Introduction

The intestinal barrier is a semi-permeable structure in the gut that plays a crucial role in both nutrient absorption and protection against harmful microorganisms and toxins. Defects in the intestinal barrier have been linked to a range of diseases, such as gastrointestinal disorders (e.g., celiac disease, IBD, colon cancer) and extraintestinal conditions (e.g., chronic liver disease, type 1 diabetes, obesity)1. Several disorders that are brought on by unhealthy eating habits may have their roots in the disturbance of the intestinal barrier. Martinez-Medina’s study found that feeding mice a diet high in fat and sugar caused changes in the microbiota’s composition. These changes included a decrease in the mucus layer’s thickness, an increase in intestinal permeability, a decrease in the expression of goblet cells and mucins, and an increase in inflammatory markers2. Furthermore, drinking alcohol can lead to an imbalance of intestinal flora and damage to the intestinal barrier. This enables pathogen-associated molecular patterns to enter the bloodstream and move to the liver, where they cause hepatic steatosis and worsen alcoholic hepatitis3,4. Therefore, maintaining a good diet and an intact intestinal barrier plays an important role in maintaining health and preventing disease.

Both sodium intake and processed food sales are quite high in the Western diet, where the average processed meal has 100 times more salt than homemade food5, and most people already consume considerably more salt than is considered normal. An HSD is intimately linked to chronic inflammatory illnesses like IBD and causes gut microecological dysbiosis. By controlling nutrient uptake, gut homeostasis, host immunity, and metabolic pathways, the gut microbiota largely contributes to the preservation of host health6. Strong epidemiologic evidence supports that HSD underlies the increase in chronic inflammatory diseases such as IBD7. In Miranda’s report, HSD ingestion in mice promotes colonic inflammation by decreasing Lactobacillus levels and butyrate production8. Qu et al. noted that disruption of the balance of the intestinal microbiota leads to the overgrowth of harmful bacteria and the production of deleterious substances (e.g., endotoxin), which can compromise the integrity of the intestinal barrier9. A compromised intestinal barrier promotes increased permeability, facilitating the transfer of harmful substances and bacteria into the bloodstream and ultimately triggering systemic inflammation10. Therefore, we propose that a high-salt diet may directly damage the intestinal barrier and promote an inflammatory response that leads to a variety of diseases.

HSD has been demonstrated to possess pro-inflammatory properties within the gut microenvironment. Such pro-inflammatory effects can disrupt the normal physiological balance and function of the intestine, ultimately leading to the impairment of intestinal health. Therefore, this study will explore the mechanism of long-term HSD damage to intestinal barrier function in rats. Immunofluorescence and PAS staining were used to assess intestinal damage in HSD rats. This study may provide theoretical support for the prevention and treatment of intestinal diseases.

Materials and methods

Materials and reagents

Commercial ELISA kits for D-lactate, SIgA, interferon (IFN)-γ, IL-4, IL-10, and IL-22 were purchased from Elabscience (Wuhan, China). β-defensin was obtained from Jiangsu Jingmei Biotechnology Co. All other reagents were of analytical grade.

Antibodies including Claudin-1, Occludin, ZO-1, and MUC2 were purchased from Proteintech (USA).

Animals and experimental design

In this study, we strictly follow the requirements of the ARRIVE Guide11 (Animal Research: Reporting of In Vivo Experiments) to ensure the transparency, quality, and repeatability of animal experiments. We confirm that all experiments were performed in accordance with relevant guidelines and regulations. The use of experimental animals has been reviewed by the Experimental Animal Welfare Ethics Association of Chengdu University of Traditional Chinese Medicine (review number:2024078). Twelve SD rats, male, 6 weeks old, 180 ± 20 g, were obtained from Beijing SPF(Beijing) Biotechnology Co., LTD, under standard laboratory conditions (temperature 25 ± 2 ℃, humidity 50 ± 5%. light/dark cycle of 12 h/12 h). They were kept in isolated cages and given food and water ad libitum. After 1 week of domestication, 12 SD rats were randomly divided into NSD and HSD groups (6 rats in each group). The rats in the NSD group were given 0.5% normal chow, and the rats in the HSD group were given 8% high salt chow. The regular and high-salt diets were obtained from the same feed company (Beijing Ke’ao Hip Lik Feed Co., Ltd.) and had the same nutritional composition except for the salt content.

