Signaling pathways involved in anti-inflammatory effects of Pulsed Electromagnetic Field in microglial cells.

Literature studies suggest important protective effects of low-frequency, low-energy pulsed electromagnetic fields (PEMFs) on inflammatory pathways affecting joint and cerebral diseases. However, it is not clear on which bases they affect neuroprotection and the mechanism responsible is yet unknown. Therefore the aim of this study was to identify the molecular targets of PEMFs anti-neuroinflammatory action. The effects of PEMF exposure in cytokine production by lipopolysaccharide (LPS)-activated N9 microglial cells as well as the pathways involved, including adenylyl cyclase (AC), phospholipase C (PLC), protein kinase C epsilon (PKC-ε) and delta (PKC-δ), p38, ERK1/2, JNK1/2 mitogen activated protein kinases (MAPK), Akt and caspase 1, were investigated. In addition, the ability of PEMFs to modulate ROS generation, cell invasion and phagocytosis, was addressed. PEMFs reduced the LPS-increased production of TNF-α and IL-1ß in N9 cells, through a pathway involving JNK1/2. Furthermore, they decreased the LPS-induced release of IL-6, by a mechanism not dependent on AC, PLC, PKC-ε, PKC-δ, p38, ERK1/2, JNK1/2, Akt and caspase 1. Importantly, a significant effect of PEMFs in the reduction of crucial cell functions specific of microglia like ROS generation, cell invasion and phagocytosis was found. PEMFs inhibit neuroinflammation in N9 cells through a mechanism involving, at least in part, the activation of JNK MAPK signalling pathway and may be relevant to treat a variety of diseases characterized by neuroinflammation.

Pulsed electromagnetic field and relief of hypoxia-induced neuronal cell death: The signaling pathway.

Low-energy low-frequency pulsed electromagnetic fields (PEMFs) exert several protective effects, such as the regulation of kinases, transcription factors as well as cell viability in both central and peripheral biological systems. However, it is not clear on which bases they affect neuroprotection and the mechanism responsible is yet unknown. In this study, we have characterized in nerve growth factor-differentiated pheochromocytoma PC12 cells injured with hypoxia: (i) the effects of PEMF exposure on cell vitality; (ii) the protective pathways activated by PEMFs to relief neuronal cell death, including adenylyl cyclase, phospholipase C, protein kinase C epsilon and delta, p38, ERK1/2, JNK1/2 mitogen-activated protein kinases, Akt and caspase-3; (iii) the regulation by PEMFs of prosurvival heat-shock proteins of 70 (HSP70), cAMP response element-binding protein (CREB), brain-derived neurotrophic factor (BDNF), and Bcl-2 family proteins. The results obtained in this study show a protective effect of PEMFs that are able to reduce neuronal cell death induced by hypoxia by modulating p38, HSP70, CREB, BDNF, and Bcl-2 family proteins. Specifically, we found a rapid activation (30 min) of p38 kinase cascade, which in turns enrolles HSP70 survival chaperone molecule, resulting in a significant CREB phosphorylation increase (24 hr). In this cascade, later (48 hr), BDNF and the antiapoptotic pathway regulated by the Bcl-2 family of proteins are recruited by PEMFs to enhance neuronal survival. This study paves the way to elucidate the mechanisms triggered by PEMFs to act as a new neuroprotective approach to treat cerebral ischemia by reducing neuronal cell death.

Synthesis, Pharmacological Evaluation, and Docking Studies of Novel Pyridazinone-Based Cannabinoid Receptor Type†2 Ligands.

In recent years, cannabinoid type 2 receptors (CB2 R) have emerged as promising therapeutic targets in a wide variety of diseases. Selective ligands of CB2 R are devoid of the psychoactive effects typically observed for CB1 R ligands. Based on our recent studies on a class of pyridazinone 4-carboxamides, further structural modifications of the pyridazinone core were made to better investigate the structure-activity relationships for this promising scaffold with the aim to develop potent CB2 R ligands. In binding assays, two of the new synthesized compounds [6-(3,4-dichlorophenyl)-2-(4-fluorobenzyl)-cis-N-(4-methylcyclohexyl)-3-oxo-2,3-dihydropyridazine-4-carboxamide (2) and 6-(4-chloro-3-methylphenyl)-cis-N-(4-methylcyclohexyl)-3-oxo-2-pentyl-2,3-dihydropyridazine-4-carboxamide (22)] showed high CB2 R affinity, with Ki values of 2.1 and 1.6 nm, respectively. In addition, functional assays of these compounds and other new active related derivatives revealed their pharmacological profiles as CB2 R inverse agonists. Compound 22 displayed the highest CB2 R selectivity and potency, presenting a favorable in silico pharmacokinetic profile. Furthermore, a molecular modeling study revealed how 22 produces inverse agonism through blocking the movement of the toggle-switch residue, W6.48.

