Sildenafil (Viagra) protective effects on neuroinflammation: the role of iNOS/NO system in an inflammatory demyelination model

We recently demonstrated that sildenafil reduces the expression of cytokines, COX-2, and GFAP in a demyelinating model induced in wild-type (WT) mice. Herein, the understandings of the neuroprotective effect of sildenafil and the mediation of iNOS/NO system on inflammatory demyelination induced by cuprizone were investigated. The cerebella of iNOS(-/-) mice were examined after four weeks of treatment with cuprizone alone or combined with sildenafil. Cuprizone increased GFAP, Iba-1, TNF- α , COX-2, IL-1 β , and IFN- γ expression, decreased expression of glutathione S-transferase pi (GSTpi), and damaged myelin in iNOS(-/-) mice. Sildenafil reduced Iba-1, IFN- γ , and IL-1 β levels but had no effect on the expression of GFAP, TNF- α , and COX-2 compared to the cuprizone group. Sildenafil elevated GSTpi levels and improved the myelin structure/ultrastructure. iNOS(-/-) mice suffered from severe inflammation following treatment with cuprizone, while WT mice had milder inflammation, as found in the previous study. It is possible that inflammatory regulation through iNOS-feedback is absent in iNOS(-/-) mice, making them more susceptible to inflammation. Sildenafil has at least a partial anti-inflammatory effect through iNOS inhibition, as its effect on iNOS(-/-) mice was limited. Further studies are required to explain the underlying mechanism of the sildenafil effects.

Figures

Western blotting for GFAP. (a)…

Western blotting for GFAP. (a) GFAP immunoblot of iNOS −/− control (Cont), cuprizone…

Western blotting for GFAP. (a) GFAP immunoblot of iNOS −/− control (Cont), cuprizone (CPZ), and cuprizone plus sildenafil (CPZ + Sild) groups. (b) β-actin immunoblot. (c) Graph represents quantification and statistical analysis. The control group showed basal expression of GFAP. CPZ treatment induced a significant increase of this protein and sildenafil plus CPZ did not reduce GFAP expression, which remained higher in relation to the control group. The experiment was performed in triplicate (n = 5 animals/group). The results were expressed as mean ± SE. *P < 0.05, **P < 0.01 compared to control.

Immunofluorescence for GFAP. (a) and…

Immunofluorescence for GFAP. (a) and (d) show expression and physiological locations of GFAP…

Immunofluorescence for GFAP. (a) and (d) show expression and physiological locations of GFAP in mice without iNOS and without treatment. CPZ administration ((b), (e)) induced reactive gliosis, with thicker and more numerous astrocytic processes, compared to control. GFAP remained high in relation to control, after application of sildenafil plus CPZ ((c), (f)). Arrows show astrocytic processes and arrowheads point to processes around vessels and other cells. W: white matter, M: molecular layer, P: purkinje layer, G: granular layer, and v: vessel. Bars: 20 μm.

Western blotting for TNF- α…

Western blotting for TNF- α and COX-2. ((a), (c)) Immunoblots of iNOS −/−…

Western blotting for TNF-α and COX-2. ((a), (c)) Immunoblots of iNOS −/− control (Cont), cuprizone (CPZ), and cuprizone plus sildenafil (CPZ + Sild) groups. ((b), (d)) Graphs represent quantification and statistical analysis. The control group showed basal expression of TNF-α. CPZ treatment caused a significant increase of this cytokine, and sildenafil plus CPZ did not decrease its expression, which remained higher in relation to the control group. Only minimum amounts of COX-2 were present in the control group. CPZ and CPZ + Sild caused a significant increase of this enzyme, compared to control. The experiment was performed in triplicate (n = 5 animals/group). The results were expressed as mean ± SE. *P < 0.05, **P < 0.01 compared to control.