The rats were continuously fed for 12 weeks, their feed intake and water intake were recorded daily and their weight was recorded weekly. After overnight fasting, rats were euthanized by intraperitoneal injection of 150 mg/kg sodium pentobarbital, followed by collection of blood, and colon tissue for future experiments. The blood was centrifuged at 4 °C and 3000 rpm for 20 min to obtain serum, which was then stored at -80 °C for further analysis. The colon tissue was divided into two parts, one part of the colon tissue was fixed in 4% paraformaldehyde for histological analysis and immunofluorescence analysis, and the other part of the colon tissue was frozen in liquid nitrogen and stored at -80 °C for further biochemical analysis.

Histological analysis

The colon tissues were fixed in 4% paraformaldehyde for more than 24 h, then dehydrated with different concentrations of ethanol, embedded in paraffin, cut into 3-µm-thick sections, and finally stained with hematoxylin and eosin (H&E). Goblet cells in the colon were identified using periodate-schiff (PAS) staining as described above12. Micrographs were taken using a microscope and images were analyzed using Image-pro plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA).

Immunofluorescence (IF) analysis

4% paraformaldehyde-fixed colon samples were rinsed in PBS after dehydration with graded ethanol, followed by antigen repair in EDTA (pH 8.0) buffer. Immunofluorescence was then assessed using primary antibodies (ZO-1, Occludin, Claudin-1, MUC2). Sections were incubated with fluorescein isothiocyanate isomer (FITC)-labeled secondary antibody, and then sections were stained for nuclear re-staining with 4’,6-diamidino-2-phenylindole (DAPI) (Servicebio, Chengdu, China). Images were captured under an Olympus confocal microscope, and fluorescence intensity was analyzed by Image J.

Biochemical evaluation

Serum D-lactate levels, colon tissue SIgA, β-defensin, and cytokines IL-4, IL-10, IL-22, and IFN-γ were determined by appropriate ELISA kits according to the manufacturer’s instructions.

Statistical analysis

All data are expressed as mean ± standard error (SEM). In SPSS (26.0, IBM), the normal distribution and homogeneity of variance of the data were checked using Shapiro-Wilke and Levin tests, respectively. Two-tailed unpaired T-tests or Mann-Whitney U tests for normally distributed or non-normally distributed datasets were used to assess statistical differences in biological parameters between the NSD and HSD groups, respectively. Use GraphPad Prism 9.5 to generate histograms. P < 0.05 was considered statistically significant. P values were expressed as follows: * P < 0.05, * * P < 0.01, * ** P < 0.001, and ns had no statistical significance (P > 0.05).

Results

Effect of HSD on body weight of rats

During the 12-week dietary intervention experiment, the weight of rats in the NSD group basically increased steadily. From week 4 to week 8, the weight of rats in the HSD group began to decrease significantly and then began to increase gradually from week 10 to week 12, but the weight of rats in the HSD group was significantly lower than that of rats in the NSD group (P < 0.05; Fig. 1).

Fig. 1
figure 1

Effects of HSD on body weight.

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Effect of HSD on the intestinal epithelial barrier

In this study, histologic examination showed that inflammatory cell aggregates were seen in multiple locations in the lamina propria of the colon in the HSD group compared to the NSD group (Fig. 2A). The paracellular barrier function of the intestinal epithelium is thought to be regulated by tight junction proteins including Claudin-1, Occludin and ZO-113, and according to our current results, rats in the HSD group showed a significantly (P < 0.05) lower expression of Occludin, and the mean values of both Claudin-1 and ZO-1 expression were reduced compared to those in the NSD group (Fig. 2B-G). Serum D-lactate is a recognized marker for monitoring changes in intestinal permeability14. In this work, serum D-lactate concentration was significantly elevated in the HSD group of rats compared to the NSD group of rats (Fig. 2H). These results clearly indicate that HSD impairs the intestinal epithelial barrier.

