4.
Results
4.1.1 Toxicity of probiotic bacteria in the intestinal model
MTT assay was performed to investigate the dose dependent inhibitory effect in Caco2, HT-29 and Caco-2/HT-29 in ratio 9:1 by the treatment with heat-inactivated Lactobacillus plantarum S2, Lactobacillus rhamnosus GG and Escherichia coli Nissle 1997 cells at varying doses, after 72 h. For pure Caco2 cell line, the IC50 of bacterial suspension was approximately > 1 x 108 cells in the suspension (figure 4). The HT-29 cell line was slightly more sensitive and the cells started developing toxicity at the highest degreee of suspension tested (fig hhhh). The measured IC50 value was 9,14 ± 0,86 x 107 for Lactobacillus rhamnosus GG, > 1 x 108 for Lactobacillus plantarum S2 and 9,06 ± 0,93 x 107 for Escherichia coli Nissle 1917. Lactobacillus rhamnosus
GG and Escherichia coli Nissle 1917 thus seems to be slightly more toxic towards HT-29 than the other strain. However, for cell mixture Caco-2/HT-29 used in the expression experiment, no toxicity was seen (figure 4). Thus, there were no signs of any toxicity of the bacterial cells towards the model even during 72h.
Table 3 Toxicity of heat-inactivated bacterial suspensions towards the cell lines used in the model
IC50 (CFU/ml)
Caco-2 HT29-MTX Caco-2 + HT29-MTX
Lactobacilus ramnosus GG >1*108 9,14 ± 0,86*107 >1*108
Lactobacilus plantarum S2 >1*108 >1*108 >1*108
Figure 4 Effect of Lactobacillus plantarum S2, Lactobacillus rhmanosus GG and Escherichia coli Nissle 1917 heat-inactivated cell suspensions on Caco-2 cell (A), HT29-MTX cell line (B) and 1:9 mixture (C) proliferation in a 72h MTT toxicity test
4.1.2 Gene expression of mucin
Mucins gene expression in Caco-2/HT29-MTX cells were evaluated using a Real-time RT-PCR. MUC 2, MUC 3, MUC 13 and MUC 17 are highly up-regulated after treatment with the previously heat-inactivated suspension of Lactobacillus rhamnosus GG in Caco-2 cells/HT-29 after 12h (Fig. 5). In contrast, Lactobacillus plantarum S2 treatment rather induced down regulation of mucins (non significant), while treatment with E. coli Nissle 1917 did not exert any significant changes. The results show a high strain-specific cross-talk between the bacteria and epithelium that may have an effect on mucin expression and mucin profile.
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Figure 5 Comparison of the effects of various bacterial strain on MUC gene expression.Mean ± S.E.M., N =3. p < 0.05 by ANOVA test with Bonferroni correction to multiple means comparison: *, P < 0,01; **P < 0,05 compared with control cells.
4.1.3 ELISA assay
The ELISA test did not show any significative differences. However, the averages for L. plantarum S2 and L. rhamnosus GG treatments showed lower mean MUC production, while it was slightly non-significantly elevated after E. coli Nissle 1917 treatment (figure 6).
Figure 6 Effects of two Lactobacillus strains and Escherichia coli on MUC 2 production in Caco-2/HT29-MTX cells mixture compared with control cells. Data obtained from the enzyme-linked immune assay (ELISA). Each group consists of two replicates.
4.2.1 Toxicity of polyphenols in the intestinal model
Toxicity of Chlorogenic acid, epicatechin gallate and quercetin was evaluated towards the Caco-2, HT29-MTX and Caco-2/HT29-MTX co-culture. A two-fold dilution series of 500; 250; 125; 62.5; 31.3 and 15.6) and 72h treatments were used for the test. The percentage of mortality for each extract concentration was plotted and fitted with a sigmoidal curve used to determine the 50% growth inhibitory concentration (IC50).
In this case, Caco-2 cells were more sensitive that HT29-MTX (Fig. 8) The IC50 for chlorogenic acid, epicatechin gallate and quercetin were 73.9, 65.0 and 33,7 µg/mL for Caco-2 cell line, 207.1, 83.5 and 83.9 µ g/mL for the HT29-MTX cell line and 119.6, 90.4 and 50.7 µ g/mL for Caco-2 and HT29-MTX cell mixture. These values correspond to 0.20, 0.22 and 0.11 µmol/L for Caco-2 cell line, 0.58, 0.29 µmol/L and 0.27 for HT29-MTX cell line and 0.33, 0.31 and 0,16 µmol/L for Caco-2 and HT29-MTX cell mixture of chlorogenic acid, epicatechin and quercetin. Chlorogenic acid seem to be least toxic compared to the other compounds, however, the toxicity was seen at very high concentrations, quercetin seems to be relatively more toxic and is exerting some low but significant effect on the cell viability even at the lowest concentration tested (15.6 µg/ml).
