Hyperglycemia impairs the expression of inflammatory mediators in rat intestine: an implication for intestinal inflammation and inflammatory bowel disease

TL;DR

This study shows that high blood sugar from diabetes can worsen gut inflammation, linking diabetes to inflammatory bowel disease through immune and metabolic interactions.

Contribution

The study reveals how chronic hyperglycemia affects intestinal inflammation and macrophage activity, linking diabetes to inflammatory bowel disease.

Findings

Chronic hyperglycemia causes intestinal inflammation and barrier disruption in both small and large intestines.

Macrophage activation and increased expression of CD68, iNOS, TNF-α, and IL-6 were observed in diabetic rats.

Female rats showed greater susceptibility to gut inflammation caused by diabetes.

Abstract

Diabetes mellitus and inflammatory bowel disease are chronic inflammatory disorders characterized by immune dysregulation and rising global prevalence. Epidemiological studies increasingly suggest a bidirectional association between the two conditions, linked through shared mechanisms of intestinal barrier dysfunction, microbial dysbiosis, and sustained innate immune activation. Activated macrophages play a central role in driving mucosal inflammation through polarization toward a pro-inflammatory M1 phenotype, accompanied by increased production of inflammatory cytokines. These mediators disrupt tight junctions, induce epithelial apoptosis, and perpetuate cycles of immune activation and tissue injury. This macrophage–cytokine axis not only amplifies local inflammation but also sustains chronic barrier dysfunction, creating a pathogenic overlap between diabetes mellitus-associated intestinal injury and intestinal bowel disease. In this study, we used a low dose streptozotocin and high-fat diet-induced diabetic Sprague–Dawley rat model in both sexes to investigate the effects of chronic hyperglycemia on intestinal inflammation, with particular emphasis on macrophage activation and pro-inflammatory cytokine responses. We found inflammation in both small and large intestines with mucosal injury and barrier disruption, and immune activation involving macrophages and enhanced expression of CD68, iNOS, TNF-α, and IL-6. Female rats were more susceptible to gut-related inflammatory changes due to diabetes. These findings suggest a complex interplay between epithelial stress, immune signaling, and microbial factors supporting the role of intestinal inflammation in the immune–metabolic interaction in diabetes-associated intestinal changes, which may contribute to the pathogenesis of inflammatory bowel disease.

Figures

Effects of chronic hyperglycemia on cellular inflammation and intestinal homeostasis

Histological alterations in small intestinal tissues of control and diabetic rats, stained with hematoxylin and eosin (H&E) at 20x magnification. Panel a - The photomicrograph shows a normal tissue section of a female control (FC) rat small intestine with all the wall layers and sublayers preserved. Panel b - The photomicrograph shows a cross-section of intestinal tissue of female diabetic (FD) rats. Mucosal villi are shortened and flattened with surface epithelium desquamation (black arrowhead) and shallowing of the Lieberkühn’s crypts with reduced number of goblet cells in comparison to the

Histological changes in the large intestinal tissues of control and diabetic rats. Panels a and d - Representative H&E-stained sections (50× magnification) of large intestinal tissue from female (FC) and male (MC) control rats display well-preserved intestinal architecture. The mucosa is lined by simple columnar epithelium rich in goblet cells (1a), with densely arranged crypts of Lieberkühn embedded in the lamina propria (1b). The lamina muscularis mucosae appears structurally preserved (1c). No pathological alterations are observed in the submucosa (2), tunica muscularis (3), or serosa (4).

Fold change in the expression of pro-inflammatory markers in intestinal tissues of control and diabetic rats. Red bars represent data from female rats; blue bars represent data from male rats. Group abbreviations: *CF* control female, *DF* diabetic female, *CM* control male, *DM* diabetic male. SI indicates the small intestine, and LI the large intestine. a *CD68* mRNA expression levels in the small intestine, b *CD68* mRNA expression levels in the large intestine c *iNOS* mRNA expression levels in the small intestine, d *iNOS* mRNA expression levels in the large intestine, e *TNF-α* mRNA expr

Immunohistochemical (IHC) staining and quantitative analysis of CD68, iNOS, TNF-α and IL-6 expression in the small and large intestines of control and diabetic rats. Panels a–d show representative IHC images of CD68 expression in the small intestine: a female control, b female diabetic, c male control, and d male diabetic rats. Panel e displays the quantification of mean optical density for CD68 in small intestinal sections. Panels f–i show representative images from the large intestine: f female control, g female diabetic, h male control, and i male diabetic rats. Panel j presents the corresp

Full text

Introduction

Diabetes mellitus (DM) and inflammatory bowel disease (IBD) are chronic, multifactorial diseases characterized by immune dysregulation and systemic or localized inflammation. DM refers to a group of metabolic diseases marked by chronic hyperglycemia, primarily resulting from insulin resistance (type 2 diabetes) or autoimmune destruction of pancreatic β-cells (type 1 diabetes) [1]. IBD, comprising Crohn’s disease and ulcerative colitis, is defined by relapsing inflammation of the gastrointestinal tract, driven by aberrant immune responses to intestinal microbiota in genetically susceptible individuals [2, 3].

The global burden of both DM and IBD continues to rise, particularly in industrialized countries, where environmental exposures, lifestyle factors, and alterations in gut microbiota are increasingly implicated in their pathogenesis [4, 5]. Notably, recent epidemiological data suggest a bidirectional association between DM and IBD [6, 7]. These may include low-grade systemic inflammation, impaired intestinal barrier integrity, microbial dysbiosis, and persistent activation of innate immune pathways.

