α-Tocopherol Protects Lipopolysaccharide-Activated BV2 Microglia

Abstract

Microglia, the resident macrophage-like population in the central nervous system, play a crucial role in the pathogenesis of many neurodegenerative disorders by triggering an inflammatory response that leads to neuronal death. Neuroprotective compounds to treat or prevent neurodegenerative diseases are a new field of study in modern medicine. Microglia are activated in response to inflammatory stimuli. The pathogenesis of various neurodegenerative diseases is closely related to the constant activation of microglia due to their fundamental role as a mediator of inflammation in the brain environment. α-Tocopherol, also known as vitamin E, is reported to possess potent neuroprotective effects. The goal of this study was to investigate the biological effects of vitamin E on BV2 microglial cells, as a possible neuroprotective and anti-inflammatory agent, following stimulation with lipopolysaccharide (LPS). The results showed that the pre-incubation of microglia with α-tocopherol can guarantee neuroprotective effects during microglial activation induced by LPS. α-Tocopherol preserved the branched morphology typical of microglia in a physiological state. It also reduced the migratory capacity; the production of pro-inflammatory and anti-inflammatory cytokines such as TNF-α and IL-10; and the activation of receptors such as TRL4 and CD40, which modulate the PI3K-Akt signaling pathway. The results of this study require further insights and research, but they present new scenarios for the application of vitamin E as an antioxidant for the purpose of greater neuroprotection in vivo for the prevention of possible neurodegenerative diseases.

Keywords: antioxidant action; central nervous system; inflammation; microglia; neuroprotective effects; vitamin E; α-tocopherol.

Figure 1

Cell viability analysis of vitamin E using an MTT assay. BV2 cells were treated with a vitamin E dose–response curve from 50 μM to 400 μM (A). Vitamin E at a concentration of 100 μM was used to treat cells in the absence or presence of 1 µg/mL LPS (B). Data are reported as percentages compared to control values and are expressed as means ± SDs. * p < 0.05 compared to the control of the same time point.

Figure 2

Morphological analysis after administration of vitamin E with or without LPS. Morphological analysis of BV2 cells in the control condition (A) and after 1 µg/mL LPS (B), 100 μM vitamin E (C), or 100 μM vitamin E in the presence of 1 µg/mL LPS (D). Bar: 75 µm (20 × objective). In the images, the arrows indicate cells that have undergone a morphological change. Cell areas (µm²) were quantified using ImageJ software bounded with Java8 64-bit (E). Data are expressed as the means ± SDs of the cell areas. * p < 0.05 compared to control. # p < 0.05 compared to LPS.

Figure 3

Analysis of the migratory capacity of microglia following the administration of vitamin E with or without LPS. A wound was procured from a sub-confluent layer of BV2 cells, and the resulting space was imaged at the time of the wound and 24 h after the treatment: BV2 cells at time 0 (A), 24 h after the cut in the control condition (B), with 1 µg/mL LPS (C), with 100 μM vitamin E (D), and with 100 μM vitamin E in the presence of 1 µg/mL LPS (E). The images are representative of an experiment with three independent replicates. The percentage of the wound gap was analyzed using ImageJ software and subsequently plotted and statistically analyzed as the percentage of wound closure compared to the 0 time condition (F). Values are presented as means ± SDs. Bar: 100 µm (10× objective). * p < 0.05 compared to control. # p < 0.05 compared to LPS condition.

Figure 4

Evaluation of cytokine concentrations following administration of vitamin E with or without LPS. Analysis of cytokine concentrations (TNF-α (A) and IL-10 (B)) with stimulation of 100 μM vitamin E with or without LPS (1 µg/mL). Data are expressed as means (pg/mL) ± SDs. (A) *** p < 0.001 compared to the control condition. ### p < 0.001 compared to the condition of LPS-stimulated microglia. (B) * p < 0.05 compared to the CTR. # p < 0.05 compared to the LPS condition.

Figure 5

Evaluation of CD40, CD206, TLR4, and p-AKT expression following administration of vitamin E with or without LPS. Western blotting detection (A) and densitometric analysis of the expression of the pro-inflammatory CD40 (B), anti-inflammatory CD206 (C), TLR4 (D), and p-AKT (E) in control cells (CTR), BV2 cells treated with vitamin E (VIT E), BV2 cells treated with LPS (LPS), and BV2 cells treated with vitamin E + LPS (VIT E + LPS). Protein expression values are expressed in arbitrary units after normalization against β-actin. Data are presented as means ± SDs (* p < 0.05 vs. CTR; # p < 0.05 vs. LPS).