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

 产品说明：酒精性肝病（ALD）是美国肝脏移植的第二大原因，但乙醇诱导的肝损伤的机制仍然知之甚少。ALD的临床研究认识到肠功能障碍是ALD的关键因素。 疾病的发展慢性乙醇消耗诱导肠屏障的瞬时损失，这与循环微生物副产物如脂多糖（LPS）的显著增加相关。LPS的增加与Toll样受体4（TLR 4）的活化相关，TLR 4刺激炎性细胞因子从肠和肝驻留巨噬细胞释放，并导致肝实质功能障碍。钴原卟啉（CoPP）是血红素加氧酶-1（HO-1）的全球诱导剂，可抑制枯否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也诱导肝细胞和肠上皮中的HO-1。caco-2细胞的初步数据表明，上皮HO-1表达保护紧密连接。我们推测HO-1在肠上皮内的诱导通过在乙醇喂养期间保持肠紧密连接的功能来保护肝脏免受炎症和损伤。为了研究HO-1在维持肠屏障中的作用，将确定空肠、回肠、近端和远端科洛中紧密连接的定位。改良乳果糖-甘露醇试验将用于评估前肠、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诱导对内毒素移位的影响。为了确定上皮HO-1对肝脏保护的影响，将产生肠上皮的条件性HO-1敲除小鼠，并在慢性乙醇饮食期间用CoPP诱导HO-1。将产生肝细胞和单核细胞条件性HO-1敲除，以确定HO-1在肝实质中的影响。将在有或没有CoPP的条件下测定caco-2单层对乙醇的渗透性，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处理来确定。HO-1催化血红素降解为一氧化碳、胆绿素和分子铁，这与铁蛋白重链的上调有关。将使用药理学抑制剂、过表达质粒、shRNA和乱序对照确定这些产品对紧密连接定位和屏障通透性的影响。在这些实验期间，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。

 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.