Fig. 2
figure 2

Effects of HSD on intestinal epithelial barrier: (A) Photomicrographs of HE-stained colon Sect. (200× magnification); (B–G) Relative expression of tight junction protein in the colon (B, C, Claudin-1; D, E, Occludin; F, G, ZO-1); (H) Level of d-lactic acid in serum.

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Effect of HSD on the intestinal mucus barrier

Since the mucus barrier also plays an important role in maintaining intestinal barrier homeostasis, we subsequently investigated the effect of HSD on intestinal mucus barrier function in rats. The results of PAS staining showed that the colons of rats in each group were enriched with goblet cells and displayed intact mucosa and normal glands (Fig. 3A). However, the mucopolysaccharide-expressing area per goblet cell in the intestinal tissues of rats in the HSD group was significantly lower compared with the NSD group (P < 0.05), and the mean mucopolysaccharide area of the mucus layer in the HSD group (18.26 ± 3.94) was lower than that in the NSD group (22.73 ± 6.08; Fig. 3B-C). In particular, we further examined the expression level of MUC2, a major member of the intestinal mucin family secreted by goblet cells and involved in the formation of the intestinal mucus layer15. The level of MUC2 in the intestinal mucus of rats in the HSD group (0.78 ± 0.75%) was significantly lower compared to that in the NSD group (3.58 ± 2.35%; Fig. 3D-E). In addition, we found that β-defensin levels were also significantly reduced in rats in the HSD group compared to the NSD group (Fig. 3F).

Fig. 3
figure 3

Effects of HSD on intestinal mucus barrier function: (A) Photomicrographs of PAS-stained colon Sect. (200× magnification); (B) Mucopolysaccharide area/Goblet cell; (C) Mucopolysaccharide area ratio (%); (D, E) Relative expression of MUC2 in the colon; (F) Levels of β-defensins in mucus layer.

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Effect of HSD on intestinal mucosal immunity

SIgA secretion was measured to investigate the effect of HSD supplementation on intestinal mucosal immunity in rats. As shown in Fig. 4A, the secretion level of SIgA in the intestinal tissues of rats in the HSD group ((25.46 ± 2.86) ng/mL) was significantly lower than that in the NSD group ((35.25 ± 5.87) ng/mL) (P < 0.01). In addition, the levels of several immune-related cytokines, including IL-4, IL-10, IL-22, and IFN-γ, were also examined, and the results showed that the levels of IL-10, IL-22, and IFN-γ were all significantly lower in the HSD group compared with the NSD group (Fig. 4B-E).

Fig. 4
figure 4

Effects of HSD intestinal immune cytokine profiles: (A) to (E) present the results of SIgA, IL-4, IL-10, IL-22, and IFN-γ, respectively.

