The Antioxidant Effects of Thymoquinone in Activated BV-2 Murine Microglial Cells

Abstract

Both neuroinflammation and microglial activation are pathological markers of a number of central nervous system (CNS) diseases. During chronic activation of the microglial cells, the induced release of excessive amounts of reactive oxygen species (ROS) and pro-inflammatory cytokines have been implicated in several neurodegenerative diseases such as Alzheimer's disease. Thymoquinone (TQ), a major bioactive compound of the natural product Nigella sativa seed, has been shown to be effective against numerous oxidative stress-induced and inflammatory disorders as well as possess neuroprotective properties. In this study, we investigated the antioxidant effects of TQ on LPS/IFNγ or H₂O₂-activated BV-2 microglia by assessing the levels of specific oxidative stress markers, the activities of selected antioxidant enzymes, as well as profiling 84 key genes related to oxidative stress via real-time reverse transcription (RT²) PCR array. Our results showed that in the LPS/IFNγ-activated microglia TQ significantly decreased the cellular production of both superoxide and nitric oxide fourfold (p < 0.0001) and sixfold (p < 0.0001), respectfully. In the H₂O₂-activated microglia, TQ also significantly decreased the cellular production of superoxide threefold (p < 0.0001) and significantly decreased hydrogen peroxide levels ~20 % (p < 0.05). Moreover, ΤQ treatment significantly decreased the levels oxidative stress in the activated BV-2 as evidenced by the assessed levels of lipid hydroperoxides and glutathione. TQ significantly decreased the levels of lipid hydroperoxides twofold (p < 0.0001) and significantly increased the levels of antioxidant glutathione 2.5-fold (p < 0.0001) in the LPS/IFNγ-activated BV-2 cells. In the H₂O₂-activated microglia, TQ significantly decreased lipid hydroperoxides eightfold (p < 0.0001) and significantly increased glutathione 15 % (p < 0.05). Activities of antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT), in the TQ-treated microglial cells also reflected a reduced oxidative stress status in the cellular environment. SOD and CAT activities were sixfold (p < 0.0001) and fivefold (p < 0.0001) lower, respectfully, for the LPS/INFγ-activated microglia treated with TQ in comparison to those that were not. For the H₂O₂-activated microglia treated with TQ, SOD and CAT activities were fivefold (p < 0.0001) and threefold (p < 0.01) lower, respectfully, compared to the untreated. Furthermore, RT² PCR array profiling of the selected 84 genes related to oxidative stress confirmed that TQ treatment in the LPS/IFNγ-activated microglia downregulates specific pro-oxidant genes, upregulates specific anti-oxidant genes, and enhances the up- or downregulation of specific genes related to the cells' natural antioxidant defense against LPS/IFNγ activation. These findings suggest that TQ may be utilized as an effective therapeutic agent for delaying the onset and/or slowing/preventing the progression of microglia-derived neurodegeneration propagated by excessive oxidative stress in the CNS.

Keywords: Microglia; Neurodegenerative disease; Oxidative stress; Thymoquinone.

FIGURE 1. Effect of thymoquinone on the viability of BV-2 cells

Cell viability was evaluated using resazurin dye (7-hydroxy-3H-phenoxazin-3-one 10-oxide), 24 h after treatment. Values expressed as mean ± SEM. **** P ≤ 0.0001.

FIGURE 2

FIGURE 2A | Effect of thymoquinone on the levels of superoxide, nitric oxide, and hydrogen peroxide in BV-2 cells stimulated with the combination of LPS (500 ng/mL) and IFNγ (0.5 ng/mL) (LPS/IFNγ) or H₂O₂ (75 μM) for 24 h. (A) The intracellular concentration of superoxide was measured using nitroblue tetrazolium (NBT) 24 h after treatment. Values expressed as mean ± SEM. ****P≤0.0001. FIGURE 2B | Effect of thymoquinone on the levels of superoxide, nitric oxide, and hydrogen peroxide in BV-2 cells stimulated with the combination of LPS (500 ng/mL) and IFNγ (0.5 ng/mL) (LPS/IFNγ) or H₂O₂ (75 μM) for 24 h. (B) Nitric oxide production was measured using Griess reagent (1% sulfanilamide, 0.1% N - (1-naphthyl) - ethylenediamine hydrochloride in 5% phosphoric acid (H₃PO₄)) 24 h after treatment. Values expressed as mean ± SEM. ****P≤0.0001. FIGURE 2C | Effect of thymoquinone on the levels of superoxide, nitric oxide, and hydrogen peroxide in BV-2 cells stimulated with the combination of LPS (500 ng/mL) and IFNγ (0.5 ng/mL) (LPS/IFNγ) or H₂O₂ (75 μM) for 24 h. (C) Hydrogen peroxide was measured using the highly sensitive and stable H₂O₂ probe ADHP (10-acetyl-3,7-dihydroxyphenoxazine) 24 h after treatment. Values expressed as mean ± SEM. *P≤0.05.

