Fig 1.
The effect of V. cholerae, S. sonnei and L. acidophilus on cell viability.
(A) cell viability of Caco-2 cells infected by V. colarae/ S. sonnei was determined by MTT assay, Each value represents the mean ±SEM (n = 3). ***p < 0.001 compared with control (untreated Caco-2cells) by one way ANOVA with Tukey’s post hoc test. ###p < 0.001 significantly different of L.a+V.c+Caco-2 group from V.c+Caco-2 group. †††p < 0.001 significantly different between L.a+S.s+Caco-2 group with S.s+Caco-2 (by unpaired two-tailed Student’s t test). All experiments were repeated three times. L.a: Lactobacillus acidophilus, V.c: Vibrio cholerae and S.s: Shigella sonnei, ns: none significant. (B) Morphological evaluation of Caco-2 cells under the above-mentioned treatment by light microscopic observation. L.a: Lactobacillus acidophilus, V.c: Vibrio cholerae and S.s: Shigella sonnei.
Fig 2.
(A and B) Effect of pretreatment of L. acidophilus on its ability to inhibit V. colarae/ S. sonnei adherence and invasion to Caco-2 cells. ND means Not Detectable. Each value indicates the mean±SEM from three independent experiments and three replicates (n = 3). ###p < 0.001 significantly different of L.a+V.c+Caco-2 group from V.c+Caco-2 group. †††p < 0.001 significant difference between L.a+S.s+Caco-2 group and S.s+Caco-2 (by unpaired two-tailed Student’s t test). L.a: Lactobacillus acidophilus, V.c: Vibrio cholerae and S.s: Shigella sonnei.
Fig 3.
Annexin V-FITC/PI staining and flow cytometry assay to determine the proportion of apoptosis after pretreatment of Caco-2 cells with L. acidophilus and then infected by V. colarae/ S. sonnei.
Images showed the typical morphologies of cells in the different regions. (A) Q1: Necrotic cells, Q2: Late apoptotic cells, Q2: Early apoptotic cells and Q4: Viable cells. L.a: Lactobacillus acidophilus, V.c: Vibrio cholerae and S.s: Shigella sonnei. (B)The pretreatment with L. acidophilus on Caco-2 cells exposed to V. colarae/ S. sonnei resulted in decreased apoptosis. Statistical analysis of representative images was performed with three independent experiments. The statistical significances were achieved when P <0.05 (***P <0.001). *** significantly different from control cells. ### significantly different of L.a+V.c+Caco-2 from V.c+Caco-2 cells. ††† significantly different of L.a+S.s+Caco-2 from S.s+Caco-2 cells L.a: Lactobacillus acidophilus, V.c: Vibrio cholerae and S.s: Shigella sonnei. ns: none significant.
Fig 4.
Expression of selected genes (IL-8. TNF-α and IL-1β) was determined by real-time quantitative polymerase chain reaction analysis in experimental cell groups.
The fold-changes in expression of (A)IL-8, (B)TNF-α and (C)IL-1β after treatment by L. acidophilus was normalized to GAPDH and compared in infected and uninfected Caco-2 cells. The fold-changes in genes expression were represented from at least 3 independent experiments. The statistical results were remarkable when P <0.05. (***P <0.001) significantly different from control cells. ## or ### significant difference between L.a+V.c+Caco-2 and V.c+Caco-2 cells. ††† or †††† significant difference between L.a+S.s+Caco-2 and S.s+Caco-2 cells L.a: Lactobacillus acidophilus, V.c: Vibrio cholerae and S.s: Shigella sonnei.
Fig 5.
NO level in Caco-2 cells after L. acidophilus pretreatment and followed by V. cholerae/ S. sonnei.
NO production after 24h, significantly decreased in comparison with V.cholerae/ S. sonnei infected cells alone. The mean of three independent experiments is shown. The significant differences were notabled when P <0.05. (***P <0.001) significantly different from control cells. ##significantly different of L.a+V.c+Caco-2 from V.c+Caco-2 cells. ††† significantly different of L.a+S.s+Caco-2 from S.s+Caco-2 cells L.a: Lactobacillus acidophilus, V.c: Vibrio cholerae and S.s: Shigella sonnei. ns: none significant.
Fig 6.
Effect of treatment with L. acidophilus on PGE2 secretion in Caco-2 epithelial cells.
PGE2 secretion was significantly elevated following infection with V. cholerae/ S. sonnei and this effect was abolished when the cells were pretreated by L. acidophilus. The data are presented as mean ±SEM. The statistical data (by unpaired two-tailed Student’s t test) were signed when P <0.05. ##significantly different of L.a+V.c+Caco-2 from V.c+Caco-2 cells. ††† significantly different of L.a+S.s+Caco-2 from S.s+Caco-2 cells L.a: Lactobacillus acidophilus, V.c: Vibrio cholerae and S.s: Shigella sonnei. ND: None Detectable.
Table 1.
Oligonucleotide primers used for Real-time PCR.