Elsevier

Journal of Neuroimmunology

Volume 320, 15 July 2018, Pages 87-97
Journal of Neuroimmunology

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

https://doi.org/10.1016/j.jneuroim.2018.04.018Get rights and content

Highlights

  • Thymoquinone (TQ) treatment in the activated BV-2 microglial cells increased the expression of antioxidant and neuroprotective proteins.

  • Proteins increased include biliverdin reductase-A, 3-mercaptopyruvate sulfurtransferase, glutaredoxin-3, and mitochondrial lon protease.

  • TQ also reduced the expression of several inflammatory cytokines in activated BV-2 microglial cells.

  • TQ modulated the expression of genes involved in the NFkB signaling pathway, which play a key role in neuroinflammation.

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 (Csingle bondC motif) ligand 3 (CXCL3), chemokine (Csingle bondC) 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.

Section snippets

List of abbreviations

3-MST3 mercaptopyruvate sulfurtransferase
ADAlzheimer's disease
ANOVAanalysis of variance
ATPadenosine triphosphate
BVR-Abiliverdin reductase A
C3complement component 3
CCL5chemokine (Csingle bondC) motif ligand 5
CFBcomplement factor B
CNScentral nervous system
CXCL3chemokine (Csingle bondC motif) ligand 3
Grxglutaredoxin
GSHglutathione
IFNγinterferon gamma
IκBinhibitor of kappa B
ILinterleukin
LC-MS/MSliquid chromatography with tandem mass spectrometry
LONMlon protease, mitochondrial homolog
LPSlipopolysaccharide

Materials

High glucose Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 4 mM GlutaMAX™, penicillin-streptomycin (10,000 U/ml), interferon gamma recombinant mouse protein (IFNγ), and trypsin/EDTA (0.25%) with phenol red were purchased from Thermo Fisher Scientific. Heat-inactivated fetal bovine serum (FBS) was purchased from Atlanta Biologicals. TQ (99% purity), lipopolysaccharides from Escherichia coli (LPS), and the reagents and Microcon-30 kDa centrifugal filter units used in the sample

Results

Comparative quantitative proteomic analysis of LPS/IFNγ-activated BV-2 cells with and without TQ treatment revealed 35 differentially expressed proteins (>95% identification confidence). Amongst these differentially expressed proteins, TQ treatment (12.5 μM for 24 h) of the LPS/IFNγ-activated microglia compared to the untreated, activated microglia resulted in the increased expression of 4 neuroprotective proteins: glutaredoxin-3 (21 fold), biliverdin reductase A (15 fold), 3-mercaptopyruvate

Discussion

Neuroinflammation has been increasingly implicated in the onset and progression of multiple neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD) and Multiple Sclerosis (MS), despite their differing pathologies (Liu and Hong, 2003; Block and Hong, 2005; Gao and Hong, 2008; Chen et al., 2016; Kempuraj et al., 2016). Neuroinflammation is an innate, and initially, protective response mechanism in the brain, facilitated mostly by microglia and astrocytes producing a

Conclusion

Our findings are the first to show that TQ treatment in the activated BV-2 microglial cells increased the expression of antioxidant and neuroprotective proteins, biliverdin reductase-A, 3-mercaptopyruvate sulfurtransferase, glutaredoxin-3, and mitochondrial lon protease. TQ also reduced the expression of several inflammatory cytokines in the LPS/IFNγ activated BV-2 microglial cells. Furthermore, our studies showed TQ modulated the expression of genes involved in the NFκB signaling pathway,

Acknowledgments

This research was supported by NIH-National Institute on Minority Health and Health Disparity Grants G12 MD007582 and P20 MD 006738.

Conflict of interest

None declared.

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