Bioactive components have been extensively studied for their beneficial activities in preventing widespread human diseases, including cerebrovascular dysfunctions [1
]. Bioactive components can modulate different epigenetic targets, including the histone acetylation/deacetylation balance, through the regulation of histone acetyltransferase (HAT) and histone deacetylase (HDAC) activities [3
]. Acetylation of histones by HATs typically enhances transcription by reducing the affinity between DNA and histones, while the removal of acetyl groups by HDACs results in chromatin condensation, leading to the repression of gene transcription [6
]. Notably, aberrant acetylation status resulting from the misregulation of HAT and/or HDAC activity has been associated with different pathologies, including brain ischemia [7
Besides histones, HATs and HDACs can also modulate the acetylation state of a number of transcription factors, including NF-κB [8
]. In the central nervous system, NF-κB plays a crucial role in regulating genes controlling either cell survival [9
] or the apoptosis and inflammation associated with neurodegeneration [10
]. We previously demonstrated that mechanisms affecting the acetylation state of the NF-κB/RelA subunit in brain ischemia can discriminate between protective and neurotoxic activation of the transcriptional factor [11
]. Protective ischemic preconditioning and harmful ischemia induce similar levels of p50/RelA activation, but only the ischemic injury induces atypical RelA acetylation. The RelA that translocates to the nucleus in primary cortical neurons exposed to preconditioning oxygen and glucose deprivation (OGD) or in cortices of mice subjected to preconditioning middle cerebral artery occlusion (MCAO) shows a general lysine deacetylation. Conversely, as demonstrated by mutagenesis analysis, the RelA activated in neurons exposed to lethal OGD or in the cortices of mice subjected to noxious ischemia displays a general lysine deacetylation, but a site-specific acetylation at the lysine 310 (K310) residue [12
]. The aberrant RelA acetylation in neurons subjected to OGD was associated with reduced histone acetylation and with the transcription of the pro-apoptotic Bim
gene, as indicated by increased H3 acetylation at the Bim
]. The aberrantly acetylated RelA co-immunoprecipitated with the HAT p300/CBP, suggesting a role of p300/CBP in K310 acetylation during the lethal exposure of neuronal cells to OGD [12
In the present work, we investigated the neuroprotective activity of a polyphenol-enriched micronutrient mixture (PMM), containing (-)-epigallocatechin-3-gallate (EGCG), quercetin, resveratrol, α-lipoic acid (LA), vitamins, amino acids and other micronutrients in primary mouse cortical neurons exposed to OGD. In addition, we studied the epigenetic mechanisms involved in the activity of the bioactive components mixture by focusing on the acetylation state of either HAT or HDAC target proteins.
Here, we demonstrated the neuroprotective effect of a polyphenol-enriched nutrient supplement in a cell-based model of ischemic stroke involving the primary cortical neurons exposed to lethal OGD.
Neuroprotection was associated with changes in the acetylation state of NF-κB/RelA. In our experimental setting, we confirmed the aberrant acetylation of RelA after the lethal OGD exposure. The PMM treatment was able to reduce the ac-RelA(K310) acetylation without improving the deacetylation of the other lysine residues. It can be inferred that PMM could induce a switch of RelA from the “lethal pro-apoptotic” form to the “preconditioning protective” form [12
]. By undergoing the aberrant acetylation, RelA binds the pro-apoptotic Bim
], an event followed by specific histone acetylation at the target gene promoter. The increased H3 acetylation at the Bim
promoter is an early marker of the triggered apoptotic process [16
]. In the present work, we demonstrated that the treatment of cells with PMM was able to limit the H3 acetylation at the Bim
promoter, suggesting that the mixture of bioactive components could regulate the expression of this pro-apoptotic gene (Figure 5
In addition to changing the acetylation state of RelA, lethal ischemia produces a significant reduction of H3 and H4 histone acetylation [16
]. Our data confirmed the general reduction of H3 and H4 histone acetylation after OGD exposure. In contrast to the effect produced by HDAC inhibitors in models of brain ischemia [16
], PMM did not restore the general histone acetylation. This suggests that the supplement mixture acts as a HAT inhibitor, rather than a HDAC inhibitor.
