Dietary Polyphenols Decrease Chemokine Release by Human Primary Astrocytes Responding to Pro-Inflammatory Cytokines

Abstract

Astrocytes are considered to be the dominant cell fraction of the central nervous system. They play a supportive and protective role towards neurons, and regulate inflammatory processes; they thus make suitable targets for drugs and supplements, such as polyphenolic compounds. However, due to their wide range, knowledge of their anti-inflammatory potential remains relatively incomplete. The aim of this study was therefore to determine whether myricetin and chrysin are able to decrease chemokine release in reactive astrocytes. To assess the antioxidant and anti-inflammatory potential of polyphenols, human primary astrocytes were cultured in the presence of a reactive and neurotoxic astrocyte-inducing cytokine mixture (TNF-α, IL-1a, C1q), either alone or in the presence of myricetin or chrysin. The examined polyphenols were able to modify the secretion of chemokines by human cortical astrocytes, especially CCL5 (chrysin), CCL1 (myricetin) and CCL2 (both), while cell viability was not affected. Surprisingly, the compounds did not demonstrate any antioxidant properties in the astrocyte cultures.

Keywords: astrocytes; chemokines; chrysin; dietary polyphenols; inflammation; myricetin.

Figure 1

The effect of chrysin (CHR) (A) and myricetin (MYR) (B) on human astrocyte viability. The results were obtained from 4 separate experiments performed for each of the 3 donors. Cells were cultured on 96-well plates for 24, 48 h or 6 days with each polyphenol at a 0.1 nM, 1 nM, and 5 nM concentration and in non-stimulatory conditions (culture medium). Data are shown as mean viability ± SD. Normality of the distribution was checked with the Shapiro–Wilk test. For comparisons between groups, the Mann–Whitney U-test or Student’s t-test was used; differences were considered significant for p values < 0.05.

Figure 2

Effect of chrysin (CHR) on cell viability in conditions of oxidative stress. The data were acquired from 4 separate experiments performed for each of the 3 donors: D1 (A), D2 (B), D3 (C). Cells were cultured on 96-well plates for 48 h in non-stimulatory conditions (culture medium), with 1 mM H₂O₂, and with 0.1, 1, or 5 nM chrysin (CHR) with the subsequent addition of 1 mM H₂O₂. Results are shown as mean viability ± SD. Normality of the distribution was checked with the Shapiro–Wilk test. For comparisons between groups, the Mann–Whitney U-test was used; differences were considered significant for p values < 0.05.

Figure 3

Effect of myricetin (MYR) on cell viability in conditions of oxidative stress. The data were acquired from four separate experiments performed for each of the 3 donors: D1 (A), D2 (B), D3 (C). Cells were cultured on 96-well plates for 48 h in non-stimulatory conditions (culture medium), with 1 mM H₂O₂, and with 0.1, 1 nM or 5 nM myricetin (MYR) with the subsequent addition of 1 mM H₂O₂. Results are shown as mean viability ± SD. Normality of the distribution was checked with the Shapiro–Wilk test. For comparisons between groups, the Mann–Whitney U test was used; differences were considered significant for p values < 0.05.

Figure 4

Production of CCL1 by human astrocytes in response to chrysin (A) or myricetin (B). The results were acquired from at least 4 separate experiments performed for the 3 donors. Cells were cultured on 48-well plates for 6 days in proinflammatory conditions (TNF-α/IL-1a/C1q, 24 results), with chrysin (A) or myricetin (B) stimulation (respectively, 0.1 nM, 1 nM, 5 nM; 12 results for every point), or under proinflammatory conditions with the addition of chrysin (CHR + TNF-α/IL-1a/C1q, 12 results) or myricetin (MYR + TNF-α/IL-1a/C1q, 12 results) or the culture medium alone (24 results). Data are shown as mean chemokine concentration ± SD. The normality of the distribution was checked with the Shapiro–Wilk test. The groups were compared using the Mann–Whitney U-test; significant differences were assumed for p < 0.05. The black dots show the distribution of results within groups.

Figure 5

Production of CCL2 by human astrocytes in response to chrysin (A) or myricetin (B). The results were acquired from at least 4 separate experiments performed for the 3 donors. Cells were cultured on 48-well plates for 6 days in proinflammatory conditions (TNF-α/IL-1a/C1q, 24 results), with chrysin (A) or myricetin (B) stimulation (respectively, 0.1 nM, 1 nM, 5 nM; 12 results for every point), or under proinflammatory conditions with the addition of chrysin (CHR + TNF-α/IL-1a/C1q, 12 results) or myricetin (MYR + TNF-α/IL-1a/C1q, 12 results) or the culture medium alone (24 results). Data are shown as mean chemokine concentration ± SD. Normality of the distribution was checked with the Shapiro–Wilk test. For comparisons between groups, the Mann–Whitney U-test was used, and differences were considered significant for p < 0.05. Black dots show the distribution of results within groups.

Figure 6

Production of CCL5 by human astrocytes in response to chrysin (A) or myricetin (B). The results were acquired from at least 4 separate experiments performed for the 3 donors. Cells were cultured on 48-well plates for 6 days in proinflammatory conditions (TNF-α/IL-1a/C1q, 24 results), with chrysin (A) or myricetin (B) stimulation (respectively, 0.1 nM, 1 nM, 5 nM; 12 results for every point), or under proinflammatory conditions with the addition of chrysin (CHR + TNF-α/IL-1a/C1q, 12 results) or myricetin (MYR + TNF-α/IL-1a/C1q, 12 results) or the culture medium alone (24 results). Data are shown as mean chemokine concentration ± SD. The normality of the distribution was checked with the Shapiro–Wilk test. For comparisons between groups, the Mann–Whitney U-test was used, and differences were considered significant for p < 0.05. Black dots show the distribution of results within groups.