Life stress may influence symptom onset and severity in certain gastrointestinal disorders in association with a dysregulated intestinal barrier. It has been widely accepted that stress triggers the hypothalamus-pituitary-adrenal (HPA) axis, releasing corticosterone, which promotes intestinal permeability. In response, colonic inflammation alters mucosal immune homeostasis and destroys the colonic architecture, leading to severe intestinal diseases. Endogenous substance P (SP) does not inhibit the initial extent of the HPA axis response to restraint stress, but it reduces the duration of the stress, suggesting that SP plays an important role in the transition between acute and chronic stress. The present study aimed to investigate the effect of two groups of mice exposed to stress, including acute and chronic stress. The corticosterone was evaluated by ELISA, colon samples were obtained to detected polymorphonuclear cells by hematoxylin and eosin staining, goblet and mast cells were identified by immunocytochemistry and cytokine-producing CD4+ T cells were analyzed by flow cytometry assays, adhesion proteins in the colon epithelium by western blotting and serum SP levels by ELISA. The results demonstrated an increase in the number of polymorphonuclear, goblet and mast cells, a decrease in claudin-1 expression and an elevation in E-cadherin expression during acute stress. Increased E-cadherin expression was also detected during chronic stress. Moreover, it was found that acute stress caused a shift towards a predominantly anti-inflammatory immune response (T helper 2 cells), as shown by the increase in the percentage of CD4+/IL-6+ and CD4+/IL4+ lymphocytes in the lamina propria and the increase in serum SP. In conclusion, this response promoted colonic protection during acute stress.
It is well established that cortisol is the main hormone that regulates stress in humans and it plays an equivalent role in rodents (
While observations have documented that mast cells are recruited by high levels of corticosterone (
The current study used a stress model in which mice were subjected to repeated movement restriction, including one session (acute) or three repeated sessions over 2 h (chronic). The objective of the present study was to evaluate the effects of these two stress protocols on mucosal components and immune response parameters in the colon, as well as on serum SP levels. The results of the present study could provide a useful reference for the management of therapies that regulate the effects of stress on intestinal bowel disease dysfunction.
A total of 45 male BALB/c mice (weight, 20-30 g; age, 10-12 weeks) were used in this study. The mice were allowed free access to food and water (Labdiet 5013; LabDiet). All mice were kept on a 12-h light/dark cycle (lights on at 6:00 a.m.) at room temperature (RT) at 20°C, with a relative humidity of 55%. The current protocol was developed based on the ARRIVE guidelines for reporting animal research (
The animals were randomly divided into three groups (n=15): i) Control group without stress; ii) acute stress group (2 h of movement restriction repeated once); and iii) chronic stress group (2 h of movement restriction repeated for 3 days). Mice were placed in cylindrical plexiglass containers according to a previously described method (
Male BALB/c mice were anaesthetized by an intraperitoneal injection of a lethal dose of 100 mg/kg body weight pentobarbital sodium salt (cat. no. P3761; Sigma-Aldrich; Merck KGaA). Blood was obtained via cardiac puncture (0.7-1 ml), and serum was obtained via centrifugation at 1,660 × g for 7 min at 4°C. The serum samples were stored at −20°C until use. After exsanguination the large intestines were dissected, a colon segments were cut (1 cm), fixed with 4% paraformaldehyde at RT for 24 h and processed for paraffin embedding. Sections (7-µm thick) were generated with a microtome (Rotatory Microtome; Leica Microsystems GmbH), placed on coverslips and stained with H&E or Alcian Blue (AB) or toluidine blue at RT.
Plasma corticosterone concentrations were determined using a commercially available ELISA kit for corticosterone analysis according to the manufacturer's instructions (cat. no. ADI-901-097; Enzo Life Sciences, Inc.). The corticosterone concentrations in the plasma samples were calculated based on a standard curve and are expressed in ng/ml.