Chronic hyperglycemia, a hallmark of DM, is increasingly recognized as a potent driver of intestinal inflammation [8]. Multiple studies have shown that long-term elevations in blood glucose levels alter the composition and diversity of the gut microbiota, reducing beneficial species while promoting the expansion of opportunistic and pro-inflammatory bacteria [9, 10]. This dysbiosis compromises intestinal homeostasis, impairs epithelial barrier integrity, exacerbates oxidative stress, and increases mucosal exposure to microbial products, which in turn triggers macrophage activation and the release of pro-inflammatory cytokines, thereby amplifying mucosal inflammation [11, 12]. Central to these processes are pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6), which are critically involved in the initiation and progression of inflammation in DM [13], and the pathogenesis of IBD [14] (Fig. 1). In both conditions, these mediators can contribute to mucosal injury by disrupting tight junction architecture, inducing epithelial apoptosis, and sustaining local immune cell activation. This cascade compromises barrier integrity, facilitates microbial translocation, and maintains chronic mucosal inflammation. The resulting pro-inflammatory environment further amplifies cytokine signaling, perpetuates barrier dysfunction, and promotes recurrent tissue injury with impaired mucosal healing [15–17].

Fig. 1. Effects of chronic hyperglycemia on cellular inflammation and intestinal homeostasis

Inflammatory cytokines contribute to the development of diabetes by triggering insulin resistance (IR) in tissues such as the liver, muscle, and fat, and by causing beta-cell dysfunction in the pancreas. This chronic inflammation, often linked to obesity, leads to a reduced body’s ability to regulate blood sugar, ultimately contributing to the development of Type 1 (T1D) and Type 2 diabetes (T2D) [18]. Adipocytes secrete pro-inflammatory mediators, including TNF-α, IL-6, and IL-1β, particularly in the setting of obesity, thereby sustaining low-grade inflammation through activation of intracellular signaling cascades and recruitment of additional immune cells [19]. TNF-α, IL-6, and IL-1β are key pro-inflammatory cytokines that are secreted by macrophages, especially when they are activated by inflammatory stimuli (Fig. 1). Macrophages play a key role in diabetes by becoming pro-inflammatory in a hyperglycemic environment, contributing to insulin resistance and the progression of T2D. These immune cells can be metabolically reprogrammed, shifting from a glucose-reliant pro-inflammatory phenotype to one that exacerbates IR and chronic low-grade inflammation known as “metaflammation”. Macrophages also influence pancreatic beta-cell function and injury in diabetes [20]. Further, they also play a role in IBD by regulating intestinal immune homeostasis, maintaining tissue repair, and participating in the inflammatory processes that characterize these chronic conditions [21]. These inflammatory stimuli also act as potent chemoattractants, driving monocyte recruitment to the intestinal lamina propria and promoting their differentiation into activated macrophages. Within the inflamed mucosa, macrophages commonly polarize toward a pro-inflammatory (M1) phenotype, amplifying cytokine production and perpetuating the inflammatory cascade that sustains the pathophysiology of both diabetes-associated intestinal inflammation and IBD [22].

Considering these shared inflammatory pathways, we aimed to characterize the effects of chronic hyperglycemia on the intestinal mucosa using a streptozotocin-induced diabetic Sprague–Dawley rat model. This experimental model enables the assessment of hyperglycemia-associated barrier dysfunction, immune activation, and macrophage polarization under controlled conditions. By delineating these mechanisms, particularly the cytokine–macrophage axis, we seek to identify convergent targets for therapeutic intervention that may mitigate intestinal complications of diabetes and reduce susceptibility to IBD-like inflammation.

Materials and methods

Experimental design, animals, and tissue collection

All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Western University of Health Sciences, Pomona, CA, USA (protocol #R24IACUC013). A total of twenty-two Sprague Dawley rats (male and female), aged 6–8 weeks and weighing approximately 180 g, were used in this study. The animals were purchased from Charles River Laboratories (USA) and housed under standard laboratory conditions (22 °C, 12-hour light/dark cycle) in the Animal Resource Facility at Western University of Health Sciences. Rats were randomly assigned to four experimental groups: female control (n = 5), male control (n = 5), female diabetic (n = 6), and male diabetic (n = 6). Control animals were maintained on a normal diet (ND; 20% protein, 70% carbohydrate, 10% fat; D12450B, Research Diet Inc.) with ad libitum access to water, while diabetic rats were fed a high-fat diet (HFD; 35% carbohydrate, 20% protein, 45% fat; 5.7 kcal/g; D12451, Research Diet Inc.), also with ad libitum access to water. Following six weeks on the assigned diet, rats in the diabetic groups received a low-dose intraperitoneal (IP) injection of streptozotocin (STZ; 25 mg/kg, dissolved in 0.1 M sodium citrate buffer, pH 4.4; Sigma-Aldrich, St. Louis, MO) to induce type 2 diabetes. Control animals received an equivalent volume of citrate buffer (0.25 mL/kg). To enhance diabetes induction, a second STZ injection (25 mg/kg) was administered seven days later, while control animals received citrate buffer. Blood glucose levels were monitored via tail vein sampling using an AlphaTrak glucometer. While this two-dose protocol was sufficient to induce diabetes in most male rats, female rats remained below the diabetic threshold. Therefore, a third STZ injection (25 mg/kg) was administered to female rats one week after the second dose. Diabetes was confirmed based on blood glucose levels consistently exceeding 250 mg/dL.

