The soluble guanylate cyclase stimulator IWP‐550 inhibits neuroinflammation in vitro and in vivo

Abstract

ObjectiveIWP‐550 is a brain‐penetrant soluble guanylate cyclase (sGC) stimulator. The objective of this work was to evaluate the effects of IWP‐550 on neuroinflammation in vitro in rat primary neurons, mouse microglial cells, rat brain 3D microtissues (MT, InSphero™), and in an in vivo quinolinic acid (QA)‐induced neuroinflammation rat model.MethodsMouse microglial SIM‐A9 cells, rat primary neurons and rat brain MT were stimulated with IWP‐550 for 30 min at 37°C. cGMP levels were measured by LC/MS. Phosphorylation of vasodilator‐stimulated phosphoprotein (VASP) and CREB was assayed using CisBio HTRF kits. For cytokine studies, cells or brain MT were treated with LPS and IWP‐550 for 24 h at 37°C. IL‐6 and TNFα levels in SIM‐A9 cells were determined by ELISA. Cytokines from isolated rat primary neurons and brain MT were measured by MSD multiplex assay. Neuroinflammation was induced in vivo by injecting QA in the rat striatum followed by treatment with IWP‐550 (10 mg/kg, p.o. QD) for 7 days. In the striatum, expression of ionized calcium‐binding adapter molecule 1 (Iba1), a marker of microglial activation, and glial fibrillary acidic protein (GFAP), a marker of astrogliosis, was quantified by IHC staining. Gene expression of TNFα, CD40, and MIP1α was determined by QuantiGene MultiPlex Assay (Affymetrix).ResultsIWP‐550 dose‐dependently stimulated cGMP formation (EC50 = 16 nM) and phosphorylation of CREB in isolated rat primary neurons. IWP‐550 also stimulated cGMP formation and phosphorylation of VASP in SIM‐A9 cells in synergy with the nitric oxide donor, DETA‐NONOate (DETA). In rat primary neurons, IWP‐550 decreased secreted proinflammatory cytokines, including IL‐1β, TNFα and MCP‐1 that were elevated by LPS treatment (1 μg/ml). In SIM‐A9 cells, IWP‐550 dose‐dependently decreased LPS‐mediated IL‐6 and TNFα elevation. In brain MT containing a mix of neurons, astrocytes, microglial cells and oligodendrocytes, IWP‐550 stimulated cGMP formation and phosphorylation of CREB. Moreover, IWP‐550 (1 and 10 μM) in the presence of DETA (10 μM) reduced LPS‐induced elevation of IL‐6, IFNγ, TNFα, IL‐1β, IL‐13 and CXCL1. In rat, QA administered unilaterally in the striatum caused local brain tissue injury and led to the activation of microglia and astrocytes, as demonstrated by increases in IHC staining for Iba1 and GFAP compared to the control side of striatum. Gene expression of pro‐inflammatory cytokines TNFα, CD40 and MIP1α were also increased following QA injection. Treatment with IWP‐550 (10 mg/kg) for 7 days significantly reduced Iba1 and GFAP staining, as well as gene expression of TNFα, CD40 and MIP1α, indicating a reduction of QA‐induced astrogliosis and inflammation in vivo.ConclusionIWP‐550 suppressed neuroinflammation in vitro in primary neurons, microglial cells, and brain microtissues, as well as in vivo in QA‐treated rats. These data suggest that sGC stimulation may provide potential neuroprotective effect by reducing neuroinflammation, a hallmark of neurodegenerative diseases.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.