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

Abstract

Neuroinflammation and microglial activation are pathological markers of a number of central nervous system (CNS) diseases. Chronic activation of microglia induces the release of excessive amounts of reactive oxygen species (ROS) and pro-inflammatory cytokines. Additionally, chronic microglial activation has been implicated in several neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. Thymoquinone (TQ) has been identified as one of the major active components of the natural product Nigella sativa seed oil. TQ has been shown to exhibit anti-inflammatory, anti-oxidative, and neuroprotective effects. In this study, lipopolysaccharide (LPS) and interferon gamma (IFNγ) activated BV-2 microglial cells were treated with TQ (12.5 μM for 24 h). We performed quantitative proteomic analysis using Orbitrap/Q-Exactive Proteomic LC-MS/MS (Liquid chromatography-mass spectrometry) to globally assess changes in protein expression between the treatment groups. Furthermore, we evaluated the ability of TQ to suppress the inflammatory response using ELISArray™ for Inflammatory Cytokines. We also assessed TQ's effect on the gene expression of NFκB signaling targets by profiling 84 key genes via real-time reverse transcription (RT²) PCR array. Our results indicated that TQ treatment of LPS/IFNγ-activated microglial cells significantly increased the expression of 4 antioxidant, neuroprotective proteins: glutaredoxin-3 (21 fold; p < 0.001), biliverdin reductase A (15 fold; p < 0.0001), 3-mercaptopyruvate sulfurtransferase (11 fold; p < 0.01), and mitochondrial lon protease (>8 fold; p < 0.001) compared to the untreated, activated cells. Furthermore, TQ treatment significantly (P < 0.0001) reduced the expression of inflammatory cytokines, IL-2 = 38%, IL-4 = 19%, IL-6 = 83%, IL-10 = 237%, and IL-17a = 29%, in the activated microglia compared to the untreated, activated which expression levels were significantly elevated compared to the control microglia: IL-2 = 127%, IL-4 = 151%, IL-6 = 670%, IL-10 = 133%, IL-17a = 127%. Upon assessing the gene expression of NFκB signaling targets, this study also demonstrated that TQ treatment of activated microglia resulted in >7 fold down-regulation of several NFκB signaling targets genes, including interleukin 6 (IL6), complement factor B (CFB), chemokine (CC motif) ligand 3 (CXCL3), chemokine (CC) motif ligand 5 (CCL5) compared to the untreated, activated microglia. This modulation in gene expression counteracts the >10-fold upregulation of these same genes observed in the activated microglia compared to the controls. Our results show that TQ treatment of LPS/IFNγ-activated BV-2 microglial cells induce a significant increase in expression of neuroprotective proteins, a significant decrease in expression inflammatory cytokines, and a decrease in the expression of signaling target genes of the NFκB pathway. Our findings are the first to show that TQ treatment increased the expression of these neuroprotective proteins (biliverdin reductase-A, 3-mercaptopyruvate sulfurtransferase, glutaredoxin-3, and mitochondrial lon protease) in the activated BV-2 microglial cells. Additionally, our results indicate that TQ treatment decreased the activation of the NFκB signaling pathway, which plays a key role in neuroinflammation. In conclusion, our results demonstrate that TQ treatment reduces the inflammatory response and modulates the expression of specific proteins and genes and hence potentially reduce neuroinflammation and neurodegeneration driven by microglial activation.

Keywords: Microglia; NFκB; Neuroinflammation; Neuroprotection; Thymoquinone.

Fig. 1

Multi-Analyte ELISArray™ for Inflammatory Cytokines & Chemokines protein expression fold change amongst the control, LPS/INFγ, TQ, and TQ + LPS/IFNγ groups. Data represent protein expression as the mean ± S.E.M (n = 3). Statistical significance of LPS/INFγ vs. TQ + LPS/IFNγ was evaluated by one-way ANOVA followed by Tukey's post hoc multiple comparisons test, p**** ≤0.0001.

Fig. 2

Scatter Plot of the Normalized Expression of NFκB Signaling Targets PCR Array in the Control, TQ, LPS/IFNγ, and TQ + LPS/IFNγ Experimental Groups. (A) TQ vs. Control, (B) LPS/IFNγ vs. Control, (C) TQ + LPS/IFNγ vs. LPS/IFNγ.