Two weeks after diabetes confirmation, rats were euthanized using isoflurane gas, and small and large intestinal tissues were collected. Samples were preserved in 10% formalin (6764254, Thermo Fisher Scientific, Waltham, MA, USA) for histological analysis and in RNAlater (AM7021, Thermo Fisher Scientific, Waltham, MA, USA) for RNA extraction.

Tissue processing, staining, and histological analysis

Following fixation in 10% formaldehyde for 48 h, tissue samples were processed for histological analysis using a Tissue-Tek VII tissue processor (Sakura Finetek, Torrance, CA, USA). Samples were passed through a graded series of ethanol, xylene, and paraffin wax solutions, then embedded in paraffin blocks. Serial sections, 7 μm thick, were obtained using a Leica RM2265 rotary microtome (Leica™, Wetzlar, Germany). The sections were mounted onto glass slides and incubated at 60 °C for one hour to ensure proper adhesion.

Before staining and immunohistochemistry, paraffin-embedded sections were deparaffinized in xylene and rehydrated through a descending ethanol series according to standard protocols established in our laboratory. Hematoxylin and eosin (H&E) staining was performed by incubating the slides in hematoxylin for 60 s and in eosin for 2–3 min. After staining, the sections were mounted using Cytoseal 60, a xylene-based mounting medium (23-244257, Thermo Fisher Scientific, Waltham, MA, USA).

Slides were scanned using a Leica DM6 light microscope (Leica™, Wetzlar, Germany) at a scale of 100 μm. For each sample, at least three adjacent sections were evaluated, with 3–5 fields per section examined at 5**×** and 20**×** magnification. All scanned sections were independently assessed by two blinded observers for histopathological features, including inflammation, epithelial disruption, and apoptosis.

Quantitative real-time polymerase chain reaction (RT-qPCR)

Approximately 50 mg of intestinal tissue from each sample was used for total RNA extraction using TRIzol reagent (T9424, Millipore Sigma, Burlington, MA, USA), following the manufacturer’s protocol. The resulting RNA pellet was resuspended in 30 µL of nuclease-free water (BP561-1, Thermo Fisher Scientific, Waltham, MA, USA), and RNA concentration and purity were measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). For complementary DNA (cDNA) synthesis, 2 µg of total RNA were reverse-transcribed using the AzuraQuant™ cDNA Synthesis Kit (AZ-1996, Azura Genomics Inc., Raynham, MA, USA) according to the manufacturer’s protocol, employing a T100™ Thermal Cycler (Bio-Rad Laboratories, Hercules, CA, USA). Quantitative real-time PCR was performed in triplicate for each sample in a final reaction volume of 10 µL using AzuraView™ GreenFast qPCR Blue Mix LR (AZ-2350, Azura Genomics Inc., Raynham, MA, USA). Amplification was conducted on a C1000™ Thermal Cycler (Bio-Rad Laboratories, Hercules, CA, USA) under the following cycling conditions: initial denaturation at 95 °C for 3 min, followed by 39 cycles of 95 °C for 10 s and 60 °C for 30 s, ending with a melting curve analysis to assess amplification specificity. Primers for the genes of interest and the housekeeping gene were obtained from Integrated DNA Technologies (Coralville, IA, USA), with sequences provided in Table 1. Relative gene expression was calculated using the 2^−ΔΔCT method, with normalization to 18 S rRNA as the reference gene.

Table 1. The forward and reverse oligonucleotide sequences used for RT-qPCR amplification of target genesGene nameForward primerReverse primer CD68 5′- TCT GAC CTT GCT GGT ACT GC −3′5′- TCC GTG AAG GAT GGC AGA AG-3′ iNOS 5′- CAC CAC CCT CCT TGT TCA AC −3′5′- CAA TCC ACA ACT CGC TCC AA −3′ TNF-α 5′- TCT GCT TGG TGG TTT GCT ACG AC −3′5′- AAA TGG GCT CCC TCT CAT CAG TTC − 3′ IL-6 5′- TCC TAC CCC AAC TTC CAA TGC TC −3′5′- TTG GAT GGT CTT GGT CCT TAG CC −3′ 18s 5′- GTA ACC CGT TGA ACC CCA TT −3′5′- CCA TCC AAT CGG TAG TAG CG −3′The 18 S gene served as the housekeeping control for normalization. The analyzed genes included cluster of differentiation 68 (CD68), inducible nitric oxide synthase (iNOS), tumor necrosis factor alpha (TNF-α), and interleukin-6 (IL-6)

Immunohistochemistry (IHC)

