Prognostic value of neurotensin in experimental terminal ileitis

Abstract

Background

Terminal ileitis (TI) is an inflammatory condition of the distal portion of the ileum.

Objective

To investigate the changes and significance of neurotensin (NT) in the course of experimental terminal ileitis (ETI).

Methods

A total of 90 Sprague Dawley (SD) rats were randomly divided into the control, model, and suture groups (30 rats in each group). After 2, 4, and 8 weeks of surgery, 10 TI tissues were taken for endoscopic observation in each group. Hematoxylin-eosin (HE) staining was used to detect the pathological changes. The NT levels in serum and terminal ileum mucosa were detected by Enzyme-linked immunosorbent assay (ELISA).

Results

Acute inflammatory changes were observed in the suture and the model groups after 2-week of operation. At 4 weeks, compared to the control group, the inflammatory damage in the model group became heavier, but it was reduced in the suture group. At 8 weeks, the model group showed chronic inflammation. However, there was no obvious inflammatory cell infiltration in both the suture group and control group. NT levels were increased in the suture and model groups at 2 weeks, particularly in the model group, and they were significantly higher in the suture and model groups than those in the control group (P < 0.05). At 4 weeks, the NT levels in the model group continued to rise, while they decreased in the suture group (P < 0.05). After 8 weeks, NT levels in the model group were significantly higher than those in the suture and control groups (P < 0.05). Furthermore, from 2 weeks to 8 weeks, the NT concentration in the model group gradually increased, and the suture group increased for 2 weeks, followed by a downward trend.

Conclusions: In the ETI, the changes in NT concentration in serum and TI mucosa were positively correlated with the degree of inflammation.

Keywords

neurotensin experimental terminal ileitis serum tissue

1. Introduction

Terminal ileitis (TI) is a group of non-specific inflammatory reactions of the terminal ileal mucosa, mostly involving the mucosal and submucosal layers, occurring within 20 cm of the terminal ileum.¹ It is divided into acute terminal ileitis (ATI) and chronic terminal ileitis (CTI), which is mainly associated with infectious factors, immune factors, colo-ileal reflux, food allergies, and medications.² The main clinical manifestations are abdominal pain, diarrhea, blood in the stool, pressure in the right lower abdomen, and even central nervous system dysfunction.³ TI is mainly diagnosed by colon endoscopy, with an endoscopic detection rate of about 10%-50%.⁴

Neurotensin (NT), a gut hormone secreted by specific endocrine cells, plays an important role in regulating intestinal digestion, small intestinal motility, and intestinal inflammation in synergy with substance P, histamine, and growth inhibitors.^5–8 Especially, NT has a variety of biological tissue effects on the gastrointestinal tract, including cell proliferation, intestinal motility, mucus secretion, and intestinal mucosa function.^6,7 The involvement of NT has also been reported in the intestinal inflammatory response.⁸ In this study, we aimed to explore the correlation between NT concentration and the inflammation of TI in a rat ETI model and to further understand the pathogenesis of ETI.

2. Materials and methods

2.1 Animals

All the experimental procedures were performed according to the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The study was approved under the regulations of the Committee on the Ethics of Animal Experiments. A total of 90 male Sprague Dawley (SD) rats weighting 250–350 g were purchased from Shanghai Laboratory Animal Center (SLAC) and randomly divided into three groups: control group, model group, and suture group (n = 30 in each group). After 6-week of feeding, an ETI model was established based on previous research.⁹ Model group was given the abdominal surgery under aseptic conditions to expose ileocecal junction to perform ileum-cecum side-to-side anastomosis in terminal ileum, suture group was treated the same as the model group except performing surgical suture at terminal ileum, and control group accepted no special treatment.¹⁰ At 2, 4, and 8 weeks postoperatively, venous blood samples of rats were collected in standard laboratory EDTA tubes containing aprotinin (ICN Pharmaceuticals, Costa Mesa, CA, USA) and stored on ice immediately. EDTA tubes were then centrifugated and the serum was collected and placed at −70°C.

