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. 2015 May 22:5:10121.
doi: 10.1038/srep10121.

Increased production of BDNF in colonic epithelial cells induced by fecal supernatants from diarrheic IBS patients

Peng Wang  1 , Fei-Xue Chen  1 , Chao Du  1 , Chang-Qing Li  1 , Yan-Bo Yu  1 , Xiu-Li Zuo  1 , Yan-Qing Li  1
Affiliations

Increased production of BDNF in colonic epithelial cells induced by fecal supernatants from diarrheic IBS patients

Peng Wang et al. Sci Rep. .

Abstract

Colonic brain-derived neurotrophic factor (BDNF) plays an essential role in pathogenesis of abdominal pain in diarrhea-predominant irritable bowel syndrome (IBS-D), but regulation on its expression remains unclear. We investigated the role of fecal supernatants (FSN) from IBS-D patients on regulating BDNF expression in colonic epithelial cells of human and mice. Using human Caco-2 cells, we found that IBS-D FSN significantly increased BDNF mRNA and protein levels compared to control FSN, which were remarkably suppressed by the serine protease inhibitor. To further explore the potential mechanisms, we investigated the impact of protease-activated receptor-2 (PAR-2) on BDNF expression. We found a significant increase in PAR-2 expression in Caco-2 after IBS-D FSN stimulation. Knockdown of PAR-2 significantly inhibited IBS-D FSN-induced upregulation of BDNF. Moreover, we found that phosphorylation of p38 MAPK, not NF-κB p65, contributed to PAR-2-mediated BDNF overexpression. To confirm these results, we intracolonically infused IBS-D or control FSN in mice and found that IBS-D FSN significantly elevated colonic BDNF and visceral hypersensitivity in mice, which were both suppressed by the inhibitor of serine protease or antagonist of PAR-2. Together, our data indicate that activation of PAR-2 signaling by IBS-D FSN promotes expression of colonic BDNF, thereby contributing to IBS-like visceral hypersensitivity.

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Figures

Figure 1
Figure 1. Serine protease activity was increased in fecal supernatants (FSN) from IBS-D patients.
The serine protease inhibitor FUT-175 markedly inhibited protease activity released from IBS-D FSN (n = 17 for HC FSN, n = 22 for IBS-D FSN. ***P < 0.001).
Figure 2
Figure 2. IBS-D fecal supernatants (FSN) elevated expression of BDNF in Caco-2 cells.
(a) BDNF mRNA levels. (b) BDNF protein levels. Preincubation of IBS-D FSN with FUT-175 significantly attenuated the effect of IBS-D FSN on BDNF expression in Caco-2 cells. (**P < 0.01, ***P < 0.001 vs. saline; ##P < 0.01, ###P < 0.001 vs. saline).
Figure 3
Figure 3. Protease-activated receptor-2 (PAR-2) was involved in IBS-D fecal supernatants (FSN)-induced BDNF overexpression in Caco-2 cells
(a and b) Western blotting of PAR-2 in Caco-2 cells (*** P < 0.001). (c) siRNA-mediated PAR-2 knockdown in Caco-2 cells. (d) BDNF protein levels secreted by Caco-2 cells after PAR-2 knockdown. (e) Effect of IBS-D FSN on PAR-2 expression in siRNA-transfected Caco-2 cells. (f) BDNF protein levels secreted by Caco-2 cells after PAR-2 knockdown followed by IBS-D FSN stimulation (** P < 0.01, PAR-2 siRNA- vs. control siRNA-transfected cells after IBS-D FSN treatment). The gels were run under the same experimental conditions. Cropped gels/blots are presented (full-length gels/blots are shown in suppl. Fig. S1 with indicated cropping lines).
Figure 4
Figure 4. Phosphorylation of p38 MAPK but not p65 NF-κB signaling contributed to IBS-D fecal supernatants (FSN)-triggered BDNF expression in Caco-2 cells.
(a b and c) Western blotting of phospho-p38, phospho-p65, total p38 and total p65 protein levels in Caco-2 cells. (d) ELISA analysis of BDNF levels in Caco-2 cells after treatment with IBS-D FSN, and preadministration of SB203580 or PDTC followed by IBS-D FSN stimulation (** P < 0.01). The gels were run under the same experimental conditions. Cropped gels/blots are presented (full-length gels/blots are shown in suppl. Fig. S1 with indicated cropping lines).
Figure 5
Figure 5. IBS-D fecal supernatants (FSN) induced colonic BDNF overexpression and visceral hypersensitivity in mice.
(a and b) Western blotting of BDNF expression in colon of mice (***P < 0.001). (c) Immunohistochemical staining of BDNF in epithelium (Black arrowheads) and mucosa in colon of mice (Bars: 50 μm). (d) Measurement of visceral sensitivity to colorectal distension in mice by using electromyography recording (*P < 0.05, ***P < 0.001 IBS-D FSN vs. control FSN; †††P < 0.001, IBS-D FSN + FUT-175 or ENMD-1068 or TrkB/Fc vs. IBS-D FSN; n = 8 per group; AUC: area under curve). The gels were run under the same experimental conditions. Cropped gels/blots are presented (full-length gels/blots are shown in suppl. Fig. S1 with indicated cropping lines).
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