Search

Astrocytes are effectively involved in the pathophysiological processes in the central nervous system (CNS) and may contribute to or protect against development of inflammatory and degenerative diseases. Sildenafil is a potent and selective phosphodiesterase-5 (PDE-5) inhibitor, which induces cyclic GMP accumulation. However, the mechanisms of actions on glial cells are not clear. The aim of the present work is to evaluate the role of sildenafil in lipopolysaccharide (LPS)-stimulated astrocytes. The cytoskeleton integrity and Ca(2+) waves were assessed as indicators of inflammatory state. Two main groups were done: (A) one prevention and (B) one restoration. Each of these groups: A1: control; A2: LPS for 24h; A3: sildenafil 1 or 10μM for 4h and then sildenafil 1 or 10μM+LPS for 24h. B1: control; B2: LPS for 24h; B3: LPS for 24h and then LPS+sildenafil 1 or 10μM for 24h. Cytoskeleton integrity was analyzed through GFAP immunolabeling and actin labeling with an Alexa 488-conjugated phalloidin probe. Calcium responses were assessed through a Ca(2+)-sensitive fluorophore Fura-2/AM. The results show that both preventive and restorative treatments with sildenafil (in both concentrations) reduced the Ca(2+) responses in intensity and induced a more organized actin fiber pattern, compared to LPS treated cells. This work demonstrated for the first time that astrocytes are a key part of the sildenafil protective effects in the CNS., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

While it has recently been shown that sildenafil (Viagra®) has a protective effect on myelination/remyelination, the mechanism of this protection is still unknown. In general, cytokines, chemokines and metalloproteinases have a pro-inflammatory action, but can also exert a role in modulating glial cell activation, contributing to the balance of cell response. Investigating these molecules can contribute to clarifying the mechanisms of sildenafil neuroprotection. In addition, it is not known whether sildenafil is able to restore an already installed neurodegenerative process or if the treatment period is critical for its action. The aim of the present study was to evaluate, in a cuprizone (CPZ)-induced demyelination model, the effects and mechanisms of time-dependent treatment with sildenafil (beginning 15 days after neurodegeneration and continuing for 15 days, or starting concomitantly with neurodegeneration and continuing for 30 days) on neuroinflammation and remyelination. Neuroinflammation and demyelination induced by CPZ in rodents has been widely used as a model of multiple sclerosis (MS). In the present study, five male C57BL/6 mice aged 7-10 weeks were used per group. For four weeks, the groups received either cuprizone (CPZ) 0.2% mixed in feed or CPZ combined with the administration of sildenafil (Viagra®, Pfizer, 25 mg/kg) orally in drinking water, starting concurrently with (sild-T0) or 15 days (sild-T15) after the start of CPZ treatment. Control animals received pure food and water. The cerebella were dissected and processed for immunohistochemistry, immunofluorescence (frozen), Western blotting, Luxol fast blue staining and transmission electron microscopy. Magnetic resonance was performed for live animals, after the same treatment, using CPZ 0.3%. CPZ induced an increase in the expression of IL-1β and a decrease in MCP-1, CCR-2, MBP and GST-pi, as well as promoting damage in the structure and ultra-structure of the myelin sheath. Interestingly, the administering of sild-T0 promoted a further increase of MMP-9, MCP-1, and CCR-2, possibly contributing to changes in the microglia phenotype, which becomes more phagocytic, cleaning myelin debris. It was also observed that, after sild-T0 treatment, the expression of GST-pi and MBP increased and the myelin structure was improved. However, sild-T15 was not efficient in all aspects, probably due to the short treatment period and to starting after the installation of the degenerative process. Therefore, the present study shows that sildenafil modulates inflammation, with the involvement of MMP-9, MCP-1, and CCR-2, and also contributes to myelin repair. These protective effects were dependent on the therapeutic strategy used. This clarification can strengthen research proposals into the mechanism of action of sildenafil and contribute to the control of neurodegenerative diseases such as MS., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Sildenafil (Viagra®) has recently been found to have a neuroprotective effect, which occurs through the inhibition of inflammation and demyelination in the cerebellum. However, the mechanism of action of sildenafil remains unknown. AMPK, the regulatory protein of the lipid and glucose metabolism, plays a protective role by activating the eNOS enzyme. The production of a nanomolar concentration of NO by eNOS has an anti-inflammatory effect through the cGMP signaling pathway and plays an important role in the regulation of the nuclear transcription factor (NFkB), preventing the expression of inflammatory genes. The present study investigated whether AMPK-eNOS-NO-cGMP-IКβα-NFkB is involved in the mechanism of action of sildenafil in a cuprizone-demyelination model. Neuroinflammation and demyelination induced by cuprizone in rodents have been widely used as a model of MS. In the present study, five male C57BL/6 mice (7-10 weeks old) were used. Over a four week period, the groups received: cuprizone (CPZ) 0.2% mixed in feed; CPZ in the diet, combined with the administration of sildenafil (Viagra®, Pfizer, 25mg/kg) orally in drinking water, starting concurrently (sild-T0) or 15 days (sild-T15) after the start of CPZ. Control animals received pure food and water. The cerebella of the mice were dissected and processed for immunohistochemistry, immunofluorescence (frozen), western blotting and dosage of cytokines (Elisa). CPZ induced an increase in the expression of GFAP, IL-1β TNF-α, total NFkB and inactive AMPK, and prompt microglia activation. CPZ also induced a reduction of IKβα. The administration of sildenafil reduced the expression of the pro-inflammatory cytokines IL-1β and TNF-α and increased the expression of the anti-inflammatory cytokine IL-10. In addition, the administration of sildenafil reduced expression of GFAP, NFkB, inactive AMPK and iNOS, and increased IKβα. Interestingly, sildenafil also reduced levels of NGF. In general, the sild-T0 group was more effective than sild-T15 in improving clinical status and promoting the control of neuroinflammation. The present study offers evidence that sildenafil has anti-inflammatory and neuroprotective effects, which are probably achieved through modulation of AMPK-IKβα-NFκB signaling. In addition, eNOS may play a role in the sildenafil neuroprotective mechanism, contributing to the activation of AMPK. However, other pathways such as MAPK-NFkB and the downstream proteins AMPK (AMPK-SIRT1-NFκB) should also be further investigated. An understanding of these mechanisms of action is critical for the clinical use of sildenafil to control neuroinflammation in neurodegenerative diseases such as MS., (Copyright © 2015 Elsevier B.V. All rights reserved.)