To evaluate protein expression in tissue sections, IHC was performed on formalin-fixed, paraffin-embedded samples. Sections were deparaffinized in xylene and rehydrated through a graded ethanol series, followed by antigen retrieval in 1% citrate buffer (C9999-1000ML, Millipore Sigma, Burlington, MA, USA) using a commercial steamer for 30 min. Slides were then cooled at room temperature for an additional 45 min and washed in 1× phosphate-buffered saline (PBS) (BP39920, Thermo Fisher Scientific, Waltham, MA, USA) for 5 min. To block endogenous peroxidase activity, sections were incubated in 3% hydrogen peroxide (H1009, Millipore Sigma, Burlington, MA, USA) for 15 min at room temperature, followed by two washes in PBS. Non-specific binding was prevented by incubating the sections for 1 h at room temperature with species-specific blocking solutions: Normal Goat Serum (S-1000-20) for rabbit primary antibodies. Following blocking, sections were incubated overnight at 4 °C with primary antibodies, applied at optimized dilutions determined by prior titration. The next day, slides were washed twice in PBS and incubated for 1 h at room temperature with secondary antibodies (Table 2). After two additional PBS washes, signal amplification was performed using the VECTASTAIN^®^ ABC-HRP Kit (PK-4000, Vector Laboratories, Newark, CA, USA) for 30 min. Chromogenic detection was carried out using the AEC Substrate Kit (3-amino-9-ethylcarbazole; SK-4200, Vector Laboratories, Newark, CA, USA) for 5 min, or until the desired staining intensity was achieved. The reaction was stopped by rinsing the slides in tap water. Counterstaining was performed with hematoxylin (2 dips), and sections were mounted using ADVANTAGE Mounting Media (NB300A, Innovex Biosciences, Pinole, CA, USA). Positive and negative controls were included for each antibody during the staining process. The stained sections were scanned using a Leica DM6 light microscope (Leica™, Wetzlar, Germany) with a scale bar set at 100 μm. For semi-quantitative analysis, at least three randomly selected fields per section were captured and analyzed using Fiji ImageJ software (version 1.54 J, NIH, USA) to quantify mean staining intensity and the percentage of positively stained area.

Table 2. The table provides detailed information on the primary and secondary antibodies used for immunohistochemistry (IHC), including their catalog numbers and Dilution ratiosAntibodyCatalog numberIHC dilutionPrimary antibody CD68ab283654 1:100  iNOSab3523 1:100  TNF-αMBS820357 1:100  IL-6MA5-51298 1:100 Secondary antibody Anti-rabbitBP-9100-50 Ready-to-use The primary antibodies target cluster of differentiation 68 (CD68), inducible nitric oxide synthase (iNOS), tumor necrosis factor alpha (TNF-α), and interleukin 6 (IL-6), with corresponding secondary antibodies (anti-rabbit or anti-mouse) selected based on the host species of each primary antibody

Statistical analysis

Data were analyzed using GraphPad Prism 10 for Windows (version 10.1.1) and are presented as mean ± standard deviation (SD). Statistical comparisons between the control (nondiabetic) and diabetic groups were performed using one-way ANOVA followed by Tukey’s post hoc test. A p-value of less than 0.05 was considered statistically significant. For pairwise comparisons, significance levels were indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Results

Histological evaluation

Histological assessment revealed marked contrasts between control and diabetic groups in both the small and large intestines (Figs. 2 and 3). Control animals of both sexes displayed well-preserved intestinal architecture, with intact villi, organized crypts, and normal layering of the mucosa, submucosa, muscularis, and serosa. In diabetic rats, however, the intestinal wall exhibited clear pathological alterations, including epithelial injury, reduction of goblet cells, inflammatory infiltration of the lamina propria, and early signs of submucosal and muscular remodeling. These changes were evident across both intestinal regions, reflecting the detrimental impact of chronic hyperglycemia on intestinal structure and tissue homeostasis.

Fig. 2. Histological alterations in small intestinal tissues of control and diabetic rats, stained with hematoxylin and eosin (H&E) at 20x magnification. Panel a - The photomicrograph shows a normal tissue section of a female control (FC) rat small intestine with all the wall layers and sublayers preserved. Panel b - The photomicrograph shows a cross-section of intestinal tissue of female diabetic (FD) rats. Mucosal villi are shortened and flattened with surface epithelium desquamation (black arrowhead) and shallowing of the Lieberkühn’s crypts with reduced number of goblet cells in comparison to the FC (*) (1a). The lamina propria exhibits increased lymphocytic cellularity, characterized by a dense infiltrate of small, round mononuclear cells with scant cytoplasm and round, hyperchromatic nuclei (indicated by white arrows) (1b). There is discrete edema present in the submucosa (black arrows) (2). Muscularis is visibly thinner in comparison to the FC (+) (3). Serosal surface appears normal (4). Panel c - Photomicrograph of a normal small intestine cross-section from a male control (MC) rat, showing a well-preserved mucosal structure with intact villi and epithelial lining. The submucosa, muscularis, and serosa appear structurally unaltered. Panel d - The photomicrograph presents a section of male diabetic (MD) rat intestinal tissue. Mucosa showed moderate alterations. The villi are shortened and atrophic with apical disintegration (black arrowhead). Lymphocytic aggregates are visible in the hypercellular lamina propria (white arrows). Submucosa and muscularis presented with mild perivascular edema (black arrows). Serosa demonstrated no visible changes. Panel (e) - Quantification of small intestinal tissue injury across control and diabetic groups in both females and males

