Effects of soybean agglutinin on mechanical barrier function and tight junction protein expression in intestinal epithelial cells from piglets

Abstract

In this study, we sought to investigate the role of soybean agglutinin (SBA) in mediating membrane permeability and the mechanical barrier function of intestinal epithelial cells. The IPEC-J2 cells were cultured and treated with 0, 0.5, 1.0, 1.5, 2.0, 2.5, or 3.0 mg/mL SBA. Transepithelial electrical resistance (TEER) and alkaline phosphatase (AP) activity were measured to evaluate membrane permeability. The results showed a significant decrease in TEER values (p < 0.05) in a time- and dose-dependent manner, and a pronounced increase in AP activity (p < 0.05). Cell growth and cell morphology were used to evaluate the cell viability. A significant cell growth inhibition (p < 0.05) and alteration of morphology were observed when the concentration of SBA was increased. The results of western blotting showed that the expression levels of occludin and claudin-3 were decreased by 31% and 64% compared to those of the control, respectively (p < 0.05). In addition, immunofluorescence labeling indicated an obvious decrease in staining of these targets and changes in their localizations. In conclusion, SBA increased the membrane permeability, inhibited the cell viability and reduced the levels of tight junction proteins (occludin and claudin-3), leading to a decrease in mechanical barrier function in intestinal epithelial cells.

Figure 1

Effects of SBA on TEER in IPEC-J2 cells. Cells were treated with various concentration of SBA for 24 h, 48 h, or 72 h, TEER values are expressed in Ω × cm² as the mean ± standard error from 3 independent experiments and presented a significant decline in a time and dose-dependent (p < 0.05). The control group was treated with 0 mg/mL SBA for the indicated times (24, 48, or 72 h). Treatment of 0.5, 1.0 or 1.5 mg/mL SBA group had a significant decrease compared with control (p < 0.05), in addition, 2.0, 2.5 or 3.0mg/mL had a significant decrease compared with 0.5,1.0 or 1.5 mg/mL SBA treatment groups (p < 0.05) (48 h or 72 h). However no significant differences were observed among 0.5, 1.0 and 1.5 mg/mL treatment, or between the 2.0 and 2.5 mg/mL SBA treatment (p > 0.05).

Figure 2

Effects of SBA on AP activity in IPEC-J2 cells. Cells were treated with various concentration of SBA for 72 h, culture supernatants were collected, and AP activity was measured. The control cells were treated with 0 mg/mL SBA for 72 h. Values are the mean ±SD from 4 independent experiments. Treatment of 0.5, 1.0 or 1.5 mg/mL SBA was significantly higher than control (p < 0.05), and the 2.0, 2.5 or 3.0 mg/mL SBA treatment were even higher than treatment of 0.5, 1.0 or 1.5 mg/mL (p < 0.05). However, no significant differences were observed among 0.5, 1.0 and 1.5 mg/mL treatment; or 2.0, 2.5 and 3.0 mg/mL SBA treatment (p > 0.05).

Figure 3

Linear correlations between AP activity and TEER value after 72 h treatment with 0, 0.5, 1.0, 1.5, 2.0, 2.5, or 3.0 mg/mL SBA. The formula was obtained by SPSS 17.0 software (SPSS Inc, Chicago, IL, USA. n_AP = 28, n_TEER = 21, y = 60.976 − 13.251x, R² = 0.883, R² = 0.883, p < 0.01, TEER values were dependent variable, AP activity were independent variable.

Figure 4

MTT assays showing the viability of IPEC-J2 cells after treatment with different concentrations of SBA for 24 h, 48 h, or 72 h. The data are the mean ± SD from 4 independent experiments and presented a significant decline trend (p < 0.05). The control group was treated with 0 mg/mL SBA for the indicated times (24, 48, or 72 h). Treatment with 0.5, 1.0 or 1.5 mg/mL SBA resulted in a significant decrease compared with control (p < 0.05), in addition, 2.0, 2.5 or 3.0mg/mL led to a significant decrease when compared with 0.5,1.0 or 1.5 mg/mL SBA treatment groups (p < 0.05) (48 h or 72 h). However no significant differences were observed among 0.5, 1.0 and 1.5 mg/mL treatment, or between the 2.0 and 2.5 mg/mL SBA treatment (p > 0.05).

Figure 5

Representative microphotographs demonstrating the effects of SBA on the morphology and growth of IPEC-J2 cells. Cells were cultured to confluence and incubated with the 0 mg/mL (a), 0.5 mg/mL (b), 2.0 mg/mL (c) of SBA for 48 h; 0 mg/mL of SBA treated cells are shown as the control. Magnification, 200×.

Figure 6

Effects of SBA on the distribution of tight junction proteins. The distributions of occludin treated with 0 mg/mL (a) or treated with 0.5 mg/mL (b) and claudin-3 treated with 0 mg/mL (c) or treated with 0.5 mg/mL (d), were determined by immunofluorescence. IPEC-J2 cells were grown on glass slides within 6-well plates until reaching complete confluence and were then treated with 0 or 0.5 mg/mL SBA for 48 h. The cells treated with 0 mg/mL SBA for 48 h were used as a control. Representative images are shown at a magnification of 400×.

Figure 7

Western blotting of tight junction proteins following SBA treatment. Fifteen microliters of 0 (Control) or 0.5 mg/mL SBA-treated total protein extracts were analyzed by immunoblotting with antibodies for occludin and claudin-3. β-actin was used as a loading control. Representative western blots from 4 independent experiments are shown.