Effect Of Corticotropin Releasing Factor On The Intestinal Barrier

BackgroudIrritable bowel syndrome (IBS) is chronic functional gastrointestinal disease associated with recurrent abdominal pain and changing bowel habits, lacking the presence of organic pathologic changes. IBS is one of the most studied functional gastrointestinal disorders (FGIDs), which may be related to psychological stress and brain-gut axis dysfunction. Stress can disturb the intestinal barrier and increase the intestinal permeability by damaging the brain gut axis, which leading to intestinal diseases, such as stress can aggravate the symptoms of IBS. Corticotropin releasing factor (CRF), one of the hormones in the brain gut axis, increased in the process of stress. The intestinal barrier is including intestinal mucus barrier and intestinal epithelial barrier, which are very important to maintain the function of intestine. Some secretory products synthesed by goblet cells are important compositions of intestinal mucosal barrier. Tight junction proteins among the epithelial cells are also important to the intestinal epithelial barrier. LS174T cells are human colon adenocarcinoma cells, which have phenotype and characteristics of goblet cells and can synthesize and secrete mucin and other products related to mucin secretion. LS174T cells can be used to study the function of goblet cells. LS174T cells are also epithelial cells, and can be used to study the function of intestinal epithelial barrier. Mental stress is a dangerous factor which disturbing the intestinal barrier. So how mental stress affect intestinal barrier by regulating the function of goblet cells and tight junction proteins via CRF remains unclear. In our study, we speculated that CRF could damage intestinal barrier by regulating the function of goblet cells and tight junction proteins among the epithelial cells.ObjectiveTo investigate the effects of CRF on the function of intestinal barrier by regulating the secretory products of intestinal goblet cells and tight junction proteins among the epithelial cells.Methods1. LS174T cells were cultured in our experiment and were treated with different concentration (10-9,10-10,10-11,10-12 mol/L) of corticotropin releasing factor (CRF) for 6 h to determine the optimum concentration, PBS were used as control group. LS174T cells were treated with 10-10 mol/L CRF and PBS for 6 h and 24 h, PBS were used as control group.2. The mRNA levels of LS174T cells secretory products, including MUC2, FUT2 and TFF3, were determined by Real-Time qPCR.3. Real-Time qPCR was used to detect the effect of CRF on the mRNA expression levels of MUC2, FUT2, TFF3, claudin-1, claudin-2, occludin and ZO-1 in LS174T cells.4. Western blot were used to detect the effect of CRF on the protein levels of FUT2, TFF3, claudin-1, claudin-2, occludin and ZO-1 in LS174T cells.5. Immunofluorescent and ELISA kit were used to measure the effect of CRF on the protein levels of MUC2 in LS174T cells.Results1. Real-Time qPCR results showed that different concentration of CRF produced different effects on MUC2, FUT2 and TFF3 of LS174T cells. CRF at 10-10 mol·L-1 significantly decreased goblet cell secretory products, compared with the PBS control group.2. Real-Time qPCR results showed that 10-10 mol·L-1 CRF decreased the mRNA expression levels of MUC2 (P< 0.01), FUT2 (P< 0.001) and TFF3 (P< 0.0001) in LS174T cells at 6 h, compared with the PBS control group. The mRNA expression of MUC2 and FUT2 had no significant difference (NS) in CRF treated LS174T cells for 24 h, while the mRNA expression of TFF3 were still decreased (P< 0.001).3. Cell immunofluorescence and ELISA results showed that CRF reduced the protein expression of MUC2 (P< 0.05) in LS174T cells for 6 h, while the protein expression of MUC2 had no significant difference (NS) in LS174T cells for 24 h, compared with the PBS control group.4. Western blot results showed that the protein expression of TFF3 is declining (P< 0.001) in CRF treated LS174T cells for 6 h, and FUT2 and TFF3 protein levels were decreased (P< 0.001) in CRF treated LS174T cells for 24 h, compared with the PBS control group.5. Real-Time qPCR results showed that the mRNA expression of claudin-2 increased under the 10-10 mol/L CRF at 6 h (P< 0.001), while the mRNA expression of occludin and ZO-1 decreased in LS174T cells at 24 h (P< 0.001).6. Western blot analysis also demonstrated that CRF increased abundance of claudin-2 at 6 h (P< 0.001) as well as decreased occludin and ZO-1 at 24 h (P< 0.001) in LS174T cells.7. In addition, both the mRNA and protein levels of claudin-1 had no significant difference in LS174T cells at 6 h (NS) compared to PBS control groups under the effect of CRF.Conclusion1. CRF can disturb the composition of the intestinal mucosal barrier by downregulating the expression levels of MUC2, FUT2 and TFF3, which go against maintaining the function of the intestinal mucosal barrier.2. CRF can break the composition of tight junction proteins among cells by increasing claudin-2 and decreasing occludin and ZO-1, which are harmful for keeping the integrity of the intestinal epithelial barrier.3. Mental stress can damage the functions of the intestinal barrier, which may due to the effects of CRF on the intestinal goblet cell secretory products and the disordered tight junction proteins among the epithelials.