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在酗酒者的肠道中积累，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。我们的初步研究还表明，表皮生长因子（EGF）和l -谷氨酰胺阻止乙醛介导的内毒素通透性增加。基于我们的研究结果，我们进一步假设：a)乙醛诱导的PTP1B和β -catenin磷酸化破坏e- cadherin - β -catenin -PTP1B复合物，b) EGF和l -谷氨酰胺通过PLCgamma和MAP激酶介导的信号阻止乙醛诱导的连接破坏，c) l -谷氨酰胺通过EGF受体依赖机制阻止乙醛诱导的连接破坏。使用肠上皮细胞培养模型和人类结肠活检，我们将确定：1) E-cadherin和PTP1B之间的相互作用被PTP1B的thr磷酸化所减少，2)酪氨酸残基上的β -catenin磷酸化阻止了它与E-cadherin的结合，3)PTP1B上特定苏氨酸残基和β -catenin上酪氨酸残基的突变阻止了乙醛诱导的连接破坏，4)EGF和谷氨酰胺阻止了乙醛诱导的PTP1B和β -catenin的磷酸化，以及E-cadherin/ β -catenin/PTP1B复合物的破坏。5) PLCgamma和MAP激酶信号通路介导EGF-和谷氨酰胺诱导的PTP1B和β -catenin的磷酸化，以及E-cadherin/ β -catenin/PTP1B复合物的破坏；6)l -谷氨酰胺通过EGF受体依赖机制阻止乙醛诱导的PTP1B和β -catenin的磷酸化，以及E-cadherin/ β -catenin/PTP1B复合物的破坏；7)EGF受体激活参与谷氨酰胺介导的乙醛诱导的AJ和TJ破坏的预防。9)谷氨酰胺通过Src激酶依赖机制阻止乙醛诱导的AJ和TJ的破坏，10)谷氨酰胺激活EGF受体是通过细胞外释放金属蛋白酶和TGFalpha介导的。从这项研究中获得的信息将扩大我们对乙醛介导的肠上皮损伤和内毒素吸收的理解。研究表皮生长因子和l -谷氨酰胺对屏障功能的保护作用，为制定酒精性肝病的预防和治疗策略提供依据。

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, is accumulated in the intestine of alcoholics and 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. Our preliminary studies also indicated that epidermal growth factor (EGF) and L-glutamine prevent acetaldehyde-mediated increase in permeability to endotoxins. On the basis of our results it is further hypothesized that: a) acetaldehyde-induced phosphorylation of PTP1B and beta-catenin disrupts E-Cadherin-beta-Catenin-PTP1B complex, b) EGF and L-glutamine prevent acetaldehyde induced disruption of junctions by PLCgamma and MAP kinase mediated signals, and c) L-glutamine prevents acetaldehyde-induced disruption of junctions by EGF receptor-dependent mechanism. Using a cell culture model of the intestinal epithelium and human colonic biopsies we will to determine that: 1) interaction between E-cadherin and PTP1B is reduced by Thr-phosphorylation of PTP1B, 2) phosphorylation of beta-catenin on tyrosine residues prevents its binding to E-cadherin, 3) mutation of specific threonine residues on PTP1B and tyrosine residues in beta-catenin prevents the acetaldehyde-induced disruption of junctions, 4) EGF and glutamine prevent acetaldehyde-induced phosphorylation of PTP1B and beta-catenin, and disruption of E-cadherin/beta-catenin/PTP1B complex, 5) PLCgamma and MAP kinase signaling pathways mediate EGF- and glutamine-induced prevention of phosphorylation of PTP1B and beta-catenin, and disruption of E-cadherin/beta-catenin/PTP1B complex, 6) L-glutamine prevents acetaldehyde-induced phosphorylation of PTP1B and beta-catenin, and disruption of E-cadherin/beta-catenin/PTP1B complex by EGF receptor-dependent mechanism, 7) EGF receptor activation is involved in glutamine-mediated prevention of acetaldehyde-induced disruption of AJ and TJ, 9) glutamine prevents acetaldehyde-induced disruption of AJ and TJ by Src kinase dependent mechanism, and 10) EGF receptor activation by glutamine is mediated by extra cellular release of metalloproteinase and TGFalpha. Information derived from this study will expand our understanding of acetaldehyde-mediated injury in intestinal epithelium and endotoxin absorption in alcoholics. The studies on the protection of barrier function by epidermal growth factor and L-glutamine have the potential to provide the basis for the development of preventive and therapeutic strategies for alcoholic liver disease.