EGCG, one of the main components of the mixture, has been proposed to act as either an HDAC inhibitor or a HAT inhibitor [29
]. In accordance with the hypothesis that in our model EGCG could work as a HAT inhibitor, Choi and colleagues reported that EGCG reduced ac-RelA(K310) acetylation by directly inhibiting the activity of HAT enzymes, p300 and CPB [29
]. However, it cannot be excluded that, at concentrations higher than those used in our experimental setting, the EGCG inhibition of HAT can lead to the reduction of histone acetylation at the HDAC promoter, resulting in the inhibition of HDAC expression. Several bioactive components of the mixture could act as HAT inhibitors, thus contributing to the observed neuroprotective effect. Besides EGCG, among the compounds present at higher concentrations, quercetin and LA inhibit p300/CBP [33
] and have been reported to exert neuroprotective effects in cellular and animal models of brain ischemia [17
]. Therefore, one of the possible mechanisms involved in the RelA regulation could very well be the inhibition of p300/CBP by PMM (Figure 5
Recently, we showed that the combination of MS-275 or valproate, two class-I HDAC inhibitors, with resveratrol, an activator of the AMP-activated protein kinase (AMPK)–sirtuin 1 pathway, could restore normal RelA acetylation and elicit neuroprotection in cortical neurons exposed to OGD and in animal models of brain ischemia [16
] or amyotrophic lateral sclerosis (ALS) [42
]. In light of these findings, we can speculate that the resveratrol present in the PMM, by activating the AMPK-sirtuin 1 pathway, could participate in ac-RelA(K310) deacetylation and work synergistically with other compounds to limit NF-κB-mediated apoptosis [12
] (Figure 5
). Recent findings indicate that also other molecules of the mixture, including EGCG, quercetin, LA, vitamin C, vitamin E, vitamin B6 and N-acetyl-L-cysteine (NAC) are able to activate the AMPK–sirtuin 1 pathway [44
], suggesting that these compounds could amplify the action of resveratrol. Future experiments will investigate the effects of sirtuin-1 and AMPK inhibitors in blocking the neuroprotective activity of PMM.
The broccoli extract present in the PMM could also contribute to the observed neuroprotective effect. The isothiocyanate compound sulforaphane (1-isothiocyanato-4-methylsulfinylbutane), which is highly produced in a variety of cruciferous vegetables (Brassica
; e.g., broccoli), has been found to protect hippocampal neurons against OGD at a concentration of 0.5 μM [56
] and to inhibit HDAC activity in mouse cortical neurons at 10–20 μM [57
]. However, the lack of HDAC inhibitor-like effects of PMM, i.e., the failure to increase the general acetylation of RelA and H3 (K9–18) and H4 (K16) histones [16
], might very well exclude the participation of sulforaphane to the synergistic effects of the other compounds in modulating RelA acetylation.
In our experimental setting, the expected concentrations of the compounds present in the mixture were thousands-fold lower than those found to be protective when individual compounds were tested in neurons exposed to OGD [16
]. Similarly, the compounds’ concentrations in the PMM were remarkably lower than those able to inhibit HATs or activate the AMPK–sirtuin 1 pathway when the bioactive components were tested individually [16
]. The neuroprotective effect and the epigenetic activity displayed by these molecules, despite their low concentrations, indicate that a synergistic interaction between different bioactive components may occur in the PMM. Notably, the beneficial effect occurs at concentrations in the nanomolar range, compatible with the predicted brain levels of most of the bioactive components reachable at the recommended daily assumption dose of PMM [63
]. Although the approach to studying PMM has the limitation of not discriminating the role of each specific compound separately, it offers the valuable advantage of evaluating the final result of multiple nutritional supplements when administered in combination.