Nature-Identical Compounds and Organic Acids Ameliorate and Prevent the Damages Induced by an Inflammatory Challenge in Caco-2 Cell Culture

Abstract

Bioactive compounds, such as organic acids (OA) and nature-identical compounds (NIC), can exert a role in the protection of intestinal mucosa functionality due to their biological properties. The aim of this study was to understand the role of 2 OA (citric and sorbic acid) and 2 NIC (thymol and vanillin), alone or combined in a blend (OA + NIC), on intestinal barrier functionality, either during homeostatic condition or during an inflammatory challenge performed with pro-inflammatory cytokines and lipopolysaccharides (LPS). The study was performed on the human epithelial cell line Caco-2, a well-known model of the intestinal epithelial barrier. The results showed how OA and NIC alone can improve transepithelial electrical resistance (TEER) and mRNA levels of tight junction (TJ) components, but OA + NIC showed stronger efficacy compared to the single molecules. When an inflammatory challenge occurred, OA + NIC blend was able both to ameliorate, and prevent, damage caused by the pro-inflammatory stimulus, reducing or preventing the drop in TEER and improving the TJ mRNA expression. The data support the role of OA + NIC in modulating gut barrier functionality and reducing the negative effects of inflammation in intestinal epithelial cells, thereby supporting the gut barrier functionality.

Keywords: Caco-2 cells; barrier function; inflammatory response; nature-identical compound; organic acid.

Figure 1

TEER of Caco-2 cells cultured with single OA and NIC. Data in the graph are represented as percentage over the initial TEER value and given as means (n = 6) ± SEM, represented by vertical bars. Means with different letters indicate statistical significance with p < 0.05 (a, b); means with at least one common letter are not significantly different (ab), colors of the letters refer to the different treatments. TEER = transepithelial electrical resistance (Ω·cm²). (a) Cells treated with citric acid; CTR = control group; 260 = treated group with 260 μM of citric acid; 455 = treated group with 455 μM of citric acid; 651 = treated group with 651 μM of citric acid. (b) Cells treated with sorbic acid; CTR = control group; 298 = treated group with 298 μM of sorbic acid; 521 = treated group with 521 μM of sorbic acid; 745 = treated group with 745 μM of sorbic acid. (c) Cells treated with thymol; CTR = control group; 23 = treated group with 23 μM of thymol; 40 = treated group with 40 μM of thymol; 58 = treated group with 58 μM of thymol. (d) Cells treated with vanillin; CTR = control group; 13 = treated group with 13 μM of vanillin; 23 = treated group with 23 μM of vanillin; 35 = treated group with 35 μM of vanillin.

Figure 2

PCP of Caco-2 cells grown with single OA and NIC. Data in the graph are means (n = 6) ± SEM represented by vertical bars. Means with different letters indicate statistical significance with p < 0.05 (a, b); means with at least one common letter are not significantly different (ab). PCP = paracellular permeability (excitation 485 nm, emission 520 nm). (a) Cells treated with citric acid; CTR = control group; 260 = treated group with 260 μM of citric acid; 455 = treated group with 455 μM of citric acid; 651 = treated group with 651 μM of citric acid. (b) Cells treated with sorbic acid; CTR = control group; 298 = treated group with 298 μM of sorbic acid; 521 = treated group with 521 μM of sorbic acid; 745 = treated group with 745 μM of sorbic acid. (c) Cells treated with thymol; CTR = control group; 23 = treated group with 23 μM of thymol; 40 = treated group with 40 μM of thymol; 58 = treated group with 58 μM of thymol. (d) Cells treated with vanillin; CTR = control group; 13 = treated group with 13 μM of vanillin; 23 = treated group with 23 μM of vanillin; 35 = treated group with 35 μM of vanillin.

