RESUMEN
The arachidonic acid (AA) metabolic pathway, plays a vital role in the production of eicosanoids by the action of pro-inflammatory secretory phospholipase A2 (PLA2 ). Release of eicosanoids is known to be involved in many inflammatory diseases. Identification of the inhibitory molecules of this AA pathway enzyme along with the regulation of intracellular signaling cascades may be a finer choice to develop as a powerful anti-inflammatory drug. In this regard, we have screened few cell-permeable antioxidant molecules Tempo, Mito-TEMPO, N,N'-Bis(salicylideneamino)ethane-manganese(II) (EUK)-134, and EUK-8 against pro-inflammatory sPLA2 s. Among these, we found EUK-8 is a potent inhibitor with its IC50 value ranges 0.7-2.0 µM for sPLA2 s isolated from different sources. Furthermore, docking studies confirm the strong binding of EUK-8 towards sPLA2 . In vivo effect of EUK-8 was studied in HSF-sPLA2 -induced edema in mouse paw model. In addition to neutralizing the edema, EUK-8 significantly reduces the phosphorylation level of inflammatory proteins such as p38 member of MAPK pathway, Akt, and p65 along with the suppression of pro-inflammatory cytokine (interleukin-6) and chemokine (CXCL1) in edematous tissue. This shows that EUK-8 not only inhibits the sPLA2 activity, it also plays an important role in the regulation of sPLA2 -induced cell signaling cascades. Apart from the sPLA2 inhibition, we also examine the regulatory actions of EUK-8 with other downstream enzymes of AA pathway such as 5-LOX assay in human polymorphonuclear leukocytes (PMNs) and COX-2 expression in carrageenan-λ induced paw edema. Here EUK-8 significantly inhibits 5-LOX enzyme activity and downregulates COX-2 expression. These data indicate that EUK-8 found to be a promising multitargeted inhibitory molecule toward inflammatory pathway. In conclusion, mitochondrial targeted antioxidant EUK-8 is not only the powerful antioxidant, also a potent anti-inflammatory molecule and may be a choice of molecule for pharmacological applications.
Asunto(s)
Fosfolipasas A2 Secretoras , Ratones , Humanos , Animales , Fosfolipasas A2 Secretoras/efectos adversos , Fosfolipasas A2 Secretoras/metabolismo , Antioxidantes/uso terapéutico , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ciclooxigenasa 2/metabolismo , Regulación hacia Abajo , Antiinflamatorios/uso terapéutico , Edema/inducido químicamenteRESUMEN
The role of basophils in an anaphylactic response is well recognized but is usually masked by mast cells, which contain similar mediators for the induction of generalized vasodilatation and laryngeal constriction. The rapid onset of systemic anaphylactic symptoms, particularly in insect stings and ingested food, suggest that basophils, a circulating pool of cells containing histamine and other potent mediators such as leukotrienes, may be more involved in systemic anaphylaxis than originally thought. We wished to examine if secretory phospholipase A2, a systemic allergen found in honey bee venom (HBV-sPLA2) may activate basophils directly leading to rapid systemic mediator release. Basophils were isolated from human blood and stimulated with increasing concentrations of HBV-sPLA2. We found that physiological concentrations of HBV-sPLA2 induce rapid leukotriene C4 production from purified human basophils within 5 min, while interleukin (IL)-4 expression and production was induced at later time-points. Histamine release was not induced, signifying that HBV-sPLA2 did not induce generalized degranulation. Surface expression of CD63, CD69 and CD11b were up-regulated following HBV-sPLA2 treatment. Stimulation of basophils with anti-immunoglobulin E (IgE) following treatment with HBV-sPLA2 did not induce more leukotriene release. To investigate the mechanism of leukotriene production, 9-12 octadecadiynioc acid, a cyclooxygenase-1 (COX-1) and 15-lipoxygenase inhibitor, was used and this abrogated leukotriene production. These results indicate that HBV-sPLA2 can directly activate human basophils in vitro to induce leukotriene production.