Effects of topical application of resveratrol on tight junction barrier and antimicrobial compound production in lactating goat mammary glands

Abstract

In mammary glands, the formation of less-permeable tight junctions (TJs) and the production of antimicrobial compounds like lactoferrin and defensins are important for preventing mastitis. Resveratrol, a polyphenol contained in red grapes, is known to protect mammary epithelial cells (MECs) from oxidative stress; however, oral administration of resveratrol causes a decrease in certain biological processes through conjugation and metabolic conversion. In this study, we determined the beneficial effects of resveratrol on TJs and antimicrobial compounds in cultured goat MECs by adding it to the medium, and in lactating goat mammary glands by topical application for percutaneous absorption. TJ barrier function was evaluated by transepithelial resistance and expression or localization pattern of claudins for culture model in vitro and by somatic cell count, Na⁺, albumin, and IgG in milk for topical application in vivo. Concentrations of antimicrobial compounds and cytokines were measured using ELISA. Activation of STAT3 was evaluated by Western blotting. Resveratrol strengthened TJ barrier function by upregulating claudin-3 in cultured MECs and topical application to udders reduced somatic cell count, Na⁺, albumin, and IgG in milk. Resveratrol increased β-defensin and S100A7 levels in cultured MECs and milk. In addition, resveratrol down-regulated cytokine production and STAT3 pathway. These findings suggest that the topical application of resveratrol to udders may be effective in preventing mastitis.

Keywords: Antimicrobial compound; mammary gland; resveratrol; tight junction.

Figure 1

Effect of resveratrol on the tight junction barrier of goat mammary epithelial cells (GMECs). Western blot analysis of claudin-3 and claudin-4 in GMECs treated with resveratrol at various concentrations for 3 days (A) or treated with 10 μM resveratrol each day (B). The graphs show the results of densitometry analysis; α-tubulin was the internal control. C The images show the localization of claudin-3 (green) and occludin (red; tight junction marker) in GMECs treated with 10 μM resveratrol for 4 days. Scale bar 10 µm. D Western blot analysis of Triton X-100-soluble and -insoluble fractions of claudin-3. The graph shows the ratio of insoluble/soluble claudin-3. E The graph shows transepithelial resistance in GMECs. Control GMECs were treated with 0.1% dimethyl sulfoxide. The data are presented as the mean ± SEM (n = 8–12). Asterisks show significant differences (p < 0.05 versus control).

Figure 2

Effect of resveratrol on antimicrobial compound production in goat mammary epithelial cells (GMECs). β-defensin-1, S100A7, and lactoferrin levels as measured by ELISA in GMECs treated with resveratrol at various concentrations for 3 days (A) or treated with 10 μM resveratrol each day (B). C The expression of BD1 (β-defensin-1), S100A7, and LTF (lactoferrin) mRNA was detected using qPCR in GMECs treated with 10 μM resveratrol for 3 days. D β-defensin-1, S100A7, and lactoferrin levels as measured by ELISA in the medium of the upper chamber of inserts treated with 10 μM resveratrol for 4 days. Control GMECs were treated with 0.1% dimethyl sulfoxide. The data are presented as the mean ± SEM (n = 8–12). Asterisks show significant differences (p < 0.05 versus control).

Figure 3

Influence of a topical application of resveratrol to udders on milk yield, somatic cell count (SCC), and blood-derived components in milk. The udders of lactating goats were topically treated with 1 mL of 100 μM resveratrol in 70% ethanol containing 0.2% dimethyl sulfoxide for 7 days. The upper graphs show changes in milk yield (A), SCC (B), Na⁺ (C), albumin (D), and IgG (E) in individual udders and the lower graphs show changes in the relative values compared with the average of the pre-treated (days −1 and 0) controls. The data are presented as the mean ± SEM (n = 9). Asterisks show significant differences (p < 0.05 versus control).

Figure 4

Influence of a topical application of resveratrol to udders on antimicrobial compound concentration in milk. The udders of lactating goats were topically treated with 1 mL of 100 μM resveratrol in 70% ethanol containing 0.2% dimethyl sulfoxide for 7 days. The upper graphs show changes in β-defensin-1 (A), S100A7 (B), lactoferrin (C), cathelicidin-2 (D), S100A8 (E), and IgA (F) levels in individual udders and the lower graphs show the changes in the relative values compared with the average of pre-treated (days −1 and 0) controls. The data are presented as the mean ± SEM (n = 9). Asterisks show significant differences (p < 0.05 versus control).

Figure 5

Effect of resveratrol on cytokine production in goat mammary epithelial cells (GMECs) and concentration in milk. The udders of lactating goats were topically treated with 1 mL of 100 μM resveratrol in 70% ethanol containing 0.2% dimethyl sulfoxide for 7 days. The upper graphs show the changes in IL1β (A), TNFα (B), and IL8 (C) levels in individual udders and the lower graphs show the changes in the relative values compared with the average of pre-treated (days −1 and 0) controls. The data are presented as the mean ± SEM (n = 9). IL1β, TNFα, and IL8 levels as measured by ELISA in GMECs treated with resveratrol at various concentrations for 3 days (D) or treated with 10 μM resveratrol each day (E). Control GMECs were treated with 0.1% dimethyl sulfoxide. The data are presented as the mean ± SEM (n = 8). Asterisks show significant differences (p < 0.05 versus control).

Figure 6

Effect of resveratrol on STAT3 signaling in goat mammary epithelial cells (GMECs). Western blot analysis of phosphorylated-STAT3 (pSTAT3) and STAT3 in GMECs treated with resveratrol at various concentrations for 3 days (A) or treated with 10 μM resveratrol each day (B). The graphs show the results of densitometry analysis; α-tubulin was the internal control. Control GMECs were treated with 0.1% dimethyl sulfoxide. The data are presented as the mean ± SEM (n = 8). Asterisks show significant differences (p < 0.05 versus control).