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高原因，但是基于乙醇引起的肝脏损伤的机制仍然很少了解。 ALD的临床研究识别肠道功能障碍是关键参与者 疾病的进展。慢性乙醇消耗会引起肠道屏障的瞬时损失，这与循环微生物副产品（如脂多糖）（LPS）的显着增加相关。 LPS的增加与受体4（TLR4）等收费的激活有关，这刺激了肠道和肝脏居民巨噬细胞释放炎症性细胞因子的释放，并导致肝实质功能障碍。血红素氧酶-1（HO-1）的全球诱导剂钴原晶酸酯（COPP）抑制库普弗细胞中炎性细胞因子的产生，并显着降低了肝损伤的标志物。但是，COPP还诱导HO-1在肝细胞和肠上皮中。 CACO-2细胞中的初步数据表明上皮HO-1表达可保护紧密连接。我们假设在肠上皮内HO-1的诱导可以通过保留乙醇进食过程中肠紧密连接的功能来保护肝脏免受炎症和损害。为了研究HO-1在维持肠道屏障中的作用，将在空肠，回肠，近端和盘状Colo中确定紧密连接的定位。修饰的乳糖甘露醇测试将用于评估前，中和后肠的渗透性。将将屏障测量与循环内毒素进行比较，以确定全球HO-1诱导对内毒素易位的影响。为了确定上皮HO-1对肝脏保护的影响，将为肠上皮产生有条件的HO-1敲除小鼠，并且在慢性乙醇饮食期间将用COPP诱导HO-1。将产生肝细胞和单核细胞有条件的HO-1敲除，以确定HO-1在肝实质中的影响。 Caco-2单层对乙醇的渗透性将在有或没有COPP的条件下确定，并与紧密连接蛋白的定位和相对表达进行比较。 HO-1对紧密连接的影响将通过用HO-1表达或酶活性的抑制剂治疗。 HO-1催化血红素降解为一氧化碳，双立菌素和分子铁，这与铁蛋白重链的上调有关。这些产品对使用药物抑制剂，过表达质粒，shRNA和扰流对照的对照器确定这些产品对紧密连接和屏障渗透率的定位的影响。在这些实验中，将使用旋转电阻，免疫组织化学和FITC-脱离渗透性评估来评估屏障功能。