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

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 (5-40µM) for 24 hours. ELISAs and western blotting were used to detect pro-inflammatory cytokines, nitric oxide, PGE2, iNOS, COX-2 and mPGES-1. BACE-1 activity and Aβ levels were measured with ELISA kits. Protein levels of targets in NF-κB and p38 MAPK signalling, as well as HO-1, NQO1 and Nrf2 were also measured with western blot. NF-κB binding to the DNA was investigated using EMSA. MTT, DNA fragmentation and 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, TNFα, and 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 p38MAPK 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. 3 KeywordsArtemether, neuroinflammation, BV2 microglia, HT22 hippocampal neurons, NF-κB, Nrf2 4 BackgroundIn the central nervous system (CNS), the microglia plays an important role in immune defence and tissue repair [1]. Under normal conditions, these cells provide surveillance whilst maintaining homeostasis in the brain [2]. In response to injury, harmful toxins, infection or inflammation, microglial cells become activated, secreting pro-inflammatory mediators such as nitric oxide (NO), prostaglandin E2 (PGE2), reactive oxygen/nitrogen species and pro-inflammatory cytokines including IL-6 and TNFα [3,4]. These pro-inflammatory mediators are mainly regulated by the transcription factor NF-κB [5]. NF-κB binds to the DNA and its transcriptional activity regulates several genes, which promote neuroinflammation. The p38 mitogenactivated protein kinase (p38 MAPK) signalling pathway also plays an important role in the expression and activity of pro-inflammatory cytokines in microglial cells [6][7][8].Excessive production o...

The p38-MAPK Pathway Is Involved in Neuroprotection of Artemisinin against H2O2-induced Apoptosis in PC12 Cells

“…Our present study here further supported the antioxidant effects of artemisinin and indicated the possibility that artemisinin administration might be a potential therapeutic strategy for the treatment of AD. Biologically, it has been found that artemisinin-mediated neuroprotection against neuronal apoptosis is regulated by several pathways including ERK1/2, p38MAPK and Nrf2/HO-1 survival/apoptotic signaling pathway [17,19,21,35]. Our results provided mechanistic evidences to support that artemisinin protected PC12 cells from H 2 O 2 -induced oxidative damage via p38MAPK activation.…”

“…In clinical relevance, artemisinin is among the most effective therapies for malaria with great safety and the preferred first-line treatment for the disease [15]. Beside these effects, previous studies including some from our group, have shown that artemisinin and its derivatives have neuroprotective effects suggesting that artemisinin might be potential candidate for AD therapy [16][17][18][19]. It has been recognized that artemisinin-mediated neuroprotection against neuronal apoptosis from various insults is regulated by ERK1/2 survival/apoptotic signaling pathway rather than the Akt pathway [16,18,19].…”

Artemisinin protects PC12 cells against β-amyloid-induced apoptosis through activation of the ERK1/2 signaling pathway

“…Firstly, artemether, a lipid-soluble derivative of artemisinin has been reported to possess anti-inflammatory properties by activated Nrf2 activity [16]. As we know, neuroinflammation played a major role in the formation of Aβ; besides, activated Nrf2 activity also shows benefits to AD in reports [42].…”

“…At present, artemisinins are among the most effective therapies for malaria with great safety and the preferred first-line treatment for the disease [15]. Except these effects, accumulated studies indicate that artemisinin and its derivatives have neuroprotective effect, therefore be potential candidates for treatment of AD [16], [17], [18]. Artemisinin easily penetrate the blood–brain barrier and we have previously showed that artemisinin was able to protect PC12 cells and cortical neurons from free radical/oxidative stress induced by hydrogen peroxide and sodium nitroprusside (SNP) [17], [19].…”

Agathisflavone isolated from Anacardium occidentale suppresses SIRT1‐mediated neuroinflammation in BV2 microglia and neurotoxicity in APPSwe‐transfected SH‐SY5Y cells

“…2.11 | NF-κB DNA binding assay NF-κB DNA binding assay was carried out as earlier described (Okorji, Velagapudi, El-Bakoush, Fiebich, & Olajide, 2016). DNA binding assay was carried on nuclear extracts using the TransAM NF-κB transcription factor EMSA kit (Activ Motif, Belgium), which employs a 96-well plate to which an oligonucleotide containing the NF-κB consensus site (5′GGGACTTTCC-3′) has been immobilized.…”