Creatine Preserves Colonic Epithelial and Vascular Barrier Integrity and Epithelial Scutoid Geometry Across Active and Remission Phases of Chronic Colitis with Sex-Specific Effects

1. Introduction

Ulcerative colitis (UC) is a chronic inflammatory bowel disease (IBD) affecting the colon and rectum, characterized by alternating periods of relapse and remission. UC is believed to develop in individuals who have genetic susceptibility after being exposed to certain environmental factors [1]. It is strongly associated with defects in the gut barrier, alterations in the microbiota, and an abnormal immune response which indicate the importance of the precise regulation of epithelial barrier integrity in the intestine to avoid the excessive activation of mucosal immune cells linked to the development of this disease [2]. During the active disease, excessive production of pro-inflammatory mediators disrupts epithelial and vascular barrier integrity, causing diarrhea, rectal bleeding, abdominal pain, and weight loss that markedly reduces quality of life. However, even during clinical remission, many patients continue to experience persistent symptoms and impaired well-being, likely reflecting ongoing low-grade inflammation and the chronic, relapsing nature of the disease [3,4]. The prevalence of UC differs slightly between men and women and varies by geographic region. In Western countries, the incidence of UC is generally similar between men and women before the age of 45. However, after 45, men exhibit a higher risk of developing UC than women. In contrast, in Eastern countries, UC shows a male predominance from adolescence until approximately 65 years of age, after which the incidence rates become comparable between sexes [5]. Previous studies conducted in colitis animal models, including our own, have shown that males exhibited more severe colitis [6,7,8].

Current treatment options for UC include chemotherapy, biological agents, and surgery. However, these therapies often fail to achieve long-term remission, highlighting the need for new approaches that could effectively induce and sustain health [4,9]. Therapeutic strategies focused on protecting vulnerable epithelial cells and restoring gut epithelial homeostasis by preserving intestinal barrier integrity represent a plausible and rational approach to achieving durable remission in UC [2].

The intestinal barrier is a dynamic semipermeable interface that facilitates nutrient absorption while restricting the passage of luminal pathogens and harmful molecules, thereby maintaining intestinal homeostasis. This function depends on the coordinated interaction of multiple components, including the mucus layer, secretory immunoglobulin A, antimicrobial peptides and proteins, the intestinal epithelium sealed by intercellular junctions, and the gut-associated lymphoid tissue [10]. The mucus layer serves as the first line of protection of the intestine, coating the epithelial cells prevents direct contact between these cells and intestinal bacteria. This layer is primarily composed of high-molecular-weight glycoproteins known as mucins, which are synthesized and secreted by goblet cells. In the colon, where the high microbial burden necessitates a stronger barrier function, the mucus layer is thicker, and mucin-2 (MUC2) is the predominant secreted mucin. The main function of mucins is to lubricate the epithelium and protect it from harmful substances. Under normal physiological conditions, goblet cells continuously produce mucins to sustain the mucus layer [11]. However, various factors can compromise mucus layer integrity, such as microbial agents, microbial toxins, and cytokines by altering mucin synthesis and secretion, changing its composition, or accelerating its degradation. These disruptions may result in a weakened mucus barrier, contributing to the development of chronic inflammatory diseases [10,12,13]. A reduced mucus layer is a hallmark feature of UC and constitutes a factor involved in mechanisms underlying the pathogenesis of this disease. Active UC is associated with a structurally weakened and penetrable mucus layer, accompanied by reduced MUC2 levels as a result of goblet cell depletion and/or defective MUC2 secretion [14,15,16].

