Interferon-γ-Induced Intestinal Epithelial Barrier Dysfunction by NF-κB/HIF-1α Pathway

Interferon-? (IFN-?) plays an important role in intestinal barrier dysfunction. However, the mechanisms are not fully understood. As hypoxia-inducible factor-1 (HIF-1) is a critical determinant response to hypoxia and inflammation, which has been shown to be deleterious to intestinal barrier function, we hypothesized that IFN-? induces loss of barrier function through the regulation of HIF-1α activation and function. In this study, we detected the expressions of HIF-1α and tight junction proteins in IFN-?-treated T84 intestinal epithelial cell line. IFN-? led to an increase of HIF-1α expression in time- and dose-dependent manners but did not change the expression of HIF-1?. The IFN-?-induced increase in HIF-1α was associated with an activation of NF-?B. Treatment with the NF-?B inhibitor, pyrolidinedithiocarbamate (PDTC), significantly suppressed the activation of NF-?B and the expression of HIF-1α. In addition, IFN-? also increased intestinal epithelial permeability and depletion of tight junction proteins; inhibition of NF-?B or HIF-1α prevented the increase in intestinal permeability and alteration in tight junction protein expressions. Interestingly, we demonstrated that a significant portion of IFN-? activation NF-kB and modulation tight junction expression is mediated through HIF-1α. Taken together, this study suggested that IFN-? induced the loss of epithelial barrier function and disruption of tight junction proteins, by upregulation of HIF-1α expression through NF-?B pathway.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/140108/1/jir.2013.0044.pd

The jagged-2/notch-1/hes-1 pathway is involved in intestinal epithelium regeneration after intestinal ischemia-reperfusion injury.

Notch signaling plays a critical role in the maintenance of intestinal crypt epithelial cell proliferation. The aim of this study was to investigate the role of Notch signaling in the proliferation and regeneration of intestinal epithelium after intestinal ischemia reperfusion (I/R) injury.Male Sprague-Dawley rats were subjected to sham operation or I/R by occlusion of the superior mesenteric artery (SMA) for 20 min. Intestinal tissue samples were collected at 0, 1, 2, 4, and 6 h after reperfusion. Proliferation of the intestinal epithelium was evaluated by immunohistochemical staining of proliferating nuclear antigen (PCNA). The mRNA and protein expression levels of Notch signaling components were examined using Real-time PCR and Western blot analyses. Immunofluorescence was also performed to detect the expression and location of Jagged-2, cleaved Notch-1, and Hes-1 in the intestine. Finally, the γ-secretase inhibitor DAPT and the siRNA for Jagged-2 and Hes-1 were applied to investigate the functional role of Notch signaling in the proliferation of intestinal epithelial cells in an in vitro IEC-6 culture system.I/R injury caused increased intestinal crypt epithelial cell proliferation and increased mRNA and protein expression of Jagged-2, Notch-1, and Hes-1. The immunofluorescence results further confirmed increased protein expression of Jagged-2, cleaved Notch-1, and Hes-1 in the intestinal crypts. The inhibition of Notch signaling with DAPT and the suppression of Jagged-2 and Hes-1 expression using siRNA both significantly inhibited the proliferation of IEC-6 cells.The Jagged-2/Notch-1/Hes-1 signaling pathway is involved in intestinal epithelium regeneration early after I/R injury by increasing crypt epithelial cell proliferation

siRNA for Jagged-2 inhibited proliferation of IEC-6 cells.

