Role Of Epidermal Growth Factor On The Formation Of Viceral Hypersensitivity And Its’ Possible Mechanism Regulation Of GLP-1Receptor Agonist Exendin-4on SERT Expression And Its’ Possible Mechanism In IEC-6Cells

BackgroundIrritable bowel syndrome (IBS) is a common chronic functional gastrointestinal disease characterized by abdominal pain and discomfort with altered bowel habits. Now the mechanism of IBS still not completely understood. It is believed that visceral hypersensitivity and alterations in gut motility is responsible for the motor and sensory abnormalities in IBS patients. Recent reports indicate high levels of serotonin (5-HT) were related to the formation of visceral hypersensitivity. The serotonin transporter (SERT) is mainly localized to the apical membrane of intestinal epithelial cells and mediate reuptake of5-HT, so it plays an important role in terminating transmitter action and maintaining transmitter homeostasis. The abnormal expressions and function of the SERT may contribute to the pathophysiology of these gastrointestinal disorders, but the underlying mechanisms are not fully understood. Previous studies demonstrated that epidermal growth factor(EGF) can up-regulate the reuptake5-HT by increasing SERT expression, but the underlying mechanism is unknown. To date, there are no published studies elucidating the possible effect of EGF on visceral hypersensitivity by SERT mediation. Above all these, we hypothesized that EGF regulated SERT expression via EGFR and SERT consequently mediated the reuptake5-HT, which may be involved in visceral hypersensitivity. In this study, first, we established a rodent model of visceral hypersensitivity by colonic infusion of0.5%(v/v) acetic acid as previously described, and then evaluated the alterations in SERT expression in visceral hypersensitivity rat model. Finally, we analyzed the association of EGF level and SERT expressions in visceral hypersensitivity model rats. Furthermore, we investigate the effect of EGF on SERT expression and its’ function via EGFR in intestinal epithelial cells. We preliminarily explore the relationship of EGF and SERT protein expression and visceral hypersensitivity.Aims1.To observe the level of EGF in plasma and colonic tissues in established rats model of viceral hypersensitivity and analysis its relationship with the SERT protein expression in colon.2. To explore the effects of EGF on the expression and reuptake function of SERT in IEC-6cells.Methods1. Group:Twenty rats were randomly divided into two groups, including control and model group (n=10in each group).2. Establishment of a rat model:Male10-day-old rats (n=10) received colonic infusion of0.2ml0.5%(v/v) acetic acid solution in saline into the colon2cm from the anus. As a matched control group, the same aged pups (n=10) were infused with an equal volume of saline alone. To avoid the influence of circadian rhythms, All of the experiments were conducted at8:00am.3. Verification of the rat model:We assessed the visceral sensitivity of the rats by observing the abdominal withdrawal reflex (AWR) and recording electromyography (EMG) activity of the external oblique muscle in response to colorectal distension (CRD).4. Detection index:The visceral sensitivity of the rats were assessed by observing the abdominal withdrawal reflex (AWR) and recording electromyography (EMG) activity of the external oblique muscle in response to colorectal distension (CRD). The levels of EGF/5-HT in plasma and intestinal tissues were measured by ELISA. The expression of the serotonin transporter (SERT) protein detected by Western blotting.5. Cell culture:Rat intestinal epithelial crypt (IEC-6) cells were grown in Dulbecco’s Modified Eagle Medium supplemented with10%FBS.6. IEC-6cell mono-layers were treated with different concentrations of EGF for various times or pre-treated with10μM EGFR specific kinase inhibitor PD153035to block EGFR action, then SERT protein and mRNA were examined by western blotting and qPCR. SERT function was examined by tritiated serotonin (5-hydroxytryptamine) uptake experiments.Results1. Compared to control rats, a higher AWR scores and AUC were detected in the model rats (P<0.05). In addition, No evidence of inflammation or structural abnormalities was found both in the control and acetic acid-treated rats, these indicated that visceral hypersensitivity was