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Antimalarial Drug Artemether Inhibits Neuroinflammation in BV2 Microglia Through Nrf2-Dependent Mechanisms

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Abstract

Artemether, a lipid-soluble derivative of artemisinin has been reported to possess anti-inflammatory properties. In this study, we have investigated the molecular mechanisms involved in the inhibition of neuroinflammation by the drug. The effects of artemether on neuroinflammation-mediated HT22 neuronal toxicity were also investigated in a BV2 microglia/HT22 neuron co-culture. To investigate effects on neuroinflammation, we used LPS-stimulated BV2 microglia treated with artemether (5–40 μM) for 24 h. ELISAs and western blotting were used to detect pro-inflammatory cytokines, nitric oxide, prostaglandin E2 (PGE2), inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2 and microsomal prostaglandin E synthase-1 (mPGES-1). Beta-site amyloid precursor protein cleaving enzyme 1 (BACE-1) activity and Aβ levels were measured with ELISA kits. Protein levels of targets in nuclear factor kappa B (NF-κB) and p38 mitogen-activated protein kinase (MAPK) signalling, as well as heme oxygenase-1 (HO-1), NQO1 and nuclear factor-erythroid 2-related factor 2 (Nrf2) were also measured with western blot. NF-κB binding to the DNA was investigated using electrophoretic mobility shift assays (EMSA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), DNA fragmentation and reactive oxygen species (ROS) assays in BV2-HT22 neuronal co-culture were used to evaluate the effects of artemether on neuroinflammation-induced neuronal death. The role of Nrf2 in the anti-inflammatory activity of artemether was investigated in BV2 cells transfected with Nrf2 siRNA. Artemether significantly suppressed pro-inflammatory mediators (NO/iNOS, PGE2/COX-2/mPGES-1, tumour necrosis factor-alpha (TNFα) and interleukin (IL)-6); Aβ and BACE-1 in BV2 cells following LPS stimulation. These effects of artemether were shown to be mediated through inhibition of NF-κB and p38 MAPK signalling. Artemether produced increased levels of HO-1, NQO1 and GSH in BV2 microglia. The drug activated Nrf2 activity by increasing nuclear translocation of Nrf2 and its binding to antioxidant response elements in BV2 cells. Transfection of BV2 microglia with Nrf2 siRNA resulted in the loss of both anti-inflammatory and neuroprotective activities of artemether. We conclude that artemether induces Nrf2 expression and suggest that Nrf2 mediates the anti-inflammatory effect of artemether in BV2 microglia. Our results suggest that this drug has a therapeutic potential in neurodegenerative disorders.

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Abbreviations

AD:

Alzheimer’s disease

ANOVA:

Analysis of variance

BACE-1:

Beta-site amyloid precursor protein cleaving enzyme 1

CNS:

Central nervous system

COX:

Cyclooxygenase

DMSO:

Dimethyl sulfoxide

FBS:

Foetal bovine serum

IκB:

Inhibitor of kappa B

IL:

Interleukin

iNOS:

Inducible nitric oxide synthase

LPS:

Lipopolysaccharide

MAPK:

Mitogen-activated protein kinase

HO-1:

Heme oxygenase-1

NO:

Nitric oxide

Nrf2:

Nuclear factor-erythroid 2-related factor 2

NF-κB:

Nuclear factor kappa B

TNFα:

Tumour necrosis factor-alpha

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Acknowledgments

Uchechukwu P Okorji and Ravikanth Velagapudi are funded by a partial PhD scholarship from the University of Huddersfield. Abdelmeneim El-Bakoush was funded by a PhD scholarship from the Libyan Government. This study was partially funded by the University of Huddersfield (University Research Fund/International Networking Fund) awarded to Dr Olumayokun Olajide.

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    Authors and Affiliations

    1. Department of Pharmacy, School of Applied Sciences, University of Huddersfield, Queensgate, Huddersfield, West Yorkshire, HD1 3DH, UK

      Uchechukwu P. Okorji, Ravikanth Velagapudi, Abdelmeneim El-Bakoush & Olumayokun A. Olajide

    2. Neurochemistry Research Laboratory, Department of Psychiatry and Psychotherapy, University of Freiburg Medical School, Hauptstrasse 5, 79104, Freiburg, Germany

      Bernd L. Fiebich

    3. VivaCell Biotechnology GmbH, Ferdinand-Porsche-Str. 5, 79211, Denzlingen, Germany

      Bernd L. Fiebich

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    1. Uchechukwu P. Okorji
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    Correspondence to Olumayokun A. Olajide.

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    Uchechukwu P. Okorji and Ravikanth Velagapudi contributed equally to this work.

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    Okorji, U.P., Velagapudi, R., El-Bakoush, A. et al. Antimalarial Drug Artemether Inhibits Neuroinflammation in BV2 Microglia Through Nrf2-Dependent Mechanisms. Mol Neurobiol 53, 6426–6443 (2016). https://doi.org/10.1007/s12035-015-9543-1

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