Analysis of intestinal motility function regarding the combined effect of mosapride and trehalose in a paralytic ileus animal model

Abstract

Postoperative ileus (POI), commonly induced by intestinal manipulation (IM) during laparotomy, results in intestinal wall injury, inflammation, adhesion, and impaired gastrointestinal motility. This study evaluated the effects of mosapride citrate (Mos), a 5-HT₄ receptor agonist, and trehalose (Tre), a cytoprotective disaccharide, on gastrointestinal transit in an IM-induced POI mouse model. Mature male C57BL/6J mice were subjected to IM and allocated into five groups: IM + vehicle (control), IM + Mos (0.2 mg/kg, two times), IM + Tre (100 mg/kg, two times), IM + combined Mos & Tre, and IM + sequential Tre + Mos treatment. Gastrointestinal transit was quantified using phenol red method. All four treatment groups exhibited a significant increase in phenol red recovery in the lower small intestine compared with the control group (P<0.05). Notably, sequential administration (Tre + Mos) resulted in greater dye progression beyond the manipulated region than that of simultaneous (Mos & Tre) treatment (P<0.05). These findings suggest that although both agents individually promote gastrointestinal transit, the therapeutic effect is enhanced when Tre is administered first, followed by Mos. Thus, the timing and sequence of administration play a critical role in the treatment of POI. Sequential Tre + Mos therapy may represent a promising strategy for accelerating the functional restoration of gastrointestinal transit after intestinal injury.

INTRODUCTION

Postoperative ileus (POI) is a common complication of abdominal surgery and primarily caused by inflammation, adhesion formation, and gastrointestinal motility disorders resulting from intraoperative intestinal manipulation (IM). In terms of ileus classification, approximately 90% of cases are caused by postoperative adhesions or mechanical ileus due to cancer, and the remaining 10% are classified as functional ileus [28]. Paralytic ileus, characterized by impaired intestinal motility, may be induced by drugs (e.g., opioids, anticancer agents, and immunosuppressants), inflammation (e.g., leukocytosis), or metabolic disturbances (e.g., electrolyte imbalances) [28]. Early detection and prompt intervention are crucial. The pathophysiology of paralytic ileus involves initial neurogenic processes such as sympathetic nerve activation and inhibition of intestinal nerve reflex pathways due to IM [25], followed by inflammatory processes as a result of persistent leukocyte infiltration [19] and postoperative use of analgesics (e.g. μ-opioid drugs) [3], among other factors contributing to delayed motility disorders. Additionally, intraoperative ischemia-reperfusion injury [13] and oxidative stress [27] exacerbate intestinal damage and adhesion. Currently, research is underway on the prevention of POI using prokinetic agents, anti-inflammatory drugs, and antioxidants, which have a lower potential to impair intestinal motility.

Mosapride citrate is a selective serotonin 5-HT₄ receptor agonist that promotes the release of acetylcholine from the myenteric plexus and improves peristalsis in the gastrointestinal tract [43]. Mosapride citrate is widely used as a gastrointestinal stimulant for patients with functional gastrointestinal disorders (FD) who complain of nonspecific symptoms, such as nausea, without accompanying organic disorders such as bleeding or ulcers [14, 17]. In a POI model, mosapride citrate has been reported to not only restore gastrointestinal motility but also exhibit anti-inflammatory effects mediated by the cholinergic anti-inflammatory immune mechanism [42] in the gastrointestinal muscle layer [41]. In addition, it has also been reported to exhibit anti-ulcer effects in an experimental gastric ulcer model [9]. Clinical studies and case reports have also shown that administration of 5-HT4 agonist in patients with postoperative or paralytic ileus improves defecation and helps shorten hospital stay [12]. Thus, 5-HT4 receptor stimulation is considered an effective therapeutic strategy in paralytic ileus in both experimental and clinical settings.

Trehalose is a non-reducing disaccharide widely distributed in nature and is involved in desiccation tolerance and moisture retention in plants [5] and animals [32], as well as in the maintenance of blood glucose levels. Currently, artificially synthesized trehalose is used as an additive in food [4] and cosmetics [26]. Although vertebrates are unable to biosynthesize trehalose, its physiological effects, including anti-inflammatory [22], antioxidant [38], cell-protective, stress resistance properties, and autophagy induction, have been reported in these organisms [22]. Clinically, trehalose has been reported to improve the quality of life (QOL) of patients with lysosomal storage diseases, such as mucopolysaccharidosis [23], promote wound healing, suppress inflammation in fracture patients [44], and improve oral dryness [31] and metabolic function [30]. Therefore, trehalose is considered a safe and promising adjunct for the management of various diseases. In recent years, trehalose has also been shown to effectively suppress intra-abdominal adhesions after open abdominal surgery [8, 35], and is expected to contribute to the prevention of POI by improving postoperative gastrointestinal motility. However, few studies have evaluated the effects of trehalose alone or in combination with mosapride citrate on intestinal motility.

