https://orcid.org/0000-0002-8962-0649
Figures & data
Table 1. Bacteria and medium used in this study.
Figure 1. Lactobacillus delbrueckii subsp. bulgaricus 2038 and Streptococcus thermophilus 1131 improved the intestinal barrier dysfunction by tumor necrosis factor (TNF)-α and interferon (IFN)-γ.
L. bulgaricus 2038, S. thermophilus 1131, TNF-α, and IFN-γ were simultaneously added to the Caco-2 monolayers. (A) Trans-epithelial electrical resistance (TEER) percentage of initial value and (B) the permeability of fluorescein isothiocyanate-dextran with an average molecular weight of 4,000 (FD-4) after 18 h in the experiments using live lactic acid bacteria (LAB) are shown (n = 5). (C) TEER value (n = 5–6) and (D) FD-4 (n = 4–7) permeability in the experiments using heat-treated LAB are shown. Data are expressed as the mean ± standard error (SE). *P < 0.05, †P < 0.1.
Figure 2. L. bulgaricus 2038 and S. thermophilus 1131 suppressed the decrease in the gene expression levels of tight junctions (TJs) by TNF-α and IFN-γ.
Live L. bulgaricus 2038, S. thermophilus 1131, TNF-α, and IFN-γ were simultaneously added to the Caco-2 monolayers. Caco-2 cells were collected after 18 h of incubation. Gene expression levels of (A) occludin (OCLN) and (B) zonula occludens-1 (ZO-1/TJP1) were evaluated via real-time polymerase-chain reaction (PCR). Data were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression and are shown as relative expression levels. Data are expressed as the mean ± SE (n = 8–9). *P < 0.05.
Figure 3. L. bulgaricus 2038 and S. thermophilus 1131 suppressed the destruction of TJ proteins by TNF-α and IFN-γ.
Live L. bulgaricus 2038, S. thermophilus 1131, TNF-α, and IFN-γ were simultaneously added to the Caco-2 monolayers. After 18 h of incubation, TJ structures (claudin [CLDN]-4, OCLN, junctional adhesion molecule [JAM]-A, and ZO-1) were visualized and analyzed via immunofluorescence microscopy (scale bar = 20 μm). White arrows show the internalization of TJ proteins.
Figure 4. L. bulgaricus 2038 and S. thermophilus 1131 promoted the assembly of TJ proteins in a calcium switch assay.
Caco-2 monolayers were cultured in S-MEM for 16 h. Then, S-MEM was replaced with normal medium, and L. bulgaricus 2038, S. thermophilus 1131, 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), and dorsomorphin were added simultaneously. (A) TEER values were measured every hour (n = 6–8). TEER value was calculated before changing to S-MEM at 100%. (B) Permeability of FD-4 after 8 h was evaluated (n = 6–8). (C) Immunofluorescence staining of ZO-1 at calcium switch (-Ca2+) and 8 h was analyzed via immunofluorescence microscopy (n = 3–4; scale bar = 20 μm). White arrows show clear formation of cell shape. (D) Fluorescence intensity of ZO-1 was quantified (n = 3–4). (E) Caco-2 cells were collected at 8 h and the expression of phosphorylated AMP-activated protein kinase (p-AMPK) was evaluated via western blotting. Expression of p-AMPK was normalized to total AMPK expression, and the relative expression levels are shown (n = 4). Data are expressed as the mean ± SE. *P < 0.05, †P < 0.1.
Figure 5. L. bulgaricus 2038 and S. thermophilus 1131 increased the expression of p-AMPK.
Caco-2 monolayers were stimulated with L. bulgaricus 2038 or S. thermophilus 1131 for 9 h. Caco-2 cells were collected and the expression of p-AMPK was evaluated via western blotting. Expression of p-AMPK was normalized to total AMPK expression, and the relative expression levels are shown. Data are expressed as the mean ± SE (n = 6–7). *P < 0.05.
Figure 6. Protective effect on FD-4 permeability increased by TNF-α and IFN-γ was strain-dependent.
Bacteria, TNF-α, and IFN-γ were simultaneously added to Caco-2 monolayers. Permeability of FD-4 after 18 h is shown. Data are expressed as the mean ± SE (n = 3). *P < 0.05 compared to TNF-α and IFN-γ group. Dashed line shows the values for the control group. L.b, L. bulgaricus; S.t, S. thermophilus; L.c, Lacticaseibacillus paracasei; P.f, Propionibacterium freudenreichii; L.l, Lactococcus lactis subsp. lactis; L.g, Lactobacillus paragasseri; B.b. Bifidobacterium bifidum; B.l., Bifidobacterium longum.
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