Table 4 Toxicity of dietary polyphenols used in our test towards selected cell lines
IC50 (µ g/ml)
Caco-2 HT29-MTX Caco-2/HT29-MTX
Chlorogenic acid 73.94 ± 7.95 207,06 ± 12,56 119,60 ± 20,72
Epicatechin gallate 65.06 ± 9.67 83,51 ± 14,39 90,39 ± 5,14
Figure 7 Effect of quercetin (A), chlorogenic acid (B) and epicatechin on Caco-2/HT-29 cells lines.
Figure 8 Effect of quercetin, chlorogenic acid and epicatechin on Caco-2 (A), HT-29 (B) and Caco-2/HT-29 (C) cell viability using MTT assay. Values are expressed as means of the percentage of cell viability with respect to control cells ± standard deviation (SD), N = 3
4.2.2 Mucin gene expression as modulated by the polyphenols
Real-time RT-PCR was used to determine the level of trascripts of MUC 2, MUC 3, MUC 13, MUC 17 in Caco-2/HT29-MTX cells treated with quercetin, epicatechin and chlorogenic acid. This data showed that each compound exert different gene expression patterns in Caco-2/HT29-MTX cell mixture. Quercetin did not have any significant effect on the expression of MUC 2, MUC 3, MUC 13 or MUC 17, only showing a statistically non-significant but remarkable 4-foldrise in MUC17. Epicatechin significantly increased MUC 13 but also showed elevation in other membrane-bound mucins expression but not to MUC 2 remained more or less costant. Chlorogenic acid lead to decrease in MUC 2 gene expression, while MUC 3, MUC 13 and MUC 17 remained constant. Thus, MUC 17 expression was elevated after all treatments, others showed a compound-specific patterns.
Figure 9. Comparison of the effects of various polyphenols on MUC gene expression..Mean ± SD, N =3. p < 0.05 by ANOVA: *, P < 0,01; **P < 0,05
4.2.3 ELISA assay of polyphenols
MUC 2 was quantified by ELISA after 48 h of treatment with each polyphenol. There were no significant differences in protein levels released to the medium.
Fig. 10 ELISA determination of MUC 2 protein in CaCo-2/HT29-MTX cells treated with 10 µM of epicatechin, quercetin and chlorogenic acid.
4.3.1 Mucin gene expression as modulated by milk hydrolysate
Caco-2/HT-29, Caco-2 and HT-29 cells were treated with fermented milk hydrolysates, which exerted toxicity in these cells when undiluted (caused a shedding of the cells
monolayer), whereas the IC50 of milk protein in the hydrolysate was found to be 1.4 µg/mLl for Caco-2 cell line, 1.3 for MTX cell line and 1.2 µg/mL for the Caco-2 and HT29-MTX cell culture mixture. (Fig. 11). The concentration of milk hydrolysate in the test of the mucin expression was 1.5 and thus borderline of the IC50. However, after the 48h treatment the monolayer did not show any visual aberrations.
Figure 11 Effect of milk hydrolysate on Caco-2 (A), HT-29 (B) and Caco-2/HT-29 (C). Value are expressed as means of the percentage of cell viability with respect to control cells ± standard deviation (SD), N = 3. Concentrations correspond to the concentration of protein in the hydrolysate. Dry matter concentrations are higher, accordingly.
4.3.2 Mucins gene expression as regulated by the milk hydrolysate
The expression levels of MUC 2, MUC 3, MUC 13 and MUC 17 mRNA were evaluated with Real-time RT-PCR in Caco-2/HT-29 cell lines. This data showed that treatment with 1.5 µg ml–1 of milk protein in the milk hydrolysate for 48 h enhanced mucins expression compared with control cells.
Figure 12 Real-time RT-PCR analysis of four mucins gene. Values are means ± SD of 3 separate experiments. *P < 0.005
4.3.3 ELISA assay
The ELISA test was also used to investigate the MUC 2 expression. There was not a significant change in mucin secretion of cells in the medium.