For the quantification of leukocyte infiltration, colon sections were stained with H&E. After deparaffinization with xylol and rehydration in a descending alcohol gradient, the samples were immersed in Harris hematoxylin solution (cat. no. H3136; Sigma-Aldrich; Merck KGaA) and incubated for 20 min at RT. After incubation in an eosin solution for 2 min at RT (cat. no. E4009; Sigma-Aldrich; Merck KGaA), the samples were then washed with distilled water. Finally, the sections were dehydrated and mounted with Entellan® (cat. no. 1079610500; Merck KGaA). The samples were analyzed via optical microscopy, and the PMN numbers were determined. The PMNs in the inflammatory infiltrates were counted (12 per slide/3 slides per animal). The percentage of PMNs in each sample was calculated. The number of cells counted was evaluated using Image-Pro Plus version 5.1 software (Media Cybernetics, Inc.) and an E600 microscope (Eclipse E-600; Nikon Corporation) at a ×40 magnification, and the total number of PMNs was quantified (
Colon samples were fixed and stained for acidic mucins as previously described (
Mast cells were observed using toluidine blue staining (cat. no. 198161; Sigma-Aldrich; Merck KGaA). After deparaffinization with xylol and rehydration in a descending alcohol gradient, the samples were immersed in a 0.5% toluidine blue solution and incubated for 30 min at RT. Then, the samples were washed with distilled water, dehydrated and mounted with Entellan. The numbers of mast cells in the intestinal lamina propria of each group were determined. The mast cells were identified and counted randomly via light microscopy at a ×40 magnification with an E600 microscope (Eclipse E-600; Nikon Corporation). The average numbers of mast cells per section per group were determined (
Claudin-1 and E-cadherin expression in isolated epithelial cells from the large intestine was determined. Briefly, fragments of the large intestine were incubated in RPMI-1640 medium (cat. no. R7388; Sigma-Aldrich; Merck KGaA) with 1 mM dithiothreitol (cat. no. 20290; Thermo Fisher Scientific, Inc.) and 1.5 mM EDTA (cat. no. E6511; Sigma-Aldrich; Merck KGaA), with continuous shaking at 415 × g for 30 min at 37°C. The cell suspension was passed through organza to remove the mucus and centrifuged at 415 × g for 10 min at 4°C. The pellet was suspended in 15 ml RPMI-1640 medium, passed through an organza filter and washed twice with 15 ml RPMI-1640 medium followed by centrifugation at 415 × g for 10 min at 4°C. The washed pellet was suspended in 20% Percoll® (cat. no. P1644; Sigma-Aldrich; Merck KGaA) and centrifuged over a discontinuous Percoll gradient at 1,160 × g for 30 min at 25°C. Epithelial cells were recovered from the interphase between 20 and 40%. The cells were washed with PBS and centrifuged as aforementioned. The cells were resuspended in RPMI-1640 medium. The purity of the samples was analyzed via light microscopy based on the normal morphology of epithelial cells. Cell viability was determined using a Neubauer chamber and an optical microscope (magnification, ×20). Then 10 µl of cell suspension were added to an equal volume of 0.4% trypan blue. The number of cells were counted and their viability (viable cells excluding trypan blue; cat. no. T8154; Sigma-Aldrich; Merck KGaA) was determined to be 90%. The samples contained up to 85% epithelial cells (
The protein expression levels of claudin-1 and E-cadherin were determined via western blotting. Samples were homogenized in 100 µl lysis buffer [10 mmol Tris pH 7.4, containing 1% SDS (cat. no. 1610301; Bio-Rad Laboratories, Inc.), 2 mmol/l sodium orthovanadate (cat. no. S6508; Sigma-Aldrich; Merck KGaA) and 12.55 µg/ml phenylmethylsulphonyl fluoride (cat. no. P7626; Sigma-Aldrich; Merck KGaA)]. The samples were sonicated four times for 15 sec with 30 sec of rest at 4°C (
After treatment with EDTA 30 min at 37°C, the large intestines were washed twice with 25 ml RPMI-1640 medium and then transferred to 50-ml tubes containing 25 ml RPMI-1640 medium with 60 U/ml type IV collagenase (cat. no. C5138; Sigma-Aldrich; Merck KGaA), 1% fetal calf serum and 50 g/ml gentamicin (cat. no. G1397; Sigma-Aldrich; Merck KGaA). The tubes were incubated horizontally for 30 min at 37°C in a shaking water bath. The contents of each tube were then transferred to Petri dishes. The intestinal mucosa samples were filtered through an organza filter with a syringe plunger; then, the single cell suspensions containing lamina propria cells were filtered and centrifuged at 415 × g for 10 min at 4°C. The cells were resuspended, collected and centrifuged in a discontinuous 40/75% Percoll gradient at 1,160 × g for 30 min at 4°C. The cells were recovered from the interphase and then washed with RPMI-1640 medium. Viability was determined using trypan blue exclusion, and it was found to be >90% (
The isolation of lymphocytes from the lamina propria of the large intestine was carried out as previously described with some modifications (
Serum SP concentrations were measured using a competitive ELISA kit according to the recommendations of the manufacturer (cat. no. 583751; Cayman Chemical Company). A total of 50 µl per well of serum samples from the acute, chronic and control groups were added, and the assay was performed in triplicate. The plate was read with a microplate reader (BioTek Instruments, Inc.) at 420 nm. The SP concentrations were calculated based on a standard curve and are expressed in pg/ml.
