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

Wang P, Chen FX, Du C, Li CQ, Yu YB, Zuo XL, Li YQ - Sci Rep (2015)

Bottom Line: 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.We found a significant increase in PAR-2 expression in Caco-2 after IBS-D FSN stimulation.Moreover, we found that phosphorylation of p38 MAPK, not NF-κB p65, contributed to PAR-2-mediated BDNF overexpression.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Gastroenterology, Qilu Hospital, Shandong University, Jinan 250012, P.R. China [2] Laboratory of Translational Gastroenterology, Qilu Hospital, Shandong University, Jinan 250012, P.R. China.

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.

No MeSH data available.


Related in: MedlinePlus

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).
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f4: 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).

Mentions: According to the available literature, PAR-2 activation in Caco-2 cells activates p38 MAPK to facilitate IL-8 expression without directly activating NF-κB12. Thus, to further identify the downstream pathway of PAR-2 activation in inducing BDNF overexpression in Caco-2 cells, we focused on MAPK p38 and NF-κB p65. We found that IBS-D FSN induced a rapid phosphorylation of p38 MAPK after 15 min of stimulation, although total p38 MAPK appeared unchanged. Preadministration of Caco-2 with PAR-2 selective inhibitor ENMD-1068 clearly blocked the effect of IBS-D FSN on p38 phosphorylation (Fig. 4a,b). However, we did not observe a significant change in levels of either phosphorylated p65 or total p65 before and after administration of IBS-D FSN (Fig. 4c). Furthermore, pretreatment with specific inhibitor of p38-MAPK SB203580, but not NK-κB specific blocking agent PDTC, prevented IBS-D FSN elevating BDNF protein levels (Fig. 4d).


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

Wang P, Chen FX, Du C, Li CQ, Yu YB, Zuo XL, Li YQ - Sci Rep (2015)

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).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4441152&req=5

f4: 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).
Mentions: According to the available literature, PAR-2 activation in Caco-2 cells activates p38 MAPK to facilitate IL-8 expression without directly activating NF-κB12. Thus, to further identify the downstream pathway of PAR-2 activation in inducing BDNF overexpression in Caco-2 cells, we focused on MAPK p38 and NF-κB p65. We found that IBS-D FSN induced a rapid phosphorylation of p38 MAPK after 15 min of stimulation, although total p38 MAPK appeared unchanged. Preadministration of Caco-2 with PAR-2 selective inhibitor ENMD-1068 clearly blocked the effect of IBS-D FSN on p38 phosphorylation (Fig. 4a,b). However, we did not observe a significant change in levels of either phosphorylated p65 or total p65 before and after administration of IBS-D FSN (Fig. 4c). Furthermore, pretreatment with specific inhibitor of p38-MAPK SB203580, but not NK-κB specific blocking agent PDTC, prevented IBS-D FSN elevating BDNF protein levels (Fig. 4d).

Bottom Line: 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.We found a significant increase in PAR-2 expression in Caco-2 after IBS-D FSN stimulation.Moreover, we found that phosphorylation of p38 MAPK, not NF-κB p65, contributed to PAR-2-mediated BDNF overexpression.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Gastroenterology, Qilu Hospital, Shandong University, Jinan 250012, P.R. China [2] Laboratory of Translational Gastroenterology, Qilu Hospital, Shandong University, Jinan 250012, P.R. China.

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.

No MeSH data available.


Related in: MedlinePlus