2.2 TI tissue sample preparation

After the execution of the rats by CO₂ asphyxiation, the abdominal cavity was opened along the originally abdominal wound location. In model group, the intestinal tube about 2 cm long at 1 cm was taken from the ileostomy wound. In both suture and control groups, the intestinal tube about 2 cm long at 1 cm was taken from the ileal wound. After rinsing with physiological saline, the tissue was divided into two parts: one for microscopic observation and the other for homogenization. After centrifugation (12000 × g for 15 min), the supernatant of homogenized tissues was collected and placed at −70°C for a further study.

2.3 Enzyme-linked immunosorbent assay (ELISA)

Rat NT Enzyme-Linked Immunoassay Kit (Nanjing Senbeijia Biotechnology Co., Ltd, Nanjing, China) was used to detect the NT levels in serum and tissues according to the manufacturer's instructions, as described in a previous study.¹¹

2.4 Preparation of visual and microscopic specimens and criteria

Gross and histomorphological scoring of TI tissue damage was performed by Hematoxylin-eosin (HE) staining assessment. The tissue was first observed by the naked eye in general. A portion of the tissue was then immediately fixed in a 10% formaldehyde solution and subjected to HE staining. Electron microscopic inflammation score was obtained according to the previous studies.^12,13

2.5 Statistical analysis

Statistical Package for the Social Sciences (SPSS) 26.0 software (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. Data were expressed as mean ± standard deviation (SD). Comparisons between the two groups were made using t-tests. Comprehensive comparisons between multiple samples were performed using one-way analysis of variance (ANOVA) in a completely randomized design. Correlations were analyzed by linear correlation analysis. α = 0.05 was used as the test level. The correlation between NT concentration in serum or TI tissues and pathology scores of TI tissue was analyzed by Spearman's correlation. P < 0.05 indicated a statistically significant difference.

3. Results

3.1 TI tissue microscopic observation

The images of TI tissues in three groups were shown in Figure 1, and the scores were demonstrated in Table 1. The TI mucosal tissues in the model and suture groups were observed microscopically at 2 weeks after surgery. It was found that the lesions mainly invaded the mucosal layer and accompanied by various inflammatory cell infiltrations, mainly neutrophils, with a small number of lymphocytes and plasma cells. The difference in microscopic scores of TI mucosal tissue between the model group and the suture group was statistically significant (P < 0.05). At 4 weeks postoperatively, inflammation of the TI mucosa was increased in the model group, with damage to the mucosa and submucosa, dominated by lymphocyte and plasma cell infiltration. Additionally, lymphoid follicles proliferated, eroded, ulcerated, and hemorrhaged. Mucosal glands were reduced and villi structures were indistinct or absent. The differences in TI hysteroscopic scores in the model group were statistically significant when compared with both the suture and control groups (P < 0.05). At 4 weeks postoperatively, the degree of inflammation in the TI mucosal tissues in the suture group was less than that at 2 weeks. The differences in the inflammation scores in the suture group were statistically significant compared with the control group or the model group (P < 0.05). At 8 weeks postoperatively, the inflammation in the model group further increased compared to that at 4 weeks. Furthermore, the degree of inflammation in TI tissues in the suture group was reduced compared with that at 4 weeks, but the difference in microscopic scores was not statistically significant when compared with the control group (P > 0.05).

Figure 1.

Microscopic images of TI tissue from various groups of rats.

Table 1.

Microscopic inflammation score of rat TI tissues.

		Electron microscopic inflammation score (points)
Groups	Cases (n)	2W	4W	8W
Control group	10	0.30 ± 0.48^•▾	0.20 ± 0.42^•▾	0.40 ± 0.52^•
Suture group	10	4.90 ± 0.58	2.80 ± 0.79^▪	0.70 ± 0.48^▪
Model group	10	5.30 ± 0.48	7.30 ± 0.95	9.40 ± 1.07
F		293.557	227.706	473.497
P		0.001	0.001	0.001

Note: ^•P < 0.05 for a model group vs control group; ^▾P < 0.05 for suture group vs control group; ^▪P < 0.05 for model group vs suture group.