Fig. 3. Histological changes in the large intestinal tissues of control and diabetic rats. Panels a and d - Representative H&E-stained sections (50× magnification) of large intestinal tissue from female (FC) and male (MC) control rats display well-preserved intestinal architecture. The mucosa is lined by simple columnar epithelium rich in goblet cells (1a), with densely arranged crypts of Lieberkühn embedded in the lamina propria (1b). The lamina muscularis mucosae appears structurally preserved (1c). No pathological alterations are observed in the submucosa (2), tunica muscularis (3), or serosa (4). Panels b and e - The photomicrographs present with cross-section of female (FD) and male (MD) diabetic rat large intestinal tissue (H&E, 50x), revealing mild mucosal epithelial desquamation (black arrowhead), accompanied by increased mononuclear cell infiltration within the lamina propria (white arrows). The inflammatory infiltrate breaches the muscularis mucosae, forming prominent basophilic mononuclear aggregates in the edematous submucosa (black arrows). The muscularis and serosal layers remain unaffected. Panels c and f - Low magnification images (H&E, 20×) highlight transmuscular lymphoid infiltration - protrusion of lymphocytes, plasma cells, and macrophages from the hypercellular lamina propria into the submucosal loose connective tissue (black arrows), along with intravascular lymphoid congestion (white arrowheads). Panel (g) - Quantification of large intestinal tissue injury across control and diabetic groups in both females and males

Gene expression analysis

To investigate the impact of chronic hyperglycemia on intestinal inflammation, we assessed the expression of key inflammatory markers, including Cluster of Differentiation 68 (CD68), Inducible Nitric Oxide Synthase (iNOS), Tumor Necrosis Factor-α (TNF-α), and Interleukin-6 (IL-6), in the small and large intestines of male and female rats. These markers were chosen to evaluate macrophage activation and pro-inflammatory signaling within the intestinal mucosa under diabetic conditions.

Analysis of CD68, a well-established marker of macrophage activation, revealed clear sex- and tissue-specific differences. In the small intestine, diabetic females exhibited a pronounced increase relative to controls (p < 0.0001), indicating enhanced macrophage recruitment. Diabetic males showed no significant difference from their controls, although a modest, non-significant increase was noted. Expression levels were significantly higher in diabetic females than in diabetic males in this region (p < 0.0001) (Fig. 4a). In the large intestine, a similar trend was observed. Diabetic females displayed a robust elevation compared to controls (p < 0.0001), while diabetic males also showed a significant upregulation (p < 0.01). Consistent with findings in the small intestine, levels remained significantly higher in diabetic females than in males (p < 0.0001), further underscoring a sex-dependent inflammatory response associated with diabetes (Fig. 4b).

Fig. 4. Fold change in the expression of pro-inflammatory markers in intestinal tissues of control and diabetic rats. Red bars represent data from female rats; blue bars represent data from male rats. Group abbreviations: CF control female, DF diabetic female, CM control male, DM diabetic male. SI indicates the small intestine, and LI the large intestine. a CD68 mRNA expression levels in the small intestine, b CD68 mRNA expression levels in the large intestine c iNOS mRNA expression levels in the small intestine, d iNOS mRNA expression levels in the large intestine, e TNF-α mRNA expression levels in the small intestine, f TNF-α mRNA expression levels in the large intestine, g IL-6 mRNA expression levels in the small intestine, h IL-6 mRNA expression levels in the large intestine. Gene expression was quantified using qPCR and normalized to the housekeeping gene. Statistical comparisons between experimental groups were performed using one-way ANOVA followed by Tukey’s post hoc test. Data are presented as mean ± SD. Statistical significance: p < 0.05, p < 0.01, p < 0.001, p < 0.0001

iNOS is an important effector of M1 macrophages, and its overexpression under hyperglycemic conditions amplifies intestinal inflammation by promoting nitric oxide-mediated mucosal injury. In the small intestine, diabetic females exhibited a highly significant increase in iNOS compared to their controls (p < 0.0001), while diabetic males showed a more moderate but significant elevation (p < 0.01). Levels were markedly higher in females than in males (p < 0.0001) (Fig. 4c). In the large intestine, iNOS was again strongly upregulated in diabetic females relative to controls (p < 0.0001), whereas no significant change was detected in males. As in the small intestine, protein expression in the large intestine remained significantly higher in females than in males (p < 0.0001) (Fig. 4d), again suggesting stronger nitric oxide–driven inflammation in females under hyperglycemic conditions.

TNF-α, a central mediator of inflammation and a hallmark of M1 macrophage activation, also showed a distinct distribution. In the small intestine, females demonstrated compared to their control counterparts (p < 0.0001), while diabetic males exhibited a modest but statistically significant increase relative to control males (p < 0.05). A highly significant difference was also observed between diabetic females and males (p < 0.0001) (Fig. 4e), again suggesting enhanced pro-inflammatory signaling in female diabetic rats. In the large intestine, TNF-α expression remained significantly elevated in diabetic females compared to controls (p < 0.0001), with no significant change detected in diabetic males. However, TNF-α levels in diabetic females were again significantly higher than in their male counterparts (p < 0.0001) (Fig. 4f), reinforcing a potential sex-dependent disparity in intestinal inflammatory responses under hyperglycemic conditions.