Another important component of the intestinal barrier is the epithelium that forms a continuous monolayer whose cells are attached through junctional complexes. In the colon, this epithelium covers the surface and is distributed forming deep invaginations called crypts. Surface epithelium is mainly formed by absorptive cells or colonocytes which contribute to intestinal barrier function by forming the apical junctional complex, composed of tight and adherens junctions. Both junctions consist of transmembrane proteins that connect adjacent cells and tether them to the cytoskeleton. Their dynamic nature, continuously undergoing disassembly and reassembly, is essential for maintaining epithelium integrity while regulating paracellular permeability, cell proliferation, and migration to preserve its homeostasis [2]. The most apical are the tight junctions (TJs) and the primary regulators of paracellular permeability to solutes and macromolecules (gate function), while polarizing the colonocytes into apical and basolateral regions that establish a gradient between the intestinal lumen and basolateral membrane (fence function), making them essential for barrier function. TJs are composed of several proteins, including occludin and claudins which are anchored to the actin cytoskeleton through cytoplasmic scaffold proteins such as zonula occludens-1 (ZO-1) [2]. Among the claudins, claudin-5 contributes to intestinal epithelial barrier integrity by reinforcing tight junctions and limiting paracellular permeability [17]. This tightening claudin is enriched in the distal intestine and it has been identified as a critical effector of signaling pathways that link inflammation to intestinal barrier dysfunction and disease progression in UC [18]. Adherens junctions (AJs) are located beneath TJs and mechanically anchor neighboring cells by linking their cytoskeletons. Since the formation of TJs is both preceded and dependent on AJs, these structures also contribute to epithelial integrity, paracellular permeability, and cell polarity. AJs are composed of transmembrane cadherins, predominantly E-cadherin in the intestinal epithelium, which mediates cell–cell adhesion through interactions with cytoplasmic scaffold proteins such as β-catenin, thereby regulating AJ assembly, stability, and epithelial integrity [2,19]. Beyond its structural role, β-catenin also functions regulating epithelial proliferation and mucosal repair, processes that are frequently dysregulated in inflammatory conditions [20]. Extensive evidence demonstrates that intestinal inflammation disrupts the expression and localization of epithelial TJ proteins, leading to impaired epithelial barrier integrity, increased intestinal permeability, and the progression of UC [10,19]. Although the role of AJ proteins in the disruption of the intestinal barrier during inflammation remains poorly understood, in clinical studies and animal models demonstrate disruption of AJ architecture, which may significantly contribute to its functional defects [19].

Beyond the mucus and epithelial layers, intestinal defense extends to a deeper level through the vascular barrier at the level of the endothelial lining. This barrier functions as a critical gatekeeper, representing the final obstacle preventing microorganisms and deleterious substances from accessing the systemic circulation [21]. It plays a central role in whole-body protection and in mediating communication between the intestine and distant organs. Consequently, the increase in vascular permeability associated with inflammation undermines its protective function by promoting the translocation of microorganisms and proinflammatory mediators into circulation. This permeability is tightly regulated by fenestral and stomatal diaphragms, key structures whose formation and integrity require the plasmalemma vesicle–associated protein-1 (PV-1/PLVAP). Thus, PV-1 is considered an intestinal vascular barrier permeability marker, and its levels increase in inflammatory or vascular damage conditions [22]. Another fenestral diaphragm implicated in vascular permeability are caveolae, plasma membrane invaginations whose major structural component is caveolin-1 (Cav-1). This protein plays a crucial role in preserving endothelial cell function and controls the selective vesicular transport of vital blood macromolecules. Elevated levels of Cav-1 indicate enhanced transendothelial cell transport and vesicular traffic [23].

To preserve the integrity of self-renewing epithelia, in which cells undergo constant junctional remodeling, the scutoids represent a geometric solution to three-dimensional packing of epithelia and a way to accommodate cell turnover without compromising barrier function. Scutoid is a geometric shape adopted by epithelial cells in highly proliferative tissues that was first described in invertebrates, such as Drosophila, in salivary gland epithelium [24]. This geometric shape is characterized by the presence of at least one vertex between two parallel surfaces and is adopted particularly by cells forming simple columnar epithelia enabling tissue curvature while maintaining optimal cell packing and barrier integrity [24]. Such curvature allows proliferating cells to be properly integrated into the epithelial layer without creating gaps, as it was seen in sea star embryo [25]. Although scutoids have been primarily identified in epithelia of in vitro models, invertebrates, and in mouse embryos, theoretical and computational studies suggest that they are expected to form in any epithelium where the mechanical stresses on the apical and basal surfaces are anisotropic (not uniform) [26]. This condition is typical in tubular structures, such as the intestine, where curvature causes differential forces between the inner and outer surfaces of epithelial cells. Cell proliferation drives three-dimensional reorganization of epithelial packing and, consequently, the number of scutoids within a tissue may adjust to minimize cell surface tension, facilitate a balanced energetic state and promote stable architecture, thereby preserving epithelial integrity and reducing the risk of barrier function loss [25].

