The Intestinal Role of Heme Oxygenase-1 in Alcoholic Liver Disease

血红素加氧酶 1 在酒精性肝病中的肠道作用

• 批准号: 8983229
• 负责人: Damien Bellos
• 金额: $ 3.32万
• 依托单位: CLEVELAND CLINIC LERNER COM-CWRU
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-16 至 2018-07-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8983229
• 关键词: A Mouse; Affect; Alcohol abuse; Alcohol consumption; Alcoholic Liver Diseases; Alcoholic liver damage; American; Animal Model; Biliverdine; Biological Assay; Biological Preservation; Caco-2 Cells; Carbon Monoxide; Cell Death; Cessation of life; Chronic; Clinical Research; Cobalt; Colon; Data; Development; Diet; Disease Progression; Distal; Electrical Resistance; Endotoxins; Enzymes; Epithelial; Epithelial Cells; Ethanol; Ethanol Metabolism; Extravasation; Ferritin; Foundations; Functional disorder; Future; Goals; Heme; Hepatocyte; Hindgut; Human; Immune Cell Activation; Immunohistochemistry; Inflammation; Inflammatory; Intestines; Investigation; Iron; Knock-out; Knockout Mice; Kupffer Cells; Lactulose; Lead; Lipopolysaccharides; Liver; Liver parenchyma; Mannitol; Measurement; Modeling; Molecular; Morbidity - disease rate; Mus; Oxidative Stress; Pathology; Patients; Permeability; Physiological; Plasmids; Production; Proteins; Relative (related person); Research; Research Project Grants; Role; Signal Transduction; Spatial Distribution; Stress; Surface; Testing; Therapeutic Intervention; Tight Junctions; Tissues; United States; Up-Regulation; Work; alcohol exposure; alcohol response; cell type; chemokine; chronic alcohol ingestion; cobaltiprotoporphyrin; cytokine; feeding; fluorescein isothiocyanate dextran; heme oxygenase-1; ileum; inhibitor/antagonist; intestinal epithelium; jejunum; liver inflammation; liver injury; liver transplantation; macrophage; microbial; monocyte; monolayer; mortality; mouse model; overexpression; problem drinker; protective effect; public health relevance; research study; response; small hairpin RNA; toll-like receptor 4; villin

 DESCRIPTION: Alcoholic liver disease (ALD) is the second highest cause of liver transplants in the United States, yet the mechanisms that underlie ethanol-induced liver damage remain poorly understood. Clinical studies of ALD recognize intestinal dysfunction as a critical player in the progression of disease. Chronic ethanol consumption induces transient losses in the intestinal barrier, which correlate with significant increases in circulating microbial byproducts such as lipopolysaccharide (LPS). Increases in LPS are associated with activation of toll like receptor 4 (TLR4), which stimulates the release of inflammatory cytokines from intestinal and liver resident macrophages, and results in the dysfunction of the liver parenchyma. Cobalt protoporphyrin (CoPP), a global inducer of heme oxygenase-1 (HO-1), inhibits the production of inflammatory cytokines in Kupffer cells and significantly reduces markers of liver injury. However, CoPP also induces HO-1 in the hepatocytes and the intestinal epithelium. Preliminary data in caco-2 cells suggest that epithelial HO-1 expression protects tight junctions. We hypothesize that the induction of HO-1 within the intestinal epithelium protects the liver from inflammation and damage by preserving the function of the intestinal tight junctions during ethanol feeding. To investigate the role of HO-1 in maintaining the intestinal barrier, the localization of tight junctions will be determined in the jejunum, ileum, proximal, and distal colo. The modified lactulose-mannitol test will be used to assess permeability of the fore, mid, and hindgut. Barrier measurements will be compared with circulating endotoxin to determine the impact of global HO-1 induction on endotoxin translocation. To determine the impact of epithelial HO-1 on liver protection, conditional HO-1 knockout mice will be generated for the intestinal epithelium, and the HO-1 will be induced with CoPP during the chronic ethanol diet. Hepatocyte, and monocyte conditional HO-1 knockouts will be generated to determine the impact of HO-1 in the liver parenchyma. The permeability of caco-2 monolayers in response to ethanol will be determined under conditions with or without CoPP and compared with the localization and relative expression of tight junction proteins. The impact of HO-1 on tight junctions will be determined by treating with inhibitors of HO-1 expression or enzymatic activity. HO-1 catalyzes the degradation of heme into carbon monoxide, biliverdin, and molecular iron, which is associated with upregulation of ferritin heavy chain. The impact of these products on the localization of tight junctions and barrier permeability will be determined using pharmacologic inhibitors, overexpression plasmids, shRNA and scrambled controls. During these experiments, the transepithelial electrical resistance, immunohistochemistry and FITC-dextran permeability assays will be used to assess barrier function.

 描述：酒精性肝病 (ALD) 是美国肝移植的第二大原因，但酒精引起的肝损伤的机制仍知之甚少，ALD 的临床研究认为肠道功能障碍是导致肝移植的关键因素。 慢性乙醇消耗会导致肠道屏障的短暂损失，这与脂多糖 (LPS) 等循环微生物副产物的显着增加有关。LPS 的增加与 Toll 样受体 4 (TLR4) 的激活有关，从而刺激 TLR4。肠道和肝脏巨噬细胞释放炎症细胞因子，导致肝实质功能障碍，钴原卟啉（CoPP）是一种全局性的物质。血红素加氧酶-1 (HO-1) 诱导剂可抑制 Kupffer 细胞中炎症细胞因子的产生，并显着减少肝损伤标志物。然而，CoPP 还可诱导肝细胞和肠上皮细胞中的 HO-1。 2 细胞表明上皮 HO-1 表达可以保护紧密连接，我们勇敢地认为，肠上皮内 HO-1 的诱导可以通过保护肝脏免受炎症和损伤。乙醇喂养期间肠道紧密连接的功能 对于 HO-1 在维持肠道屏障中的作用，将确定空肠、回肠、近端和远端结肠中紧密连接的定位。用于评估前肠、中肠和后肠的渗透性，将屏障测量值与循环内毒素进行比较，以确定整体 HO-1 诱导对内毒素的影响。为了确定上皮 HO-1 对肝脏保护的影响，将产生肠上皮条件性 HO-1 敲除小鼠，并在长期乙醇饮食期间用 CoPP 诱导 HO-1。将产生 HO-1 敲除以确定 HO-1 对肝实质的影响 将在一定条件下确定 caco-2 单层对乙醇的渗透性。有或没有CoPP，并与紧密连接蛋白的定位和相对表达进行比较。通过用HO-1表达抑制剂处理或HO-1催化血红素降解来确定HO-1对紧密连接的影响。转化为一氧化碳、胆绿素和分子铁，这与铁蛋白重链的上调有关。这些产品对紧密连接定位和屏障通透性的影响将通过药理学来确定。抑制剂、过表达质粒、shRNA 和乱序对照 在这些实验中，将使用跨上皮电阻、免疫组织化学和 FITC-葡聚糖渗透性测定来评估屏障功能。