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

大量摄入芸苔类蔬菜与不同类型的癌症呈负相关，可能是由于其硫代葡萄糖苷和水解产物（如异硫氰酸酯）的含量。除了显示化学预防作用外，异硫氰酸酯还具有抗炎作用。众所周知，不同类型的异硫氰酸酯诱导氧化还原敏感转录因子核因子（红细胞衍生2）样2 （Nrf2）在基础条件下在细胞质中与其抑制剂蛋白Keap1结合。亲电化合物，如萝卜硫素，一种从西兰花中提取的异硫氰酸盐，从Nrf2- keap1复合体中释放Nrf2。因此，Nrf2易位到细胞核，与DNA上的抗氧化反应元件（ARE）结合，并启动其靶基因的表达，包括不同的II期酶和抗氧化酶。第一次Nrf2反应被称为“早期反应”，持续约4小时。4-8小时后，“晚期反应”发生，核Nrf2蛋白被不同的激酶磷酸化，导致其输出到细胞质和随后的蛋白酶体降解。一些出版物表明，次生植物代谢物也通过表观遗传靶点（如microRNAs）发挥作用。此外，最近的研究指出了microrna受表观遗传调控的概念。我们自己在RAW264.7巨噬细胞中的研究表明，烯丙基异硫氰酸酯（AITC）治疗可以剂量依赖性地下调脂多糖诱导的促炎microRNA-155。此外，我们观察到萝卜硫素的抗炎作用。在应用葡聚糖硫酸钠后发生急性结肠炎的C57BL/6小鼠中，与未处理的对照组相比，萝卜硫素预处理减轻了结肠炎症。本项目旨在阐明AITC抗炎作用的分子机制。因此，我们想利用RAW264.7小鼠单核细胞分析microRNA-155的抑制、对Nrf2早期或晚期反应的影响、转录因子Nrf2与NFkB之间的直接蛋白-蛋白相互作用或microRNA-155的表观遗传调控是否介导了AITC的抗炎作用。将进行不同的细胞和分子方法，包括实时PCR， western blotting，共免疫沉淀，甲基化分析，siRNA和microrna抑制实验以及硅分析。

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.