Creatine (Cr) (methylguanidine-acetic acid) is a derivative of amino acids and one of the most widely used dietary supplements among athletes due to its ability to enhance muscle mass and strength. Creatine, obtained through dietary intake, is absorbed in the small intestine via the apical creatine transporter (CrT), which was first identified by our group [27]. Subsequently, we demonstrated [28] that CrT-mediated creatine uptake occurs also in the large intestine of rats, confirming its functional activity and highlighting that dietary creatine is actively transported into the epithelial cells along the entire intestinal tract, including the colon. Creatine can also be synthesized endogenously via a two-step process mostly in the kidneys and liver, involving the enzymes arginine: glycine amidinotransferase (AGAT) and guanidinoacetate methyl transferase (GAMT) [29]. It can be reversibly phosphorylated by the creatine kinases (CKs), and creatine/phosphocreatine (PCr) system forms a cycle that plays a crucial role in spatial energy distribution across all cells. This system functions as an energy buffer by transferring one phosphate group from PCr to adenosine diphosphate (ADP) to regenerate adenosine triphosphate (ATP) during periods of increased energy demand. This process is tightly regulated in polarized cells, where the differential localization of the CKs, together with easily diffusible PCr and Cr, facilitate efficient energy transfer from ATP generation site to regions with the highest ATP demand. In the intestinal epithelium these high-demand regions include the sites of assembly of intercellular junctions [30]. According to this, in intestinal epithelial cell cultures, either the loss of CrT [31] or the CK inhibition [32] altered the junctions’ proteins indicating that creatine is necessary to maintain structure and function of intestinal barrier. The importance of Cr/PCr system in the intestinal epithelial barrier is supported by studies in deficient mice in the AGAT enzyme or the CKs, which are more susceptible to the development of colitis and exhibit decreased proliferation and increased metabolic stress in the colonic epithelium [33,34]. Additionally, we and other authors found reduced colonic levels of creatine in rats with colitis, where barrier was disrupted [35,36], and with the creatine supplementation, these low levels were normalized together with the preservation of colonic epithelial integrity [32,35]. Despite these encouraging findings, only a few studies, including ours, have proposed creatine as an adjuvant therapy for ulcerative colitis, reporting improvements in clinical symptoms and/or intestinal inflammation [32,33,35,37,38]. However, the mechanisms underlying its protective effects, particularly its role in preserving intestinal barrier integrity under inflammatory conditions, remain largely unexplored.

To the best of our knowledge, studies investigating the effects of creatine on the intestinal barrier during inflammation have not comprehensively examined its impact on epithelial cell geometry, key intercellular junction proteins, or the intestinal vascular barrier, nor have they considered disease phase or sex differences. Therefore, our objective was to investigate these issues as potential mechanisms underlying the protective effects of creatine on preserving the intestinal barrier integrity in chronic colitis. For that, we used a dextran sulfate sodium (DSS)-induced chronic colitis model in male and female rats, as previously described [8,35], and integrated classical readouts with novel structural and vascular parameters. Thus, we analyzed clinical symptoms, histopathological alterations, proinflammatory cytokine expression, MUC2 abundance, and the expression and spatial distribution of epithelial intercellular junction proteins. As innovative aspects of intestinal barrier assessment, we also evaluated epithelial scutoid architecture and the expression and distribution of key proteins involved in vascular barrier integrity. We assessed the effects of creatine on all these parameters during the active and remission phases of chronic colitis, while also determining potential sex-dependent differences.