successfully established.2. The concentrations of5-HT in plasma and colonic tissues were significantly increased in model rats compared with the control (P<0.05). Plasma and colonic tissues EGF levels in visceral sensitized rats were significantly decreased compared with control rats (P<0.01). SERT protein expressions in colon tissues were decreased compared with the control (P<0.05). Furthermore, we analyzed the relationship between EGF and SERT levels in colon tissues, and found that they were positively correlated (r=0.820, P<0.001).3. EGF up-regulated SERT expressions in a dose-dependent manner, and peaked at the dose of40ng/ml. SERT expression was up-regulated after treatment with EGF for12hours, and maximal effects were seen after treatment with EGF for24-48hours. Uptake of [3H]-5-HT was significantly higher in cells pre-treated with EGF (40ng/ml) for24h than in the solvent control (P=0.021).4. The EGF-induced up-regulation of SERT expression was blocked by PD153035.ConclusionEGF down-regulated SERT-mediated5-HT uptake into enterocytes and this maybe contribute to the development of visceral hypersensitivity. BackgroundIrritable bowel syndrome (IBS) is one of the most common functional gastrointestinal disorders, characterized by the abdominal pain or discomfort with altered bowel habits. The pathgenetic mechanism of IBS is still not completely understood. But therapeutic options of IBS are currently limited and are often disappointing in efficacy. Recently, some studies demonstrated that glucagon-like peptide-1(GLP-1) analogue was effective at relieving pain in IBS patients. However the mechanism was still remains unknown. A GLP-1receptor agonist exendin-4exhibits biologic actions similar to GLP-1and has a longer half-life. In addition,5-hydroxytryptamine (5-HT) as one of important neurotransmitters plays vital role in the transmission of viceral sensation and its’ regulation. Moreover in our previous study, we found exendin-4produced viceral analgesia, which might be mediated in part by endogenous5-HT pathways, is associated with increasing the colon SERT expressions in rats with viceral hypersensitivity. Previous study demonstrated that SERT gene expression was significantly regulatable by activation via PKA pathway. So in this experiment, we speculated whether exendin-4regulate SERT expression via cAMP-PKA pathways in intestinal epithelial cells.AimRat intestinal epithelial cell line IEC-6cells were used to investigate the effect of exendin-4on SERT expression and5-HT reuptake function, and further analysis the possible intracellular molecular mechanisms.Methods1.Cell culture:IEC-6cells were grown in Dulbecco’s Modified Eagle Medium supplemented with10%FBS.2.Effect of exendin-4on the expression of SERT and5-HT reuptake function:IEC-6cells were stimulated with different concentrations of exendin-4for12h and at various times by given in optimal concentration exendin-4. SERT protein、mRNA and5-HT reuptake function were evaluated by western blot, qRT-PCR and tritiated serotonin (5-hydroxytryptamine) uptake experiments.3.Influence of GLP-1R inhibitor exendin-9on SERT expression:IEC-6cells were pre-treated with exendin-9, then SERT protein expression was examined by western blot.4. IEC-6cells were treated with signaling pathways associated factors. The expressions of SERT were detected by western blotting. The PKA activity assay kit was used to detect the intracellular PKA activity.Results1. The expressions of SERT were up-regulated and5-HT reuptake function was enhanced in IEC-6cells after treated with exendin-4in dose-and time-dependently manner. The peak expression of SERT was showed at the12th hour after stimulating with10nM exendin-4, which was considered as the most effective concentration in vitro.2. The expression of SERT was down regulated by exendin-4, an inhibitor of GLP-1R.3. Forskolin up-regulated the SERT protein expression, as well as the effect of exendin-4, but all the effect could be abolished by pre-stimulated with SQ22536and H89.4. The intracellular PKA activity increased in the treatment of exendin-4and Forskolin. However the effect was absolutely disappeared by treated with SQ22536and H89in IEC-6cells.ConclusionExendin-4can not only enhance the expression of the SERT protein in intestinal epithelial cells by the AC-cAMP-PKA signaling pathway, but also can promote5-HT reuptake.