Immunohistochemistry for COX-2 ((a)–(c)) and…

Immunohistochemistry for COX-2 ((a)–(c)) and immunofluorescence for Iba-1 ((d)–(f)). iNOS −/− control (a)…

Immunohistochemistry for COX-2 ((a)–(c)) and immunofluorescence for Iba-1 ((d)–(f)). iNOS −/− control (a) showed very low COX-2 expression (arrow). After CPZ treatment (b), COX-2 labeling significantly increased, mainly in white matter, in relation to control. Animals treated with CPZ + Sild (c) also increased COX-2, comparing to the control group. A basal expression of Iba-1 was seen in control animals without iNOS (d). CPZ increased Iba-1 and induced an activated phenotype (arrowheads) of microglia (e). Sildenafil plus CPZ decreased Iba-1 and induced latent phenotype (arrows) of microglia (f).

Western blotting for IL-1 β…

Western blotting for IL-1 β and IFN- γ . ((a), (c)) Immunoblots of…

Western blotting for IL-1β and IFN-γ. ((a), (c)) Immunoblots of iNOS −/− control (Cont), cuprizone (CPZ), and cuprizone plus sildenafil (CPZ + Sild) groups. ((b), (d)) Graphs represent quantification and statistical analysis. The control group showed basal expression of IL-1β and IFN-γ. CPZ treatment induced a significant increase of these cytokines, compared to control. Sildenafil plus CPZ significantly decreased IL-1β and IFN-γ expression, in relation to the CPZ group. The experiment was performed in triplicate. The results were expressed as mean ± SE. **P < 0.01, ***P < 0.001, compared to control; ## P < 0.01, ### P < 0.001, compared with CPZ.

Western blotting for eNOS. (a)…

Western blotting for eNOS. (a) Immunoblot of wild-type mice control (WT cont), iNOS…

Western blotting for eNOS. (a) Immunoblot of wild-type mice control (WT cont), iNOS −/− mice control (iNOS −/− cont), iNOS −/− animals treated with cuprizone (iNOS −/− CPZ), and iNOS −/− animals treated with cuprizone plus sildenafil (iNOS −/− CPZ + Sild). (b) Graph represents quantification and statistical analysis. eNOS was physiologically expressed in WT mice. Animals without iNOS without treatment showed a significant increase of this enzyme, compared to WT control. After CPZ and CPZ plus sildenafil, iNOS −/− animals also showed a significant increase of eNOS, compared to WT animals, but no significant difference was identified between iNOS −/− control and treated animals. The experiment was performed in triplicate. The results were expressed as mean ± SE. *P < 0.05, ***P < 0.001, compared to control.

Western blotting for GSTpi. (a)…

Western blotting for GSTpi. (a) Immunoblot of iNOS −/− control (Cont), cuprizone (CPZ),…

Western blotting for GSTpi. (a) Immunoblot of iNOS −/− control (Cont), cuprizone (CPZ), and cuprizone plus sildenafil (CPZ + Sild) groups. (b) Graph represents quantification and statistical analysis. iNOS −/− control showed basal expression of GSTpi. After CPZ, GSTpi decreased significantly, compared to iNOS −/− cont. CPZ + Sild treatment increased GSTpi expression in relation to the CPZ group, but the levels of this protein remained significantly decreased compared to control. The experiment was performed in triplicate. ***P < 0.001 compared to control; ### P < 0.001 compared to CPZ.

Luxol Fast Blue (LFB) staining…

Luxol Fast Blue (LFB) staining ((a)–(c)) and electron micrographs ((d)–(i)). (a), (d), and…

Luxol Fast Blue (LFB) staining ((a)–(c)) and electron micrographs ((d)–(i)). (a), (d), and (g) represent iNOS −/− control group; (b), (e), and (h) represent CPZ-treated animals; (c), (f), and (i) represent CPZ + Sild-treated animals. Arrows in (a), (c): vacuoles in the white matter; arrowhead in (b): spaces between fibers; arrows in (d), (e), (g), and (h): damaged myelin sheath; arrowheads in (f), (i): preserved myelin. Bars = 20 μm ((a)–(c)); 2 μm ((d)–(f)); 0.5 μm ((g)–(i)).

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