Postoperative ileus (POI) refers to impaired gastrointestinal (GI) transit as a response to surgical stress.^1,2 It commonly occurs after GI surgery, affecting up to 30% of patients, although it can also manifest after other surgeries.³ POI contributes to patient discomfort, complications, increased morbidity, and prolonged hospital stays, leading to significant healthcare costs, particularly for patients undergoing surgery.^4-6 Since knowledge of effective therapies for POI remains limited, it represents a major challenge in surgery, especially abdominal surgery.¹

Numerous studies have investigated the underlying mechanisms of POI, revealing the involvement of inflammatory, pharmacological, hormonal, and neurogenic factors. However, the precise pathophysiology of POI is still not completely understood.⁷

Intestinal permeability, closely associated with intestinal inflammation, is regulated by tight junction (TJ) proteins in the GI epithelium. Alterations in intestinal permeability and TJ proteins have been observed in various diseases, including inflammatory bowel disease, tumoral disease, irritable bowel syndrome, metabolic diseases, and autoimmune diseases.^8-12 In a previous study on an animal model of POI, increased intestinal inflammation and permeability were accompanied by changes in TJ proteins such as claudin-1 and claudin-2.^3,4

The gut microbiota plays a crucial role in influencing bowel diseases and various aspects of host physiology, including nutrient, xenobiotic, and drug metabolism, maintenance of the gut mucosal barrier’s structural integrity, immunomodulation, and protection against pathogens.^15-19 Dysbiosis of the gut microbiota, a major concern for multiple diseases, is associated with the pathogenesis of both intestinal and extraintestinal conditions.^20-22 Prolonged stress or severe injury induced by surgery can hinder the recovery of intestinal microbiota, potentially leading to the emergence of lethal pathogens or the dominance of more virulent healthcare-associated ones.^23-25 A previous study on an animal model of POI demonstrated the induction of gut bacterial dysbiosis following surgery. Administration of probiotics before surgical intervention prevented a decrease in beneficial intestinal bacteria, butyrate production, and bowel movement.²⁶ However, the mechanisms connecting the improvement in colonic transit time and gut microbiota dysbiosis through probiotic pretreatment have not been investigated.

Therefore, this study aimed to investigate the mechanisms by which probiotic pretreatment improves POI. We measured intestinal permeability, intestinal inflammation, TJ protein expression in the GI epithelium, and plasma interleukin (IL)-17 levels in a guinea pig model of POI.

1. Preparation of animals

Adult male Hartley guinea pigs (Orient Bio Inc., Seoul, Korea) weighing 250 to 350 g were acclimatized to controlled breeding conditions for at least 1 week prior to the surgical intervention. The conditions were a temperature of 20 to 22°C, humidity of 50%±10%, and a 12-hour light/dark cycle commencing at 7 AM. The guinea pigs had ad libitum access to water and feed consisting of ≥16% crude protein, ≥2.0% crude fat, ≤20% crude fiber, ≥0.8% calcium, and ≥0.52% phosphorus. All experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee, Department of Laboratory Animal Resources, Yonsei Biomedical Research Institute, Yonsei University College of Medicine, with an Institutional Review Board (protocol number: 2020-0271).

2. Group settings

The guinea pigs were randomly assigned to control, POI, and probiotic groups, with eight to nine animals per group. The control group did not receive any manipulation or drugs before tissue or serum collection. The POI and probiotic groups underwent the following surgical procedure: after a 24-hour fast (except for water) prior to the procedure, a mixture of Zoletil, Rompun, and saline was administered as an intraperitoneal injection. After 15 minutes, the abdomen was shaved and disinfected with an alcohol swab. A minimal peritoneal incision was made after incising the abdominal skin and muscle layers. The cecum was extracted, gently rubbed with wet gauze for 1 minute using the fingers, and then sutured.

The probiotic group received 50 mg/kg probiotics (5 mg 2.5×10⁵ colony forming unit [CFU] Enterococcus faecalis, 25 mg 2.5×10⁵ CFU Bacillus mesentericus, and 25 mg 5×10⁵ CFU Clostridium butyricum) mixed with buffered saline via an intragastric tube once daily for 5 days before the surgical procedure. The POI group guinea pigs received buffered saline for 5 days before the surgical procedure. The purpose was to determine the effect of the buffer on the efficacy of probiotics.

