Mechanism of Endotoxin absorption in alcoholism

描述(申请人提供)：来自临床和实验研究的证据表明，肠道对内毒素的通透性增加以及由此引起的内毒素血症在酒精性肝病的发病机制中起着至关重要的作用。到目前为止，我们进行的研究表明，乙醇的代谢产物乙醛破坏了肠道上皮屏障功能，并增加了对内毒素的通透性。这种乙醛诱导的上皮屏障功能破坏的机制包括抑制蛋白酪氨酸磷酸酶PTP1B，连接蛋白的酪氨酸磷酸化，破坏连接蛋白之间的相互作用(决定屏障功能)，以及失去连接复合体的完整性。此外，我们的研究表明，表皮生长因子和L-谷氨酰胺通过PLC？、PKC？、PKC？I和钙依赖机制来抑制乙醛介导的内毒素通透性增加。我们的初步研究表明，乙醛诱导PP2A转位，导致occludin和claudin-4去磷酸化，乙醇通过Src激酶和MLCK依赖机制放大乙醛的作用，而益生菌，植物乳杆菌阻止乙醛诱导的屏障破坏。在这些结果的基础上，进一步假设：a)乙醇代谢和肠道菌群在乙醇诱导的肠屏障功能障碍中起关键作用，b)依赖PP2A的occludin和cldn-4的去磷酸化参与乙醛诱导的肠屏障功能的破坏，c)乙醇通过c-Src介导的MLCK激活来协同乙醛诱导的屏障破坏，以及d)植物益生菌通过EGF受体、p38MAPK和rac1依赖的机制来阻止乙醇和乙醛引起的屏障功能的破坏。利用肠上皮细胞培养模型和人结肠活检组织，我们将确定：1)ADH1B和ALDH2调节乙醇诱导的TJs和屏障功能的破坏。2)ALDH2基因缺陷小鼠对乙醇诱导的屏障功能障碍更为敏感。3)肠道菌群在乙醇代谢和乙醇对TJs的破坏中起一定作用。4)乙醛诱导的PP2A甲基化和易位导致TJ蛋白去磷酸化和屏障功能的破坏。5)特异性丝氨酸和苏氨酸残基上的occludin和cldn-4去磷酸化与乙醛诱导的TJs破坏和屏障功能障碍有关。6)PP2A易位在乙醛诱导的小鼠肠道TJ断裂中起一定作用。7)乙醇介导的c-Src激活与乙醛诱导的TJ破坏有协同作用。8)MLCK介导乙醇对乙醛诱导的TJ破坏的协同作用。9)乙醇通过c-Src和MLCK依赖机制使小鼠结肠对乙醛引起的屏障功能障碍增敏。10)植物乳杆菌通过p38MAPK依赖机制阻止乙醇/乙醛诱导的连接中断。11)植物乳杆菌对乙醇/乙醛诱导的紧密连接断裂具有保护作用，而rac1的激活和稳定与肌动球蛋白环的稳定有关。12)植物乳杆菌可改善乙醇/乙醛诱导的小鼠和人结肠黏膜的肠屏障功能障碍。这些研究的结果直接关系到我们对酒精性肝和胰腺疾病的发病机制的理解，并有可能为未来新的治疗策略的发展做出贡献。 公共卫生相关性：根据我们在过去几年的研究，我们假设肠道微生物群将乙醇代谢为乙醛通过诱导细胞间连接蛋白的磷酸化来破坏肠上皮屏障功能，而益生菌植物乳杆菌可以防止乙醛对细胞的这种损害。我们建议进行研究，以揭示参与这些过程的细胞和分子机制，并确定益生菌在减轻酒精诱导的组织损伤中的保护作用。这些研究的结果有望为开发治疗酒精性肝病和酒精性胰腺和肺组织损伤的新疗法提供知识。

DESCRIPTION (provided by applicant): Evidence from clinical and experimental studies indicates that elevated intestinal permeability to endotoxins and the resulting endotoxemia play a crucial role in the pathogenesis of alcoholic liver disease. Our studies conducted so far have shown that acetaldehyde, the metabolic product of ethanol, disrupts the intestinal epithelial barrier function and increases the permeability to endotoxins. The mechanism of this acetaldehyde-induced disruption of epithelial barrier function involves inhibition of a protein tyrosine phosphatase, PTP1B, tyrosine phosphorylation of junctional proteins, disruption of the interactions among the junctional proteins (that determine the barrier function), and loss of integrity of the junctional complexes. Furthermore, our studies demonstrated that epidermal growth factor (EGF) and L-glutamine prevent acetaldehyde-mediated increase in permeability to endotoxins by a PLC?, PKC?, PKC?I and calcium-dependent mechanism. Our preliminary studies indicate that acetaldehyde induces translocation of PP2A leading to dephosphorylation of occludin and claudin-4, ethanol amplifies the effect of acetaldehyde by a Src kinase and MLCK-dependent mechanism and that probiotic, L. plantarum prevents acetaldehyde- induced barrier disruption. On the basis of these results it is further hypothesized that: a) ethanol metabolism and gut microflora play crucial roles in ethanol-induced intestinal barrier dysfunction, b) PP2A-dependent