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Discussion

The effects of high salt intake have long been focused mostly on its mechanisms of action on blood pressure and cardiovascular effects. However, there is also a correlation between high salt dietary intake and obesity. There is still some controversy about the impact of HSD on body weight in rats, which may be related to the duration of HSD intake. Several short-term experimental studies of high salt intake showed no significant differences in body weights between rats fed a normal salt diet and rats fed a high salt diet16,17. This is consistent with the results of the present study, in which HSD for less than 4 weeks did not change the body weight of the rats compared to the rats in the NSD group. The experiment by Kitada et al. explains that a short-term HSD leads to hyperphagia in mice and humans, but in order to maintain fluid homeostasis, high salt intake re-adjusts the body’s metabolism and energy expenditure, and the body energy of the mice remains in a high catabolic state, and their body weights remain stable under the ad libitum dietary conditions18. With the prolongation of high salt intake, HSD may enhance the ameliorative effect of insulin resistance by reducing the number of intestinal probiotics, and lead to weight loss in rats. In an 8-week study conducted by Kerem et al. for obese type 2 diabetes mellitus (T2DM) mice19, the body weight of mice in the high-salt-high-fat diet (HSD-HFD) group was significantly reduced compared to the high-fat diet (HFD) alone group, suggesting that HSD inhibits weight gain in obese T2DM mice. In our study, the body weight of rats in the HSD group decreased significantly from week 4 to week 8. It has been shown that prolonged high salt intake reduces the number of intestinal probiotics8, which are known to have a positive effect on reducing insulin resistance and ameliorating diseases such as hepatic steatosis20. In addition, other studies have shown that high salt intake reduces HRD (high-rice diet)-induced weight gain and white adipose tissue (WAT) weight gain. HSD has also been found to modulate HRD-induced increases in peroxisome proliferator-activated receptor-γ (PPAR-γ) and lipid metabolism-related protein expression21. The study considered by Do and colleagues further concluded that chronically high salt intake reduces body weight and fat accumulation in animals, probably because the increased salt load promotes energy expenditure and fatty acid oxidation processes in animals22. In the current study, we observed that from the 10th week onwards, the rats started to show a slow increase in body weight. This phenomenon may be related to the activation of the aldose reductase-fructose kinase pathway in the liver and hypothalamus by high salt intake. Studies by Miguel Lanaspa and colleagues have demonstrated that high salt intake activates this pathway, which in turn triggers endogenous fructose production and the development of leptin resistance23. Specifically, in the experiments of Miguel Lanaspa et al., insulin resistance occurred in mice that began to gain weight at week 13 of high salt intake. In view of the complexity of the association between HSD and body weight, it is necessary to further investigate the specific mechanisms and interrelationships between the duration of HSD intake and factors such as intake, energy metabolism, osmolality, as well as the intestinal microbiota and its metabolites in the subsequent studies, and to clarify the effects of HSD on body weight under different conditions by using a comprehensive combination of multi-histology techniques, animal model experiments, and clinical observations, in order to provide a scientific basis for the development of rational dietary strategies and the prevention of related diseases. This will provide a scientific basis for the formulation of rational dietary strategies and the prevention of related diseases.

The intestine plays a crucial role in the digestion and absorption of nutrients while also providing a robust mucosal barrier that fulfills its protective function within the body24. The mucus layer is the first physical line of defense encountered by external molecules reaching the intestinal lumen, and it prevents bacteria from directly contacting the epithelial cells25. The main components of the mucus layer are highly glycosylated mucins, which form a gel-like sieve structure covering the intestinal epithelium26. Mucins are secreted by intestinal goblet cells and form a barrier capable of restricting bacterial entry and screening bacterial metabolites or toxins from entering the epithelium27. The results of the present study clearly show that ingestion of HSD significantly reduces the area of mucopolysaccharide expression per goblet cell as well as the mucin area ratio in the colon, thus suggesting that the reduction of mucin synthesis in the intestine attenuates the mucosal barrier function. MUC2 is the most abundant mucin secreted by goblet cells, and MUC2 expression is essential for epithelial protection. Studies have shown that mice with knockout of MUC2 spontaneously develop colitis28. Chronic HSD intake has been shown to reduce MUC2 levels in intestinal contents, which attenuates intestinal mucus barrier function and impairs intestinal health. MUC2 is a skeletal component of the mucus layer, and MUC2 also cooperates with other constituents, such as antimicrobial peptides (AMPs), to consolidate the mucosal barrier structure and regulate local microenvironmental homeostasis. AMPs are a class of biologically active peptides with antimicrobial properties induced in organisms and released in mucus gels to enhance the physical separation of the gradient from the epithelial cells to the lumen29. AMPs not only have antimicrobial and immunomodulatory roles but also increase the body’s resistance to pathogenic infections by enhancing intestinal epithelial barrier function30. β-Defensins are a major type of AMPs31. Studies have reported that β-defensins can act as chemokines to drive leukocytes to the site of infection, thereby inhibiting the progression of inflammation and promoting mucosal repair32.In this study, we found that long-term intake of HSD reduced the levels of β-defensins. From all the previously mentioned evidence, it can be concluded that long-term intake of HSD attenuates the intestinal mucus barrier function, and therefore, HSD may have a damaging effect on the intestinal mucosa.