The experimental assays were repeated for ≥3 independent assays (n=5 mice per group). The data are expressed as the mean ± SD, and multiple comparisons between groups were analyzed using one-way ANOVA, and the means of the respective groups were compared using the post hoc Tukey's multiple comparison test. Statistical analysis was performed using GraphPad Prism Version 9 software (GraphPad Software, Inc.). P<0.05 was considered to indicate a statistically significant difference.
The serum corticosterone concentration is an indicator of stress, and different stressors are associated with increases in serum corticosterone concentrations (
The acute stress group (
Acute stress altered goblet cell numbers, and an increase was observed in the acute stress group compared with the control group (
The presence of metachromatic mast cells in colon was detected via toluidine blue staining. The number of mast cells was markedly increased in the acute stress group (
Western blot analysis demonstrated different protein expression patterns under stress conditions. Densitometric analysis revealed lower levels of claudin-1 expression in the acute and chronic stress groups compared with those in the control group (P<0.001). Claudin-1 expression was also significantly higher in the acute stress group compared with in the chronic stress group (P<0.05;
Compared with the control, acute and chronic stress diminished the percentages of CD4+ T cells and CD4+ Treg cells in the lamina propria (
Stimulation of the sensory nerve causes an axonal reflex, producing neuropeptides, especially SP, that activate inflammatory cells. Next, the effect of stress on serum SP concentration was examined. As shown in
The present study identified an association between plasma corticosterone and the stress type in the mouse model, and similar results have been reported previously under different stress conditions (
Despite the decreased claudin-1 protein expression in the TJ region during acute and chronic stress, the current study observed that the mucosa in those experimental groups was similar to that in the control group. A previous study (
The present results revealed an increase in the CD4+/IL-4+ lymphocyte percentage under acute stress. Although it is well established that stress in the colon increases the number of CD4 lymphocytes (
The delicate balance between pro- and anti-inflammatory mechanisms, essential for intestinal immune homeostasis, is regulated by Treg cells that express the transcription factor FoxP3 and play role in limiting inflammatory responses in the intestine (
Additionally, in the current study, the serum SP concentration after repeated exposure to restriction stress increased during acute stress, and this observation differed from the results after exposure to chronic stress. This phenomenon could be consistent with a study showing that SP reduces the duration of acute stress, and therefore, SP plays an essential role in the transition between acute and chronic stress (
The increase in the serum SP concentration during acute stress may be associated with two factors that are not necessarily contradictory. First, a previous study has proposed that SP does not inhibit the initial activation of the HPA axis in response to restraint stress, but does act via NK1R at a central level (
An important limitation of the present study was to analyze substance P in serum and not in colonic tissue, this would have allowed the present study to relate
In conclusion, the parameters evaluated in the current study suggest that acute stress may facilitate stress management therapy to benefit the resolution of intestinal diseases.
