Resveratrol Activates Antioxidant Protective Mechanisms in Cellular Models of Alzheimer's Disease Inflammation

Abstract

Resveratrol is a natural phenolic compound with known benefits against neurodegeneration. We analyzed in vitro the protective mechanisms of resveratrol against the proinflammatory monomeric C-reactive protein (mCRP). mCRP increases the risk of AD after stroke and we previously demonstrated that intracerebral mCRP induces AD-like dementia in mice. Here, we used BV2 microglia treated with mCRP for 24 h in the presence or absence of resveratrol. Cells and conditioned media were collected for analysis. Lipopolysaccharide (LPS) has also been implicated in AD progression and so LPS was used as a resveratrol-sensitive reference agent. mCRP at the concentration of 50 µg/mL activated the nitric oxide pathway and the NLRP3 inflammasome pathway. Furthermore, mCRP induced cyclooxygenase-2 and the release of proinflammatory cytokines. Resveratrol effectively inhibited these changes and increased the expression of the antioxidant enzyme genes Cat and Sod2. As central mechanisms of defense, resveratrol activated the hub genes Sirt1 and Nfe2l2 and inhibited the nuclear translocation of the signal transducer NF-ĸB. Proinflammatory changes induced by mCRP in primary mixed glial cultures were also protected by resveratrol. This work provides a mechanistic insight into the protective benefits of resveratrol in preventing the risk of AD induced by proinflammatory agents.

Keywords: BV2; inflammation; lipopolysaccharide; monomeric C-reactive protein; oxidative stress; primary mixed glial cultures; resveratrol.

Figure 1

TNFα release by BV2 microglial cells activated with the proinflammatory agents mCRP (a) or LPS (b), in the absence or presence of resveratrol. Statistics: two-way ANOVA, significance of factors is indicated in graph; post hoc test, *** p < 0.001 compared to control, ### p < 0.001 compared to mCRP in the absence of resveratrol. RV, resveratrol.

Figure 2

Nitrite levels in the culture media of BV2 microglial cells that indicate nitric oxide generation induced by mCRP (a) or LPS (b), in the absence or presence of resveratrol. Statistics: two-way ANOVA, significance of factors is indicated in graph; post hoc test, ** p < 0.01, *** p < 0.001 compared to control, ## p < 0.01, ### p < 0.001 compared to mCRP or LPS in the absence of resveratrol. Nitrite levels induced by LPS at 1 µg/mL without resveratrol or with resveratrol at 1 µM were higher than the corresponding treatments with LPS at 0.1 µg/mL (p < 0.05, not shown). RV, resveratrol.

Figure 3

Protein levels of iNOS enzyme in BV2 microglial cells, induced by mCRP (a) or LPS (b), in the absence or presence of resveratrol. Statistics: two-way ANOVA, significance of factors is indicated in graph; post hoc test, *** p < 0.001 compared to control, # p < 0.05 compared to mCRP in the absence of resveratrol. Results and representative blots are displayed in the upper and lower panels, respectively. RV, resveratrol.

Figure 4

Protein levels of NLRP3 in BV2 microglial cells, induced by mCRP (a) or LPS (b), in the absence or presence of resveratrol. Statistics: two-way ANOVA, significance of factors is indicated in graph; post hoc test, ** p < 0.01, *** p < 0.001 compared to control, # p < 0.05 compared to LPS in the absence of resveratrol. Results and representative blots are displayed in the upper and lower panels, respectively. RV, resveratrol.

Figure 5

Immunofluorescence detection of NF-ĸB p65 in BV2 microglial cells. (a) Quantification of NF-ĸB p65 in the nucleus after exposure to mCRP, in the absence or presence of resveratrol. (b) Representative confocal images used for analysis. In the upper panel, images show green fluorescence for NF-ĸB p65 and blue fluorescence for DAPI nuclear staining; in the bottom panel, images of the same cells show green fluorescence. (c) Cell cultures stained for NF-ĸB p65 shown at lower magnification. Statistics in (a): two-way ANOVA, significance of factors is indicated in graph; post hoc test, ** p < 0.01 compared to control, ### p < 0.001 compared to mCRP in the absence of resveratrol. RV, resveratrol, A.U., arbitrary units.

Figure 6

Expression levels of proinflammatory genes in BV2 microglial cells, induced by mCRP in the absence or presence of resveratrol. (a) Nos2. (b) Cox2. (c) Clec7a. (d) Il6. Statistics: two-way ANOVA, significance of factors is indicated in graph; post hoc test, * p < 0.05, *** p < 0.001 compared to control, ## p < 0.01, ### p < 0.001 compared to mCRP in the absence of resveratrol. RV, resveratrol.

Figure 7

Expression levels of antioxidant genes induced by resveratrol in BV2 microglial cells, either control or mCRP treated. (a) Sirt1. (b) Nfe2l2. (c) Cat. (d) Sod2. Statistics: two-way ANOVA, significance of factors is indicated in graph; post hoc test, * p < 0.05, ** p < 0.01, *** p < 0.001 compared to control, # p < 0.05, ## p < 0.01, ### p < 0.001 compared to the absence of resveratrol. RV, resveratrol.

Figure 8

Proinflammatory changes induced by mCRP in primary mixed glial cultures in the presence or absence of resveratrol. (a) Microphotographs of cultures showing astrocytes (GFAP, red fluorescence) and microglia (lectin, green fluorescence) submitted to treatments as indicated in the figure. (b) Nitrite levels in the culture media indicative of nitric oxide generation. (c) IL1ß release to the culture media. Statistics: two-way ANOVA, significance of factors is indicated in graph; post hoc test, * p < 0.05, *** p < 0.001 compared to control, # p < 0.05, ## p < 0.01, ### p < 0.001 compared to mCRP in the absence of resveratrol. RV, resveratrol.

Figure 9

Schematic representation of the protective mechanisms of resveratrol against the proinflammatory agent mCRP and LPS.