Banan A, Choudhary S, Zhang Y, Fields J Z, Keshavarzian A
Division of Digestive Disease, Rush University Medical Center, Department of Internal Medicine, Chicago, IL 60612, USA.
Free Radic Biol Med. 2000 Mar 1;28(5):727-38. doi: 10.1016/s0891-5849(00)00160-x.
Reactive oxygen metabolites (ROM) are increased in the inflamed mucosa of inflammatory bowel disease (IBD) and may contribute to loss of intestinal barrier function in this disorder. Growth factors (GF) are protective. But the mechanisms of disruption and protection remain elusive. In the present investigation, we hypothesized that the microtubules (a critical cytoskeletal element) play a key role in the molecular mechanism of intestinal barrier dysfunction induced by ROM and in GF-mediated protection. Utilizing monolayers of a human colonic cell line (Caco-2), we evaluated the effects of ROM (H(2)O(2) or HOCl), in the presence or absence of GF (epidermal growth factor [EGF]; transforming growth factor-alpha [TGF-alpha]), on intestinal barrier function, tubulin (microtubule structural protein), and microtubule stability. Monolayers were also processed for two highly sensitive western immunoblots: fractionated polymerized tubulin (S2; an index of stability); monomeric tubulin (S1; an index of disruption) to detect the oxidation and disassembly/assembly of tubulin. ROM exposure led to a significant increase in the oxidation of tubulin, decrease in the stable S2 polymerized tubulin, and increase in the unstable S1 monomeric tubulin. In concert, each ROM in a dose dependent manner damaged the microtubule cytoskeleton and disrupted barrier function. GF pretreatment not only increased the S2 stable tubulin and decreased tubulin oxidation but also, concomitantly, prevented the disruption of microtubules and loss of barrier function in monolayers exposed to ROM. Antibody against the GF-receptor and inhibitors of GF-receptor tyrosine kinase abolished GF protection, indicating the involvement of epidermal growth factor receptor (EGFR) signaling pathway. As predicted, colchicine, an inhibitor of microtubule assembly, caused barrier dysfunction and prevented GF protection whereas taxol, a microtubule-stabilizing agent, mimicked the protective effects of GF. Thus, organization and stability of the microtubule cytoskeleton appears to be critical to both oxidant-induced mucosal barrier dysfunction and protection of intestinal barrier mediated by GF. Therefore, microtubules may be useful targets for development of drugs for the treatment of IBD.
活性氧代谢产物（ROM）在炎症性肠病（IBD）的炎症黏膜中增加，可能导致该疾病肠道屏障功能丧失。生长因子（GF）具有保护作用。但破坏和保护的机制仍不清楚。在本研究中，我们假设微管（一种关键的细胞骨架成分）在ROM诱导的肠道屏障功能障碍的分子机制以及GF介导的保护作用中起关键作用。利用人结肠癌细胞系（Caco-2）单层细胞，我们评估了在有或没有GF（表皮生长因子[EGF]；转化生长因子-α[TGF-α]）存在的情况下，ROM（过氧化氢[H₂O₂]或次氯酸[HOCl]）对肠道屏障功能、微管蛋白（微管结构蛋白）和微管稳定性的影响。单层细胞还进行了两种高灵敏度的蛋白质免疫印迹分析：分级聚合微管蛋白（S2；稳定性指标）；单体微管蛋白（S1；破坏指标），以检测微管蛋白的氧化和分解/组装。暴露于ROM导致微管蛋白氧化显著增加，稳定的S2聚合微管蛋白减少，不稳定的S1单体微管蛋白增加。同时，每种ROM均以剂量依赖性方式破坏微管细胞骨架并破坏屏障功能。GF预处理不仅增加了S2稳定微管蛋白并减少了微管蛋白氧化，而且同时防止了暴露于ROM的单层细胞中微管的破坏和屏障功能的丧失。针对GF受体的抗体和GF受体酪氨酸激酶抑制剂消除了GF的保护作用，表明表皮生长因子受体（EGFR）信号通路参与其中。如预期的那样，微管组装抑制剂秋水仙碱导致屏障功能障碍并阻止GF的保护作用，而微管稳定剂紫杉醇模拟了GF的保护作用。因此，微管细胞骨架的组织和稳定性似乎对于氧化剂诱导的黏膜屏障功能障碍和GF介导的肠道屏障保护均至关重要。因此，微管可能是开发治疗IBD药物的有用靶点。