FIGURE 2

FIGURE 2A | Effect of thymoquinone on the levels of superoxide, nitric oxide, and hydrogen peroxide in BV-2 cells stimulated with the combination of LPS (500 ng/mL) and IFNγ (0.5 ng/mL) (LPS/IFNγ) or H₂O₂ (75 μM) for 24 h. (A) The intracellular concentration of superoxide was measured using nitroblue tetrazolium (NBT) 24 h after treatment. Values expressed as mean ± SEM. ****P≤0.0001. FIGURE 2B | Effect of thymoquinone on the levels of superoxide, nitric oxide, and hydrogen peroxide in BV-2 cells stimulated with the combination of LPS (500 ng/mL) and IFNγ (0.5 ng/mL) (LPS/IFNγ) or H₂O₂ (75 μM) for 24 h. (B) Nitric oxide production was measured using Griess reagent (1% sulfanilamide, 0.1% N - (1-naphthyl) - ethylenediamine hydrochloride in 5% phosphoric acid (H₃PO₄)) 24 h after treatment. Values expressed as mean ± SEM. ****P≤0.0001. FIGURE 2C | Effect of thymoquinone on the levels of superoxide, nitric oxide, and hydrogen peroxide in BV-2 cells stimulated with the combination of LPS (500 ng/mL) and IFNγ (0.5 ng/mL) (LPS/IFNγ) or H₂O₂ (75 μM) for 24 h. (C) Hydrogen peroxide was measured using the highly sensitive and stable H₂O₂ probe ADHP (10-acetyl-3,7-dihydroxyphenoxazine) 24 h after treatment. Values expressed as mean ± SEM. *P≤0.05.

FIGURE 2

FIGURE 2A | Effect of thymoquinone on the levels of superoxide, nitric oxide, and hydrogen peroxide in BV-2 cells stimulated with the combination of LPS (500 ng/mL) and IFNγ (0.5 ng/mL) (LPS/IFNγ) or H₂O₂ (75 μM) for 24 h. (A) The intracellular concentration of superoxide was measured using nitroblue tetrazolium (NBT) 24 h after treatment. Values expressed as mean ± SEM. ****P≤0.0001. FIGURE 2B | Effect of thymoquinone on the levels of superoxide, nitric oxide, and hydrogen peroxide in BV-2 cells stimulated with the combination of LPS (500 ng/mL) and IFNγ (0.5 ng/mL) (LPS/IFNγ) or H₂O₂ (75 μM) for 24 h. (B) Nitric oxide production was measured using Griess reagent (1% sulfanilamide, 0.1% N - (1-naphthyl) - ethylenediamine hydrochloride in 5% phosphoric acid (H₃PO₄)) 24 h after treatment. Values expressed as mean ± SEM. ****P≤0.0001. FIGURE 2C | Effect of thymoquinone on the levels of superoxide, nitric oxide, and hydrogen peroxide in BV-2 cells stimulated with the combination of LPS (500 ng/mL) and IFNγ (0.5 ng/mL) (LPS/IFNγ) or H₂O₂ (75 μM) for 24 h. (C) Hydrogen peroxide was measured using the highly sensitive and stable H₂O₂ probe ADHP (10-acetyl-3,7-dihydroxyphenoxazine) 24 h after treatment. Values expressed as mean ± SEM. *P≤0.05.

FIGURE 3

FIGURE 3A | Effect of thymoquinone on lipid hydroperoxides and glutathione in stimulated BV-2 microglial cells. (A) Lipid hydroperoxides were measured in BV-2 microglial cells 24 h after treatment. Values expressed as mean ± SEM. ****P≤0.0001. FIGURE 3B | Effect of thymoquinone on lipid hydroperoxides and glutathione in stimulated BV-2 microglial cells. (B) Glutathione was measured in BV-2 microglial cells 24 h after treatment. Values expressed as mean ± SEM. ****P≤0.0001; *P≤0.05.

FIGURE 3

FIGURE 3A | Effect of thymoquinone on lipid hydroperoxides and glutathione in stimulated BV-2 microglial cells. (A) Lipid hydroperoxides were measured in BV-2 microglial cells 24 h after treatment. Values expressed as mean ± SEM. ****P≤0.0001. FIGURE 3B | Effect of thymoquinone on lipid hydroperoxides and glutathione in stimulated BV-2 microglial cells. (B) Glutathione was measured in BV-2 microglial cells 24 h after treatment. Values expressed as mean ± SEM. ****P≤0.0001; *P≤0.05.

FIGURE 4

FIGURE 4A | Effect of thymoquinone on superoxide dismutase and catalase enzyme activities in stimulated BV-2 microglial cells. (A) SOD enzyme activity was measured in BV-2 microglial cells 24 h after treatment. Values expressed as mean ± SEM. ****P≤0.0001. FIGURE 4B | Effect of thymoquinone on superoxide dismutase and catalase enzyme activities in stimulated BV-2 microglial cells. (B) Catalase enzyme activity was measured in BV-2 microglial cells 24 h after treatment. Values expressed as mean ± SEM. ****P≤0.0001; **P≤0.01.

FIGURE 4

FIGURE 4A | Effect of thymoquinone on superoxide dismutase and catalase enzyme activities in stimulated BV-2 microglial cells. (A) SOD enzyme activity was measured in BV-2 microglial cells 24 h after treatment. Values expressed as mean ± SEM. ****P≤0.0001. FIGURE 4B | Effect of thymoquinone on superoxide dismutase and catalase enzyme activities in stimulated BV-2 microglial cells. (B) Catalase enzyme activity was measured in BV-2 microglial cells 24 h after treatment. Values expressed as mean ± SEM. ****P≤0.0001; **P≤0.01.

FIGURE 5. Effect of thymoquinone on the expression of key genes related to oxidative stress in stimulated BV-2 microglial cells

(A) The fold regulation change of Control vs LPS/IFNγ and LPS/IFNγ vs TQ+LPS/IFNγ. Values expressed as fold regulation. P values listed in the table. (B) Gene name for the symbol listed in the table.