Stress is one of the primary factors associated with the onset, exacerbation, and reactivation of many GI disorders.1011 The detail mechanisms by which stress leads to disordered GI functions still remain to be discovered. However, strong experimental evidence suggests that corticotropin releasing factor (CRF) and its receptors in the brain facilitate stress-related alterations in GI motility.12131718 Central injection of CRF ligands in experimental animals reproduces stress related alterations of gut motor function, whereas CRF antagonist administration prevents the effects of various stressors.12 In addition to the brain, CRF ligands and receptors are widely expressed in enteric nervous system neurons and other cells of the GI tract in animals and humans.19202122 Remarkably, peripheral administration of CRF ligands was found to mimic changes similar to that induced by stress, while its antagonists alleviate stress-induced alteration of GI function.2324 This indicates the co-existence of a peripheral and central CRF signaling pathway, and together, they might play critical role in stress-related alterations of gut function. Several studies reported differential action of CRF ligands upon interaction with different CRF receptor subtypes on upper and lower GI motility.2526

Apart from CRF induced stress, water avoidance stress (WAS) is also used as a model to study psychological stress.14151627 Enck, et al.28 reported that WAS delays gastric emptying, while Nozu, et al.16 demonstrated enhanced gastric contractions are mediated by peripheral CRF1 receptors. However, several studies have shown that WAS augments lower GI transit.141528 In the colon, WAS stimulates motility mediated by the central CRF pathway.141528 However, the relationship between stress and impairment of GI motor activity has not been fully elucidated. Therefore, the aim of the present study was to determine the effect of peripheral CRF peptide and water avoidance induced stress on the colonic and the gastric transit in the guinea pig model.

Adult male Hartley guinea pigs (250–350 g, Orient Bio, Inc., Seoul, Korea) were acclimatized to their holding room. A standard guinea pig diet and drinking water were provided ad libitum. All experiments were conducted in accordance with the guidelines of animal laboratory Ethics Committees of the Department of Laboratory Animal Medicine, Medical Research Center, Yonsei University College of Medicine. This study was approved by the Institutional Animal Care and Use Committee of Yonsei University College of Medicine with IRB protocol number 2015-0221.

The following chemicals and drugs were used: isotonic sodium chloride solution (Dai Han Pharmaceuticals, Seoul, Korea), charcoal (Sigma, Milwaukee, WI, USA), barium sulfate (Taejoon Pharmaceuticals, Seoul, Korea), r/h CRF (Peptide Institute, Inc., Osaka, Japan) and Astressin (Sigma, St. Louis, MO, USA).

The effect of CRF treatment on upper GI transit was tested by charcoal transit assay in fasted guinea pigs. r/h CRF peptides of different concentrations (1, 3, and 10 µg/kg) and vehicle (saline) were injected intraperitoneal (IP) in guinea pigs. CRF receptor blockade by astressin and the effect of CRF on the upper GI transit was measured by charcoal transit assay. Guinea pigs were IP injected with different doses of astressin (1, 3, and 10 µg/kg) and vehicle (saline) and incubated for 15 minutes. Subsequently, each of these groups was injected with 10 µg/kg of IP CRF. After 30 minutes of stabilization, 3 mL of charcoal mixture was administered via orogastric cannula. 10 minutes after incubation with charcoal mix, guinea pigs were sacrificed to evaluate the charcoal migration. The effect of CRF treatment on lower GI transit was measured by fecal pellet output (FPO) assay in non-fasted guinea pigs. r/h CRF peptides of different concentrations (1, 3, and 10 µg/kg) and vehicle were injected IP in guinea pigs. Guinea pigs were IP injected with different concentrations of astressin (1, 3, and 10 µg/kg), and after 15 minutes of stabilization, 10 µg/kg of CRF was injected IP in each group. Each guinea pig was incubated into an individual experimental cage, and cumulative number and weight of fecal pellets expelled were measured and recorded for 3 hours. WAS was performed as described by Bonaz and Taché14 and Miwa, et al.27 Stress model guinea pigs were acclimatized on the platform in a box 1 hour daily for 3 days, and on the next 2 days, were given WAS. Sham stressed group was acclimatized on the platform for 1 hour daily for 5 days. A control group was not subjected to any kind of stress and kept in housing cages only. After WAS exposure for 1 hour on day 5, guinea pigs were administered charcoal mix, incubated for 15 minutes, and then sacrificed and evaluated for upper GI transit. Lower GI transit in stress model was measured by cumulative fecal pellets in the course of the 4th and 5th days of WAS. Statistical analysis was performed using repeated measures ANOVA with post hoc comparison. In all tests, statistical significance was assigned at p<0.05. All data were analyzed using SPSS software, version 12.0 for Windows (SPSS Inc., Chicago, IL, USA).