Figure 3

Gene expression in Caco-2 cells after 15 days of treatment with single OA and NIC. Values are least square means (n = 6) ± SEM represented by vertical bars. A modification of the 2^–ΔΔCT method [16] was used to analyze the relative expression (fold changes), calculated relative to the control group. Means with different letters indicate statistical significance with p < 0.05 (a, b); means with at least one common letter are not significantly different (ab). (a) Cells treated with citric acid; CTR = control group; 260 = treated group with 260 μM of citric acid; 455 = treated group with 455 μM of citric acid; 651 = treated group with 651 μM of citric acid. (b) Cells treated with sorbic acid; CTR = control group; 298 = treated group with 298 μM of sorbic acid; 521 = treated group with 521 μM of sorbic acid; 745 = treated group with 745 μM of sorbic acid. (c) Cells treated with thymol; CTR = control group; 23 = treated group with 23 μM of thymol; 40 = treated group with 40 μM of thymol; 58 = treated group with 58 μM of thymol. (d) Cells treated with vanillin; CTR = control group; 13 = treated group with 13 μM of vanillin; 23 = treated group with 23 μM of vanillin; 35 = treated group with 35 μM of vanillin. ZO-1 = zonula occludens.

Figure 4

TEER of Caco-2 cells cultured with OA + NIC. Data in the graph are represented as percentage over the initial TEER value and given as means (n = 6) ± SEM, represented by vertical bars. Means with different letters indicate statistical significance with p < 0.05 (a, b); means with at least one common letter are not significantly different (ab), colors of the letters refer to the different treatments. TEER = transepithelial electrical resistance (Ω·cm²); CTR = control group; OA + NIC 200 = treated group with 200 ppm of OA + NIC, composed from 260 µM of citric acid, 298 µM of sorbic acid, 23 µM of thymol, and 13 µM of vanillin; OA + NIC 1000 = treated group with 1000 ppm of OA + NIC, composed from 1300 µM of citric acid, 1490 µM of sorbic acid, 110 µM of thymol, and 70 µM of vanillin.

Figure 5

PCP of Caco-2 cells after 15 days of treatment with OA + NIC. Data in the graph are means (n = 6) ± SEM represented by vertical bars. Means with different letters indicate statistical significance with p < 0.05 (a, b); means with at least one common letter are not significantly different (ab). PCP = paracellular permeability (excitation 485 nm, emission 520 nm). CTR = control group; OA + NIC 200 = treated group with 200 ppm of OA + NIC, composed from 260 µM of citric acid, 298 µM of sorbic acid, 23 µM of thymol, and 13 µM of vanillin; OA + NIC 1000 = treated group with 1000 ppm of OA + NIC, composed from 1300 µM of citric acid, 1490 µM of sorbic acid, 110 µM of thymol, and 70 µM of vanillin.

Figure 6

Gene expression in Caco-2 cells after 15 days of treatment with OA + NIC. Values are least square means (n = 6) ± SEM represented by vertical bars. A modification of the 2^–ΔΔCT method [16] was used to analyze the relative expression (fold changes), calculated relative to the control group. Means with different letters indicate statistical significance with p < 0.05 (a, b); means with at least one common letter are not significantly different (ab). CTR = control group; OA + NIC 200 = treated group with 200 ppm of OA + NIC, composed from 260 µM of citric acid, 298 µM of sorbic acid, 23 µM of thymol, and 13 µM of vanillin; OA + NIC 1000 = treated group with 1000 ppm of OA + NIC, composed from 1300 µM of citric acid, 1490 µM of sorbic acid, 110 µM of thymol, and 70 µM of vanillin; ZO-1 = zonula occludens 1.

Figure 7

Gene expression in Caco-2 cells after 15 days of treatment with OA + NIC. Values are least square means (n = 6) ± SEM represented by vertical bars. A modification of the 2^–ΔΔCT method [16] was used to analyze the relative expression (fold changes), calculated relative to the control group. Means with different letters indicate statistical significance with p < 0.05 (a, b); means with at least one common letter are not significantly different (ab). CTR = control group; OA + NIC 200 = treated group with 200 ppm of OA + NIC, composed from 260 µM of citric acid, 298 µM of sorbic acid, 23 µM of thymol, and 13 µM of vanillin; OA + NIC 1000 = treated group with 1000 ppm of OA + NIC, composed from 1300 µM of citric acid, 1490 µM of sorbic acid, 110 µM of thymol, and 70 µM of vanillin; TRPV1 = Transient Receptor Potential Vanilloid 1; TRPV3 = Transient Receptor Potential Vanilloid 3.