dephosphorylation of occludin and Cldn-4 is involved in acetaldehyde-induced disruption of intestinal barrier function, c) ethanol synergizes acetaldehyde-induced barrier disruption by c-Src-mediated MLCK activation, and d) probiotic, L. plantarum, prevents ethanol and acetaldehyde-induced disruption of barrier function by a EGF receptor, p38MAPK and Rac1- dependent mechanism. Using a cell culture model of the intestinal epithelium and human colonic biopsies we will determine that: 1) ADH1B and ALDH2 modulate ethanol-induced disruption of TJs and barrier function. 2) ALDH2 deficient mice are more sensitive to ethanol-induced barrier dysfunction. 3) Gut microflora play a role in ethanol metabolism and ethanol-induced disruption of TJs. 4) Acetaldehyde-induced PP2A methylation and translocation leads to dephosphorylation of TJ proteins and disruption of barrier function. 5) Dephosphorylation of occludin and Cldn-4 on specific Ser and Thr residues is associated with acetaldehyde-induced disruption of TJs and barrier dysfunction. 6) PP2A translocation plays a role in acetaldehyde-induced TJ disruption in mouse intestine. 7) Ethanol-mediated c-Src activation synergizes acetaldehyde-induced TJ disruption. 8) MLCK mediates synergization of acetaldehyde-induced TJ disruption by ethanol. 9) Ethanol sensitizes mouse colon for acetaldehyde-induced barrier dysfunction by a c-Src and MLCK- dependent mechanism. 10) L. plantarum prevents ethanol/acetaldehyde-induced disruption of junctions by p38MAPK-dependent mechanism. 11) Rac1 activation and stabilization of actomyosin ring are involved in the L. plantarum-mediated prevention of ethanol/acetaldehyde-induced tight junction disruption. 12) L. plantarum ameliorates ethanol/acetaldehyde-induced intestinal barrier dysfunction in mice and human colonic mucosa. The outcome of these studies has a direct relevance to our understanding of the pathogenesis of alcoholic liver and pancreatic diseases, and has the potential to contribute to the future development of new therapeutic strategies. PUBLIC HEALTH RELEVANCE: On the basis of our research during the past several years we hypothesized that ethanol metabolism by gut microflora into acetaldehyde disrupts intestinal epithelial barrier function by inducing phosphorylation of proteins of intercellular junctions, and the probiotic L. plantarum prevents such cellular damage by acetaldehyde. We propose to conduct studies to uncover the cellular and molecular mechanisms involved in these processes and determine the protective role of a probiotic in alleviating the alcohol- induced tissue injury. The outcome of these studies is expected to provide knowledge to develop new therapies in the treatment of alcoholic liver disease and alcohol-induced tissue injury in pancreas and lung.