The effect of different doses (1, 3, and 10 µg/kg) of CRF peptide was tested in upper GI transit by charcoal transit assay. As shown in Fig. 1, the percent (%) charcoal migration (cm) in control group (55.6±19.9) was relatively similar to that of CRF 1 µg/kg (64.1±9.2) and 3 µg/kg (57.9±22.7) groups. However, IP CRF 10 µg/kg injected guinea pigs showed significant inhibition of upper GI transit, compared to control (6.4±2.2 vs. 55.6±19.9) (Fig. 1). Similarly, the effect of different doses of CRF peptide on lower GI transit was analyzed by FPO assay. As shown in Fig. 2A, the number of fecal pellets expelled in the control group was 7.8±4.4, whereas those in the CRF (1, 3, and 10 µg/kg) IP injected groups were 9.8±4.5, 19.6±1.8, and 22.1±13.8, respectively. The data clearly showed a dose dependent increase in the FPO in CRF injected guinea pigs. As compared to controls, CRF 3 and 10 µg/kg treated guinea pigs significantly increased the cumulative fecal pellets and weight (Fig. 2). Upper and lower GI transit experiments in this study confirmed 10 µg/kg of IP CRF was the most effective dose that can impact both upper and lower GI transit in the experimental model of guinea pig. Next, the effect of astressin was tested on this model. As shown in Fig. 3, pretreated guinea pigs with different doses (1, 3, and 10 µg/kg) of astressin significantly increases the upper GI transit, compared to only CRF 10 µg/kg treated model. In contrast, the effects of different doses (1, 3, and 10 µg/kg) of astressin on lower GI transit significantly reduced FPO, compared to only the CRF 10 µg/kg treated model (Fig. 4). WAS experiment was divided into three different groups: control, sham stress, and stress model. Charcoal migration assay showed that WAS had an insignificant effect on upper GI transit among different groups (Fig. 5). In contrast, WAS significantly accelerated lower GI transit (Fig. 6). The cumulative FPO of the stress model was more than 3 fold compared to sham stress and control groups (p<0.05) (Fig. 6).

Several studies demonstrated that patients with one of the GI diseases often suffer from a second overlapping disease.29303132 A recent population based study from China based on Rome III criteria showed 33% overlap of patients of IBS in FD patients and 45.9% of FD in IBS patients.30 Again a study from Japanese general population showed the prevalence of overlap of FD and/or IBS in gastroesophageal reflux disease (GERD), GERD, and/or IBS in FD, and GERD and/or FD in IBS were 46.9, 47.6, and 34.4% respectively.2 Apart from high prevalence, our previous study, as well as several other reports, have suggested a health related decline in quality of life and higher severity symptom score among patients of FD-IBS overlap.22930313233 Despite higher incidence, there is no successful animal model with face and construct validity for human syndrome. Therefore, it was imperative to develop an animal model that could mimic more than one symptom of the human overlap syndrome and could be used for the identification of molecular targets in drug development.

Emerging evidence links the activation of CRF receptors with stress-related alterations of gastric and colonic motor function.34353637 In order to establish an experimental animal model of overlap syndrome, the present study targeted CRF receptors to analyze its effects on upper and lower GI transit in conscious guinea pigs. This study showed that 10 µg/kg IP CRF significantly inhibited upper GI transit, compared to controls. In contrast, IP CRF led to dose dependent acceleration of lower GI transit, compared to controls. Therefore, our data clearly indicated that peripheral administration of CRF simultaneously exerts an inhibitory effect on upper gastric transit and stimulatory effect on colonic propulsive activity. Our findings support earlier observations that central or peripheral CRF suppresses gastric emptying and stimulates colonic transit.24252634353637

To validate whether CRF indeed alters gastric and colonic transits through modulation of peripheral CRF receptors, different doses of CRF receptor antagonist astressin were injected IP in conscious guinea pigs. Our data showed that even the lowest dose (1 µg/kg) of astressin significantly blocks exogenous CRF-induced alterations of gastric and colonic transits. Hence, our study confirms that CRF acts through peripheral CRF pathways and corroborated earlier findings indicating the differential role of non-selective CRF peptide on the gastric emptying and colonic transit.34353637 Several studies demonstrated the selective CRF action on the activation of CRF2 receptors in the inhibition of gastric emptying and CRF1 receptors in the stimulation of colonic transit.343839 Thus, these results strongly suggest that CRF receptor subtypes 2 and 1 may be activated simultaneously during stress or during central and peripheral administration of CRF.34353637

The WAS model was applied to analyze the role of central CRF pathways in GI motility. The WAS model represents psychological stress condition in guinea pigs.27 Our study clearly showed an increase in cumulative FPO in stress model, compare to sham stress and control groups. This corroborates earlier findings that WAS stimulates colonic motility mediated by the central CRF pathway.14272840 Surprisingly, there was an insignificant difference in upper GI transit among the control, sham stress, and stress models. This is in contrast to earlier findings that indicated delayed gastric emptying due to WAS.28 We speculate that acute WAS may fail to produce a certain high threshold level of CRF to modulate the upper GI motility. Our peripheral CRF data (Fig. 1) precisely indicated high dose (10 µg/kg) and inhibition of upper GI transit. In addition to vital threshold dose, the distribution of CRF receptor subtypes (1 and 2) may induce opposite actions on upper and lower GI transit.2534

In conclusion, peripheral r/h CRF was found to induce opposite actions on upper and lower gut transit in conscious guinea pigs. WAS involved central CRF signaling pathway stimulated colonic propulsive activity, but failed to inhibit upper GI transit. CRF peptides were shown to mediate through interactions with peripheral CRF receptors that have physiologic relevance in acute psychological stress-induced overlap syndrome. This overlap syndrome model may provide new venues and construct validity for the identification of molecular targets/biomarkers under stress-related activation of CRF pathways.