EGCG通过Notch信号通路抑制早期炎症的研究

发布时间：2018-01-07 04:18

本文关键词：EGCG通过Notch信号通路抑制早期炎症的研究　出处：《吉林大学》2017年博士论文　论文类型：学位论文

【摘要】：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,如何调控炎症反应逐渐成为干预这些疾病的重要关注点。炎症反应的机理已有广泛的研究,其中,NF-κB是炎症反应中关键的调节因子,MAPK信号通路在炎症的发生、发展过程中发挥着重要作用。近年的研究表明,在机体对LPS刺激产生的炎症反应过程中,本底水平的Notch活化信号对于炎症反应的信号放大是必要的,抑制Notch的活化能够有效降低炎症反应的强度。茶叶中的表没食子儿茶素没食子酸酯(EGCG)有降低炎症反应的效果,其调控炎症反应的机理已有较多的研究。然而,这些研究集中在EGCG处理6h以上的细胞生理反应上,细胞在6h或更长时间的过程中经过了多轮信号传导,且在此过程中,细胞因子的释放以及反馈调节已经发生。因此,现有的研究结果可能是EGCG与细胞转导通路的中间蛋白的相互作用,而EGCG处理的直接或最原始的机理仍不清楚。为了探究EGCG调节炎症反应的初始作用点和作用机制,我们以人急性单核细胞白血病细胞THP-1来源的人巨噬细胞为实验模型,研究EGCG即时处理对LPS诱导的早期炎症的影响。在THP-1来源的人巨噬细胞上,我们研究了EGCG即时处理对LPS诱导的早期炎症因子分泌的影响,通过Elisa和人炎症因子芯片检测巨噬细胞上清中炎症因子的分泌情况。结果表明,EGCG即时处理可以显著抑制LPS诱导的早期炎症因子分泌。现有研究认为,EGCG通过下调NF-κB,MAPK(p38,Erk,JNK)磷酸化水平来抑制LPS诱导的炎症反应。为了探究该机理是否适用于EGCG即时处理抑制LPS诱导的早期炎症。我们使用EGCG或LPS处理THP-1来源的巨噬细胞,通过Western Blot检测不同处理间NF-κB,MAPK磷酸化水平。我们发现EGCG即时处理不能下调NF-κB,MAPK磷酸化,说明EGCG即时处理抑制LPS诱导的早期炎症,不是通过NF-κB,MAPK信号通路所介导的。Notch信号通路在调节炎症反应过程中起到重要的作用。通过EGCG处理,我们发现EGCG可以显著抑制Notch蛋白的表达且这种效果具有瞬时性和浓度依赖性。蛋白酶体抑制剂MG132处理可以抑制EGCG诱导的Notch蛋白的降解,说明EGCG通过蛋白酶体途径降解Notch蛋白。67LR是EGCG在小鼠细胞膜上的受体,为了探究EGCG即时处理抑制炎症反应与67LR的关系,我们使用67LR封闭性抗体(与文献中使用的抗体、使用浓度、使用方法完全一致)阻断EGCG与67LR的结合,通过Elisa和人炎症因子芯片检测LPS诱导的炎症因子分泌情况。在THP-1来源的人巨噬细胞上,我们发现EGCG即时处理降低炎症因子分泌的效应并没有消失,甚至没有降低,说明EGCG即时处理抑制LPS诱导的炎症反应不依赖于67LR。同时这些结果提示我们EGCG在细胞膜上除67LR外,还有新的作用靶点。为了验证EGCG即时处理调节炎症反应是通过Notch信号通路所介导的必要性。我们通过RNA干扰将THP-1细胞中Notch1/2蛋白表达沉默,将其分化成巨噬细胞,通过人炎症因子芯片进行研究,发现EGCG即时处理抑制LPS诱导的炎症效应消失或显著减弱。这些结果证实了,在炎症发生的早期阶段,EGCG通过Notch信号通路调节炎症反应。THP-1细胞是人急性单核细胞白血病细胞,在调节炎症反应中,THP-1细胞可能存在着与正常细胞不同的作用机理。我们从健康人外周血中分离出单核细胞(PBMC),将其分化成巨噬细胞并进行研究,发现EGCG抑制LPS诱导的早期炎症和抑制Notch蛋白的表达在健康人来源的巨噬细胞上同样适用。综上所述,本论文阐明了表没食子儿茶素没食子酸酯(EGCG)通过Notch信号通路抑制早期炎症反应,为更深入地了解EGCG抑制炎症的作用机理提供依据。研究结果,对于人们饮茶和服用富含EGCG的茶产品提供科学指导,将为人们通过科学地饮茶进行炎症相关疾病的防治提供理论依据,同时也有助于人们以茶作为中药材的类似物,为传统中药的现代科学研究提供经验。
[Abstract]:Inflammation is diabetes, cancer, cardiovascular disease and other diseases or promoting factors, how to regulate inflammation has gradually become an important focus of intervention for these diseases. The study has extensive inflammatory reaction mechanism, NF- kappa B is a critical regulator of the inflammatory response in MAPK pathway in inflammation, play an important role in the process of development. Recent studies suggest that, in the process of inflammatory reaction in LPS induced, the level of activation of Notch signal for signal amplification of the inflammatory response is necessary, activation can effectively reduce the intensity of the inflammatory response. The inhibition of Notch in tea epigallocatechin gallate (EGCG) reduce inflammation effect, to study the regulation of inflammatory reaction mechanism more. However, these studies focused on the physiology of cells treated with EGCG above 6h reaction, cell In the process of 6h or longer after many rounds of signal transduction, and in the process, the release of cytokines and feedback regulation has occurred. Therefore, the existing research results may be the interaction among protein EGCG and cell transduction pathway, while EGCG directly or the original mechanism is not clear. In order to initial action point and to explore the mechanism of EGCG regulating inflammatory reaction, we used the human acute monocytic leukemia cell THP-1 from human macrophages as experimental model of EGCG instant effect on early inflammation induced by LPS in THP-1. The source of human macrophages, we investigated the EGCG effects on LPS induced immediate treatment the early inflammatory factor secretion, through the secretion of inflammatory cytokines Elisa and inflammatory cytokines in the supernatant of macrophage chip detection. The results showed that EGCG treatment can significantly