UCH-L1和高良姜素对LPS诱导的炎症反应的研究

发布时间：2018-05-19 06:57

本文选题：UCH-L1 + THP-1　；参考：《广州医科大学》2017年硕士论文

【摘要】：背景:脓毒症在临床上的致死率极高,多由细菌感染引起。当内毒素脂多糖释放入血液,激活炎症反应,再通过MAPK和NF-κB途径逐级扩大,最后产生瀑布效应,从而引起组织器官功能的损害。关于脓毒症目前在临床上仍缺乏有效的治疗方法。基于对脓毒症发病机制的理解,我们试图从炎症反应方面寻找治疗脓毒症的新靶点。泛素蛋白酶体系统能调节蛋白降解,影响细胞内各种活动。泛素羟基末端水解酶L1(ubiquitin C-terminal hydrolase-L1,UCH-L1)作为去泛素蛋白酶体家族中的成员存在于人类许多组织结构中,包括神经细胞,内皮细胞,平滑肌细胞等。目前许多研究证实了UCH-L1的异常表达与某些疾病有着内在联系,诸如神经疾病、局部缺血、颅脑创伤、癌症及帕金森等。UCH-L1也被证实在肾炎、血管内皮细胞炎症方面发挥一定调节作用,但UCH-L1与脓毒症炎症反应之间的关系尚未明确,有待我们进一步研究。目的:本研究通过LPS诱导人急性单核细胞白血病细胞株(THP-1)构建脓毒症体外细胞炎症模型,探讨UCH-L1与LPS诱导THP-1细胞炎症反应之间的关系及其可能的机制。内容与方法:1.利用UCH-L1的阻滞剂LDN57444抑制THP-1细胞的活性,采用ELISA法检测LDN57444(10、20、30μM)对LPS(100ng/m L)刺激THP-1细胞6、12、24小时后,其释放炎症因子TNF-α、IL-1β和IL-6水平的影响。同时,利用MTS法检测LDN57444(10、20、30μM)对LPS(100ng/m L)刺激THP-1细胞6、12、24小时后细胞活力的影响。用Western blot法检测不同浓度LPS(0-200ng/m L)刺激THP-1后UCH-L1的表达。2.用Western blot法研究LDN57444(20μM)对LPS(100ng/m L)刺激THP-1细胞后MAPK通路中蛋白p-ERK、p-P38、p-JNK、ERK、P38、JNK的表达影响。用Western blot法探索NF-κB通路中胞核p65、胞浆p65、IκBα的表达影响。用免疫荧光共聚焦显微镜分析LDN57444(20μM)对LPS(100ng/m L)刺激THP-1细胞12小时后胞核、胞浆P65的表达。3.转染UCH-L1 si RNA(40n M)进入THP-1细胞48小时后,采用Western blot法检测UCH-L1的表达。转染si RNA后,再用LPS(100ng/m L)培养12小时后提取上清,利用ELISA法检测炎症因子TNF-α、IL-1β和IL-6的水平。用MTS法检测UCH-L1 si RNA对THP-1细胞活力的影响。4.采用Western blot法测UCH-L1 si RNA(40n M)对LPS诱导的THP-1中的MAPK通路中p-ERK、p-P38、P38、p-JNK、ERK、JNK的表达及NF-κB通路中胞核p65、胞浆p65、IκBα的表达。结果:1.诱导THP-1细胞的LPS浓度增加时,UCH-L1的表达上调。与LPS组相比,LDN57444+LPS组中THP-1细胞释放的炎症因子TNF-α、IL-1β、IL-6减少(p0.05)。LDN57444对细胞活力无明显影响(p0.05)。2.与LPS组相比,LDN57444+LPS组中THP-1细胞p-ERK、p-P38、p-JNK的表达稍减少,P38的表达上调(p0.05),ERK、JNK的表达无明显变化(p0.05)。Western blot中:与LPS组相比,LDN57444+LPS组中的胞核p65减少(p0.05),胞浆p65无明显变化(p0.05),IκBα在15、30分钟增加(p0.05)。共聚焦显微镜下:与LPS组相比,LDN57444+LPS组中的胞核p65有减少。3.与negative si RNA+LPS组相比,UCH-L1 si RNA+LPS组中释放炎症因子TNF-α、IL-1β和IL-6减少(p0.05)。UCH-L1 si RNA对THP-1细胞活力无明显影响(p0.05)。4.与negative si RNA组相比,UCH-L1 si RNA组中UCH-L1的表达明显减少(p0.05)。UCH-L1 si RNA组MAPK通路中p-ERK、p-P38、p-JNK的表达稍减少(p0.05),NF-κB通路中胞核p65的表达明显减少(p0.05),IκBα的表达增加(p0.05),ERK、P38、JNK、胞浆p65的表达无明显变化(p0.05)。结论:UCH-L1促进LPS诱导的THP-1细胞炎症反应,其机制与MAPK通路蛋白及P65核转位有关。背景:脓毒症是宿主对炎症反应失控引起的多器官功能障碍。脓毒症的全球发病人数、患病率、致死率都呈逐年上升的趋势,是目前ICU中致死率最高的疾病之一。其发生机制仍未完全阐明,但始终认为炎症反应的失控是其基本机制。炎症反应是通过炎症通路逐级扩大的过程,最后促使巨噬细胞释放出大量的炎症因子,包括TNF-α、IL-1β、IL-6、基质金属蛋白酶(matrix metalloproteinases,MMPs)等。这些炎症因子增加血管内皮细胞通透性,进一步加大炎症因子的释放,最终使内皮细胞损伤、缺氧、组织坏死。当LPS刺激炎症细胞后,激活了MAPK和NF-κB通路,促使许多炎症介质基因的合成及相应的炎症介质的释放。高良姜素(galangin)作为黄酮醇类化合物的一种,在缺血性损伤,抗病毒,抗肿瘤,镇痛,抗氧化,抗炎等方面都有大量的研究。我们的前期研究表明高良姜素对脂多糖(LPS)诱导的THP-1细胞炎症反应具有一定的保护作用,但高良姜素对LPS刺激巨噬细胞的炎症反应的研究很少。本研究扩大了高良姜素对炎症细胞的范围的探究,为高良姜素干预脓毒症的炎症反应提供更多依据。目的:本研究采用LPS刺激的巨噬细胞作为体外炎症模型,探索高良姜素是否能抑制该模型的炎症反应。内容与方法:1.用PMA(乙酰豆肉蔻佛波醇,Phorbol 12-myristate 13-acetate)诱导单核细胞(THP-1)成巨噬细胞。2.酶联免疫吸附(ELISA)法检测高良姜素对LPS诱导的巨噬细胞12、24小时后释放TNF-α、IL-1β、IL-6的水平。3.MTS法(实时荧光?MT细胞活性检测)检测LPS处理12、24小时后高良姜素对巨噬细胞活性的影响。同时采用Annexin V-FITC/PI双染流式细胞术检测LPS诱导巨噬细胞6小时后巨噬细胞凋亡率。4.酶联免疫吸附(ELISA)法检测高良姜素对LPS诱导的巨噬细胞12、24小时后TIMP-1的释放水平。用免疫印迹(Western blot)法检测LPS处理12小时后高良姜素对LPS诱导的巨噬细胞MMP-2、MMP-9蛋白水平的变化。结果:1.LPS的用药浓度为500ng/m L时,与LPS组相比,高良姜素+LPS组(2.5-20μM+500ng/m L)能有效抑制炎症因子TNF-α、IL-1β、IL-6的释放(P0.05)。2.高良姜素在0-20μmol/L范围内对LPS诱导的巨噬细胞活力无明显影响(P0.05)。高良姜素在10、20μM时对LPS诱导的巨噬细胞凋亡率无明显影响(P0.05)。3.与LPS组比,10、20μM的高良姜素能够抑制LPS诱导的巨噬细胞MMP-2的表达(P0.05),20μM的高良姜素能够抑制LPS诱导的巨噬细胞MMP-9的表达(P0.05),但对TIMP-1的表达无调节作用(P0.05)。结论:高良姜素能够抑制LPS诱导的巨噬细胞炎症反应,使得TNF-α、IL-1β、IL-6、MMP-2、MMP-9的表达下调。
