The Inhibition of Inflammasome by Brazilian Propolis (EPP-AF)

Abstract

Propolis extracts have gained the attention of consumers and researchers due to their unique chemical compositions and functional properties such as its anti-inflammatory activity. Recently, it was described a complex that is also important in inflammatory processes, named inflammasome. The inflammasomes are a large molecular platform formed in the cell cytosol in response to stress signals, toxins, and microbial infections. Once activated, the inflammasome induces caspase-1, which in turn induces the processing of inflammatory cytokines such as IL-1 β and IL-18. So, to understand inflammasomes regulation becomes crucial to treat several disorders including autoinflammatory diseases. Since green propolis extracts are able to regulate inflammatory pathways, this work purpose was to investigate if this extract could also act on inflammasomes regulation. First, the extract was characterized and it demonstrated the presence of important compounds, especially Artepillin C. This extract was effective in reducing the IL-1 β secretion in mouse macrophages and this reduction was correlated with a decrease in activation of the protease caspase-1. Furthermore, we found that the extract at a concentration of 30 μ g/mL was not toxic to the cells even after a 18-hour treatment. Altogether, these data indicate that Brazilian green propolis (EPP-AF) extract has a role in regulating the inflammasomes.

Figures

Figure 1

Fingerprint analysis of propolis alcoholic extract (EPP-AF) with four years and half of shelf-life stability conditions. Chromatograms were plotted at 275 nm, using RP-HPLC equipment with C18 (shim-pack, CLC-ODS (M), 25 cm × 4.6) column and gradient elution with methanol and acidic water (pH = 2.7). Chromatographic profile includes the compounds: (1) caffeic acid (around 15 min), (2) p-coumaric acid (around 20 min), (3) trans-cinnamic acid (around 35-36 min), (4) aromadendrin (38 min), and (5) Artepillin C (around 61-62 min).

Figure 2

Fingerprint analysis of propolis standardized extract (EPP-AF) in comparison with enrichment sample with CAPE. (a) Presentation of propolis standardized extract (EPP-AF) with CAPE (100 μg) in the same chromatographic conditions used in Figure 1, (b) propolis standardized extract showing a possible candidate to CAPE (A). To check the signs with similar retentions time of CAPE, UV spectra of CAPE and the candidate present in (b) were shown, demonstrating that propolis used does not present CAPE.

Figure 3

Propolis standardized extract (EPP-AF) reduces the secretion of IL-1β by mouse macrophages. (a) BMDMs from C57BL/6 mice were stimulated with LPS, pretreated or not with different concentrations of propolis extract and treated with nigericin for 1 h. The cell supernatant was collected and levels of IL-1β were measured by ELISA assay. (b) Cytotoxicity assay of propolis. BMDMs from C57BL/6 mice were treated with 30, 100, or 300 ug/mL of propolis during 1 h (c), 3 h (d), 6 h (e), and 18 h (f) and cells were analyzed by fluorescence microscopy. Data show average ± standard deviation and an asterisk indicates a  P  value of <0.05.

Figure 4

Propolis standardized extract (EPP-AF) inhibits the NLRP3 inflammasome. (a) BMDMs, 5.0 × 105 macrophages were plated in 48-well plates, stimulated with LPS, pretreated or not with 30 ug/mL of propolis extract, and treated with nigericin for 1 h. The cells were stained for 1 h with FAM–YVAD–fluoromethylketone (FAM–YVAD–FMK) and analyzed by Flow Cytometer on a FACS-CantoII. 30.000 events were acquired. (b) BMDMs from C57BL/6 and Caspase-1−/− mice were stimulated with LPS, pretreated or not with 30 ug/mL of propolis extract, and treated with nigericin, Gramicidin (c), and ATP (d) for 1 h. The supernatant was collected and levels of IL-1β were measured by ELISA assay. Data show average ± standard deviation and an asterisk indicates a  P  value of <0.05.

Figure 5

Propolis standardized extract (EPP-AF) reduces the IL-1β secretion in macrophages infected with L. pneumophila. (a) BMDMs from C57BL/6 mice were stimulated with LPS, pretreated or not with 30, 100, or 300 μg/mL of propolis extract, and infected with L. pneumophila during 12 hours. The supernatant was collected and levels of IL-1β were measured by ELISA assay. (b) BMDMs from C57BL/6 mice were pretreated or not with 30, 100, or 300 μg/mL of propolis extract and infected with L. pneumophila. Cultures were infected with 2 × 106 bacteria and further incubated for 12 hours. After this period, the cells were lysate with deionized water and the bacteria were plated on CYE agar for CFU determination. Data show average ± standard deviation and an asterisk indicates a  P  value of <0.05.