Background

The Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that promotes the expression of antioxidant genes, protecting cells against oxidative stress and it also exerts immunomodulatory properties. The activation of Nrf2 is one of the proposed mechanisms of action of dimethyl fumarate (DMF), an approved drug for Multiple Sclerosis (MS).

DMF is rapidly metabolized into its active metabolite monomethyl fumarate (MMF) in the intestine. However, there is a lack of studies comparing the effects of both compounds. The combination of other Nrf2 activators could be relevant as adjuvants for DMF in neuroinflammation. Cannabidiol (CBD), a cannabinoid that attenuates MS in murine models, is known to have antioxidant properties, although there are no studies on Nrf2 activation by CBD.

Objectives

The aim of this study was to evaluate the in vitro effects of DMF, MMF and CBD on the activation of Nrf2 in neurons and microglia.

Methods

Primary hippocampal neurons and the microglial cell line BV-2 were treated for 4 hours with either vehicle, DMF (1-30 µM), MMF (1-30 µM) or CBD (1-10 μM). Cells were fixed, permeabilized and stained with a Nrf2 antibody. Activation of Nrf2 was considered as nuclear translocation, measured by confocal microscopy as the mean density of nuclear fluorescence. Five fields were taken from each condition in 3 experiments. One-way ANOVA test was used, considering p<0.05 statistically significant.

Results

DMF induced Nrf2 translocation in both neurons and microglia. However, Nrf2 translocation in neurons needed a higher dose (30 µM) than microglia (10 µM), and the induction was lower in neurons (four-fold increase) than in microglia (eight-fold increase). We did not find activation of Nrf2 with MMF in neither neurons nor microglia. CBD induced a dose-dependent Nrf2 activation in neurons, statistically significant at 6 and 7 μM, with a higher increase than that of DMF (8 and 12-fold compared to vehicle, respectively). CBD did not produce any effect on Nrf2 activation in microglia.

Conclusions

Our results support the idea that DMF acts as a neuroprotective and immunomodulatory drug through the activation of the Nrf2 pathway in neurons and microglia. We also demonstrate that DMF and MMF differ in their mechanisms of action, as we did not see Nrf2 activation with MMF. CBD could be relevant in neuroprotection as an adjuvant to DMF, as it induces a higher Nrf2 activation than DMF. CBD’s mechanism of action differs between neurons and microglia.