inhibit the instant The early inflammatory factor secretion system induced by LPS. The existing studies suggest that EGCG downregulation by NF- kappa B, MAPK (p38, Erk, JNK) to inhibit the phosphorylation of LPS induced inflammation. In order to explore the mechanism of EGCG is suitable for real-time processing of early inflammation induced suppression of LPS. We use EGCG or LPS treated macrophages THP-1 the source, through the Western Blot detection among different treatments NF- kappa B, phosphorylation of MAPK. We found that EGCG treatment can not immediately down-regulation of NF- kappa B, MAPK phosphorylation, EGCG real-time early inflammation induced suppression of LPS, not by NF- kappa B,.Notch signaling pathway mediated by MAPK signaling pathway play an important the role in regulating the inflammatory reaction process. By EGCG, we found that EGCG can significantly inhibit the expression of Notch protein and this effect is transient and concentration dependent manner. The proteasome inhibitor MG132 can inhibit EG The degradation induced by CG Notch protein, EGCG Notch protein degradation via the proteasome pathway.67LR is EGCG in mouse receptors on the cell membrane, in order to explore the relationship between EGCG of immediate treatment inhibits the inflammatory reaction and 67LR, we use 67LR blocking antibody (anti body literature use concentration, using the identical method) blocking the combination of EGCG and 67LR, through the secretion of inflammatory cytokines Elisa and inflammatory factors induced by LPS chip in THP-1. The source of human macrophages, we found that EGCG treatment decreased the effect of immediate secretion of inflammatory cytokines did not disappear, even not reduced, EGCG real-time processing and inhibit the inflammatory reaction induced by LPS is not dependent on 67LR. at the same time, these results suggest that EGCG in the cell membrane in addition to 67LR, as well as new targets. In order to verify the EGCG real-time regulation of the inflammatory response through Notch signaling The necessity of road mediated by RNA interference. We will silence the expression of Notch1/2 protein in THP-1 cells to differentiate into macrophages, through the research of human inflammatory factor EGCG chip, found that immediate treatment inhibited LPS induced inflammatory effects disappeared or decreased. These results confirmed that in the early stage of inflammation, EGCG regulation of inflammation in.THP-1 cells through Notch signaling pathway is a human acute monocytic leukemia cell, in the regulation of the inflammatory response in THP-1 cells may have different mechanisms and normal cells. We isolated mononuclear cells from healthy human peripheral blood (PBMC) and its differentiation into macrophages and study, found that EGCG inhibited LPS induced early inflammation and inhibit the expression of Notch is also applicable in the sources of healthy macrophages. In summary, this paper illustrates the catechins epigallocatechin no food Sub acid (EGCG) inhibits the inflammatory reaction by Notch signal pathway, and provide the basis for a deeper understanding of the mechanism of EGCG inhibiting inflammation. Research results provide scientific guidance for people taking EGCG rich tea and tea products, will provide theoretical basis for the prevention and treatment for people through scientific drinking tea in inflammation related diseases, at the same time it also helps people with tea as analogues of Chinese medicinal materials, provide experience for modern scientific research of traditional Chinese medicine.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R364.5

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