3.2 Changes in serum NT concentrations

The serum NT concentrations in each group were shown in Table 2. At 2 weeks after surgery, the serum NT concentration was increased in both the model group and the suture group compared with the control group (P < 0.05). Compared to results at 2 weeks, the serum NT concentrations were promoted in the model group, and they were inhibited in the suture group at 4 weeks postoperatively (P < 0.05). Compared to the serum NT concentrations at 4 weeks, at 8 weeks postoperatively, they were increased in the model group. There were statistically significant differences between the model group and the control and suture groups (P < 0.05). The serum NT concentration in the suture group at 8 weeks postoperatively was lower than that at 4 weeks. The difference was not statistically significant between the suture group and the control group (P > 0.05).

Table 2.
Serum NT concentrations in each group.

Serum NT concentrations (ng/L)

Groups Cases (n) 2W 4W 8W

Control group 10 38.74 ± 0.99^▾• 39.02 ± 1.03^▾• 38.96 ± 0.96^▾

Suture group 10 55.57 ± 1.90^▪ 43.58 ± 2.96^▪ 38.43 ± 1.13^▪

Model group 10 67.01 ± 2.07 70.53 ± 1.68 75.69 ± 2.95

F 717.142 685.848 1255.467

P 0.001 0.001 0.001

		Serum NT concentrations (ng/L)
Control group	10	38.74 ± 0.99^▾•	39.02 ± 1.03^▾•	38.96 ± 0.96^▾
Suture group	10	55.57 ± 1.90^▪	43.58 ± 2.96^▪	38.43 ± 1.13^▪
Model group	10	67.01 ± 2.07	70.53 ± 1.68	75.69 ± 2.95
F		717.142	685.848	1255.467
P		0.001	0.001	0.001

Note: ^•P < 0.05 for a model group vs control group; ^▾P < 0.05 for a suture group vs control group; ^▪P < 0.05 for a model group vs suture group.

In addition, serum NT concentrations were significantly higher in the suture and model groups at 2 weeks postoperatively than those in the control group. Serum NT concentrations in the suture group gradually decreased to normal levels between 2 and 8 weeks, while those in the model group still gradually increased. This suggests that NT is involved in the acute inflammatory response of the intestine and may be related to the chronic inflammation of the intestine and the degree of inflammation.

3.3 Changes in NT concentrations of TI tissues

The NT concentrations in the TI mucosal tissues in three groups were shown in Table 3. Two weeks after surgery, the NT concentrations in the TI mucosal tissues in both the model group and the suture group were increased compared to the control group. There were no significant differences between in the model group and in the suture group (P > 0.05). At 4 weeks postoperatively, the NT concentration in TI mucosal tissues was higher in the model group than that at 2 weeks, and it was lower in the suture group than that at 2 weeks (P < 0.05). At 8 weeks postoperatively, NT concentrations in the TI mucosal tissues were increased in the model group and decreased in the suture group compared with those at 4 weeks. At 8 weeks postoperatively, the difference between the model group and the suture group was statistically significant (P < 0.05), while the difference between the suture group and the control group was not statistically significant (P > 0.05).

Table 3.
Nt concentrations in TI mucosal tissues.