The ileum and proximal colon tissues were harvested from each guinea pig. The tissues were harvested from the POI and probiotic group animals 6 hours post-operation based on previous reports evidencing high inflammatory cell counts and intestinal permeability in the ileum and proximal colon 6 hours post-operation.^13,14

3. Intestinal permeability

To evaluate the intestinal permeability, the harvested tissues were placed in a modified Ussing chamber (Physiologic Instruments Inc., Reno, NV, USA; EM-CSYS-2). Each half of the tissue was bathed with 2 mL of Krebs-Ringer bicarbonate (KRB) solution to cover the mucosal and serosal sides of the specimens. A gas mixture of 95% O₂ and 5% CO₂ was provided to both sides at 37°C. After a 30-minute equilibration period, the KRB solution on the mucosal side was replaced with a KRB solution containing horseradish peroxidase (HRP) at a final concentration of 0.4 mg/mL. The KRB solution on the serosal side was replaced with a fresh KRB solution, and a 0.3 mL sample was collected from the serosal side and replaced with 0.3 mL KRB. The serosal samples were enzymatically analyzed using the modified Worthington method with o-dianisidine dihydrochloride (OPD; Sigma Chemical Co., St. Louis, MO, USA) as the substrate. Samples (50 μL) were transferred to microtiter plates, and 100 μL of OPD working solution (diluted 1:10 in OPD) as a stable peroxide buffer was added to each well. Subsequently, the plates were incubated by shaking at 300 rpm at room temperature. After 30 minutes, 100 μL of 2.5 M sulfuric acid was added. After 10 minutes, the permeability of the decolorized products was measured at 492 nm using a microplate reader (Model 680; Bio-Rad Laboratories Inc., Hercules, CA, USA). All samples were analyzed in duplicate, and the concentrations were calculated using a standard curve. The HRP flux was represented as ng/2 hr/mm² during steady-state permeation. Intestinal permeability tested using a Ussing chamber was expressed as a percentage change compared to the mean flux of the control group animals.

4. Occludin and ZO-1 expression

Occludin and zonula occludens (ZO)-1 expressions were determined immunohistochemically. The ileum and proximal colon tissues collected 6 hours post-operation were fixed in 4% paraformaldehyde, embedded in paraffin, and sliced into 4 μm thick sections. The sections were deparaffinized, rehydrated, and rinsed using standard methods. Subsequently, they were incubated overnight with the primary antibodies for occludin (1:100; Invitrogen, South San Francisco, CA, USA) or ZO-1 (1:500; Invitrogen) at 4°C, followed by washing and incubation with the secondary anti-rabbit IgG antibody (1:200; Santa Cruz Biotechnology, Dallas, TX, USA) for 30 minutes at 37°C. The stained samples were incubated with streptavidin-HRP for 30 minutes, treated with an AB peroxidase solution, and counterstained with hematoxylin. Images were analyzed using MetaMorph (MDS Analytical Technologies, Sunnyvale, CA, USA) microscopy automation and ImageJ (National Institutes of Health and Laboratory for Optical and Computational Instrumentation, University of Wisconsin, USA) software. Five different fields were randomly selected, and the frame was split into channels to select the stained area only. The integrated density was calculated, and then the ratio of integrated density to area was deduced.

5. Intestinal inflammation

The harvested ileum and proximal colon muscle layers were sectioned, fixed in 10% neutral-buffered formalin, and embedded in paraffin. The embedded sections were sliced into 4 μm thickness and stained with hematoxylin and eosin. Leukocyte counts were compared between the control and POI groups, as well as the POI and probiotic groups, using a semi-quantitative scoring system.

6. Calprotectin expression

Calprotectin expression was determined using immunohistochemical analysis. The paraffin-embedded ileum and proximal colon sections were deparaffinized and incubated with 3% hydrogen peroxide for 10 minutes to block endogenous peroxidase activity. The tissue sections were incubated overnight at 4°C with the primary antibody anti-calprotectin (1:250; Thermo Fisher, Waltham, MA, USA). After three washes with phosphate-buffered saline, they were incubated with the secondary antibody anti-mouse IgG (1:200; Vector Laboratories, Newark, CA, USA). Next, the sections were incubated with streptavidin-HRP for 30 minutes, treated with an AB peroxidase solution, and counterstained with hematoxylin. The images were analyzed using MetaMorph and ImageJ software. When analyzing slide images, each intestinal layer was manually designated and separately analyzed.

