Ethanol disrupts intestinal epithelial tight junction integrity through intracellular calcium-mediated Rho/ROCK activation
Ethanol disrupts intestinal epithelial tight junction integrity through intracellular calcium-mediated Rho/ROCK activation
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DOI：
10.1152/ajpgi.00236.2013
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发表时间：
2014-04-01
影响因子：
4.5
通讯作者：
Jonkers, Daisy
中科院分区：
文献类型：
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作者：
Elamin, Elhaseen;Masclee, Ad;Jonkers, Daisy
[10]李晓，李晓，李晓.乙醇通过细胞内钙介导的Rho/ROCK激活破坏肠上皮紧密连接的完整性Am J Physiol Gastrointest Liver Physiol 306：G677-G685，2014.首次发表于2014年2月20日; doi：10.1152/ajpgi.00236.2013.-有证据表明，乙醇诱导的肠屏障功能障碍和随后的内毒素血症在酒精性肝病的发病机制中起着关键作用。最近，它已被证明，乙醇诱导RhoA激酶激活肠上皮细胞，从而破坏屏障的完整性。在这项研究中，在Caco-2细胞单层中研究了乙醇诱导的Rho相关卷曲螺旋形成激酶（Rho/ROCK）激活和屏障破坏中细胞内钙浓度（[Ca 2 + ]（i））升高的作用。用40 mmol/l乙醇处理Caco-2单层可诱导[Ca 2 +]（i）释放，表现为Fluo-3的相对荧光单位从0.06 +/- 0.02增加至2.27 +/- 1.96（P < 0.0001）。用1，2-双预处理（2-氨基苯氧基）乙烷-N，N，N '，N '-四乙酸（BAPTA-AM）完全抑制[Ca 2 +]（i）释放，而肌醇1，4，5-三磷酸受体（IP 3R）拮抗剂Xestospongin C部分抑制乙醇诱导的[Ca 2 +]（i）释放（分别为2.27 +/- 1.96至0.03 +/- 0.01; P < 0.0001和2.27 +/- 1.96至1.19 +/- 1.80; P < 0.001）。[Ca ~（2+）]（i）的升高与肠通透性的增加相一致，BAPTA-AM或Xestospongin C均可减弱肠通透性的增加。此外，乙醇诱导Rho/ROCK激活，如肌球蛋白结合亚基的磷酸化增加所示，这可以通过BAPTA、Xestospongin C或特异性Rho/ROCK抑制剂Y27632来防止。最后，通过Y27632抑制Rho/ROCK激酶改善了乙醇诱导的闭锁小带-1、粘附连接蛋白（包括E-钙粘蛋白和β-连环蛋白）的重新分布，以及F-肌动蛋白的解体。这些结果表明，乙醇诱导的[Ca 2 +]（i）释放，通过刺激IP 3R门控Ca 2+通道介导，激活Caco-2细胞中的Rho/ROCK，从而促进乙醇诱导的肠屏障功能障碍。
Elamin E, Masclee A, Dekker J, Jonkers D. Ethanol disrupts intestinal epithelial tight junction integrity through intracellular calcium-mediated Rho/ROCK activation. Am J Physiol Gastrointest Liver Physiol 306: G677-G685, 2014. First published February 20, 2014; doi:10.1152/ajpgi.00236.2013.-Evidence indicates that ethanol-induced intestinal barrier dysfunction and subsequent endotoxemia plays a key role in the pathogenesis of alcoholic liver disease. Recently, it has been demonstrated that ethanol induces RhoA kinase activation in intestinal epithelium, thereby disrupting barrier integrity. In this study, the role of a rise in intracellular calcium concentration ([Ca2+ ](i)) in ethanol-induced Rho-associated coiled coil-forming kinase (Rho/ROCK) activation and barrier disruption was investigated in Caco-2 cell monolayers. Treatment of Caco-2 monolayers with 40 mmol/l ethanol induced [Ca2+](i) release as indicated by increased relative fluorescent units of Fluo-3 from 0.06 +/- 0.02 to 2.27 +/- 1.96 ( P < 0.0001). Pretreatment with 1,2-bis(2-aminophenoxy) ethane-N, N, N ', N '-tetraacetic acid (BAPTA-AM) completely inhibited the release, whereas the inositol 1,4,5- triphosphate receptor ( IP3R)-antagonist, Xestospongin C, partially inhibited the ethanol-induced [Ca2+](i) release (from 2.27 +/- 1.96 to 0.03 +/- 0.01; P < 0.0001 and from 2.27 +/- 1.96 to 1.19 +/- 1.80; P < 0.001, respectively). The rise in [Ca2+](i) was paralleled with increased intestinal permeability, which could be attenuated by either BAPTA-AM or Xestospongin C. Furthermore, ethanol induced Rho/ROCK activation, as indicated by increased phosphorylation of myosin-binding subunit, which could be prevented either by BAPTA, Xestospongin C, or the specific Rho/ROCK inhibitor Y27632. Finally, inhibition of Rho/ROCK kinase by Y27632 ameliorated the ethanol-induced redistribution of zonula occluden-1, adherens junction proteins including E-cadherin and beta-catenin, and also disorganization of F-actin. These findings suggest that ethanol-induced [Ca2+](i) release, mediated by stimulating IP3R-gated Ca2+ channel, activates Rho/ROCK in Caco-2 cells, thereby contributing to ethanol-induced intestinal barrier dysfunction.