The authors thank Dr Rosa Adriana Jarillo-Luna for her laboratory facilities; Postgraduate Studies and Research Section, Superior School of Medicine, National Polytechnic Institute, Mexico City, Mexico.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
JPY and EAR conceptualized the study. IMAM, AARA, LMCJ, BMA, RFV, JMGM, MYO, JPY and EAR designed the study. IMAM, AARA, LMCJ, JPY and EAR utilised the software. IMAM, AARA, LMCJ, JPY and EAR performed formal analysis. JPY, AARA and EAR provided the resources. JPY and EAR contributed to data curation. IMAM, AARA, JPY and EAR wrote initial draft of the manuscript. IMAM, AARA, LMCJ, BMA, RFV, JMGM, MYO, JPY and EAR wrote, reviewed and edited the manuscript. JPY and EAR supervised the study. JPY and EAR performed project administration and acquired the funding. IMAM and EAR confirm the authenticity of all the raw data. All authors have read and approved the final version of the manuscript.
The study was conducted according to the guidelines the Mexican Federal Regulations for animal experimentation and care (Regulation-062-ZOO-1999; Ministry of Agriculture, Mexico City, México) and was approved by the Superior School of Medicine, National Polytechnic Institute.
Not applicable.
Drs Judith Pacheco-Yépez, Edgar Abarca-Rojano and Aldo A. Reséndiz-Albor are fellows of Commission for the Operation and Promotion of Academic Activities of the National Polytechnic Institute (COFAA) and Stimulus to the Performance of Researchers-National Polytechnic Institute (EDI–IPN). Professor Ivonne Maciel Arciniega-Martínez is a fellow of EDI–IPN.
The authors declare that they have no competing interests.
Corticosterone serum concentrations. Mice were subjected to different experimental conditions, and then, peripheral blood was collected and analyzed using a colorimetric-enzymatic assay. The data are expressed as the mean ± SD (n=5). *P<0.05, ***P<0.001 vs. control group; ***P<0.001 (umbrella line comparison).
Representative photomicrographs obtained via light microscopy. (A) control, (B) acute stress and chronic stress (C) Colon samples stained with H&E to detect infiltrating inflammatory cells (arrows). (D) control, (E) acute stress and chronic stress (F) Colon samples stained with Alcian blue to visualize goblet cells (arrows). (G) control, (H) acute stress and chronic stress. (I) Colon samples stained with toluidine blue for the detection of mast cells (arrows). Scale bar, 25 µm. (J) Quantification of the polymorphonuclear infiltrate in mouse colon. (K) Goblet cell quantification in mouse colon. (L) Mast cell quantification in mouse colon. Data are expressed as the mean ± SD (n=5). *P<0.05, **P<0.01 vs. control group; **P<0.01, ***P<0.001 (umbrella line comparison).
Relative protein expression levels of claudin-1 and E-cadherin, as analyzed via western blotting. (A) Claudin-1 expression. (B) E-cadherin expression. Data are expressed as the mean ± SD for each group (n=5). (C) Intensities of the bands were measured via densitometric analysis and normalized to the intensities of the corresponding β-actin bands. **P<0.01, ***P<0.001 vs. control group; *P<0.05, ***P<0.001 (umbrella line comparison).
Serum substance P concentration. Data are expressed as the mean ± SD (n=5). *P<0.05 vs. control group; **P<0.01 (umbrella line comparison).
Cytokine/IL CD4+ T and Treg cell responses in the lamina propria of the large intestine under acute stress and chronic stress.
Cytokine/IL | Control | Acute stress | Chronic stress |
---|---|---|---|
IFN-γ | 2.77±0.15 | 0.50±0.10 |
0.23±0.06 |
TNF-α | 1.93±0.15 | 0.73±0.12 |
0.70±0.10 |
IL-1β | 1.47±0.15 | 0.43±0.15 |
0.30±0.10 |
IL-12 | 4.20±0.20 | 4.10±0.20 | 0.57±0.12 |
IL-6 | 2.60±0.20 | 11.60±1.18 |
0.70±0.10 |
IL-4 | 2.03±0.25 | 8.03±0.15 |
0.23±0.12 |
IL-10 | 2.70±0.20 | 0.63±0.15 |
0.13±0.06 |
FoxP3 | 5.17±0.21 | 1.07±0.31 |
0.93±0.15 |
Percentage of cytokine/IL CD3+/CD4+ T and FoxP3+/CD3+/CD4+ Treg cell from lamina propria is expressed as mean ± SD of 5 mice per group from three independent experiments.
P<0.05 vs. control group. Treg, regulatory T cells.