NT concentrations in TI mucosal tissues (ng/L)

Groups Cases (n) 2W 4W 8W

Control group 10 42.64 ± 1.02^•▾ 42.46 ± 1.40^•▾ 42.83 ± 1.21^▾

Suture group 10 64.49 ± 2.70^▪ 54.00 ± 2.64^▪ 43.76 ± 1.66^▪

Model group 10 70.90 ± 1.38 76.27 ± 1.50 81.81 ± 1.90^•▾

F 644.583 792.341 1900.061

p 0.001 0.001 0.001

		NT concentrations in TI mucosal tissues (ng/L)
Control group	10	42.64 ± 1.02^•▾	42.46 ± 1.40^•▾	42.83 ± 1.21^▾
Suture group	10	64.49 ± 2.70^▪	54.00 ± 2.64^▪	43.76 ± 1.66^▪
Model group	10	70.90 ± 1.38	76.27 ± 1.50	81.81 ± 1.90^•▾
F		644.583	792.341	1900.061
p		0.001	0.001	0.001

Note: ^▾P < 0.05 for a model group vs. control group; ^▪P < 0.05 for suture group vs. control group; ^•P < 0.05 for model group vs suture group.

Also, as shown in Table 3, the NT concentration in the mucosal tissues in the model group and the suture group increased significantly at 2 weeks after surgery. The NT concentration in the mucosal tissues in the suture group gradually decreased to normal levels from 2 weeks to 8 weeks, while the NT concentration in the model group gradually increased. It can be concluded that NT is involved in the acute inflammation of the terminal ileum and plays a role in the persistence of chronic inflammation.

3.4 Correlation between the changes in NT concentration and the degree of inflammation in TI mucosal tissue in the model group

Correlation between the changes in NT concentration and the degree of inflammation in TI mucosal tissue in the model group was conducted and shown in Table 4. In the model group, a lateral anastomosis between the end of the ileum and the beginning of the colon was taken. The changes of NT concentrations in serum and tissues and inflammation scores showed a positive correlation between NT concentrations and the degree of TI tissue inflammation (R-values of 0.948 and 0.979, respectively; P < 0.05; Table 4).

Table 4.
Correlation analysis between NT concentration and inflammation score of TI tissue in the model group.

NT concentration (ng/L)

Inflammatory scores Cases (n) Serum TI tissues

5 7 66.77 ± 1.69 70.29 ± 0.73

6 5 69.09 ± 1.24 73.37 ± 1.39

7 4 70.18 ± 1.01 75.64 ± 0.40

8 5 71.72 ± 1.01 78.10 ± 1.55

9 5 74.69 ± 1.69 80.78 ± 0.93

10 2 77.39 ± 0.66 82.75 ± 0.39

11 2 79.32 ± 0.71 84.70 ± 0.35

R 0.948 0.979

P 0.001 0.001

		NT concentration (ng/L)
5	7	66.77 ± 1.69	70.29 ± 0.73
6	5	69.09 ± 1.24	73.37 ± 1.39
7	4	70.18 ± 1.01	75.64 ± 0.40
8	5	71.72 ± 1.01	78.10 ± 1.55
9	5	74.69 ± 1.69	80.78 ± 0.93
10	2	77.39 ± 0.66	82.75 ± 0.39
11	2	79.32 ± 0.71	84.70 ± 0.35
R		0.948	0.979
P		0.001	0.001

4. Discussion

According to the currently recognized possible factors and main mechanisms of CTI pathogenesis,¹⁴ the ETI model was constructed in SD rats based on the basic principle of denuded ileal valve function - bacterial translocation - ileal gut-associated lymphoid tissue (GALT) immune response - ileal mucosal damage - CTI in this study. The NT concentrations in the serum and TI mucosa were measured by ELISA. The significance of NT in the development of ETI was further confirmed, and a simpler, more convenient and sensitive test was explored for the diagnosis of the disease, providing a theoretical basis for the treatment and monitoring of the efficacy of the ETI disease.