Figure 8

TEER of Caco-2 cells cultured with OA + NIC and subjected to an inflammatory challenge. Data in the graph are represented as percentage of the initial TEER value and given as means (n = 6) ± SEM, represented by vertical bars. Means with different letters indicate statistical significance with p < 0.05 (a, b); means with at least one common letter are not significantly different (ab), colors of the letters refer to the different treatments. TEER = transepithelial electrical resistance (Ω·cm²); CTR = control group; OA + NIC 200 = treated group with 200 ppm of OA + NIC, composed from 260 µM of citric acid, 298 µM of sorbic acid, 23 µM of thymol, and 13 µM of vanillin; OA + NIC 1000 = treated group with 1000 ppm of OA + NIC, composed from 1300 µM of citric acid, 1490 µM of sorbic acid, 110 µM of thymol, and 70 µM of vanillin. (a) Cells challenged at day 0 of the study. (b) Cell challenged at day 14 of the study.

Figure 9

PCP of Caco-2 cells after 15 days of treatment with OA + NIC and subjected to an inflammatory challenge. Data in the graph are means (n = 6) ± SEM represented by vertical bars. Means with different letters indicate statistical significance with p < 0.05 (a, b); means with at least one common letter are not significantly different (ab). PCP = paracellular permeability (excitation 485 nm, emission 520 nm). CTR = control group; OA + NIC 200 = treated group with 200 ppm of OA + NIC, composed from 260 µM of citric acid, 298 µM of sorbic acid, 23 µM of thymol, and 13 µM of vanillin; OA + NIC 1000 = treated group with 1000 ppm of OA + NIC, composed from 1300 µM of citric acid, 1490 µM of sorbic acid, 110 µM of thymol, and 70 µM of vanillin. (a) Cells challenged at day 0 of the study. (b) Cell challenged at day 14 of the study.

Figure 10

Gene expression in Caco-2 cells after 15 days of treatment with OA + NIC and subjected to an inflammatory challenge. Values are least square means (n = 6) ± SEM represented by vertical bars. A modification of the 2^–ΔΔCT method [16] was used to analyze the relative expression (fold changes), calculated relative to the control group. Means with different letters indicate statistical significance with p < 0.05 (a, b); means with at least one common letter are not significantly different (ab). CTR = control group; OA + NIC 200 = treated group with 200 ppm of OA + NIC, composed from 260 µM of citric acid, 298 µM of sorbic acid, 23 µM of thymol, and 13 µM of vanillin; OA + NIC 1000 = treated group with 1000 ppm of OA + NIC, composed from 1300 µM of citric acid, 1490 µM of sorbic acid, 110 µM of thymol, and 70 µM of vanillin; ZO-1 = zonula occludens. (a) Cells challenged at day 0 of the study. (b) Cell challenged at day 14 of the study.

Figure 11

Gene expression in Caco-2 cells after 15 days of treatment with OA + NIC and subjected to an inflammatory challenge. Values are least square means (n = 6) ± SEM represented by vertical bars. A modification of the 2^–ΔΔCT method [16] was used to analyze the relative expression (fold changes), calculated relative to the control group. Means with different letters indicate statistical significance with p < 0.05 (a, b); means with at least one common letter are not significantly different (ab). CTR = control group; OA + NIC 200 = treated group with 200 ppm of OA + NIC, composed by 260 µM of citric acid, 298 µM of sorbic acid, 23 µM of thymol, and 13 µM of vanillin; OA + NIC 1000 = treated group with 1000 ppm of OA + NIC, composed by 1300 µM of citric acid, 1490 µM of sorbic acid, 110 µM of thymol, and 70 µM of vanillin; TRPV1 = Transient Receptor Potential Vanilloid 1; TRPV3 = Transient Receptor Potential Vanilloid 3. (a) Cells challenged at day 0 of the study. (b) Cell challenged at day 14 of the study.