Inhibition of A2A Adenosine Receptor Signaling in Cancer Cells Proliferation by the Novel Antagonist TP455.

Several evidences indicate that the ubiquitous nucleoside adenosine, acting through A1, A2A, A2B, and A3 receptor (AR) subtypes, plays crucial roles in tumor development. Adenosine has contrasting effects on cell proliferation depending on the engagement of different receptor subtypes in various tumors. The involvement of A2AARs in human A375 melanoma, as well as in human A549 lung and rat MRMT1 breast carcinoma proliferation has been evaluated in view of the availability of a novel A2AAR antagonist, with high affinity and selectivity, named as 2-(2-furanyl)-N5-(2-methoxybenzyl)[1,3]thiazolo[5,4-d]pyrimidine-5,7-diammine (TP455). Specifically, the signaling pathways triggered in the cancer cells of different origin and the antagonist effect of TP455 were investigated. The A2AAR protein expression was evaluated through receptor binding assays. Furthermore, the effect of A2AAR activation on cell proliferation at 24, 48 and 72 hours was studied. The selective A2AAR agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride (CGS21680), concentration-dependently induced cell proliferation in A375, A549, and MRMT1 cancer cells and the effect was potently antagonized by the A2AAR antagonist TP455, as well as by the reference A2AAR blocker 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM241385). As for the signaling pathway recruited in this response we demonstrated that, by using the specific inhibitors of signal transduction pathways, the effect of A2AAR stimulation was induced through phospholipase C (PLC) and protein kinase C-delta (PKC-δ). In addition, we evaluated, through the AlphaScreen SureFire phospho(p) protein assay, the kinases enrolled by A2AAR to stimulate cell proliferation and we found the involvement of protein kinase B (AKT), extracellular regulated kinases (ERK1/2), and c-Jun N-terminal kinases (JNKs). Indeed, we demonstrated that the CGS21680 stimulatory effect on kinases was strongly reduced in the presence of the new potent compound TP455, as well as by ZM241385, confirming the role of the A2AAR. In conclusion, the A2AAR activation stimulates proliferation of A375, A549, and MRMT1 cancer cells and importantly TP455 reveals its capability to counteract this effect, suggesting selective A2AAR antagonists as potential new therapeutics.

A2B adenosine receptors stimulate IL-6 production in primary murine microglia through p38 MAPK kinase pathway.

The hallmark of neuroinflammation is the activation of microglia, the immunocompetent cells of the CNS, releasing a number of proinflammatory mediators implicated in the pathogenesis of neuronal diseases. Adenosine is an ubiquitous autacoid regulating several microglia functions through four receptor subtypes named A1, A2A, A2B and A3 (ARs), that represent good targets to suppress inflammation occurring in CNS. Here we investigated the potential role of ARs in the modulation of IL-6 secretion and cell proliferation in primary microglial cells. The A2BAR agonist 2-[[6-Amino-3,5-dicyano-4-[4-(cyclopropylmethoxy)phenyl]-2-pyridinyl]thio]-acetamide (BAY60-6583) stimulated IL-6 increase under normoxia and hypoxia, in a dose- and time-dependent way. In cells incubated with the blockers of phospholipase C (PLC), protein kinase C epsilon (PKC-ε) and PKC delta (PKC-δ) the IL-6 increase due to A2BAR activation was strongly reduced, whilst it was not affected by the inhibitor of adenylyl cyclase (AC). Investigation of cellular signalling involved in the A2BAR effect revealed that only the inhibitor of p38 mitogen activated protein kinase (MAPK) was able to block the agonist's effect on IL-6 secretion, whilst inhibitors of pERK1/2, JNK1/2 MAPKs and Akt were not. Stimulation of p38 by BAY60-6583 was A2BAR-dependent, through a pathway affecting PLC, PKC-ε and PKC-δ but not AC, in both normoxia and hypoxia. Finally, BAY60-6583 increased microglial cell proliferation involving A2BAR, PLC, PKC-ε, PKC-δ and p38 signalling. In conclusion, A2BARs activation increased IL-6 secretion and cell proliferation in murine primary microglial cells, through PLC, PKC-ε, PKC-δ and p38 pathways, thus suggesting their involvement in microglial activation and neuroinflammation.

Synthesis, molecular modeling and SAR study of novel pyrazolo[5,1-f][1,6]naphthyridines as CB2 receptor antagonists/inverse agonists.