7. Plasma IL-17 level measurement

Blood samples were collected via cardiac puncture before euthanizing the animals. The plasma was separated by centrifugation and then stored at −70°C until the assay. Plasma IL-17 levels were determined using an enzyme-linked immunosorbent assay kit (MyBioSource, San Diego, CA, USA) according to the manufacturer’s instructions.

8. Statistical methods

The data are expressed as the mean±standard error. Statistical analysis was performed using non-parametric tests because the number of guinea pigs per group was <10, so a normal distribution could not be assumed. The Kruskal-Wallis H test was used to determine whether there was a significant difference between the three groups, followed by the Mann-Whitney U test for post hoc testing between the two groups, and the Bonferroni method was used to correct the p-value. SPSS version 26.0 (IBM Corp., Armonk, NY, USA) was used for statistical analysis. A two-tailed p<0.05 indicated statistical significance.

1. Intestinal permeability

Intestinal permeability was assessed by measuring HRP permeability in the ileal and proximal colonic samples of the control, POI, and probiotic groups (eight, nine, and eight samples, respectively) (Fig. 1).

Figure 1. Intestinal permeability in the ileum and proximal colon of the control, POI, and probiotic groups. HRP permeability measured 6 hours after the operation. Bars indicate the mean±SEM. POI, postoperative ileus; HRP, horseradish peroxidase; SEM, standard error of mean. *p<0.05 means statistical significance compared with POI group.

The HRP permeability in the ileal tissues of the POI group animals was significantly higher than that in the control group animals (p=0.046 and p=0.13 in the ileum and proximal colon, respectively), and pre-administration of probiotics decreased the permeability of the ileum (p=0.021 and p=0.236 in the ileum and proximal colon, respectively).

2. Occludin and ZO-1 expression

Occludin and ZO-1 expression levels were measured by analyzing images of the immunohistochemically stained ileum and proximal colon tissues. In the proximal colon, occludin expression was lower in the POI group animals than in the control group animals (p=0.431); however, no significant differences were observed in the ileal tissue (Fig. 2). Administration of probiotics improved the expression of occludin in the proximal colon (p=0.064), while no significant differences were observed in the ileum.

Figure 2. (A) Occludin expression in the ileum and proximal colon of the control, POI, and probiotic groups. (B) Representative immunohistochemical occludin staining (×200) in the ileum and proximal colon of the control, POI, and probiotic groups 6 hours after the operation. Bars indicate the mean±SEM. POI, postoperative ileus; SEM, standard error of mean.

Similar results were observed for ZO-1 expression levels. No significant differences were observed in the ileal tissue among the three groups. However, ZO-1 expression in the proximal colon of the POI group was significantly lower than that in the control group (p=0.047). ZO-1 expression was greater in the probiotic group than in the POI group (p=0.002) (Fig. 3).

Figure 3. (A) Expression of ZO-1 in the control, POI, and probiotics group. (B) Representative immunohistochemical ZO-1 staining (×200) in the ileum and proximal colon of the control, POI, and probiotic groups 6 hours after the operation. Bars indicate the mean±SEM. ZO-1, zonula occludens 1; POI, postoperative ileus; SEM, standard error of mean. *p<0.05 indicated significance with respect to that in the POI group.

3. Intestinal inflammation

Analysis of leukocyte counts revealed that intestinal inflammation in both the ileal and proximal colonic muscle layers of the POI group was significantly higher than in the control group (p=0.001). In contrast, the leukocyte count in both the ileal and proximal colonic muscle layers of the probiotic group was significantly lower than that in the POI group (p=0.001 and p=0.028 in the ileum and proximal colon, respectively (Fig. 4).

Figure 4. Leukocyte count per field (×400) in the ileum and proximal colon of the control, POI, and probiotic groups. Bars indicate the mean±SEM. POI, postoperative ileus; SEM, standard error of mean. *p<0.05 indicated significance with respect to that in the POI group.

