The Effect of Alcohol on NADPH oxidase structure in pancreatic stellate cells

酒精对胰腺星状细胞NADPH氧化酶结构的影响

• 批准号: 7476472
• 负责人: KYM Francis FAULL
• 金额: $ 7.75万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-01 至 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7476472
• 关键词: Acute; Alcohol abuse; Alcohol consumption; Alcoholic Pancreatitis; Alcohols; Angiotensin II; Antibodies; Antioxidants; Arts; Buffers; Cell Proliferation; Cells; Cellular biology; Chemicals; Chronic; Cicatrix; Complex; Condition; Coupled; Data; Data Set; Databases; Development; Digestion; Dimensions; Disease; Environmental Risk Factor; Enzymes; Ethanol; Event; Extracellular Matrix Proteins; Fibrosis; Gel; Genetic; Genomics; Goals; Growth Factor; Hepatic Stellate Cell; Host Defense; Human; Individual; Inflammation; Inflammatory; Inflammatory Response; Injury; Invaded; Knowledge; Lead; Lipids; Liquid Chromatography; Liver; Liver Fibrosis; Malignant neoplasm of pancreas; Mass Spectrum Analysis; Mediating; Membrane; Messenger RNA; Metabolic; Methods; Molecular; Morbidity - disease rate; Multienzyme Complexes; NADP; Open Reading Frames; Oxidases; Oxidative Stress; Pancreas; Pancreatitis; Participant; Pathologic; Patients; Phagocytes; Platelet-Derived Growth Factor; Play; Process; Production; Protein Precursors; Proteins; Proteomics; Rate; Rattus; Reactive Oxygen Species; Recurrence; Regulation; Reporting; Research; Research Design; Risk; Role; Sentinel; Signal Transduction; Staging; Structure; System; Techniques; Testing; Therapeutic Agents; Therapeutic Intervention; Tissues; Translations; Vitamin A; acute pancreatitis; alcohol abuse therapy; alcohol effect; chronic pancreatitis; cytokine; design; mortality; neutrophil; pathogen; research study; response; stellate cell; tandem mass spectrometry; two-dimensional

DESCRIPTION (provided by applicant): Approximately 80% of chronic pancreatitis cases are associated with alcohol consumption. Analogous to liver fibrosis and hepatic stellate cells, there is increasing evidence for a key role in the activation of the pancreatic stellate cell in pancreatitis. As in the liver, the central gateway for this process may be NADPH oxidase. When the reactive oxygen species generated by NADPH oxidase overwhelm the innate anti-oxidant system in cells, then tissue injury can result. In susceptible individuals these injuries can lead to chronic activation of the inflammatory response and ultimately pancreatitis. Although much is known about the composition and activation of the NADPH oxidase system in neutrophils, little is known about the complex in other tissues. For example, this proposal contains the first report of the presence of components of this reactive oxygen species generating complex in activated pancreatic stellate cells. Current knowledge is limited by the available antibodies that target NADPH oxidase subunits, and mRNA experiments that show the presence of protein precursors. Because of these limitations and the importance of this system in alcoholic pancreatitis, we propose to identify the components of the NADPH oxidase complex from pancreatic stellate cells by state-of-the-art methods. The specific hypothesis we will be testing is that alcohol and growth factors alter the subunit composition of the NADPH oxidase system in pancreatic stellate cells, leading to a synergistic increase in the activity of the NADPH oxidase, an effect that mediates several pathologic responses in the pancreas. Pancreatic stellate cell cultures will be prepared from rat pancreas by established methods and treated with PDGF and ethanol, alone and in combination. Membrane fractions will be isolated from these cultures and analyzed by blue native two-dimensional gels. For the first dimension electrophoretic separation will be used with a non-denaturing buffer that enables separation of complexes. The second dimension utilizes SDS-PAGE denaturing conditions to separate the component parts of each complex isolated in the first dimension of the gel. In-gel tryptic digestion followed by microbore liquid chromatography coupled to tandem mass spectrometry with data dependant acquisition will be used to collect mass spectra and tandem mass spectra of the digest components. Data sets will be screened against human genomic databases of predicted or known open reading frame translations. Significant matches will be examined manually to confirm assignments. In this way components of the complex will be identified by mass and sequence information derived from in-gel digests. These data are essential for the successful development of targeted therapeutic agents for treatment of alcohol related pancreatitis. By far the most common cause of chronic pancreatitis is alcohol abuse, estimated to cause 70- 90% of the cases. The processes of chronic pancreatitis include inflammation and scarring with the loss of tissue. Of particular note, the processes of this condition continue even after cessation of alcohol intake, indicating that the processes are self-sustaining once a certain stage of the disease is reached. Adding to the morbidity and mortality of this disorder is the fact that patients with chronic pancreatitis are at a significantly increased risk for pancreatic cancer. Successful completion of the experiments described in this proposal are essential for the development of targeted therapeutic agents for treatment of alcohol related pancreatitis.

描述（由申请人提供）：大约80%的慢性胰腺炎病例与饮酒有关。与肝纤维化和肝星状细胞类似，越来越多的证据表明胰腺星状细胞的激活在胰腺炎中起关键作用。和肝脏一样，这个过程的中心通道可能是NADPH氧化酶。当NADPH氧化酶产生的活性氧压倒细胞内固有的抗氧化系统时，就会导致组织损伤。在易感个体中，这些损伤可导致炎症反应的慢性激活，最终导致胰腺炎。尽管人们对中性粒细胞中NADPH氧化酶系统的组成和激活了解甚多，但对其他组织中的NADPH氧化酶复合体知之甚少。例如，这一提议包含了在激活的胰腺星状细胞中产生这种活性氧复合物的成分的首次报告。目前的知识仅限于针对NADPH氧化酶亚基的抗体，以及显示蛋白质前体存在的mRNA实验。由于这些限制和该系统在酒精性胰腺炎中的重要性，我们建议用最先进的方法从胰腺星状细胞中鉴定NADPH氧化酶复合物的成分。我们将测试的具体假设是，酒精和生长因子改变了胰腺星状细胞中NADPH氧化酶系统的亚基组成，导致NADPH氧化酶活性的协同增加，这种效应介导了胰腺的几种病理反应。胰星状细胞培养将从大鼠胰腺中通过既定的方法制备，并用PDGF和乙醇单独或联合处理。膜组分将从这些培养物中分离出来，用蓝色原生二维凝胶进行分析。对于第一维电泳分离将使用非变性缓冲液，使复合物的分离。第二维利用SDS-PAGE变性条件分离凝胶第一维中分离的每个络合物的组成部分。凝胶内胰蛋白酶消化，然后微孔液相色谱耦合串联质谱与数据依赖采集将用于收集消化组分的质谱和串联质谱。数据集将根据预测或已知的开放阅读框翻译的人类基因组数据库进行筛选。重要的匹配将手工检查以确认分配。通过这种方法，复合物的组分将通过凝胶内酶切得到的质量和序列信息来识别。这些数据对于成功开发治疗酒精相关性胰腺炎的靶向治疗药物至关重要。到目前为止，慢性胰腺炎最常见的原因是酒精滥用，估计导致70- 90%的病例。慢性胰腺炎的过程包括炎症和瘢痕组织的损失。特别值得注意的是，这种情况的过程甚至在停止饮酒后仍在继续，这表明一旦达到疾病的某一阶段，这种过程是自我维持的。慢性胰腺炎患者患胰腺癌的风险显著增加，这一事实增加了该病的发病率和死亡率。成功完成本建议中描述的实验对于开发治疗酒精相关性胰腺炎的靶向治疗剂至关重要。