IL-6, another key pro-inflammatory cytokine involved in M1 macrophage polarization and chronic intestinal inflammation, was also significantly upregulated in diabetic animals. In the small intestine, IL-6 expression was markedly elevated in diabetic females compared to controls (p < 0.0001), while diabetic males showed a moderate but significant increase (p < 0.01). Notably, expression levels were substantially higher in diabetic females than in diabetic males (p < 0.0001), further supporting a heightened inflammatory state in female diabetic rats (Fig. 4g). A similar pattern was observed in the large intestine, where IL-6 levels were significantly increased in diabetic females (p < 0.0001) and moderately elevated in diabetic males (p < 0.01) compared to the control group. Again, diabetic females exhibited significantly greater IL-6 expression than diabetic males (p < 0.0001) (Fig. 4h), reinforcing a consistent sex-specific trend of amplified intestinal inflammation under hyperglycemic conditions.

Immunohistochemical (IHC) analysis

To further validate the transcriptional findings, we assessed protein expression of the key inflammatory markers selected in this study in the small and large intestines of male and female rats using immunohistochemistry. This analysis allowed us to determine whether the observed changes at the mRNA level were mirrored at the protein level, thereby providing additional insight into macrophage infiltration and pro-inflammatory signaling under diabetic conditions.

Evaluation of CD68 protein expression in the small intestine revealed pronounced macrophage infiltration in diabetic females, which was markedly higher compared to their controls (p < 0.0001) (Fig. 5a, b). In contrast, diabetic males showed no significant difference relative to controls, although a slight increase was observed (Fig. 5c, d). Notably, CD68 expression remained significantly elevated in diabetic females compared to diabetic males (p < 0.001) (Fig. 5e), underscoring a sex-dependent difference in macrophage activation in this region. In the large intestine, both sexes demonstrated increased CD68 expression, with diabetic females showing a robust elevation (p < 0.001) and diabetic males exhibiting a more modest but significant increase compared to their respective controls (p < 0.01) (Fig. 5f–i). However, no difference was observed between diabetic females and males, suggesting a more uniform macrophage response in the colonic mucosa (Fig. 5j).

Fig. 5. Immunohistochemical (IHC) staining and quantitative analysis of CD68, iNOS, TNF-α and IL-6 expression in the small and large intestines of control and diabetic rats. Panels a–d show representative IHC images of CD68 expression in the small intestine: a female control, b female diabetic, c male control, and d male diabetic rats. Panel e displays the quantification of mean optical density for CD68 in small intestinal sections. Panels f–i show representative images from the large intestine: f female control, g female diabetic, h male control, and i male diabetic rats. Panel j presents the corresponding quantification of mean optical density for large intestinal sections. Panels k–n show representative IHC images of iNOS expression in the small intestine: k female control, l female diabetic, m male control, and n male diabetic rats. Panel o displays the quantification of mean optical density for iNOS in small intestinal sections. Panels p–s show representative images from the large intestine: p female control, q female diabetic, r male control, and s male diabetic rats. Panel t presents the corresponding quantification of mean optical density for large intestinal sections. Panels u–x show representative IHC images of TNF-α expression in the small intestine: u female control, v female diabetic, w male control, and x male diabetic rats. Panel y displays the quantification of mean optical density for TNF-α in small intestinal sections. Panels z–ad show representative images from the large intestine: z female control, aa female diabetic, ab male control, and ac male diabetic rats. Panel ad presents the corresponding quantification of mean optical density for large intestinal sections. Panels ae–ah show representative IHC images of IL-6 expression in the small intestine: ae female control, af female diabetic, ag male control, and ah male diabetic rats. Panel a.i. displays the quantification of mean optical density for IL-6 in small intestinal sections. Panels aj–am show representative images from the large intestine: aj female control, ak female diabetic, al male control, and am male diabetic rats. Panel an presents the corresponding quantification of mean optical density for large intestinal sections. Images are representative of all animals in each group. Statistical comparisons between experimental groups were performed using one-way ANOVA followed by Tukey’s post hoc test. Data are presented as mean ± SD. Statistical significance: p < 0.05, p < 0.01, p < 0.001, p < 0.0001

Analysis of iNOS, a marker of M1 macrophage activation, showed a similar female-predominant response. In the small intestine, diabetic females displayed significantly higher protein expression than controls (p < 0.01) (Fig. 5k, l), while diabetic males exhibited only a mild but significant increase (p < 0.05) (Fig. 5m, n). No sex-related difference was found between diabetic groups in this region (Fig. 5o). In the large intestine, iNOS was strongly upregulated in diabetic females (p < 0.001), whereas diabetic males showed no significant change compared to their respective controls (Fig. 5p–s). Importantly, iNOS expression was significantly higher in diabetic females than in males (p < 0.0001), indicating a more pronounced pro-inflammatory macrophage phenotype in females under hyperglycemic conditions (Fig. 5t).

Cytokine profiling at the protein level also demonstrated increased expression of both TNF-α and IL-6, with notable differences across sexes and tissues. In the small intestine, TNF-α expression was strongly elevated in diabetic females versus controls (p < 0.0001) and moderately increased in diabetic males (p < 0.01) (Fig. 5u–x). Diabetic females also exhibited significantly higher TNF-α protein expression than their male counterparts (p < 0.01) (Fig. 5y). In the large intestine, TNF-α remained significantly elevated in diabetic females (p < 0.001), while diabetic males showed only a slight increase compared to controls (p < 0.05) (Fig. 5z–ac). Expression remained higher in females than in males (p < 0.01), indicating stronger cytokine-driven inflammation in female colonic tissue (Fig. 5ad).