Beneath the mucus layer, the epithelium is by far the strongest determinant of the physical gut barrier1. There are two main pathways for epithelial permeation: trans-epithelial and paracellular - the integrity of epithelial barrier function depends on the presence of healthy epithelial cells and a functioning paracellular pathway33. The paracellular space is sealed by tight junctions (TJs), which restrict solute flow along the paracellular pathway, which is typically more permeable than the transcellular pathway. Thus, tight junctions are the rate-limiting step in trans-epithelial transport and a major determinant of mucosal permeability25. The TJ complex consists of different members of the intramembrane, Occludin, and tight junction families of proteins, with tight junctions being the transmembrane proteins primarily responsible for intestinal barrier function. Transmembrane proteins such as ZO-1, Occludin, and Claudin-1 constitute the core tight junction complex34. In this study, we observed that HSD significantly down-regulated the expression of Occludin in the colon, and there was also a trend of down-regulation in the expression of Claudin-1 and ZO-1, suggesting that HSD can damage the TJs of IECs to disrupt mucosal integrity and impair intestinal epithelial barrier function. Serum D-lactate is a metabolite of bacterial fermentation and can be produced by a variety of intestinal bacteria. When the intestinal mucosal permeability is increased, a large amount of D-lactic acid produced by intestinal bacteria enters the bloodstream through the damaged mucosa, resulting in elevated serum D-lactic acid levels35,36. Therefore, the elevated serum D-lactate concentration in the HSD group in this study further supports the conclusion that HSD has the ability to disrupt the integrity of the intestinal barrier in rats. Although our research results suggest a potential association among HSD, increased D-lactate levels, and intestinal barrier damage, we recognize that the increase in serum D-lactate concentration may be attributed to cerebral hypoxia or diabetes mellitus. Further research is needed to establish a more definite causal relationship and rule out the confounding effects of other factors.

In addition to the luminal mucus layer and intestinal epithelium formed by epithelial cells, the intestinal barrier includes the mucosal immune system37. In the intestine, the mucosal immune system plays an important role. It not only helps in digestion and absorption of nutrients but also serves as an important barrier for the body to resist pathogenic damage38. Intestinal SIgA is an important component of the intestinal mucosal immune barrier. When pathogenic microorganisms threaten the intestinal mucosal barrier, SIgA serves as the first line of defense for intestinal-specific immunity, preventing bacterial invasion by capturing bacteria on the mucosal surface and directly binding to specific sites, inhibiting bacterial motility and adhesion to the intestinal mucosa. In addition, it neutralizes enzymes and enterotoxins in the gut through chemical neutralization, thus doubly blocking contact between pathogens and the mucosa in space and time. Localized SIgA deficiency increases susceptibility to mucosal infections39. In this study, we observed a significant reduction in SIgA secretion in the intestinal tissues of rats chronically ingesting HSD. The observations suggest that chronic HSD impairs the immune barrier function of rat intestinal mucosa and disrupts intestinal homeostasis. SIgA is an immunoglobulin induced by intestinal-associated lymphoid reticulum (GALT)-derived B cells with the help of Th1-type and Th2-type CD4 + T lymphocytes and is regulated by Th1 cytokines (e.g., IFN-γ) and Th2 cytokines (e.g., IL-4, IL- 10) regulation40. In the present study, we observed a significant reduction in IFN-γ levels and a trend toward lower IL-4 levels in the intestinal tissues of rats in the HSD group, corresponding to a significant reduction in SIgA secretion. The reduction in the levels of these cytokines suggests that HSD may exert an immunomodulatory response by altering the balance of Th1/Th2. IL-10, often considered the most important anti-inflammatory cytokine, has been shown to prevent protein misfolding and endoplasmic reticulum stress by sustaining the production of mucin and contributing to the intestinal protective mucus barrier41,42. In addition, IL-22, secreted by innate lymphocytes 3, balances intestinal immunity and inflammation43. Therefore, after chronic HSD consumption, the reduction of IL-10 and IL-22 levels in intestinal tissues disrupts intestinal immune function, which contributes to impairing intestinal barrier function and promoting intestinal inflammatory responses44. In summary, our study suggests that long-term HSD decreases the ability of intestinal mucosal immunity, which can increase the chance of invasion by various pathogens leading to intestinal damage.

Conclusion

In conclusion, this study provides clear evidence that long-term HSD impairs intestinal barrier function and affects intestinal health. Therefore, salt-restricted dietary interventions may represent a new dietary approach to promote gut health and reduce the risk of gut-related diseases.