Polyphenol-Rich Propolis Extracts Strengthen Intestinal Barrier Function by Activating AMPK and ERK Signaling

Abstract

Propolis has abundant polyphenolic constituents and is used widely as a health/functional food. Here, we investigated the effects of polyphenol-rich propolis extracts (PPE) on intestinal barrier function in human intestinal epithelial Caco-2 cells, as well as in rats. In Caco-2 cells, PPE increased transepithelial electrical resistance and decreased lucifer yellow flux. PPE-treated cells showed increased expression of the tight junction (TJ) loci occludin and zona occludens (ZO)-1. Confocal microscopy showed organized expressions in proteins related to TJ assembly, i.e., occludin and ZO-1, in response to PPE. Furthermore, PPE led to the activation of AMPK, ERK1/2, p38, and Akt. Using selective inhibitors, we found that the positive effects of PPE on barrier function were abolished in cells in which AMPK and ERK1/2 signaling were inhibited. Moreover, rats fed a diet supplemented with PPE (0.3% in the diet) exhibited increased colonic epithelium ZO-1 expression. Overall, these data suggest that PPE strengthens intestinal barrier function by activating AMPK and ERK signaling and provide novel insights into the potential application of propolis for human gut health.

Keywords: AMPK; Caco-2; ERK; propolis; tight junctions.

Figure 1

Effects of polyphenol-rich propolis extracts (PPE) on intestinal TJ permeability in Caco-2 cell monolayers. (A) Transepithelial electrical resistance (TER) in PPE-treated Caco-2 cell monolayers. Caco-2 cell monolayers were grown on 12-Costar Transwell filters for 14 days. TER was measured after treatment of Caco-2 monolayers with PPE (▲, 0 µg/mL, ◆, 5 µg/mL, ●, 25 µg/mL, ■, 50 µg/mL) at the indicated time points. The change in TER is expressed as the percentage change compared to the initial TER for each monolayer. The values represent the means ± SD (n = 3). Means sharing the same letter are not significantly different from each other (p < 0.05); (B) Unidirectional lucifer yellow (LY) flux in PPE-treated Caco-2 cell monolayers. LY flux was measured 120 min across Caco-2 cell monolayers after PPE exposure. Values represent means ± SD (n = 3) and are expressed as percentages of LY permeation. Means sharing the same letter are not significantly different from each other (p < 0.05); (C) Cellular viability was measured at 48 h post-PPE treatment using a cell counting kit-8 proliferation analysis. Results are expressed as % relative to control cells and data are given as means ± SD of three independent experiments, each performed in triplicate. Means sharing the same letter are not significantly different from each other (p < 0.05).

Figure 2

Effects of PPE treatment on gene expression, the distribution of tight junction proteins (ZO-1 and occludin) in Caco-2 cells. (A) Effect of PPE treatment on the mRNA expression of ZO-1 and occludin. Caco-2 cells were grown to confluence on 6-well plates and treated with PPE (50 μg/mL) for the indicated periods. ZO-1 and occludin mRNA expression levels were analyzed by qRT-PCR. Data are presented as means ± SD (n = 3). Means sharing the same letter are not significantly different from each other (p < 0.05); (B) Confocal microscopy images of immuno-stained tight junction proteins in confluent Caco-2 cells untreated or treated with PPE (50 µg/mL) for 48 h. Representative confocal microscopy images were obtained by fluorescent microscopy from three independent experiments after immunofluorescence staining of ZO-1 and occludin. 4′,6-Diamidino-2-phenylindole (DAPI) staining was performed to identify nuclei.

Figure 3

PPE treatment activates AMPK and ERK signaling in Caco-2 cell monolayers and selective inhibitors block PPE-induced TJ regulation. (A) Caco-2 cells were grown to confluence on six-well plates and treated with PPE (50 µg/mL) for the indicated periods. Whole cell lysates were collected and further subjected to an immunoblot analysis. Selective antibodies were used to detect the expression of phospho-AMPKα, total-AMPK, phospho-Akt, total-Akt, phospho-ERK1/2, total-ERK1/2, phospho-p38, and total-p38. β-Tubulin was used as a loading control. Representative Western blots are shown from three independent experiments; (B) The intensity of corresponding bands was measured by densitometry and normalized to β-tubulin. The values are the means ± SD (n = 3). Means sharing the same letter are not significantly different from each other (p < 0.05); (C) Caco-2 cell monolayers were grown on 12-Costar Transwell filters for 14 days. Cells were pretreated for 1 h with selective inhibitors: 5 µM DM (Dorsomorphin, for AMPK signaling), 10 µM LY (LY294002, for Akt signaling), 25 µM PD (PD98059, for ERK1/2 signaling), and 10 µM SB (SB203580, for p38 signaling) before treatment with PPE (50 µg/mL) for 24 h or 48 h. The change in TER is expressed as the percentage change compared to the initial TER for each monolayer. The values are the means ± SD (n = 3). Means sharing the same letter are not significantly different from each other (p < 0.05).

Figure 4

Effects of oral administration of PPE on rat colonic TJ mRNA expression. Distal colon tissues were collected from rats that were administered the control diet or control diet containing PPE (0.3% w/w). Effects of PPE administration on the mRNA expression of ZO-1 and occludin in rat distal colons. ZO-1 (A) and occludin (B) mRNA expression was analyzed by qRT-PCR. Data are presented as means ± SD (n = 6). *** p < 0.001, n.s., not significant.