Somatostatin attenuates intestinal epithelial barrier injury during acute intestinal ischemia-reperfusion through Tollip/Myeloiddifferentiationfactor 88/Nuclear factor kappa-B signaling

Abstract

In the process of ischemia-reperfusion injury, intestinal ischemia and inflammation interweave, leading to tissue damage or necrosis. However, oxygen radicals and inflammatory mediators produced after reperfusion cause tissue damage again, resulting in severe intestinal epithelial barrier dysfunction. The aim of this study was to determine the protective effect of somatostatin on intestinal epithelial barrier function during intestinal ischemia-reperfusion injury and explore its mechanism. By establishing a rat intestinal ischemia-reperfusion model, pretreating the rats with somatostatin, and then detecting the histopathological changes, intestinal permeability and expression of tight junction proteins in intestinal tissues, the protective effect of somatostatin on the intestinal epithelial barrier was measured in vivo. The mechanism was determined in interferon γ (IFN-γ)-treated Caco-2 cells in vitro. The results showed that somatostatin could ameliorate ischemia-reperfusion-induced intestinal epithelial barrier dysfunction and protect Caco-2 cells against IFN-γ-induced decreases in tight junction protein expression and increases in monolayer cell permeability. The expression of Tollip was upregulated by somatostatin both in ischemia-reperfusion rats and IFN-γ-treated Caco-2 cells, while the activation of TLR2/MyD88/NF-κB signaling was inhibited by somatostatin. Tollip inhibition reversed the protective effect of somatostatin on the intestinal epithelial barrier. In conclusion, somatostatin could attenuate ischemia-reperfusion-induced intestinal epithelial barrier injury by inhibiting the activation of TLR2/MyD88/NF-κB signaling through upregulation of Tollip.

Keywords: Intestinal ischemia–reperfusion injury; Tollip; intestinal epithelial barrier; somatostatin; tight junction.

Graphical abstract

Figure 1.

SST alleviated intestinal epithelial barrier injury induced by I/R and regulated the expression of inflammation-related genes. (a) Representative images of rat intestinal tissues stained with H&E. Bars ‘ – ’ indicate 50 μm. (b) The expression of the TJ proteins ZO-1 and Occludin in intestinal tissues detected by Western blotting. (c-d) Expression levels of the inflammatory factors IFN-γ and TNF-α in serum. (e) Heatmap of the differential expression of some inflammation-related genes detected by PCR array. The yellow square frames show inflammation-related Tollip/TLR2/MyD8/NF-κB gene expression changes. Sham, control group; I/RI (IIRI), ischemia–reperfusion injury group; SST (IIRI+SST), I/RI rats pretreated with SST. (a-d), n = 4 per group; (E), n = 6 per group. One‑way ANOVA followed by Bonferroni post hoc test was used to compare the differences among different groups, ‘ns’ indicated no significance. ^nsP>0.05, ****P < 0.0001 vs. sham group; ^###P < 0.001, ^####P < 0.0001 vs. I/RI group.

Figure 2.

SST inhibited IFN-γ-induced disruption of tight junctions in Caco-2 cells. (a) Changes in TER in Caco-2 monolayer cells treated with IFN-γ and/or SST. (b) Changes in FITC-dextran permeability in Caco-2 monolayer cells treated with IFN-γ and/or SST. (c) Expression of ZO-1 and Occludin in Caco-2 cells treated with IFN-γ and/or SST. (d) Representative images of Caco-2 cell immunofluorescence staining. Bars ‘ – ’ indicate 20 μm. NC, negative control; IFN-γ, IFN-γ treated only; SST, SST treated only; SST/IFN-γ, SST pretreated before IFN-γ treatment. One‑way ANOVA followed by Bonferroni post hoc test was used to compare the differences among different groups, ‘ns’ indicated no significance. ^nsP>0.05, ****P < 0.0001 vs. NC group; ^###P < 0.001, ^####P < 0.0001 vs. IFN-γ group.

Figure 3.

