Anti-inflammatory effects of allyl-isothiocyanate - mechanistic studies on the interaction of microRNA-155 and Nrf2 in murine monocytes

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A high intake of brassica vegetables has been negatively associated with different kinds of cancers, possibly due to their content of glucosinolates and hydrolysis products (e. g. isothiocyanates). Besides showing chemopreventive effects, isothiocyanates also act anti-inflammatorily. It is well known that different types of isothiocyanates induce the redox-sensitive transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) which under basal conditions is bound to its inhibitor protein Keap1 in the cytoplasm. Electrophilic compounds like sulforaphane, an isothiocyanate derived from broccoli, liberate Nrf2 from the Nrf2-Keap1-complex. Consequently, Nrf2 translocates to the nucleus where it binds to the antioxidant responsive element (ARE) on the DNA and initiates expression of its target genes including different phase II and antioxidant enzymes. This first Nrf2 reaction is called 'early response' and lasts for about 4 hours. After 4-8 hours the 'late response' takes place, where the nuclear Nrf2 protein is phosphorylated by different kinases causing its export to the cytoplasm and subsequent proteasomal degradation. Several publications suggest that secondary plant metabolites exhibit their effects also via epigenetic targets such as microRNAs. Furthermore, recent studies point to the notion that microRNAs are regulated epigenetically. Our own studies in RAW264.7 macrophages have shown that allyl-isothiocyanate (AITC) treatment can dose-dependently downregulate lipopolysaccharide induced pro-inflammatory microRNA-155. Additionally, we observed anti-inflammatory effects of sulforaphane. In C57BL/6 mice that develop acute colitis after application of sodium dextransulfate, a pre-treatment with sulforaphane attenuated colonic inflammation in comparison to the untreated controls. The aim of the present project is to elucidate the molecular mechanism involved in the anti-inflammatory action of AITC. Therefore, we would like to use RAW264.7 murine monocytes to analyse if the inhibition of microRNA-155, the influence on the early or late Nrf2-response, a direct protein-protein-interaction between the transcription factors Nrf2 and NFkB or an epigenetic regulation of microRNA-155 mediate the anti-inflammatory effects of AITC. Different cellular and molecular methods including real time PCR, western blotting, co-immunoprecipitation, methylation analysis, siRNA and microRNA-inhibition experiments as well as in silico analyses will be conducted.