Assessment of the effects of rosemary extract on mast cell-mediated allergic inflammation
The prevalence of allergic inflammatory disorders is increasing at an alarming rate, with 40-50% of school-aged children suffering today. Mast cells are immune sentinels and a driving force in both normal and pathological contexts of inflammation. Crosslinking of FcεRI by allergen-bound IgE antibodies leads to mast cell degranulation resulting in an early phase response, and the release of newly synthesized pro-inflammatory mediators, contributing to a late phase response. The mitogen-activated protein kinase (MAPK) family, phosphoinositide 3-kinase/protein kinase B (PI3K-Akt), and nuclear factor-κ-light-chain-enhancer of activated B cells (NF-κB) pathways have been established to be driving mechanisms behind mast cell-induced inflammation. Rosemary extract (RE) is rich in polyphenols and has been shown to inhibit the MAPK, PI3K-Akt, and NF-κB pathways in other cellular contexts in vitro and in in vivo. However, the effect of RE on mast cell activation has not been explored. Therefore, the aim of this study was to evaluate RE in modulating mast cell activation and FcεRI signaling via these pathways toward understanding the mechanism of action and functional outcomes. Mast cells were sensitized with anti-TNP IgE and were stimulated with the cognate allergen (TNP-BSA) under stem cell factor (SCF) potentiation and treated with 0 – 25 µg/ml RE. Samples were then collected for western blot analysis, quantitative polymerase chain reaction (qPCR), enzyme-linked immunosorbent assay (ELISA), β-hexosaminidase assay, and NFкB transcription factor activity assay. Western blot analysis demonstrated that RE treatment at both 5 and 25 µg/ml inhibited phosphorylation of p38-MAPK, and treatment with 25 µg/ml inhibited JNK. qPCR analysis showed that RE treatment at 25 µg/mL resulted in decreased gene expression of IL6, TNF, IL13, CCL1, and CCL3. It also reduced Rcan1, and NFкBIA mRNA levels. ELISA analysis further supported the qPCR data showing decreases in pro-inflammatory IL-6, TNF, IL-13, CCL1, and CCL3. The β-hexosaminidase assay demonstrated that RE treatment inhibited mast cell degranulation dose-dependently to a maximum (down to 15% of control) at 25 µg/mL RE. Finally, RE reduced NFкB activity. This work suggests that RE is capable of modulating mast cell functional outcomes, and warrants further investigation for use as a potential therapeutic.