[Abstract]:Background: sepsis is highly lethal in clinic and is caused by bacterial infection. When endotoxin lipopolysaccharide is released into the blood, it activates the inflammatory reaction, and then expands by MAPK and NF- kappa B. Finally, it produces the waterfall effect, which causes the damage of tissue and organ function. The clinical treatment of sepsis is still lacking in clinical treatment. Based on the understanding of the pathogenesis of sepsis, we try to find a new target for the treatment of sepsis from the inflammatory response. The ubiquitin proteasome system can regulate protein degradation and affect various activities in cells. The ubiquitin hydroxyl terminal hydrolase L1 (ubiquitin C-terminal hydrolase-L1, UCH-L1) is a member of the family of the ubiquitin proteasome. There are many human tissue structures, including nerve cells, endothelial cells, smooth muscle cells and so on. Many studies have confirmed that the abnormal expression of UCH-L1 has an inherent relationship with some diseases, such as nerve disease, local ischemia, craniocerebral trauma, cancer, and Parkinson, and.UCH-L1 are also confirmed in nephritis and inflammation of vascular endothelial cells. The relationship between UCH-L1 and the inflammatory response of sepsis is not clear. We need further study. Objective: to construct a human acute monocytic leukemia cell line (THP-1) to construct an inflammatory model of sepsis in vitro by LPS, and to explore the relationship between UCH-L1 and LPS induced THP-1 cell inflammation. The mechanism. Content and methods: 1. using the inhibitor LDN57444 of UCH-L1 to inhibit the activity of THP-1 cells, the effect of LDN57444 (10,20,30 M) on LPS (100ng/m L) stimulated THP-1 cell 6,12,24 hours after LPS (100ng/m L) was detected by ELISA method. L stimulated the cell viability after 6,12,24 hours in THP-1 cells. Western blot method was used to detect the expression of LPS (0-200ng/m L) stimulated by LPS (0-200ng/m L). The expression of p65, cytoplasmic p65, I kappa B alpha in NF- kappa B pathway was explored. The expression of LDN57444 (20 mu M) to LPS (100ng/m L) stimulated by LPS (100ng/m L) for 12 hours was analyzed by immunofluorescence confocal microscopy. After RNA, LPS (100ng/m L) was used to extract the supernatant after 12 hours, and the ELISA method was used to detect the level of TNF- alpha, IL-1 beta and IL-6. The expression of p65, cytoplasmic p65, I kappa B alpha in the NF- kappa B pathway. Results: 1. the expression of UCH-L1 increased when the LPS concentration increased in THP-1 cells. Compared with the LPS group, the inflammatory factor released by THP-1 cells in LDN57444+LPS group had no obvious effect on cell viability. The expression of THP-1 cells p-ERK, p-P38 and p-JNK decreased slightly in group PS, and the expression of P38 was up to up (P0.05), ERK and JNK had no obvious changes (P0.05).Western blot. Compared with the group of negative Si RNA+LPS in the group LDN57444+LPS, the release of inflammatory factors TNF- a in the UCH-L1 Si RNA+LPS group was less than that in the UCH-L1 Si RNA+LPS group. The expression of p-ERK, p-P38, p-JNK in the MAPK pathway of group RNA decreased slightly (P0.05), and the expression of p65 in the NF- kappa B pathway decreased significantly (P0.05), and I kappa B alpha expression