Mechanism of Endotoxin absorption in alcoholism

描述（由申请人提供）：来自临床和实验研究的证据表明，肠道对内毒素的通透性升高以及由此产生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，我们的研究表明，乙醇的代谢产物乙醛会破坏肠上皮屏障功能，增加对内毒素的渗透性。这种乙醛诱导的上皮屏障功能破坏的机制包括蛋白酪氨酸磷酸酶PTP1B的抑制，连接蛋白的酪氨酸磷酸化，连接蛋白之间相互作用的破坏（决定屏障功能），以及连接复合物完整性的丧失。此外，我们的研究表明，表皮生长因子（EGF）和l -谷氨酰胺可以防止乙醛介导的PLC？, PKC ?, PKC ?I和钙依赖机制。我们的初步研究表明，乙醛诱导PP2A易位，导致occludin和cludin -4的去磷酸化，乙醇通过Src激酶和mlck依赖机制放大了乙醛的作用，益生菌L. plantarum防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) pp2a依赖性occludin和Cldn-4的去磷酸化参与了乙醛诱导的肠屏障功能破坏，c)乙醇通过c- src介导的MLCK激活协同乙醛诱导的屏障破坏，d)益生菌L. plantarum通过EGF受体阻止乙醇和乙醛诱导的屏障功能破坏。p38MAPK和Rac1依赖性机制。通过肠上皮细胞培养模型和人类结肠活检，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缺陷小鼠对乙醇诱导的屏障功能障碍更敏感。3)肠道菌群在乙醇代谢和乙醇诱导的TJs破坏中发挥作用。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) occludin和Cldn-4在特定Ser和Thr残基上的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破坏中起作用。7)乙醇介导的c-Src活化协同乙醛诱导的TJ破坏。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诱导的屏障功能障碍增敏。10) L. plantarum通过p38mapk依赖机制阻止乙醇/乙醛诱导的连接破坏。11) Rac1的激活和肌动球蛋白环的稳定参与了L. plantaram介导的乙醇/乙醛诱导的紧密连接破坏的预防。12)植物乳杆菌改善乙醇/乙醛诱导的小鼠和人结肠黏膜肠道屏障功能障碍。这些研究的结果与我们对酒精性肝脏和胰腺疾病发病机制的理解直接相关，并有可能为未来新的治疗策略的发展做出贡献。

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.