4. Calprotectin expression

Calprotectin expression in each intestinal wall layer was analyzed immunohistochemically. We classified and designated the mucosal, submucosal, and muscle layers in the images for separate analysis. Calprotectin expressions in the ileal and proximal colonic mucosal and submucosal layers of the control, POI, and probiotic groups were not significantly different. However, calprotectin expression in the ileal or proximal colonic muscle layer of the POI group was significantly higher than that in the control group (p=0.001) and probiotic group (p=0.001 and p=0.028 in the ileum and proximal colon, respectively (Fig. 5).

Figure 5. Calprotectin expression in the intestinal wall mucosal, submucosal, and muscle layers of the ileum (A, C) and proximal colon (B, D) of the control, POI, and probiotics groups. (C, D) Representative immunohistochemical calprotectin staining (×200) in the ileum and colon. Bars indicate the mean±SEM. M, mucosal layer; SM, submucosal layer; Muscle, muscle layer; POI, postoperative ileus; SEM, standard
error of mean. *p<0.05 indicated significance with respect to that in POI group.

5. Plasma IL-17 level measurement

The plasma IL-17 levels did not differ significantly among the three groups (Fig. 6).

Figure 6. Plasma IL-17 levels in the control, POI, and probiotic groups. Bars indicate the mean±SEM. IL, interleukin; POI, postoperative ileus; SEM, standard error of mean.

Probiotics are used to rectify gut microbiota dysbiosis in various diseases.²⁷ In this study, we found that intestinal permeability and inflammation were significantly downregulated in the probiotic group compared to those in the POI group. A previous study found that gut microbiota composition was significantly altered before and after surgical intervention in guinea pigs as Bifidobacterium and Lactobacillus (lactic acid-producing bacteria) decreased and Bacteroides and Blautia increased; pretreatment with probiotics prevented these changes and the delay in colonic transit time.²⁶ Furthermore, intestinal manipulation in a mouse model enhanced intestinal permeability and significantly increased the translocation of aerobes and anaerobes into the tissue compared to that seen with laparotomy alone, demonstrating an important factor influencing the development and persistence of POIs.²⁸ Additionally, gut microbiome dysbiosis increases intestinal permeability in various chronic diseases, which in turn causes secondary inflammation.²⁹ HRP, a 45 kDa protein antigen, serves as a marker for protein uptake and can trigger immune responses in humans. It typically enters cells through macropinocytosis and is easily detectable via enzyme-linked immunosorbent assay. HRP is commonly employed in Ussing chambers for permeability studies. Using HRP, we previously demonstrated that increased gut paracellular permeability is strongly associated with the typical features of POI, particularly delayed contractile activity recovery and increased inflammation.¹⁴ Our findings suggest that probiotic pretreatment may reduce the incidence and degree of POI by preventing increased intestinal permeability.

Increased intestinal permeability after surgical stimulation enhances the movement of pathogen-associated molecules from the intestinal lumen to tissues, producing an inflammatory response in the intestinal muscle layer.^30,31 In this study, the increased leukocyte count in the muscle layer of the POI group animals compared to that in the control group animals supports this hypothesis. Moreover, the decreased leukocyte count in the probiotic group compared to that in the POI group appears to be caused by the prevention of the increase in intestinal permeability. Several studies found that gut microbiota dysregulation is closely associated with increased intestinal permeability in chronic diseases like obesity, diabetes, inflammatory bowel disease, irritable bowel syndrome, cirrhosis, autoimmune diseases, and prolonged psychological stress.^32-37 Moreover, intestinal permeability increases in some acute diseases, such as colitis and acute pancreatitis.^38,39 Probiotics are expected to improve altered intestinal permeability by modifying gut microflora, dietary proteins, and bacterial enzyme activity; however, most studies show that probiotics typically do not reduce the already increased intestinal permeability in colitis, and acute pancreatitis.^40-44 Only a few studies have reported that probiotic consumption improves intestinal permeability. Ait-Belgnaoui et al.^45,46 found that probiotic pretreatment prevented an increase in intestinal permeability in an acute psychological stress rat model but did not evidence a positive effect on the increase in intestinal permeability in a chronic psychological stress rat model. Liu et al.⁴⁷ reported occludin and ZO-1 restoration in an autoimmune hepatitis mouse model after compound probiotic treatment. Few animal studies, including ours, have confirmed the effect of probiotics on improving intestinal permeability. Although the reason for this improvement remains unclear, it may be related to the differences in the pathophysiology of each disease, intestinal permeability measurement methods, probiotic strains used, and probiotic intake method or dose.

