Child and Family Research Institute, British Columbia Children's Hospital, Vancouver, British Columbia, Canada; Division of Gastroenterology, British Columbia Children's Hospital, Vancouver, British Columbia, Canada; and.
Child and Family Research Institute, British Columbia Children's Hospital, Vancouver, British Columbia, Canada; Division of Gastroenterology, British Columbia Children's Hospital, Vancouver, British Columbia, Canada; and Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, British Columbia, Canada
Am J Physiol Gastrointest Liver Physiol. 2015 Mar 1;308(5):G389-402. doi: 10.1152/ajpgi.00195.2014. Epub 2014 Dec 11.
We previously showed that vasoactive intestinal peptide (VIP) protects against bacterial pathogen-induced epithelial barrier disruption and colitis, although the mechanisms remain poorly defined. The aim of the current study was to identify cellular pathways of VIP-mediated protection with use of pharmacological inhibitors during enteropathogenic Escherichia coli (EPEC) infection of Caco-2 cell monolayers and during Citrobacter rodentium-induced colitis. EPEC-induced epithelial barrier disruption involved the PKC pathway but was independent of functional cAMP, Rho, and NF-κB pathways. VIP mediated its protective effects by inhibiting EPEC-induced PKC activity and increasing expression of the junctional protein claudin-4. Short-term treatment with TPA, which is known to activate PKC, was inhibited by VIP pretreatment, while PKC degradation via long-term treatment with TPA mimicked the protective actions of VIP. Immunostaining for specific PKC isotypes showed upregulated expression of PKCθ and PKCε during EPEC infection. Treatment with specific inhibitors revealed a critical role for PKCε in EPEC-induced barrier disruption. Furthermore, activation of PKCε and loss of barrier integrity correlated with claudin-4 degradation. In contrast, inhibition of PKCε by VIP pretreatment or the PKCε inhibitor maintained membrane-bound claudin-4 levels, along with barrier function. Finally, in vivo treatment with the PKCε inhibitor protected mice from C. rodentium-induced colitis. In conclusion, EPEC infection increases intracellular PKCε levels, leading to decreased claudin-4 levels and compromising epithelial barrier integrity. VIP inhibits PKCε activation, thereby attenuating EPEC-induced barrier disruption.
我们之前曾表明，血管活性肠肽（VIP）可防止细菌病原体引起的上皮屏障破坏和结肠炎，尽管其机制仍未得到充分阐明。本研究的目的是在肠致病性大肠杆菌（EPEC）感染 Caco-2 细胞单层和柠檬酸杆菌诱导的结肠炎期间，使用药理学抑制剂来鉴定 VIP 介导的保护的细胞途径。EPEC 诱导的上皮屏障破坏涉及 PKC 途径，但与功能性 cAMP、Rho 和 NF-κB 途径无关。VIP 通过抑制 EPEC 诱导的 PKC 活性和增加连接蛋白 claudin-4 的表达来发挥其保护作用。已知可激活 PKC 的 TPA 的短期处理被 VIP 预处理所抑制，而通过 TPA 的长期处理导致 PKC 降解，模拟了 VIP 的保护作用。针对特定 PKC 同工型的免疫染色显示，在 EPEC 感染期间 PKCθ和 PKCε的表达上调。特异性抑制剂的处理表明，PKCε 在 EPEC 诱导的屏障破坏中起关键作用。此外，PKCε 的激活和屏障完整性的丧失与 claudin-4 的降解相关。相比之下，通过 VIP 预处理或 PKCε抑制剂抑制 PKCε 可维持膜结合的 claudin-4 水平和屏障功能。最后，体内用 PKCε 抑制剂治疗可保护小鼠免受柠檬酸杆菌诱导的结肠炎。总之，EPEC 感染会增加细胞内 PKCε 水平，导致 claudin-4 水平降低，破坏上皮屏障完整性。VIP 抑制 PKCε 的激活，从而减轻 EPEC 诱导的屏障破坏。
