Carotenoid Extract Derived from Euglena gracilis Overcomes Lipopolysaccharide-Induced Neuroinflammation in Microglia: Role of NF-κB and Nrf2 Signaling Pathways

Abstract

Activation of microglia results in the increased production and release of a series of inflammatory and neurotoxic mediators, which play essential roles in structural and functional neuronal damage and in the development and progression of a number of neurodegenerative diseases. The microalga Euglena gracilis (Euglena), rich in vitamins, minerals, and other nutrients, has gained increasing attention due to its antimicrobial, anti-viral, antitumor, and anti-inflammatory activities. In particular, anti-inflammatory properties of Euglena could exert neuroprotective functions in different neurodegenerative diseases related to inflammation. However, the mechanisms underlying the anti-inflammatory effect of Euglena are not fully understood. In this study, we investigated whether Euglena could attenuate microglia activation and we also studied the mechanism of its anti-inflammatory activity. Our results showed that non-cytotoxic concentrations of a Euglena acetone extract (EAE) downregulated the mRNA expression levels and release of pro-inflammatory mediators, including NO, IL-1β, and TNF-α in LPS-stimulated microglia. EAE also significantly blocked the LPS-induced nuclear translocation of NF-κB p65 subunit and increased the mRNA expression of nuclear factor erythroid 2–related factor (Nrf2) and heme oxygenase-1 (HO-1). Furthermore, the release of pro-inflammatory mediators and NF-κB activation were also blocked by EAE in the presence of ML385, a specific Nrf2 inhibitor. Together, these results show that EAE overcomes LPS-induced microglia pro-inflammatory responses through downregulation of NF-κB and activation of Nrf2 signaling pathways, although the two pathways seem to get involved in an independent manner.

Pre- and Early Post-treatment With Arthrospira platensis (Spirulina) Extract Impedes Lipopolysaccharide-triggered Neuroinflammation in Microglia

“…We recently showed very similar results obtained with the use of an acetone extract from the microalga Euglena gracilis on the same in vitro model of neuroinflammation, despite some differences in the composition of the two extracts. Euglena gracilis extract contained diadinoxanthin as the most abundant xanthophyll, followed by zeaxanthin, whereas β-carotene represented only 8% ( Piovan et al, 2021 ). Conversely, the most abundant carotenoid present in the Spirulina extract was β-carotene, whereas the amount of zeaxanthin resulted comparable in the two extracts.…”

“…However, more studies are necessary to verify a possible direct interaction of the extract components with Nrf2, as already showed for some carotenoids and their derivatives that can interact with Keap1, the negative regulator of Nrf2, by changing its physical properties ( Kaulmann and Bohn, 2014 ). However, in this regard, it should be noted that, despite Nrf2 is considered a key regulator of inflammatory processes ( Ahmed et al, 2017 ), based on the existence of a complex interaction between oxidative stress and inflammation, we recently showed that the anti-inflammatory effect of the acetone extract of Euglena gracilis is Nrf2 independent ( Piovan et al, 2021 ). Similarly, the anti-inflammatory effect of E1 could be independent of the activation of Nrf2.…”

“…Microglial cell viability was evaluated by a colorimetric method utilizing the protein-binding dye sulforhodamine B (SRB) ( Skehan et al, 1990 ; Piovan et al, 2021 ). Growth medium was replaced with serum-free medium 2 h before treatment with increasing concentrations of E1 for 16 h. After the incubation, the medium was replaced with cold 10% trichloroacetic acid, and plates were incubated for 1 h at 4°C.…”

Anti-Inflammatory Activities of Euglena gracilis Extracts

“…In our protocol, we used acetone as the solvent to obtain the crude extract. Indeed, we recently reported that acetone extract of Euglena is endowed with biological activity and modulated microglial activation by inhibiting the expression and release of pro-inflammatory molecules in LPS-induced neuroinflammation [38]. Moreover, acetone extracts photosynthetic pigments with a wide range of polarity and yields fractions enriched in carotenoids [39].…”

The Effect of C-Phycocyanin on Microglia Activation Is Mediated by Toll-like Receptor 4

“…The sulforhodamine B (SRB) assay was used to measure microglial cell viability [75,76]. Cells were plated in poly-L-lysine coated 96-well plates (50,000 cells/well) in growth medium containing 10% of serum and allowed to adhere overnight.…”