TJ proteins are integral transmembrane proteins found in the TJs of all epithelia and endothelia, which mediate cell-to-cell adhesion and seal the paracellular space between epithelial cells.⁴⁸ TJ structure regulation is influenced by various physiological and pathological stimuli, and its disruption increases intestinal permeability, which is closely related to various diseases.⁴⁹ Occludin and ZO-1 are TJ proteins localized at endothelial cell junctions, which associate with each other to create a complex compound.⁵⁰ Occludin maintains the integrity and barrier function of the TJ, and ZO-1 is an important liker protein in TJ, binding to C-terminal sequences of occludin and beta-actin and acting as a bridge between the plasma membrane and cytoskeleton proteins.⁵¹ Increased occludin and ZO-1 expression accompany reduced intestinal permeability.⁵² In this study, occludin and ZO-1 expression decreased and increased in the colonic tissues of POI and the probiotic group, respectively. This supports the hypothesis that probiotics prevent an increase in intestinal permeability by preventing TJ protein downregulation, thereby preventing POI. However, TJ protein expression in the ileal tissue was not significantly different among the three groups. It is unclear why there were differences in the changes in TJ protein expression in each organ. In a previous study, the expression of claudin-1, another TJ protein that regulates intestinal permeability, was significantly reduced in both the ileum and proximal colon of a guinea pig POI group compared to that in the control group.¹⁴ This suggests that the TJ proteins affected by the pathophysiology of POI in the proximal colon and ileum are different. Interestingly, one study showed that endotoxemia induced ileal mucosal permeability in mice and inhibited deterioration of gut mucosal barrier function in inducible NO synthase (iNOS) knockout mice, suggesting that iNOS-dependent NO production is involved in changes in intestinal mucosal permeability,⁵³ and another study in mice have shown that in normal mice, iNOS mRNA and iNOS protein are detectable only in the ileum and that lipopolysaccharide injection is required to detect iNOS mRNA in the jejunum and colon.⁵⁴ And in another study, ionizing radiation exposure in rats increases iNOS activity significantly in 2 to 6 hours in the ileum, but not in the colon.⁵⁵ This allows us to hypothesize that earlier iNOS expression in the ileum than in the colon is responsible for the increase in intestinal permeability during the early stages of POI induction, but further experiments are required to confirm this.

Leukocyte infiltration in the intestinal muscular layer increased in the POI group, indicating intestinal wall inflammation. According to several studies, POI pathophysiology consists of several steps involving various factors.^1,3,31 Neural dysfunction is predominant in the early stages, and intestinal inflammation is considered an important contributor in the late stages.^1,56 In a POI animal model, intestinal manipulation induced inflammation-mediated impaired smooth muscle contraction, which is one of the main mechanisms of POI.⁵⁶ In our study, probiotics prevented intestinal muscle layer inflammation followed by intestinal manipulation. Intestinal smooth muscle inflammation is associated with reduced smooth muscle contraction in multiple diseases and is clearly connected to the intestinal microbiota. Several studies have reported a crosstalk between the microbiota, intestinal wall adipose tissue, and muscle in intestinal inflammation,⁵⁷ and probiotics should improve intestinal inflammation in various diseases.⁵⁸ In fact, several animal and human studies have reported that probiotics improve intestinal inflammation. However, no clear mechanism for suppressing the incidence or degree of inflammation in the intestinal muscle layer is known.

