EGCG通过Notch调节炎症的分子机制

Inflammatory may play decisive roles at different stages of the development of the diabetes, tumor and cardiovascular disease. The (-)-Epigallocatechin gallate(EGCG), contains in the green tea, can attenuate the inflammatory responses effectively. The foreign reseaechers had reported the 67KD laminin receptor(LR) is the target of EGCG for the effect of inflammation reducing in mice macrophage model. In our experiments performed on the human macrophage model for the inflammation response, the inflammatory attenuating effect of EGCG didn't decrease after the LR blocked with the antibody which has been declared effectively in the LR blocking. These facts suggested there have unknown new inflammatory reducing receptor on the cell membrane for the EGCG except the 67LR. Recent years,Notch signaling pathway had been found playing essential role in the regulation of inflammation response.The ongoing experiments of this project have found: 1. The Notch signal can be inhibited rapidly by treat the cells with EGCG; 2. The effects of the attenuating inflammation by the EGCG was erased if the direct effective transcripe factor for the Notch signal pathway, CSL, was decreased by the siRNA. Basis on these fact, this project will design and perform experiments below on the human inflammation response macrophage model: 1. To clear the mechanisms of the Notch inhibited by the EGCG; 2. To reveal the function difference between 4 subtypes of Notch in the processing of inflammation reducing effects of EGCG. The achievements of this project will clarify the Notch related molecular mechanism in inflammation attenuate of EGCG, also provide evidences that prevent the inflammation related disease through the tea drinking. This project will show evidence for the interaction and the mechanisms between the human body and the compounds contains in the food.

炎症是糖尿病、肿瘤、心血管疾病等多种疾病的诱发或促进因素。绿茶中的EGCG有降低炎症反应的效果。国外报告EGCG在小鼠巨噬细胞模型上通过层粘连蛋白受体LR发挥抑制炎症效应。Notch信号途径也参与炎症反应的调节。前期研究发现：1.在人巨噬细胞炎症模型中封闭LR对EGCG的炎症抑制效果没有影响；2.EGCG能够快速抑制Notch信号；3.RNA干扰降低Notch的直接效应转录因子CSL的表达能够消除EGCG的炎症抑制效应。在此基础上，本项目拟以人巨噬细胞炎症反应模型为研究对象，研究下列内容：1.EGCG抑制Notch信号的作用方式；2.Notch亚型在EGCG抑制炎症反应中的作用差异。本项目的研究成果可以阐明EGCG抑制Notch进而抑制炎症反应的分子机理。研究结果将为人们通过科学地饮茶进行炎症相关疾病的防治提供依据，也有助于人们深入了解细胞、机体与食物成份的相互作用及其机制。

EGCG具有调节炎症的作用，但对其直接调节炎症的机制还知之甚少。本项目采用THP-1细胞经佛波酯处理48小时后形成的巨噬细胞为炎症细胞，用脂多糖（LPS）处理建立炎症反应模型，考察在极短时间内EGCG对炎症调节的作用机制。获得了如下结果：1.EGCG在早期即可显著降低LPS 激发的炎症反应。且EGCG处理早期的炎症调节效应不依赖于67LR。2. EGCG在30-90分钟内没有降低LPS引起的NF-κB磷酸化。MAPK中介分子ERK、p38的磷酸化在30分钟没有显著变化；p38在60分钟、90分钟有明显升高。EGCG降低炎症效应可能不是通过NF-κB、MAPK。3.EGCG处理细胞后的30-90分钟，Notch1-2发生了显著的降解，Notch3由于表达量低于检测限没有观测到变化，Notch4则没有发生明显的改变。EGCG对Notch的作用不依赖于LPS。EGCG处理能够在极短时间内导致细胞膜表面成熟Notch的大幅减少,细胞核内Notch活化产物NICD快速降解，表现出以关闭Notch信号为主的效应。EGCG对Notch的效应呈剂量依赖性，并在短至2分钟的时间内即可观察到，对Notch靶基因的表达也呈现出类似效应，且不依赖于67LR。4.通过RNA干扰的方法将THP-1细胞中Notch1、Notch2沉默，以沉默后的THP-1／siNotch1、THP-1／siNotch2诱导分化成的巨噬细胞为模型，研究Notch对EGCG调节炎症效应的必要性。EGCG处理没有显著降低LPS 激发的炎症因子分泌，表明Notch1/2对于EGCG调节炎症效应是必要的，且Notch1被干扰对EGCG效应影响大，Notch2影响小。5.以人外周血单核细胞分化形成人原代巨噬细胞，观察EGCG对炎症的效应及对Notch的作用，得到与传代细胞系源巨噬细胞类似的结果。6.分子互作实验证实EGCG与Notch胞外段对于Notch活化至关重要的负调节区域能够以中等亲和力结合，EGCG与Notch可以直接发生作用。7.EGCG可以通过抑制Notch信号途径减弱糖尿病小鼠皮肤伤口炎症反应，促进糖尿病小鼠伤口的愈合。完成了全部研发任务，在成果形成过程中进行了适当扩展，项目研发中发现的深层次科学问题进行了研究，获得了国家基金的进一步研究资助。项目研发成果发表SCI文章7篇，其中影响因子大于5的文章2篇。