<p>Cells were plated onto 6-well plates. Inhibition of Jagged-2 with siRNA was carried out as mentioned above (see the Materials and Methods). <i>A</i>: After 48 h of culture, protein was extracted from IEC-6 cells plated on 6-well plates. Western blot analysis showed that siRNA for Jagged-2 downregulated protein expression of Jagged-2, NICD-1, and Hes-1 of IEC-6 cells compared with the si-NC or control group. GAPDH was used as loading control. <i>B</i>: Graphic representation of relative expression of Jagged-2, NICD-1, and Hes-1 normalized to GAPDH. Data are given as the means ± SDs (<i>n</i> = 5). **<i>p</i><0.01 versus control group. <i>C</i>: Cell count of IEC-6 cells was taken for control group, si-NC, and siRNA for Jagged-2 group (magnification×400). <i>D</i>: IEC-6 cells were plated onto a 96-well plate. Inhibition of Jagged-2 with siRNA was carried out as mentioned above (see the Materials and Methods). MTT assay showed that siRNA for Jagged-2 decreased the cell number of IEC-6 cells significantly. Data are given as the means ± SDs (<i>n</i> = 10) **<i>p</i><0.01 versus control group. Control: IEC-6 cells were cultured normally. si-NC: IEC-6 cells were transfected with unrelated control siRNA. siRNA: IEC-6 cells were transfected with siRNA for Jagged-2.</p

siRNA for Hes-1 inhibited proliferation of IEC-6 cells.

<p>Cells were plated onto 6-well plates. Inhibition of Jagged-2 with siRNA was carried out as mentioned above (see the Materials and Methods). <i>A</i>: After 48 h of culture, protein was extracted from IEC-6 cells. Western blot analysis showed that siRNA for Hes-1 downregulated protein expression of Hes-1 in IEC-6 cells compared with the si-NC or control group. GAPDH was used as loading control. <i>B</i>: Graphic representation of relative expression of Hes-1 normalized to GAPDH. Data are given as the means ± SDs (<i>n</i> = 5). **<i>p</i><0.01 versus control group. <i>C</i>: Cell count of IEC-6 cells was taken for control group, si-NC, and siRNA for Hes-1 group (magnification×400). <i>D</i>: IEC-6 cells were plated onto a 96-well plate. Inhibition of Hes-1 with siRNA was carried out as mentioned above (see the Materials and Methods). MTT assay showed that siRNA for Hes-1 decreased the cell number of IEC-6 cells significantly. Data are given as the means ± SDs (<i>n</i> = 10) **<i>p</i><0.01 versus control group. Control: IEC-6 cells were cultured normally. si-NC: IEC-6 cells were transfected with unrelated control siRNA. siRNA: IEC-6 cells were transfected with siRNA for Hes-1.</p

Immunofluorescence evidence for increased Notch signaling activation in intestine after I/R injury.

<p>Frozen sections of intestine from sham operated rats or rats after I/R injury were stained with antibodies against Jagged-2 (A), NICD-1 (B), and Hes-1 (C). Nuclei were stained with DAPI (blue). Images (A: magnification×200; B, C: magnification×400) were taken by confocal microscopy.</p

Inhibition of Notch signaling by DAPT suppressed cell proliferation of IEC-6 cells.

<p><i>A</i>: IEC-6 cells with DMSO (Control) or DAPT (20 µM) for indicated times (magnification×100). <i>B</i>: IEC-6 cells were plated onto a 96-well culture plate, and DAPT was added. MTT assay was performed to examine the proliferation of IEC-6 cells. Data are given as the means ± SDs (<i>n</i> = 10). <i>C</i>: Protein was extracted from IEC-6 cells plated onto a 6-well plate, and protein expression of NICD-1, and Hes-1 was examined by Western blot. GAPDH was used as the loading control. <i>D</i>: Graphic representation of relative expression of NICD-1, and Hes-1 normalized to GAPDH. Data are given as the means ± SDs (<i>n</i> = 5). **<i>p</i><0.01 versus control group. *<i>p</i><0.05 versus control group.</p

Transcripts of Notch signaling components were increased in intestine after I/R injury.

<p><i>A</i>: The I/R and sham-operated rats were sacrificed at indicated times, mRNA of intestinal mucosa was collected, and RT-PCR was performed to detect mRNA levels of Jagged-2, Notch-1, and Hes-1. GAPDH was used to verify equivalent loading. <i>B</i>: mRNA expression of Jagged-2, Notch-1, and Hes-1 detected by Real-time PCR. Data are given as the means ± SDs (<i>n</i> = 7). **<i>p</i><0.01 versus sham group.</p