Several studies pointed to a role for NT in inflammatory reactions.^15–17 Intravenous administration of NT causes mast cell degranulation and increases vascular permeability and levels of histamine and leukotriene C4 in the plasma of rats.¹⁵ NT also interacts with immune and inflammatory cells such as leukocytes, peritoneal mast cells, and macrophages.^16,17 In this study, we investigated the changes in NT concentrations in serum and terminal ileal mucosal tissues during the development of ETI. The NT concentrations are likely to be an important indicator of the degree of mucosal damage in the mucosa of TI patients. This study showed that NT concentrations in serum and TI mucosa were significantly increased in the suture and model groups compared to the control group. From 2 weeks to 8 weeks after surgery, the NT concentration gradually increased in the model group. Additionally, the correlation between the changes in NT concentrations of serum or TI mucosal tissues and inflammation scores in the model group showed that the NT concentrations in the serum and tissues were positively correlated with the degree of inflammation in TI. Our findings suggest that NT is involved in the acute inflammatory response of the intestine and plays a role in the persistence of chronic inflammation of the intestine and the degree of inflammation.

With the development of the “neuro-immuno-endocrine network” theory, the connection between the immune system and the nervous system has been increasingly understood.^18–20 The research on neurohypophyseal peptides began in the 1970s. NT is a brain-intestinal peptide that functions independently of the central nervous system. After many foreign scholars conducted extensive studies on the role of NT in this regard, it is recognized that NT exerts a variety of biological tissue effects on the gastrointestinal mucosa.²¹ For example, it is involved in the regulation of gastrointestinal dynamics, the diastolic function of the oesophageal sphincter, and the function of the intestinal barrier.²² The immune barrier is an important component of the intestinal mucosal barrier, and there is a bidirectional regulation between the immune system and the secretory system, thus forming a neuro-immuno-endocrine network.²³ Zhang et al. have used this model to study the etiology and pathogenesis of neuropeptides through the neuro-immuno-endocrine network in CTI and its significance.²⁴ In this study, we also have applied the TI model and confirmed the value of NT.

NT is a member of a neuro-immune-endocrine network system. NT is mainly secreted and released by N cells distributed in the ileum into the intestinal mucosal tissues to protect the intestinal mucosal barrier. Once the mucosal tissue of the terminal ileum is damaged, the NT concentration in the serum and the terminal ileal mucosal tissue increases. The rupture of N cells in the intestinal mucosal tissue of the terminal ileum is considered to result in a large release of intracellular NT into the surrounding mucosal tissue and partial reflux into the blood. However, further studies are needed to clarify the exact mechanism. Further, little researches have reported on the relevance of neurohypocretin in the pathogenesis of CTI. In the present study, the pattern of NT concentration in serum and terminal ileal mucosal tissues during the development of CTI has been clarified.

Conclusion

In ETI rats, NT concentrations in the serum and terminal ileum mucosal tissues are increased significantly and are positively correlated with the degree of inflammation in the terminal ileum. It is indicated that NT concentration in the mucosal tissues and serum may be an important indicator of the degree of inflammation in the terminal ileum. However, the clinical value of NT concentration needs to be further investigated.

Footnotes

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

References

Jain

Tang

Jones

, et al. Understanding the presentation of Terminal Ileitis. Case Rep Gastroenterol 2022; 16: 675–679.

Triantafillidis

Thomaidis

Papalois

. Terminal Ileitis due to Yersinia infection: an underdiagnosed situation. Biomed Res Int 2020; 2020: 1240626.

Sachdeva

Kedia

Ahuja

. Terminal ileitis: another battle of panipat or just a skirmish? Indian J Gastroenterol 2023; 42: 598–600.

Freitas

Cúrdia Gonçalves

Boal Carvalho

, et al. From terminal ileitis to Crohn's disease: how capsule endoscopy is crucial to diagnosis. Eur J Gastroenterol Hepatol 2021; 33: 631–638.

Czepielewski

, et al. Neurotensin is an anti-thermogenic peptide produced by lymphatic endothelial cells. Cell Metab 2021; 33: 1449–1465.e6.

Zhou

. Research progress on the effect of neurotensin on the intestinal mucosal barrier. Med Rev 2016; 22: 650–653.

