Epidermal growth factor upregulates serotonin transporter and its association with visceral hypersensitivity in irritable bowel syndrome

Figure 1 Effect of neonatal acetic acid treatment on 8-wk-old rat sensitivity to colorectal distension. A: Abdominal withdrawal reflex (AWR) responses to the graded pressures of colorectal distension (CRD) in saline-treated (n = 10) and acetic acid-treated (n = 10) rats. Acetic acid-treated rats show increased AWR scores compared with the saline rats. Values are expressed as mean ± SD; B: Representative electromyogram (EMG) traces recorded in control and acetic acid-treated rats in response to CRD; C: EMG responses to CRD in rats treated with saline and acetic acid at the neonatal stage. Similar to the AWR scores, acetic acid-treated rats exhibited exaggerated EMG activity responses to CRD at different pressures compared with the saline-treated rats. Neonatal rats vs control rats, ^aP < 0.05, ^bP≤ 0.01, error bars represent the mean ± SD.

Figure 2 Epidermal growth factor levels in plasma and colon tissue of the visceral-sensitized rats. A: Plasma epidermal growth factor (EGF) levels were significantly lower in visceral-sensitized rats than in controls (visceral hypersensitive group rats vs control group rats, ^bP < 0.01); B: EGF levels in colon were significantly lower in visceral-sensitized rats than in controls (visceral-sensitized rats vs control rats, ^dP < 0.01). Error bars represent the mean ± SD (n = 10 in each group).

Figure 3 Serotonin transporter levels in colon tissues of control and visceral-sensitized rats. A: Western blot of serotonin transporter (SERT) expression in colonic tissues in both acetic acid- and saline-treated rat groups; B: Quantitation of SERT protein in rat colonic tissue in both acetic acid- and saline-treated rat groups compared to GAPDH. The SERT protein expressions in colon tissues were significantly lower in visceral-sensitized rats than in the controls (visceral-sensitized rats vs controls, ^bP < 0.01) (n = 10 in each group).

Figure 4 Effects of epidermal growth factor on serotonin transporter in rat intestinal epithelial cells. Intestinal epithelial cells (IEC-6) cells were treated with epidermal growth factor (EGF) (0, 20, 40, 60, and 80 ng/mL) for 24 h. A: Western blots were performed to detected serotonin transporter (SERT) protein expressions. GAPDH was used to verify equivalent protein loading (^aP < 0.05 vs control; ^cP < 0.05 vs 20, 80, and 160 ng/mL); B: SERT gene expression was examined by real-time PCR (^aP < 0.05 vs control; ^eP < 0.05 vs 20 and 80 ng/L). To determine the optimal time for EGF treatment, IEC-6 cells were treated with EGF (40 ng/L) for the indicated times (0, 3, 6, 12, 24, and 48 h); C: SERT protein levels were examined by Western blot (^aP < 0.05 vs control); D: SERT gene expression was examined by real-time PCR (^aP < 0.05 vs control; ^gP < 0.05 vs 48 h); E: Uptake of [³H]-serotonin in cells pre-treated with 40 ng/ml EGF for 24 h (^IP < 0.05 vs 24 h) . All values are mean ± SD of three independent experiments.

Figure 5 Role of epidermal growth factor receptor in the regulation of serotonin transporter levels and function in intestinal epithelial cells. IEC-6 cells were pre-treated with an epidermal growth factor receptor (EGFR) inhibitor (10 μmol/L PD153035) prior to stimulation with epidermal growth factor (EGF). A: Serotonin transporter (SERT) was detected by Western blot (^aP < 0.05 vs control, EGF + PD153035, and PD153035) and quantified relative to GAPDH; B: Serotonin (5-HT) reuptake was estimated by an [³H]-5-HT uptake assay. Data are mean ± SD of at least three independent experiments.