increased. Background: sepsis is the multiple organ dysfunction caused by the host's runaway inflammatory response. The global incidence, morbidity and mortality of sepsis are increasing year by year. It is one of the most fatal diseases in ICU. The mechanism of its occurrence is still not fully elucidated, but it is believed that inflammation is the basic mechanism of the disease. The disease response is a gradual expansion of the inflammatory pathway, which eventually causes macrophages to release a large number of inflammatory factors, including TNF- alpha, IL-1 beta, IL-6, matrix metalloproteinase (matrix metalloproteinases, MMPs). These inflammatory factors increase the permeability of vascular endothelial cells, further increase the release of inflammatory factors, and eventually make endothelial cells Injury, hypoxia, tissue necrosis. When LPS stimulates the inflammatory cells, it activates the MAPK and NF- kappa B pathway, which promotes the synthesis of many inflammatory mediators and the release of the corresponding inflammatory mediators. As one of the flavonols, alpinin (galangin) has a large number of ischemic damage, antivirus, anti-tumor, analgesic, antioxidant, and anti-inflammatory. Our previous study showed that alpinin has a protective effect on the inflammatory response of THP-1 cells induced by lipopolysaccharide (LPS), but alpinin has few studies on the inflammation of macrophages stimulated by LPS. This study expanded the study of Takara Jiangso's model of inflammatory cells and the intervention of alpinin in sepsis. Objective: the purpose of this study was to explore whether alpinin could inhibit the inflammatory response of the model by using LPS stimulated macrophages as an in vitro model of inflammation. Content and methods: 1. using PMA (acetyl nutmeg, phorbol, Phorbol 12-myristate 13-acetate) induced.2. enzyme linked immunosorbent (E) macrophages in mononuclear cells (THP-1) (E). LISA method was used to detect the effect of alpinin on the release of TNF- alpha, IL-1 beta, IL-6 in 12,24 hours after 12,24 hours of LPS induced macrophages. The effect of alpinin on the activity of macrophages was detected after 12,24 hours of 12,24 hours of LPS treatment. Meanwhile, 6 small macrophages were detected by Annexin V-FITC/. .4. enzyme linked immunosorbent assay (ELISA) was used to detect the release level of TIMP-1 after 12,24 hours of LPS induced macrophages. The level of MMP-2 and MMP-9 protein of macrophages induced by LPS after LPS treatment was detected by immunoblotting (Western blot) method for 12 hours after LPS treatment. 500ng/m L, compared with the LPS group, the group of alpinin +LPS (2.5-20 mu L) could effectively inhibit the inflammation factor TNF- alpha, IL-1 beta, IL-6 release (P0.05).2. algomin had no obvious effect on the viability of macrophage induced by 0-20 micron. P0.05.3. and LPS group, 10,20 mu M can inhibit the expression of LPS induced macrophage MMP-2 expression (P0.05). 20 micron alpinin can inhibit MMP-9 expression of macrophage induced by LPS (P0.05), but it has no regulatory effect on the expression of macrophage. The expression of TNF-, IL-1, IL-6, MMP-2 and MMP-9 was downregulated.
【学位授予单位】：广州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R459.7

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