IL-6 expression followed a slightly different trend. In the small intestine, both diabetic females and males exhibited a significant increase compared to their respective controls (p < 0.001), but no difference was observed between sexes (Fig. 5ae–ai). A similar pattern was observed in the large intestine, where IL-6 was significantly upregulated in diabetic females (p < 0.001) and males (p < 0.01) relative to controls, without sex-dependent differences (Fig. 5aj–an).

Taken together, the protein-level findings align with the gene expression data, indicating that chronic hyperglycemia promotes macrophage infiltration and pro-inflammatory cytokine activity in the intestinal mucosa, with female rats exhibiting a more pronounced and consistent inflammatory response.

Discussion

Type 2 diabetes is increasingly recognized as a chronic condition marked by persistent low-grade inflammation [23], which affects multiple organs, including the intestinal mucosa, and contributes to metabolic disturbances and tissue injury. Our findings show that chronic hyperglycemia is associated with a pronounced intestinal inflammatory response with clear sex- and tissue-specific differences. Across both the small and large intestines, diabetic females exhibited consistently higher expression of inflammatory macrophage markers and pro-inflammatory mediators than their male counterparts, suggesting a heightened susceptibility of the female gut to diabetes-related inflammation. These observations align with evidence that the immune system displays fundamental sex-based differences, with females generally mounting stronger innate and adaptive immune responses than males [24, 25]. This heightened immunoreactivity likely reflects the combined influence of sex hormones, immune-regulatory genes on the X chromosome, and sex-dependent differences in gut microbiota composition [26].

The healthy intestine contains a large population of tissue-resident macrophages located mainly in the lamina propria of both the small and large bowel [27]. These cells are continually renewed by circulating monocytes and play key roles in maintaining barrier integrity, clearing pathogens, and promoting immune tolerance to the commensal microbiota. In our study, we observed a distinct pattern of CD68 expression, a marker of macrophage activation, that varied by both sex and intestinal region. Female diabetic rats exhibited significant upregulation of CD68 at both the gene and protein levels in the small and large intestines, whereas males showed increased expression only in the large intestine, with no notable change in the small intestine compared to controls. The marked CD68 increase observed in the large intestine suggests that local factors, particularly the higher microbial density and the distinctive cytokine milieu of the colon, may outweigh systemic hormonal influences in driving macrophage activation. On the other hand, our findings in the small intestine are consistent with previous studies demonstrating sex-specific immune modulation, where estrogen receptor signaling promotes macrophage recruitment [28, 29], and facilitates epithelial repair in females. The minimal CD68 expression in the male small intestine may also reflect a weaker macrophage response or the involvement of alternative immune pathways. Another plausible mechanistic explanation for the low CD68 signal observed in this region involves interference from S100 proteins, calcium-binding molecules strongly associated with inflammation. These proteins are known to bind tightly to CD68 on the surface of activated macrophages, potentially obstructing antibody access during immunologic assays. This interaction could lead to reduced signal intensity and misinterpretation of CD68 expression levels [30]. Furthermore, S100 proteins, particularly S100A8 and S100A9 (which form calprotectin), play a significant role in the inflammatory cascade of IBD, contributing to immune cell activation and mucosal tissue damage. Their presence has been documented in inflamed mucosal areas, and elevated serum levels have been reported in children with Crohn’s disease and ulcerative colitis, further supporting their involvement in gut inflammation and diagnostic complexity [31].

In this study, we also analyzed the expression of inducible nitric oxide synthase (iNOS), a key enzyme involved in epithelial defense, antimicrobial activity, and oxidative signaling. Diabetic male rats exhibited significant upregulation of iNOS in the small intestine, but not in the large intestine, at both gene and protein levels. In contrast, diabetic females showed elevated iNOS expression in both intestinal regions. Activation of iNOS in the small intestine may reflect a localized response to hyperglycemia-induced oxidative stress, particularly within the crypts of Lieberkühn, which house Paneth cells, specialized epithelial cells known to express iNOS and support the intestinal stem-cell niche [32]. This regional and sex-specific variation in iNOS expression suggests that distinct epithelial and immune cell populations, along with hormonal and microbial influences, differentially regulate nitric oxide signaling in the diabetic gut.

In addition to CD68 and iNOS, we examined the expression of tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), two key pro-inflammatory cytokines secreted by inflammatory macrophages and implicated in intestinal barrier dysfunction and metabolic inflammation. Diabetic female rats again showed significant upregulation of TNF-α at both the gene and protein levels in the small and large intestines, consistent with their heightened inflammatory profile. In contrast, male diabetic rats exhibited elevated TNF-α protein in the colon, without a corresponding rise in its mRNA, indicating the involvement of post-transcriptional regulation, which should be further investigated. This pattern is consistent with a mechanism in which the dense colonic microbiota provides continuous exposure to lipopolysaccharide (LPS), a potent ligand for Toll-like receptor 4 (TLR4) [33]. Chronic hyperglycemia, combined with sustained LPS stimulation, can maintain low-grade inflammation and oxidative stress and activate signaling pathways such as p38 mitogen-activated protein kinase (p38 MAPK) and nuclear factor kappa B (NF-κB), which selectively enhance the translation of existing TNF-α transcripts [34]. LPS can also act on AU-rich elements (AREs) in the 3′ untranslated region of TNF-α mRNA by altering the activity of tristetraprolin (TTP), an RNA-binding protein that normally promotes rapid mRNA decay. Although we did not directly measure TTP activity, previous studies show that chronic inflammatory conditions can impair TTP function, stabilizing TNF-α mRNA and increasing its translational efficiency without elevating transcription [35, 36].

