Effects Of Soybean Agglutinin On Mechanical Barrier Function In Intestinal Epithelial Cells From Piglets

Soybean agglutinin (SBA) is a major antinutritional factor in soybeans. The binding of SBAcan damage the basic structure of intestinal mucosal cells, which decreases the efficiency ofsoybean protein. In this paper, we mainly studyed the effects of SBA with differentconcentration on epithelial cell membrane integrity and barrier function through threeexperiments, which would provide a basis for studying the anti-nutritional mechanism of SBA.Experiment I: Intestinal epithelial cells from piglets were separated by methods ofmechanical scrape and collagenase Ⅳ digestion, after being purified by the method of differentdigestion and adherent method, the cells were identified by cell morphological observation andimmunohistochemistry. The results showed that: we successfully obtained the intestinalepithelial cells with good condition. Observed via a microscope, the morphology of the cellswere oval or polygon, and the cellular boundaries were clear. After purification, when cellularpurity reached95%, we found a positive test results through the identification of keratin8monoclonal antibody.Experiment II: Effect of SBA with different concentrations on integrity of the intestinalepithelial cells. After treated with0、0.5、1.0、1.5、2.0、2.5and3.0mg/ml SBA for24,48and72h, changes of cell membrane permeability were measured by cell morphological observation,tras-epithelial electrical resistance (TEER), cellular viability (MTT absorbance), extracellularalkaline phosphatase activity (AP) activity.The results indicated:1) With the increase concentration of SBA and the extension of time,obvious changes of the cell morphology were observed.2) Cellular TEER values and MTTvalues had a significant decrease trend in time-dose dependent (P<0.05) when compared withcontrol group (0mg/ml). With the increase concentration of SBA, AP activity in72h treatmentgroup had a significant increased trend (P<0.05).Experiment III: Effect of SBA on distribution and relative expression of tight junctionproteins (occludin and claudin-3).After being treated with0.5mg/ml SBA for48h, cellular immunofluorescence and westernblotting were used to determine the distribution and relative expression of occludin andclaudin-3proteins. The immunofluorescence labeling indicated an obviously decrease instaining and alteration of localization. The western blotting data revealed that the expression ofoccludin and claudin-3was decreased by31%and65%respectively (P<0.05). In conclusion, different levels of SBA decreased intestinal epithelial cell membranepermeability and inhibited cell proliferation. On this basis, SBA decreased the relativeexpression of TJ proteins from a perspective of protein molecules. These results provide animportant theoretical basis for studying the anti-nutritional mechanism of soybean agglutininand improving the use of the soy protein.