Stress-Derived Corticotropin Releasing Factor Breaches Epithelial Endotoxin Tolerance

Background and aims
Loss of the endotoxin tolerance of intestinal epithelium contributes to a number of intestinal diseases. The etiology is not clear. Psychological stress is proposed to compromise the intestinal barrier function. The present study aims to elucidate the role of the stress-derived corticotropin releasing factor (CRF) in breaching the established intestinal epithelial endotoxin tolerance.
Methods
Epithelial cells of HT-29, T84 and MDCK were exposed to lipopolysaccharide to induce the endotoxin tolerance; the cells were then stimulated with CRF. The epithelial barrier function was determined using as indicators of the endotoxin tolerant status. A water-avoid stress mouse model was employed to test the role of CRF in breaching the established endotoxin tolerance in the intestine.
Results
The established endotoxin tolerance in the epithelial cell monolayers was broken down by a sequent exposure to CRF and LPS manifesting a marked drop of the transepithelial resistance (TER) and an increase in the permeability to a macromolecular tracer, horseradish peroxidase (HRP). The exposure to CRF also increased the expression of Cldn2 in the epithelial cells, which could be mimicked by over expression of TLR4 in epithelial cells. Over expression of Cldn2 resulted in low TER in epithelial monolayers and high permeability to HRP. After treating mice with the 10-day chronic stress, the intestinal epithelial barrier function was markedly compromised, which could be prevented by blocking either CRF, or TLR4, or Cldn2.
Conclusions
Psychological stress-derived CRF can breach the established endotoxin tolerance in the intestinal mucosa.

研究背景与目的
肠上皮细胞内毒素耐受的丧失可引发多种肠道疾病，但其病因目前尚不明确。有研究提出心理应激会损伤肠屏障功能。本研究旨在阐明应激源性促肾上腺皮质激素释放因子（corticotropin releasing factor, CRF）在破坏已建立的肠上皮内毒素耐受状态中的作用。

研究方法
将HT-29、T84及MDCK肠上皮细胞暴露于脂多糖（lipopolysaccharide, LPS）以诱导内毒素耐受，随后用CRF对细胞进行刺激。以肠屏障功能作为内毒素耐受状态的检测指标。本研究采用避水应激小鼠模型，验证CRF在体内破坏肠道已建立的内毒素耐受中的作用。

研究结果
上皮细胞单层中已建立的内毒素耐受可经CRF与LPS的后续暴露而被打破，具体表现为跨上皮电阻（transepithelial resistance, TER）显著下降，且对大分子示踪剂辣根过氧化物酶（horseradish peroxidase, HRP）的通透性升高。CRF暴露还可上调肠上皮细胞中Cldn2的表达，该效应可通过过表达Toll样受体4（Toll-like receptor 4, TLR4）进行模拟。过表达Cldn2会导致上皮细胞单层的TER降低以及对HRP的通透性升高。对小鼠施加10天慢性应激后，其肠上皮屏障功能显著受损，而通过阻断CRF、TLR4或Cldn2均可予以缓解。

研究结论
心理应激源性CRF可破坏肠黏膜中已建立的内毒素耐受状态。