Zhao

Pothoulakis

. Effects of NT on gastrointestinal motility and secretion, and role in intestinal inflammation. Peptides 2006; 27: 2434–2344.

Song

, et al. Absence of neurotensin attenuates intestinal dysbiosis and inflammation by maintaining Mmp7/alpha-defensin axis in diet-induced obese mice. FASEB J 2020; 34: 8596–8610.

Chen

Wang

. Research status and progress of intestinal mucosal biological barrier. Colorectal Anal Surg 2012; 18: 268–271.

10.

Zhou

Yan

Fang

, et al. Change and significance of IL-8, IL-4, and IL-10 in the pathogenesis of terminal Ileitis in SD rat. Cell Biochem Biophys 2014; 69: 327–331.

11.

Yang

Xin

, et al. Involvement of neurotensin-mediated brain-gut axis in electroacupuncture intervention induced improvement of functional dyspepsia in rats. Zhen Ci Yan Jiu 2016; 41: 35–39, 50.

12.

Negussie

Dell

Davis

, et al. Colonic fluid and electrolyte transport 2022: an update. Cells 2022; 11: 1712.

13.

Dieleman

Palmen

Akol

, et al. Chronic experimental colitis induced by dextran sulphate sodium (DSS) is characterized by Th1 and Th2 cytokines. Clin Exp Immunol 2001; 114: 385–391.

14.

Zhou

Long

, et al. Experimental study on the construction of chronic terminal ileitis in SD rats. Chin J Digestion 2002; 22: 702–703.

15.

Babu

Mohanty

. Neurotensin modulation of lipopolysaccharide induced inflammation of gut-liver axis: evaluation using neurotensin receptor agonist and antagonist. Neuropeptides 2023; 97: 102297.

16.

Liu

Wang

, et al. Neurotensin, a novel messenger to cross-link inflammation and tumor invasion via epithelial-mesenchymal transition pathway. Int Rev Immunol 2016; 35: 340–350.

17.

White

Noinaj

Shibata

, et al. Structure of the agonist-bound neurotensin receptor. Nature 2012; 490: 508–513.

18.

Iorfida

Montuori

Trovato

, et al. Fasting neurotensin levels in pediatric celiac disease compared with a control cohort. Gastroenterol Res Pract 2020; 2020: 1670479.

19.

Wilson

Naves

Saada

, et al. The implications of sortilin/vps10p domain receptors in neurological and human diseases. CNS Neurol Disord Drug Targets 2014; 13: 1354–1365.

20.

Di Fruscia

Koenig

, et al. The discovery of indole full agonists of the neurotensin receptor 1 (NTSR1). Bioorg Med Chem Lett 2014; 24(16): 3974–3978.

21.

Di Fruscia

Koenig

, et al. The discovery of indole full agonists of the neurotensin receptor 1 (NTSR1). Bioorg Med Chem Lett 2014; 24: 3974–3978.

22.

Zhu

Cheng

, et al. Ghrelin, CGRP, NT: advances in the study of gastrointestinal effects. Adv Mod Biomed 2014; 14: 3191–3197.

23.

Wang

Zhou

, et al. Glutamine and intestinal barrier function. Amino Acids 2015; 47: 2143–2154.

24.

Zhou

Zhang

, et al. Correlation analysis of changes in SP, VIP, NO and experimental terminal ileitis. South Chin J Nat Def Med 2010; 24: 341–345.

		NT concentration (ng/L)
Inflammatory scores	Cases (n)	Serum	TI tissues
5	7	66.77 ± 1.69	70.29 ± 0.73
6	5	69.09 ± 1.24	73.37 ± 1.39
7	4	70.18 ± 1.01	75.64 ± 0.40
8	5	71.72 ± 1.01	78.10 ± 1.55
9	5	74.69 ± 1.69	80.78 ± 0.93
10	2	77.39 ± 0.66	82.75 ± 0.39
11	2	79.32 ± 0.71	84.70 ± 0.35
R		0.948	0.979
P		0.001	0.001