Rho kinase inhibition maintains intestinal and vascular barrier function by upregulation of occludin in experimental necrotizing enterocolitis
Rho kinase inhibition maintains intestinal and vascular barrier function by upregulation of occludin in experimental necrotizing enterocolitis
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DOI：
10.1152/ajpgi.00357.2017
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发表时间：
2018-10-01
影响因子：
4.5
通讯作者：
Hunter, Catherine J.
中科院分区：
文献类型：
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作者：
Grothaus, Justyna S.;Ares, Guillermo;Hunter, Catherine J.
坏死性小肠结肠炎 (NEC) 是一种致命疾病，5-10% 的新生儿会发生这种疾病。尽管 NEC 已被广泛研究，但尚未确定单一的治疗靶点。 Rho 激酶 (ROCK) 是一种丝氨酸/苏氨酸激酶，影响多种细胞过程，包括紧密连接 (TJ) 功能、细胞通透性和细胞凋亡。我们假设 ROCK 抑制会降低细胞通透性、稳定 TJ 蛋白 (occludin) 并降低 NEC 的严重程度。为了检验这一假设，研究了人结肠上皮细胞 (Caco-2) 和人内皮细胞。用脂多糖处理细胞来模拟 NEC 的体外模型。通过跨上皮膜阻力 (TEER) 和细胞对 FITC-葡聚糖的通透性来测量 ROCK 抑制的效果。在NEC幼鼠模型中分析了ROCK抑制的体内效果。 NEC是通过添加坂崎克罗诺杆菌（Cronobacter sakazakii）的喂养配方在有或没有强饲ROCK抑制剂的情况下诱导的。根据组织学损伤程度对大鼠肠道进行评分。对所有 NEC 模型进行了 occludin 蛋白的 RNA 和蛋白测定。 ROCK 抑制剂治疗显着降低 Caco-2 细胞的细胞通透性并增加 TEER。肠损伤评分显示，与仅接受 NEC 治疗的幼犬相比，接受 ROCK 抑制剂治疗的幼犬评分有所下降。细胞和大鼠幼仔模型均显示 ROCK 抑制剂治疗组中 occludin 表达上调。因此，我们得出结论，ROCK 抑制通过上调 occludin 来增强屏障功能，从而防止实验性 NEC。这些数据表明 ROCK 可能是 NEC 患者的潜在治疗靶点。新的和值得注意的这些研究首次证明了 Occludin 紧密连接蛋白响应 Rho 激酶 (ROCK) 抑制而上调。此外，我们还证明，在坏死性小肠结肠炎 (NEC) 实验模型中，ROCK 抑制可在体外和体内疾病模型中预防 NEC。
Necrotizing enterocolitis (NEC) is a deadly disease that occurs in 5-10% of neonates. Although NEC has been extensively studied, no single therapeutic target has been identified. Rho kinase (ROCK) is a serine/threonine kinase that affects multiple cellular processes, including tight junction (TJ) function, cellular permeability, and apoptosis. We hypothesized that ROCK inhibition would decrease cellular permeability, stabilize TJ proteins (occludin), and decrease the severity of NEC. To test this hypothesis, human colon epithelial cells (Caco-2) and human endothelial cells were studied. Cells were treated with lipopolysaccharide to simulate an in vitro model of NEC. The effect of ROCK inhibition was measured by transepithelial membrane resistance (TEER) and cellular permeability to FITC-dextran. The effects of ROCK inhibition in vivo were analyzed in the rat pup model of NEC. NEC was induced by feeding formula supplemented with Cronobacter sakazakii with or without gavaged ROCK inhibitor. Rat intestines were scored based on histological degree of injury. RNA and protein assays for occludin protein were performed for all models of NEC. Treatment with ROCK inhibitor significantly decreased cellular permeability in Caco-2 cells and increased TEER. Intestinal injury scoring revealed decreased scores in ROCK inhibitor-treated pups compared with NEC only. Both cell and rat pup models demonstrated an upregulation of occludin expression in the ROCK inhibitor-treated groups. Therefore, we conclude that ROCK inhibition protects against experimental NEC by strengthening barrier function via upregulation of occludin. These data suggest that ROCK may be a potential therapeutic target for patients with NEC.NEW & NOTEWORTHY These studies are the first to demonstrate an upregulation of occludin tight junction protein in response to Rho kinase (ROCK) inhibition. Furthermore, we have demonstrated that ROCK inhibition in experimental models of necrotizing enterocolitis (NEC) is protective against NEC in both in vitro and in vivo models of disease.