In this study, we used a mouse model of postoperative intestinal paralysis to evaluate the effects of the co-administration of mosapride and trehalose, with the aim of restoring gastrointestinal transit in a safe and cost-effective manner and the evaluation focused on assessing intestinal transit in vivo.

MATERIALS AND METHODS

Animals

The experimental protocols and animal care complied with the guidelines outlined in the Guide to Animal Use and Care of Nippon Veterinary and Life Science University (approval codes: 2022K-52, 2023K-42, 2024K-48, 2025K-29). Animal studies were reported in accordance with the ARRIVE guidelines for reporting in vivo experiments [21, 37]. Mature male C57BL/6J mice were used in all experiments after acclimatization to standard laboratory conditions (12:12-hr light-dark cycle, temperature 22 ± 1°C). They were kept in TPX floor flat-bottom cages (W22 × D32 × H13.5 cm) containing small soft fir wood chips (Soft tip, SLC Japan, Inc., Hamamatsu, Japan). The animals were allowed free access to a standard rodent diet (EF; Oriental Yeast Co., Ltd., Tokyo, Japan) and water. They were kept in groups of up to five per cage.

Preparation and administration of drugs

The drugs/reagents used in this study were trehalose (Tre; FUJIFILM Wako Chemicals, Osaka, Japan), mosapride citrate (Mos; Sumitomo Pharma Ltd., Osaka, Japan), medetomidine hydrochloride (Nippon Zenyaku Kogyo Ltd., Fukushima, Japan), midazolam hydrochloride (Teva Takeda Pharma Ltd., Nagoya, Japan), butorphanol tartrate (Meiji Seika Pharma Ltd., Tokyo, Japan), atipamezole hydrochloride (Kyoritsu Seiyaku Ltd., Tokyo, Japan), isoflurane (Viatris Inc., Tokyo, Japan), D-glucose, phenol red (FUJIFILM Wako Chemicals), and physiological saline solution (Otsuka Pharmaceutical Factory Inc., Tokushima, Japan). Each drug was prepared before use and administered either intraperitoneally or subcutaneously to mice at a volume of 5 mL/kg body weight. Tre and Mos were diluted in a saline solution.

Experimental design

A total of 47 male C57BL/6J mice were divided into five groups: IM + physiological saline (10 mL/kg) (control), IM + Mos (0.2 mg/kg, 10 mL/kg, two times), IM + Tre (100 mg/kg, 10 mL/kg, two times) [35], IM + combined Tre & Mos (100 mg/kg + 0.2 mg/kg, 10 mL/kg, two times) (IM + Tre & Mos), IM + sequential Tre (100 mg/kg, 10 mL/kg) & Mos (0.2 mg/ kg, 10 mL/kg) (IM + Tre + Mos) group. Each solution was administered once around the surgical site at the time of surgery, followed by a second intraperitoneal dose 24 hr postoperatively. The phenol red method was used to determine intestinal transit, and group comparisons were performed at 48 hr (Fig. 1A).

Fig. 1.

Experimental protocol. Following a 12-hr fast, IM surgery was performed on the ileum, and various drugs were applied to the surgical site. Drug treatment was repeated 24 hr post-surgery, and an intestinal transition assay was performed 48 hr after surgery (A). The surgical site was located in the anterior segment of the lower small intestine (white area) (B).

Intestinal manipulation

The POI model was established using IM as previously reported [41]. The animals were anesthetized with a three-anesthetic mixture solution (medetomidine 0.75 mg/kg, midazolam 4 mg/kg, butorphanol 5 mg/kg; 0.05 mL/10 g, subcutaneously). After confirming sedation and analgesia, animals were placed in the supine position, abdominal coats were shaved (Feather Razor S double-edged blade, Feather Inc.), and the skin was disinfected with 50% isopropanol. The entire area was covered with sterile gauze (One Way Gauze Alpha, Daiei Co., Ltd., Osaka, Japan) and lightly moistened with saline solution (20 mL Otsuka Seishoku^®, Otsuka Pharmaceutical). The skin and abdominal wall were incised 1.5 cm above the midline with an ophthalmic shear and held with ring tweezers (New Vision, Tokyo, Japan) to expose the distal ileum of the small intestine approximately 10 to 15 cm from the cecum to the rostrum outside the abdominal cavity. IM treatment was applied to the serosal side of the muscle layer using a sterile cotton swab moistened with sterile physiological saline, with four round trips per site, to avoid damage to the microcirculatory system of the small intestine (Fig. 1B). Following the direct application of Mos (0.02 mg/mL, 300 μL), Tre (10 mg/mL, 300 μL), or their combination onto the surgical site, the abdomen was subsequently closed. Intraoperative and postoperative heating was performed using a hot plate, and saline solution (0.3 mL per head) was administered subcutaneously to prevent dehydration.