SST inhibited IFN-γ-mediated TLR2/MyD88/NF-κB/MLCK signal activation in Caco-2 cells. (a) Western blotting results of TLR2/MyD88/NF-κB/MLCK signaling. (b-i) The relative protein levels measured by ImageJ software. NC, negative control; IFN-γ, IFN-γ treated only; SST, SST treated only; SST/IFN-γ, SST pretreated before IFN-γ treatment. One‑way ANOVA followed by Bonferroni post hoc test was used to compare the differences among different groups, ‘ns’ indicated no significance. ^nsP>0.05, *P < 0.05, ***P < 0.001, ****P < 0.0001 vs. NC group; ^####P < 0.0001 vs. IFN-γ group; ^@@P < 0.01, ^@@@P < 0.001, ^@@@@P < 0.0001 vs. SST group.

Figure 4.

SST inhibited IFN-γ-mediated disruption of tight junctions in Caco-2 cells by upregulating Tollip. (a) Changes in TER in Caco-2 monolayer cells under different conditions. (b) Changes in FITC-dextran permeability in Caco-2 monolayer cells under different conditions. (c) The expression of proteins involved in Tollip/MyD88/NF-κB signaling. (d) The expression of ZO-1 and Occludin in Caco-2 cells under different conditions. (e) Representative images of Caco-2 cell immunofluorescence staining. Bars ‘ – ’ indicate 20 μm. IFN-γ, IFN-γ treated only; SST/IFN-γ, SST pretreated before IFN-γ treatment; IFN-γ/siTollip, siTollip transfected before IFN-γ treatment; IFN-γ/SST/siTollip, siTollip transfected and SST pretreated before IFN-γ treatment. One‑way ANOVA followed by Bonferroni post hoc test was used to compare the differences among different groups, ‘ns’ indicated no significance. ^nsP>0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. IFN-γ group; ^##P < 0.01, ^####P < 0.0001 vs. SST/IFN-γ group; ^@@P < 0.01, ^@@@P < 0.001, ^@@@@P < 0.0001 vs. IFN-γ/siTollip group.

Figure 5.

SST relieved intestinal epithelial barrier injury mediated by intestinal I/R inflammation by upregulating Tollip in rats. (a) The intestinal permeability detected by the content of FITC-dextran in serum. (b-c) Expression levels of the inflammatory factors IFN-γ and TNF-α in rat serum. (d) Representative images of rat intestinal tissues stained with H&E. Bars ‘ – ’ indicate 50 μm. (e) The expression of ZO-1 and Occludin in intestinal tissues. (f) Representative images of immunofluorescence staining of intestinal sections. Bars ‘ – ’ indicate 100 μm. I/RI, intestinal I/R injury group; I/RI/SST, I/RI with SST pretreatment; I/RI/siTollip, I/RI with siTollip injection; I/RI/SST/siTollip, I/RI with SST pretreatment and siTollip injection. n = 5 per group. One‑way ANOVA followed by Bonferroni post hoc test was used to compare the differences among different groups, ‘ns’ indicated no significance. ^nsP>0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. I/RI group; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, ^####P < 0.0001 vs. I/RI/SST group; ^@@P < 0.01, ^@@@P < 0.001, ^@@@@P < 0.0001 vs. I/RI/siTollip group.

Figure 6.

SST inhibited MyD88/NF-κB/MLCK signaling in the rat intestine by upregulating Tollip. (a) Western blotting results of Tollip/MyD88/NF-κB/MLCK signaling. (b-e) The relative protein levels measured by ImageJ software. (f) Summary of this study. I/RI, intestinal I/R injury group; I/RI/SST, I/RI with SST pretreatment; I/RI/siTollip, I/RI with siTollip injection; I/RI/SST/siTollip, I/RI with SST pretreatment and siTollip injection. n = 5 per group. One‑way ANOVA followed by Bonferroni post hoc test was used to compare the differences among different groups, ‘ns’ indicated no significance. ^nsP>0.05, *P < 0.05, ***P < 0.001, ****P < 0.0001 vs. I/RI group; ^####P < 0.0001 vs. I/RI/SST group; ^@@P < 0.01, ^@@@P < 0.001, ^@@@@P < 0.0001 vs. I/RI/siTollip group.