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 4-fold (p<0.0001) and 6 fold (p<0.0001), respectfully. In the H2O2-activated microglia, TQ also significantly decreased the cellular production of superoxide 3-fold (p<0.0001) and significantly decreased hydrogen peroxide levels ~20% (p<0.05). Moreover, TQ 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 2-fold (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 8-fold (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 6 fold (p<0.0001) and 5 fold (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 5 fold (p<0.0001) and 3 fold (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.

Dissecting the Potential Roles of Nigella sativa and Its Constituent Thymoquinone on the Prevention and on the Progression of Alzheimer's Disease

“…To this regard, the authors demonstrated that TQ and other antioxidants [e.g., EGCG, dilinoleoylphosphatidylcholine (DLPC)] were able to prevent oxidation of Aß, by acting on TNF-α-mediated signaling pathway, through the downregulation of nitric oxide (NO) and the upregulation of glutathione (GSH). Recently (Cobourne-Duval et al, 2016 ), it has been demonstrated that TQ had antioxidant effects in activated BV-2 murine microglial cells, by acting on ROS and pro-inflammatory cytokines levels whose expression are extremely higher in several neurodegenerative disorders, including AD. Basically, the authors showed that the treatment with TQ of the lipopolisaccaride (LPS)/Interferon-gamma (IFNγ) activated microglia, impaired the expression profiles of specific genes involved in the oxidation process.…”

Thymoquinone increases the expression of neuroprotective proteins while decreasing the expression of pro-inflammatory cytokines and the gene expression NFκB pathway signaling targets in LPS/IFNγ -activated BV-2 microglia cells

“…Specifically, TQ has been shown to have anti-inflammatory and antioxidant effects in different disease states/models in vivo and in vitro. Studies have shown that TQ is a ROS scavenger (Mansour et al, 2002; Badary et al, 2003) capable of reducing the levels of several ROS and lipid/protein oxidation markers in liver and kidney tissue, microglia and macrophages (El-Mahmoudy et al, 2002; Attia et al, 2010; Mahmoud et al, 2014; Cobourne-Duval et al, 2016). Likewise, the anti-inflammatory properties of TQ treatment have been demonstrated in mast cells, microglia, kidney, and liver (El Gazzar et al, 2007; Al-Malki and Sayed, 2014; Mahmoud et al, 2014; Taka et al, 2015).…”

“…The TQ stock was freshly prepared by initially dissolving in DMSO then diluting further with experimental media to the appropriate concentration so that the concentration of DMSO did not exceed 0.025%, which was used for the control (0 μM TQ). The concentration of TQ used in all the experiments was 12.5 μM, which was established by cell viability in our previous study (Cobourne-Duval, Taka, et al 2016). …”

The Neuroprotective Effects of Thymoquinone: A Review

“… 5 Thymoquinone is the main ingredient of the plant, which is effective for treatment of various diseases such as neurodegenerative disorders, coronary artery diseases, and respiratory and urinary system diseases. 6 - 13 Thymoquinone has also been indicated to possess antioxidant, anti-inflammation, anticancer, antibacterial, antimutagenic, and antigenotoxic activities. 9 , 14 - 27 Thymoquinone may be considered as a therapeutic agent for the prevention of oral supplementation of chrysin (100 mg/kg body weight) to hyperammonemic rats, which considerably restored the levels of brain ammonia, water content, and the expressions of glutamine synthetase (GS), glial fibrillary acidic protein (GFAP), tumor necrosis factor α (TNF-α), interleukin (IL) 1β, IL-6, p65, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2).…”