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

异硫氰酸烯丙酯的抗炎作用——小鼠单核细胞中 microRNA-155 和 Nrf2 相互作用的机制研究

• 批准号: 233525292
• 负责人: Professorin Dr. Anika Eva Wagner
• 金额: --
• 依托单位: Professur für Ernährung und Immunsystem
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2013
• 资助国家: 德国
• 起止时间: 2012-12-31 至 2015-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/233525292?language=en
• 关键词: Anti; inflammatory; effects; allyl; isothiocyanate

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.

大量甘蓝蔬菜的摄入量与不同种类的癌症呈负相关，这可能是由于它们的葡萄糖醇和水解产物的含量（例如异硫氰酸酯）。除了表现出化学预防作用外，异硫氰酸盐还起作用。众所周知，不同类型的异硫氰酸盐会诱导氧化还原敏感的转录因子核因子（红细胞衍生的2）类似2（NRF2），这些2（NRF2）在基础条件下与其抑制剂蛋白Keap1结合在细胞质中。诸如Sulforaphane，一种源自西兰花，从NRF2-KEAP1-COMPERX衍生的异硫氰酸硫酸硫素的化合物。因此，NRF2转移到核与DNA上与抗氧化剂响应元件（IS）结合的细胞核，并启动其靶基因的表达，包括不同的II期和抗氧化剂酶。第一个NRF2反应称为“早期响应”，持续约4小时。 4-8小时后发生“晚期反应”，其中核NRF2蛋白被不同的激酶磷酸化，导致其导出到细胞质和随后的蛋白酶体降解。一些出版物表明，二级植物代谢产物也通过表观遗传靶标（例如microRNAS）表现出其作用。此外，最近的研究表明，microRNA受到表观遗传的调节。我们对RAW264.7巨噬细胞的研究表明，烯丙基异氰酸酯（AITC）处理可以依赖性地依赖性地下调脂多糖诱导的促炎性microRNA-155。此外，我们观察到磺胺的抗炎作用。在施用脱氧硫酸钠后发生急性结肠炎的C57BL/6小鼠中，与未处理的对照相比，磺胺硫烷的预处理减弱了结肠炎症。本项目的目的是阐明AITC抗炎作用所涉及的分子机制。因此，我们想使用RAW264.7鼠单核细胞来分析MicroRNA-155的抑制是对NRF2早期或晚期NRF2反应的影响，这是转录因子NRF2和NFKB和NFKB或NFKB或MicroRNA-155的表观遗传调节的直接蛋白质 - 蛋白质交流，对MicroRONNA-155 MECRORNA-155 MECRONENA-155 MEDIDIAD MEDIRES MEDIRETIAD ATFLAMS-ATFLAMS效应。将进行不同的细胞和分子方法，包括实时PCR，Western印迹，共免疫沉淀，甲基化分析，siRNA和microRNA抑制实验以及在计算机分析中。