RESUMO
Activated macrophages upregulate inducible nitric oxide synthase (iNOS) leading to the profuse production of nitric oxide (NO) and, eventually, tissue damage. Using macrophage NO production as a biochemical marker of inflammation, we tested different parts (flower, leaf, and stem) of the medicinal plant, Spilanthes acmella. We found that extracts prepared from all three parts, especially the flowers, suppressed NO production in RAW macrophages in response to interferon-γ and lipopolysaccharide. Follow up experiments with selected bioactive molecules from the plant (α-amyrin, ß-caryophylline, scopoletin, vanillic acid, trans-ferulic acid, and spilanthol) indicated that the N-alkamide, spilanthol, is responsible for the NO-suppressive effects and provides protection from NO-dependent cell death. Spilanthol reduced the expression of iNOS mRNA and protein and, as a possible underlying mechanism, inhibited the activation of several transcription factors (NFκB, ATF4, FOXO1, IRF1, ETS, and AP1) and sensitized cells to downregulation of Smad (TF array experiments). The iNOS inhibitory effect translated into an anti-inflammatory effect, as demonstrated in a phorbol 12-myristate 13-acetate-induced dermatitis and, to a smaller extent, in cerulein-induced pancreatitis. In summary, we demonstrate that spilanthol inhibits iNOS expression, NO production and suppresses inflammatory TFs. These events likely contribute to the observed anti-inflammatory actions of spilanthol in dermatitis and pancreatitis.
Assuntos
Dermatite/tratamento farmacológico , Dermatite/metabolismo , Macrófagos/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Pancreatite/tratamento farmacológico , Pancreatite/metabolismo , Alcamidas Poli-Insaturadas/uso terapêutico , Animais , Sobrevivência Celular/efeitos dos fármacos , Dermatite/genética , Proteína Forkhead Box O1/metabolismo , Camundongos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Pancreatite/genética , Peroxidase/metabolismo , Células RAW 264.7 , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
Differentiation between active and latent TB is a diagnostic challenge in TB-endemic regions. The commercially available IFN-γ-release assays are unsuitable for achieving this discrimination. We, therefore, screened ESAT-6 and CFP-10 proteins through population coverage analysis to identify minimal sets of peptides that can discriminate between these two forms of TB in a North Indian population. Comparing the diagnostic performance of a set of 2 ESAT-6 peptides (positions: 16-36; 59-79) to that of the QuantiFERON(®)-TB Gold IT (QFTGIT) assay, we observed significant difference in IFN-γ and TNF-α levels between patients (n = 15) and their age- and sex-matched healthy household contacts (n = 15). While the mean (±SD) IFNγ titer was 241.8 (±219.24) IU/ml for patients, the same in controls was 564.2 (±334.82) IU/ml (p = 0.039). Similarly the TNFα response was significantly higher in patients, compared to controls (796.47 ± 175.21 IU/ml vs. 481.81 ± 378.72 IU/ml; p = 0.047). IL-4 response to these peptides was non- discriminatory between the two groups. The QFTGIT Assay, however, elicited no significant difference in IFN-γ, TNF-α or IL-4 levels. Hence we conclude that IFN-γ or TNF-α response to these ESAT-6 peptides has the potential to differentiate between active and latent TB in our population.