Intestinal transit determination

Of the two non-surgical groups, the nontreatment group was administered a 0.05% phenol red/5% glucose solution orally immediately after fasting. The trehalose-treated group, in contrast, administered the dye one hour after trehalose treatment. At 48 hr post-surgery of the five surgical groups, phenol red dye was administered. All groups, 1 hr after dye administration, the stomach, small intestine, cecum, and colon were excised, and the residual dye concentration in the lumen was detected and quantified at an excitation wavelength of 560 nm. The data were analyzed using Gen5^TM software (Synergy HTX, BioTek, Winooski, VT, USA). Phenol red concentration was determined in each of the 15 sections (stomach, 10 sections of the small intestine, cecum, and three sections of the large intestine). The concentrations were summed for each localized section as appropriate, and the percentage of each section was calculated based on 100%. The geometric center (GC) of distribution was calculated by referring to Sallam et al. [40]. We established two geometric centers for this analysis. One was calculated using all 15 segments (Fig. 3A), and the other was calculated using the five lower small intestine segments (Fig. 3B) as 100% to determine the GC value. The five lower small intestine segments specifically included the IM surgical site area and were designated for the purpose of evaluating surgical site recovery.

Statistical analysis

Statistical analysis was performed using Statcel4^® (OMC with one-way analysis of variance followed by post-hoc tests. A P-value of <0.05 was considered statistically significant.

RESULTS

Establishment of the POI Model

We measured how phenol red-labeled contents were transported within 1 hr after administration. For this analysis, we established two geometric centers to calculate the GC value. One used all 15 segments (Fig. 3A), while the other used the five lower small intestine segments (Fig. 3B) as 100%. The five lower segments were specifically designated to evaluate recovery at the surgical site. In the healthy mouse non-treatment group (Fig. 2A, non-IM treatment) and the Tre group (Fig. 2B), the dye was mainly concentrated in the stomach and the distal small intestine (I8-I10), with some distribution also observed in the middle segment (I4-I6). In contrast, in the IM + Veh group (Fig. 2C), an IM treatment group, the dye was dispersed from the proximal to the middle part of the small intestine (I3-I6), showing low values in the distal segment. Compared to the IM + Veh group, an increase in GC values was observed in the untreated normal mouse group and the Tre-treated group. Specifically, the Tre group had 7.37 ± 0.76, the IM + Veh group had 5.73 ± 0.42 (Fig. 3A, P<0.05), the Non group had 3.26 ± 0.05, the Tre group had 3.49 ± 0.45, and the IM + Veh group had 2.16 ± 0.29 (Fig. 3B, P<0.05). Based on these results, we estimate that we have successfully generated a POI mouse model using intestinal manipulation (IM).

Fig. 2.

Gastrointestinal transition assay. The residual amount of dye from the stomach to the rectum was set to 100%, and the area was divided into fifteen sections: stomach (St), small intestine (10 segments, from I1 to I10), Cecum, Colon (Col, 3 segments). The non-surgical groups were divided into two groups: A: Untreated group and B: Tre group. The surgical groups comprised the following five groups: C: IM + vehicle; D: IM + Tre; E: IM + Tre & Mos; and F: IM + Tre + Mos (IM: intestinal manipulation, Tre: trehalose, Mos: mosapride). Vehicle (physiological saline), Tre, Mos and Tre & Mos (mixuture of two agents) were administered intraperitoneally one hour before administration of phenol red (A, B). In the other five groups, each drug was administered intraperitoneally twice (immediately after surgery and 24 hr later), and the dye was administered at 47 hr (C−G). Each column represents the mean ± S.E. of 4–7 mice per group.

Fig. 3.

The values of geometric center (GC) of seven groups were calculated from Fig. 2. The total residual amount of dye across all 15 segments (A) and lower small intestine (B, from I6 to I10) was set to 100%, and the GC value was calculated. Each column represents the mean ± S.E. of 4–7 mice per group. *; P<0.05, **; P<0.01 v.s. IM + Veh (Non.: nontreatment control, IM: intestinal manipulation, Veh: vehicle, Tre: trehalose, Mos: mosapride).

