal epithelium Cell junction could be effective in preventing the paracellular transport of intestinal bacteria, toxins, and inflammatory mediators, maintaining the integrity of intestinal mucosal epithelial barrier. Some pathogenic microorganisms could secrete toxins specifically acting on the cell junction proteins and cytoskeletal proteins, causing destruction of intestinal mucosal barrier and promoting the transport of harmful substances across intestinal epithelium. Lactobacilli could improve the disorder of cell junction structure induced by pathogen. However, the exact mechanism was unclear. In order to study the effects of enterotoxigenic Escherichia coli K88, Salmonella typhimurium SL1344 and SARB21 on cell junction proteins claudin-1, occludin, ZO-1 and E-cadherin and detect whether lactobacilli could improve the disorder of cell junction structure, the research was performed through the Caco-2 cells monolayer model (in vitro) and rats (in vivo) tests. The results showed that E. coli K88, Salmonella typhimurium SL1344 and SARB21 induced occludin, claudin-1, ZO-1, and E-cadherin scattered in the cytoplasm and the cell gap increased. Through the Western blot test, we further proved that when Caco-2 cells or rats infected with E. coli K88 or Salmonella typhimurium SL1344 or SARB21, the expression of occludin, claudin-1, ZO-1, and E-cadherin were significantly decreased (P<0.05). The lactobacilli had strains-specific improvement effects on the disorder of cell junction structure induced by pathogen. This experiments proved that E. coli K88, Salmonella typhimurium SL1344 and SARB21 could affect the expression and distribution of cell junction proteins (occludin, claudin-1, ZO-1 and E-cadherin) and caused damage to the cell junction; while lactobacilli could improve the destruction induced by pathogenic bacteria and maintain the structure of cell junction and the function of intestinal mucosal barrier. Lactobacillus, a normal intestinal flora, closely integrated with the intestinal mucosa, constitute the biological barriers preventing the invasion of bacteria, viruses, and food antigens. The results of experiment three showed that lactobacilli could improve the phenomenon of cell junction disorder induced by pathogen. We speculated that lactobacilli might perform the protective character by inhibiting the adhesion of pathogens to intestinal epithelial cells and reducing the damage to cell junction. In order to study of antagonistic effect of lactobacilli against Escherichia coli K88, Salmonella typhimurium SL1344 and SARB21, the experiment was performed on Caco-2 cells by competition assay, exclusion assay, and displacement assay. The results showed that L.delbrueckii ssp. lactis D17, R4, Lactobacillus fructosus C2, Lactobacillus amylophilus D14, Lactobacillus delbrueckii ssp. delbrueckii D11 and Lactobacillus brevis all showed strong antagonistic effects against Escherichia coli K88, Salmonella typhimurium SL1344 and SARB21 and the adhesion number of pathogens decreased significantly (P<0.05). The lactobacilli had strains-specific antagonistic effects. Compared with the control group, in the competition assay, L.delbrueckii ssp. lactis R4 and Lactobacillus fructosus C2 significantly reduced the adhesion number of E. coli K88 (P<0.01); in the displacement assay, Lactobacillus delbrueckii ssp. delbrueckii D11 showed the antagonistic effect against salmonella SARB21 and the difference was significant (P<0.01); in the exclusion assay, L.delbrueckii ssp. lactis D17 and Lactobacillus fructosus C2 showed good antagonistic effect against the adhesion of Salmonella SL1344 (P<0.01). Compared with the competition assay and exclusion assay, the inhibiting effect on pathogenic strains adhesion was a little weaker in the displacement assay. This experiment proved that lactobacilli could significantly inhibit pathogens' adhesion to intestinal epithelial cells and simultaneously revealed that lactobacilli might perform the inhibition effect by competition with pathogenic bacteria to the receptors of epithelial cells, providing a theoretical basis for the reasonable application of lactobacilli.