Pyrazolo[5,1-f][1,6]naphthyridine-carboxamide derivatives were synthesized and evaluated for the affinity at CB1 and CB2 receptors. Based on the AgOTf and proline-cocatalyzed multicomponent methodology, the ethyl 5-(p-tolyl)pyrazolo[5,1-f][1,6]naphthyridine-2-carboxylate (12) and ethyl 5-(2,4-dichlorophenyl)pyrazolo[5,1-f][1,6]naphthyridine-2-carboxylate (13) intermediates were synthesized from the appropriate o-alkynylaldehydes, p-toluenesulfonyl hydrazide and ethyl pyruvate. Most of the novel compounds feature a p-tolyl (8a-i) or a 2,4-dichlorophenyl (8j) motif at the C5-position of the tricyclic pyrazolo[5,1-f][1,6]naphthyridine scaffold. Structural variation on the carboxamide moiety at the C2-position includes basic monocyclic, terpenoid and adamantine-based amines. Among these derivatives, compound 8h (N-adamant-1-yl-5-(p-tolyl)pyrazolo[5,1-f][1,6]naphthyridine-2-carboxamide) exhibited the highest CB2 receptor affinity (Ki=33nM) and a high degree of selectivity (KiCB1/KiCB2=173:1), whereas a similar trend in the near nM range was seen for the bornyl analogue (compound 8f, Ki=53nM) and the myrtanyl derivative 8j (Ki=67nM). Effects of 8h, 8f and 8j on forskolin-stimulated cAMP levels were determined, showing antagonist/inverse agonist properties for such compounds. Docking studies conducted for these derivatives and the reference antagonist/inverse agonist compound 4 (SR144528) disclosed the specific pattern of interactions probably related to the pyrazolo[5,1-f][1,6]naphthyridine scaffold as CB2 inverse agonists.

The activation of µ-opioid receptor potentiates LPS-induced NF-kB promoting an inflammatory phenotype in microglia.

Increased production of proinflammatory cytokines has a prominent role in tolerance to opioids. The objectives of this study were to examine whether µ-opioid receptor affects proinflammatory signalling through the activation of NF-kB in microglia. The novelty of the described research is that a low dose of morphine, exerting its effects via the µ-opioid receptor, increases the DNA-binding activity of NF-kB via PKCε, while a high dose of morphine triggers a nonopiate receptor response mediated by TLR4 and, interestingly, PKCε signalling. The identification of morphine as a crucial upstream regulator of PKCε-NF-κB signalling in microglia argues for a central role of these pathways in neuroinflammation development and progression. Therefore, the morphine-PKCε-NF-κB pathway may provide novel targets to induce neuroprotective mechanisms, thereby reducing tolerance to opioids.

A2a and a2b adenosine receptors affect HIF-1α signaling in activated primary microglial cells.

Microglia are central nervous system (CNS)-resident immune cells, that play a crucial role in neuroinflammation. Hypoxia-inducible factor-1 (HIF-1), the main transcription factor of hypoxia-inducible genes, is also involved in the immune response, being regulated in normoxia by inflammatory mediators. Adenosine is an ubiquitous nucleoside that has an influence on many immune properties of microglia through interaction with four receptor subtypes. The aim of this study was to investigate whether adenosine may affect microglia functions by acting on HIF-1α modulation. Primary murine microglia were activated with lipopolysaccharide (LPS) with or without adenosine, adenosine receptor agonists and antagonists and HIF-1α accumulation and downstream genes regulation were determined. Adenosine increased LPS-induced HIF-1α accumulation leading to an increase in HIF-1α target genes involved in cell metabolism [glucose transporter-1 (GLUT-1)] and pathogens killing [inducible nitric-oxide synthase (iNOS)] but did not induce HIF-1α dependent genes related to angiogenesis [vascular endothelial growth factor (VEGF)] and inflammation [tumor necrosis factor-α (TNF-α)]. The stimulatory effect of adenosine on HIF-1α and its target genes was essentially exerted by activation of A2A through p44/42 and A2B subtypes via p38 mitogen-activated protein kinases (MAPKs) and Akt phosphorylation. Furthermore the nucleoside raised VEGF and decreased TNF-α levels, by activating A2B subtypes. In conclusion adenosine increases GLUT-1 and iNOS gene expression in a HIF-1α-dependent way, through A2A and A2B receptors, suggesting their role in the regulation of microglial cells function following injury. However, inhibition of TNF-α adds an important anti-inflammatory effect only for the A2B subtype. GLIA 2015;63:1933-1952.