The effect of a nitro-aspirine on apoptosis of neutrophil granulocytes

Abstract

Apoptosis of neutrophil granulocytes is a critical event in the resolution of inflammation. Neutrophils have a short lifespan which can be modulated by aspirin. In this work we studied the effect of a nitroaspirin (NCX4016) on apoptosis of inflammatory granulocytes. This aspirin analogue has been synthesized to reduce the side effects of aspirin in the gastrointestinal tract. Inflammatory granulocytes have been isolated from polyvinyl sponges implanted under the skin of Albino Oxford (AO) rats. Inflammatory cells that were isolated 20 hours later were about 95% neutrophil granulocytes. The cells were cultivated 24h with different concentrations of NCX4016 ranging from 0.25 μM to 500 μM. After that period, apoptosis of neutrophils was assessed by morphological criteria, as well as by flow cytometry (after staining the cells with propidium iodide). We found that NCX4016 at concentrations from 50 to 500 μM induced the apoptosis of rat inflammatory granulocytes in a dose-dependent manner. However, at concentrations from 1 μM to 10 μM it inhibited apoptosis. In conclusion, our results suggest that anti-inflammatory properties of the NO-aspirin are additionally potentiated by the proapoptotic effect on granulocytes, which could be a novel mechanism of their action.

Neutrofilni granulociti imaju najkraći životni vek među leukocitima, koji se može modulisati citokinima i farmakološkim agensima. Efekat nitro-aspirina na apoptozu inflamatornih granulocita do sada nije ispitivan. Zbog toga smo u ovom radu ispitivali efekat nitro-aspirina (NCX-4016) na apoptozu inflamatornih neutrofilnih granulocita pacova. Inflamatorni granulociti su izolovani iz polivinilskih sunđera potkožno implantiranih pacovima Albino Oxford (AO) soja. Inflamatorne ćelije koje su izolovane 20 sati kasnije, najvećim delom (više od 95 %) su bili neutrofilni granulociti. Ove ćelije su kultivisane 24 sata sa NCX-4016 u koncentracijama od 0,25 μM do 500 μM. Supernatanti kultura su sakupljani i korišćeni za merenje koncentracije NO. Ćelije su bojene propidijum jodidom i apoptoza je analizirana na protočnom citofluorimetru, kao i pomoću morfoloških kriterijuma. Ustanovljeno je da u koncentracijama od 1 μM do 10 μM nitro-aspirin inhibiše, a u koncentracijama višim od 50 μM indukuje apoptozu ovih ćelija, na dozno zavistan način. Nitro-aspirinom indukovana apoptoza je u pozitivnoj korelaciji sa smanjenom produkcijom NO.