We evaluated calprotectin expression in each layer, which was not significantly different in the mucosal and submucosal layers; however, calprotectin expression was markedly increased in the muscle layer of the POI animal model. Calprotectin, an abundant calcium-binding protein belonging to the S100 family, is derived predominantly from neutrophils, monocytes, and macrophages. It has direct antimicrobial effects and plays a role in innate immune responses. Clinically, fecal calprotectin is a useful surrogate marker of GI inflammation.⁵⁹ Previous work by our group has already shown that intestinal wall expression of calprotectin is elevated relative to control in the POI guinea pig model,¹⁴ but in this study, we analyzed calprotectin expression in each layer to determine whether the mechanism by which probiotics inhibit POI occurs through changes in the mucosal layer, where intestinal microorganisms and their products were in direct contact, but there were no noticeable changes in the mucosal layer. If toxic substances or bacteria penetrated the intestinal wall during POI and caused an inflammatory reaction in the muscle layer, an increase in calprotectin accompanied by an inflammatory reaction would have been confirmed in the mucosal and submucosal layers. However, this hypothesis was rejected by the results. The increased calprotectin expression in the muscle layer was attributed to increased macrophage activity. Macrophages residing in the muscularis externa of the GI tract are highly specialized cells essential for tissue homeostasis during steady-state conditions as well as during disease.⁶⁰ They closely communicate with the enteric nervous system and regulate colonic peristalsis by changing the pattern of smooth muscle contractions in both the inflammatory and steady states.⁵⁷ Particularly during inflammation, muscularis macrophages secrete inflammatory cytokines and recruit inflammatory cells, which further accelerate the inflammatory process.^61-63 A previous study using a murine POI model reported that vagus nerve stimulation reduced intestinal inflammation by activating cholinergic enteric neurons in close contact with muscularis macrophages.⁶⁴ It seems that the regulatory function of probiotics in POI indirectly affects muscularis macrophages via the nervous system; however, the exact mechanism remains unclear. Previous studies have shown that short-chain fatty acids, such as butyrate, affect the central nervous system by altering the expression of brain-derived neurotrophic factor, and pre-administration of probiotics in the POI guinea pig model inhibited the decrease in fecal butyrate levels after surgery. This suggests that it may be related to the fact that short-chain fatty acids produced by pretreated probiotic-regulated gut microbiota inhibit the increased inflammatory response in the muscle layer.^26,65 In this study, we did not measure fecal calprotectin in this animal model for two reasons: first, given the low expression of calprotectin in the mucosal layer, it was unlikely that a significant increase in fecal calprotectin, which is a good reflection of inflammation in the mucosal layer, would be observed; and second, the study design required a 5-day fasting period prior to POI induction, which made it difficult to collect a sufficient amount of feces for testing at the appropriate time point.

IL-17 is a well-known proinflammatory cytokine that increases intestinal inflammation, such as in inflammatory bowel disease and colitis. Plasma IL-17 levels did not increase in this study, which appears to be caused by the minor role of systemic cytokines in the acute phase of POI generation. However, as IL-17 is also secreted by muscularis macrophages, an increase in its levels in the POI group was anticipated over the long term. This rise was not observed, likely because of the timing of the blood sampling, which occurred only 6 hours after surgical intervention. A recent study found that gut microbiota-derived short-chain fatty acids regulate IL-17 production via intestinal γδ T cells in mice and humans; however, at least in the early stages of POI development, the effect of IL-17 on pathophysiology is insignificant, and probiotics are not the direct cause of POI prevention.⁶⁶

We used mixture of E. faecalis, B. mesentericus, and C. butyricum in this study. Each of these three strains is highly viable in a variety of environments, making them suitable for oral probiotic formulations, and they have a variety of positive effects on the gut microbiota by modulating the composition of the gut microbiota in favor of upregulating lactic acid bacteria such as Lactobacillus and Bifidobacterium.^67-69

A mixture of these three strains is already widely used in clinical practice and has been used in various animal and clinical studies. In human studies, a mixture of the three strains has been shown to stimulate the Th 1 immune response, decrease proinflammatory cytokines and increase anti-inflammatory cytokines, and in studies of children hospitalized for acute infectious diarrhea, it has been shown to decrease prevalence, length of hospital stay, and increase Bifidobacterium, Lactobacillus, and increase IL-10 and decrease tumor necrosis factor-alpha.^70-72 Based on these findings, we determined that a mixture of the three strains would be a suitable probiotic strain to prevent intestinal inflammation and increased intestinal permeability, which are known to contribute to the POIs targeted in this study.

