Sildenafil (Viagra®) prevents and restores LPS-induced inflammation in astrocytes

Abstract

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.

Anti-inflammatory effects induced by pharmaceutical substances on inflammatory active brain astrocytes—promising treatment of neuroinflammation

“…Different pharmaceutical combinations were shown to exert powerful anti-inflammatory effects on the astrocytes in culture. Sildenafil in ultralow concentration showed prominent effects on the ATP-evoked intracellular Ca 2+ release in accordance with our previous results [ 19 ]. The combination of sildenafil and vitamin D3 showed even better effects.…”

“…It has turned out through our experiments the necessity to have a critical number of microglia in the astroglial cultures to obtain reactive inflammatory astrocytes after incubation with LPS. The amount of microglia increased to 5–10% when the cultures were incubated with LPS (10 ng/ml) for 24 h. When the cultures were incubated first with LPS for 24 h followed by incubation with LPS and sildenafil (1 μM) [ 19 ] for another 24 h, the number of microglial cells decreased, less than 5% (Fig. 1 ).…”

“…An extremely low concentration of sildenafil, 1 μM, was found to be optimal for ATP-evoked intracellular Ca 2+ release [ 19 ]. For anti-inflammatory restoration back to a normal physiological level, the cells were first incubated with LPS for 24 h followed by LPS + sildenafil for further 24 h. The cells were cultivated in physiological or high glucose concentrations from start.…”

“…Sildenafil can also normalize endothelial function and has been proposed for pain therapy in humans and animals [ 18 ] due to its anti-inflammatory properties. Furthermore, our group has shown that sildenafil works as an anti-inflammatory substance in lipopolysaccharide (LPS)-induced inflammatory reactive astrocytes and we observed that ATP-evoked intracellular Ca 2+ release was prominently affected [ 19 ].…”

Sildenafil Can Affect Innate and Adaptive Immune System in Both Experimental Animals and Patients

“…Recent data published by de Santana Nunes et al [20] indicate direct effect of sildenafil on cells. Sildenafil treatment of astrocytes prevents and also restores effect of LPS on actin filaments.…”