Effect of mosapride and trehalose on intestinal transit in POI mice

Compared with the surgical model control (Fig. 2C, IM + Veh), the dye distribution in the IM + Mos (Fig. 2D) and IM + Tre group (Fig. 2E) tended to migrate towards the distal small intestine and cecum. In the groups with concomitant use of Trehalose and Mosapride (Fig. 2E, 2F), dye distribution was clearly evident in the lower small intestine (I6–I10) and cecum. However, some segments showed low values of around 5%, indicating variability. In the POI model, administration of Mos and Tre increased intestinal transit potential and significantly raised GC values. Specifically, significant differences of P<0.05 or P<0.01 were observed in the IM + Mos group (7.99 ± 0.76), the IM + Tre group (8.17 ± 0.46), the IM + M & T group (7.86 ± 0.54), and the IM + M + T group (7.94 ± 0.24) (Fig. 3A). These results suggest that Mos and Tre promote intestinal transit in vivo. However, no significant difference in GC values was observed in the lower small intestine of the mixed administration group (IM + Tre & Mos) (Fig. 3B, P=0.217 vs. IM + Veh group). Furthermore, based on the results of this experiment, the Mos alone, Tre alone, Mos & Tre and Tre + Mos groups all improved IM-induced transition delay compared to the IM + Veh group (Fig. 3A, 3B). However, the direct comparison between the IM + M&T group and the IM + M + T group in Fig. 3B yielded a P-value of 0.36441, indicating no significant difference. Regarding recovery rates for the IM + M&T and IM + M + T groups, defined as the reduction from the non-treatment group to the IM + Veh group, the former recovered to 63.7% and the latter improved further to 104.1%, showing a difference of approximately 40% between them.

DISCUSSION

In this study, we investigated the potential synergistic effects of combining Tre and Mos (a 5-HT₄ receptor agonist) to enhance gastrointestinal motility. Our results demonstrated that the recovery of gastrointestinal motility was more effectively promoted when Tre was administered immediately after IM and Mos at a later stage, rather than by co-administration (Fig. 3B). In general, when trehalose is ingested orally, it is degraded into two glucose molecules by trehalase [36]. However, when trehalose is applied to the serosal side of the intestine during surgical laparotomy, as in this study, its action is likely to be due to the trehalose itself. We speculate that adding Mos’s anti-inflammatory and gastrointestinal motility-promoting effects after Tre’s repair effects on the serosal side of the gastrointestinal tract had been fully exerted would likely enhance the advantages of both Tre and Mos. A recent study has indicated that trehalose induces autophagy in an mTOR-independent manner, targeting AMPK activation, TFEB nuclear translocation, and Rubicon inhibition, and is also implicated in Nrf2-mediated antioxidant responses [7, 15, 16, 22]. However, its relationship with gastrointestinal transit remains unclear, detailed analysis of smooth muscle layer (autophagy activity (LC3-II/LC3-I ratio via immunostaining, p62 levels, etc.), oxidative stress (malondialdehyde (MDA), redox balance (GSH/GSSG ratio), antioxidant enzymes Nrf2/HO-1 etc.) of this model is warranted.

Tre possesses moisture-retaining properties that make it useful in preventing tissue dryness. Maintaining moisture on the serosal surface of the digestive tract is particularly important for preventing postoperative adhesions [8, 35]. Currently available “intestinal adhesion prevention agents” include: (I) natural or synthetic physical barrier agents (films, gels, sprays) [1]; (II) pharmacological agents (anticoagulants [2], fibrinolytic factors [33], anti-inflammatory agents [18, 24], cytokine inhibitors [29], antibiotic-containing films, antioxidant [6]); (III) surgical techniques (minimally invasive procedures: laparoscopic/robot-assisted surgery [39]); and (IV) new combination products (such as drug-eluting barriers). Tre used in this study was categorized as a physical barrier that did not interfere with the pharmacological action of Mos. Thus, it is expected to have a low risk of adverse effects and can be regarded as generally safe.

Mos enhances gastrointestinal transit through 5-HT₄ receptor stimulation and exerts anti-inflammatory effects via neuro-immune crosstalk [41]. Given its relatively low risk of adverse effects, Mos is considered a valuable agent for combination therapies. Its high gastrointestinal selectivity, minimal central actions, and distinct metabolic pathways contribute to its favorable safety profile [43]. While Mos alone exhibits potent gastrointestinal transit-promoting effects, its concomitant use with other agents, such as enemas [11], proton pump inhibitors (PPIs) [10, 34], and anti-inflammatory drugs [9, 20], may further restore gastrointestinal homeostasis through synergistic actions. The present findings on the combined use of Mos and Tre highlight its clinical potential for POI, warranting further investigation, including evaluation of the anti-inflammatory effects in subsequent studies.

In conclusion, this study confirms that both Mos and Tre promote gastrointestinal transit in mice with POI. Moreover, optimizing the timing and method of administration appeared to enhance gastrointestinal transit, suggesting a potential benefit of this combination approach.

CONFLICT OF INTEREST

The authors declare no conflicts of interest associated with this manuscript.