Guinea pigs serve as superior models for certain human medical conditions compared to other rodents. E-cadherin on the intestinal surface of guinea pigs is homologous to that of humans and serves as the primary receptor interacting with bacteria upon the initiation of intestinal invasion.⁷³ Hildebrand et al.⁷⁴ compared the intestinal metagenomes in guinea pigs and humans, which were highly similar at the phylum level. Therefore, we chose guinea pigs as an experimental model because guinea pigs may represent a suitable model for investigating the microbiota-dependent effects. In this study, only male guinea pigs were used. This decision was based on the finding that the alpha diversity of microbiota in guinea pigs does not differ between males and females.⁷⁵ Additionally, our previous study also utilized adult male Hartley guinea pigs, providing a consistent basis for comparison.²⁶

Our study had several limitations. First, while we have previously demonstrated POI in guinea pigs by observing delays in colonic transit time,²⁶ we did not verify the occurrence of POI in each individual in this study. This is because the purpose of this study was to determine whether the increased intestinal permeability and changes in TJ proteins that occur early after POI induction are closely associated with intestinal inflammation, so samples had to be collected relatively soon after POI induction (6 hours), and given the invasive manipulations involved in collecting ileum and colon samples, it was not appropriate to assess whether ileus occurred after sample collection. However, given that postoperative inflammation is strongly associated with POI and that previous studies have shown improvements in colonic transit time in the same animal model setting, we believe it is reasonable to conclude that suppression of postoperative intestinal inflammation by pre-administration of probiotics prevents POI. Consequently, we could not confirm the presence or severity of POI in the subjects used in this experiment. Although we established that preoperative probiotic administration can inhibit POI by preventing an increase in intestinal permeability, the mechanisms by which probiotics enhance intestinal permeability remain unexplored. Furthermore, while we observed that probiotics protect occludin and ZO-1 in the colon, the specific effects of probiotic administration on these TJ proteins remain unclear. Additionally, since all animals in the probiotic group received the same strain and dose, determining the most effective probiotic strain and dosage for optimal outcomes remains uncertain.

Gene expression analysis techniques, such as RNA sequencing or quantitative real time polymerase chain reaction, can be valuable for assessing the mechanism and determining the most appropriate strain and dosage of probiotics to prevent POI. Given the species differences between guinea pigs and humans, further research is also needed to optimize dosages and strains for human applications. Although many human studies have been conducted, the ideal strain, dose, and duration of probiotic use for treating specific diseases are not well defined. For instance, various meta-analyses have shown that the effectiveness of probiotics varies under different conditions. One meta-analysis indicated that a short duration of probiotic use effectively improved overall irritable bowel syndrome symptoms, yet the studies analyzed involved months of probiotic use.⁷⁶ Conversely, 65% of studies in a meta-analysis of probiotic use in acute infectious diarrhea in pediatric patients reported benefits from just 5 days of use.⁷⁷ Therefore, even if the conclusions of these animal studies hold true in humans, further research is needed to determine the most effective way to administer probiotics.

In conclusion, POI increases intestinal mucosal permeability, which seems to be closely related to the altered TJ proteins, occludin, and ZO-1 expression. Even though inflammation of the intestinal muscular layer plays an important role in POI development, the mucosal and submucosal layers were not inflamed. Preoperative probiotic administration prevents both increased intestinal permeability and intestinal muscular layer inflammation; therefore, it can be considered a preventive measure of POI.

This study was supported by research grants from Daewoong Pharmaceutical Co., Ltd.

MID (Medical Illustration & Design), as a member of the Medical Research Support Services of Yonsei University College of Medicine, provided excellent support to the medical illustration.

This study was supported by research grants from Daewoong Pharmaceutical Co., Ltd. Except for that, no potential conflict of interest relevant to this article was reported.

Study concept and design: M.J.K., H.P. Data acquisition: Z.H., Y.J.L. Data analysis and interpretation: M.J.K., Z.H., Y.J.L. Drafting of the manuscript: M.J.K. Critical revision of the manuscript for important intellectual content: M.J.K., H.P. Statistical analysis: M.J.K. Obtained funding: H.P. Administrative, technical, or material support; study supervision: M.J.K., H.P. Approval of final manuscript: all authors.