In parallel, we observed significant upregulation of IL-6 at both the gene and protein levels in the small and large intestines of diabetic rats, irrespective of sex. IL-6 is a pleiotropic cytokine involved in both acute and chronic inflammation, and its levels are consistently elevated in individuals with T2D who are at risk or in the early stages of the disease [37]. Given its ability to increase intestinal permeability, recruit immune cells, and promote insulin resistance (IR), the consistent upregulation of IL-6 across sexes and intestinal regions underscores its central role in the pathophysiology of diabetes-associated intestinal inflammation [38–40]. Hyperglycemia induces significant sex-specific changes in inflammatory mediators, with men and women exhibiting distinct inflammatory profiles in response to elevated glucose levels. Higher levels of pro-inflammatory markers such as IL-6 and leptin are frequently observed. Peripheral blood mononuclear cells (PBMCs) from men with metabolic syndrome show an increased capacity to produce cytokines, including IL-1β and IL-6, when stimulated. In addition, male pancreatic islets demonstrate a more aggressive response to pro-inflammatory cytokines compared to female islets. Women with metabolic syndrome may display lower concentrations of the anti-inflammatory mediator adiponectin. Some type 2 diabetes models suggest that females experience greater improvements in inflammatory markers and insulin resistance following certain therapies, such as those promoting regulatory T-cell expansion. Estrogens appear to exert a protective, anti-inflammatory influence, helping to modulate immune responses and potentially contributing to sex-related differences in inflammatory signaling [41–44]. Hyperglycemia also induces tissue-specific alterations in inflammatory mediators, particularly in immune cells, placental tissue, vascular structures, endothelial cells, and fibroblasts. These changes include shifts in macrophage polarization within immune tissues, increased production of inflammatory cytokines such as TNF-α and IL-1β across multiple tissue types, and impaired immune cell functions such as reduced phagocytosis. Several mechanisms drive these responses, including activation of the hexosamine biosynthetic pathway, accumulation of advanced glycation end-products (AGEs), and upregulation of inflammatory signaling pathways such as NF-κB [12, 45–47]. Our histological evaluations of both small and large intestinal tissues in diabetic rats revealed consistent patterns of mucosal injury, immune cell infiltration, and submucosal edema, with structural preservation of deeper layers such as the muscularis and serosa. These findings underscore the vulnerability of the intestinal mucosa to chronic hyperglycemia and support the notion that diabetes induces localized inflammation and barrier dysfunction in the gut. Chronic hyperglycemia has been shown to impair epithelial turnover and tight junction integrity, leading to increased permeability and susceptibility to luminal antigens [48]. The presence of lymphoid aggregates, epithelial desquamation, and goblet cell depletion across both intestinal regions suggests a sustained immune response and impaired epithelial renewal, consistent with previous reports of diabetes-associated enteropathy [49]. Inflammatory cell infiltration into the lamina propria and submucosa reflects activation of mucosal immunity, likely driven by microbial dysbiosis and translocation of bacterial products such as LPS, which are known to activate TLR4 signaling and promote cytokine release [50]. These changes may contribute to systemic inflammation and IR, forming a gut–metabolic axis that further exacerbates the pathophysiology of T2D. Overall, our findings reveal distinct sex- and region-specific inflammatory patterns that link intestinal pathology to systemic metabolic dysfunction.

Conclusion

This study highlights the impact of hyperglycemia on intestinal health, revealing inflammation, structural damage, and immune activation involving macrophages across both the small and large intestines. Sex-specific differences in immune marker expression suggest that females may be more susceptible to gut-related inflammatory changes. Elevated levels of CD68, iNOS, TNF-α, and IL-6 point to a complex interplay between epithelial stress, immune signaling, and microbial factors. Histological evidence of mucosal injury and barrier disruption further supports the role of intestinal inflammation in the broader metabolic dysfunction of diabetes. These findings reinforce the importance of the gut as a major player in immune-metabolic interaction in diabetes-associated intestinal changes, which may contribute to the pathogenesis of inflammatory bowel disease.

Study limitations

While this study provides valuable insights into diabetes-associated intestinal inflammation, certain limitations should be considered. The streptozotocin-induced rat model, although widely used, may not fully replicate the complexity of human type 2 diabetes, particularly regarding genetic and environmental factors. Functional evaluations such as intestinal permeability, microbiota composition, or hormone profiling were not included, which limits the ability to directly link observed molecular and histological changes to physiological outcomes. Additionally, mechanisms of post-transcriptional regulation, particularly those involving RNA-binding proteins like tristetraprolin, were inferred but not directly measured. Since this study focused on profiling pro-inflammatory macrophage activation, further evaluation of M2-associated and anti-inflammatory markers (such as IL-4, IL-13, or IL-10) should be considered in future experiments. Future research should also more comprehensively investigate the sex-specific and tissue-specific effects of hyperglycemia on inflammatory pathways. This includes clarifying how distinct tissues respond differentially to high glucose exposure and how biological sex modulates these responses, as suggested by evidence from studies highlighting sex differences in cytokine production and tissue-level inflammatory signaling [43, 44]. Taken together, these considerations should be kept in mind when interpreting the present findings and underscore important avenues for further investigation.