2. Results

2.1. Creatine Differently Alleviates the Chronic Colitis Clinical Symptoms and Prevents Colon Inflammation, Especially During the Active Period of the Disease, in Male and Female Rats

We started the study by examining the effect of creatine on colonic chronic inflammation during the active and the remission phases of chronic colitis. Male and female rats received DSS to induce chronic colitis or normal drinking water. Then, each of these groups, separated by sex, was further randomized into subgroups with and without creatine supplementation, following the experimental design illustrated in Figure 1A. Creatine was administered in drinking water 10 days before starting the DSS treatment and throughout the entire treatment. We performed the measurements at two time points of treatment: 2 days after the third cycle of DSS (day 37), coinciding with an active phase, and after a 9-day recovery period with normal water (day 44), coinciding with the remission phase of the disease (Figure 1A).

First, we assessed colitis-related clinical manifestations in DSS-treated male and female rats, with and without creatine, using the disease activity index (DAI) score, which includes measurements of body weight gain, stool consistency, and rectal bleeding, as described in the Methods section. Additionally, we measured the colon weight-to-length ratio, to account for size differences between males and females, and the mRNA abundance of proinflammatory cytokines, IL-1β, IL-6 and TNF.

A Three-way ANOVA was conducted to examine the effect of sex, colitis, and creatine on the parameters associated with colon inflammation (Table S2). The analysis indicated significant effects on symptoms, colonic inflammation and sex differences. In most of them, during active period the interactions between sex and colitis, and between colitis and creatine were significant (p<0.05), and also during remission phase between sex and creatine (p=0.006) in IL-6 levels, and between sex, colitis, and creatine (p=0.022) in IL-1β levels.

Post-hoc comparisons showed that, in both DSS-treated males and females, by day 37, there was an increase in the DAI score (Figure 1B), the colon weight-length ratio (Figure 1C,D), and the relative mRNA abundance of proinflammatory cytokines, except for IL-6 in females (Figure 1E,F). In the recovery phase (day 44), these elevated values had returned to levels similar to those of the control group (Figure 1B,E,F), excepting the colon weight-to-length ratio, which remained elevated in both sexes, and IL-1β and TNF levels in males (Figure 1C-F). Creatine supplementation only reduced DAI score without avoiding some symptoms, although it prevented the DSS-induced increases in colon weight-length ratio and cytokines during the active phase (day 37). However, on day 44, creatine only had an effect on the colon weight-length ratio in both sexes, and IL-1β in males, as these were the parameters that remained more elevated (Figure 1D,E).

As we previously demonstrated [8], sex differences were found in the active phase in DAI, colon weight-length ratio, and IL-1β and IL-6 mRNA levels, being the increases due to colitis higher in males (Figure 1B,D,E). In the remission phase (day 44), the only sex differences were the lowest IL-1β and TNF levels in females. Creatine supplementation reduced the values up to a similar level in both sexes. To determine whether the effect of creatine differs between males and females, we calculated the differences between the DSS and DSS-creatine groups for each parameter and compared them between sexes (Table 1), revealing that creatine effects were higher in males.

These results indicate that females experience milder colitis than males during the active phase of the disease, but both sexes recovered almost equally, except for the cytokines TNF and IL-1β that remained higher in males. Creatine alleviates colitis symptoms by preventing colonic inflammation in the active phase of the disease and in both sexes, but to a greater extent in males.

2.2. Creatine Differentially Ameliorates Colon Histopathological Changes in Male and Female Rats with Chronic Colitis

Next, we investigated whether creatine supplementation prevents or reduces colon damage and whether this effect is influenced by sex. We performed histopathological analysis using hematoxylin and eosin-stained distal colon sections on days 37 and 44 of DSS treatment. Besides evaluating the overall colon injury, we analyzed the infiltration of inflammatory cells separately, including the extension and the depth of the cell infiltrate.

Three-way ANOVA (Table S2) revealed significant effects on total colon injury score, on both days 37 and 44, including sex differences, as shown by interactions between sex and colitis (p<0.01) and between colitis and creatine (p=0.0001), and also between sex, colitis, and creatine on day 44 (p=0.022). Moreover, there were significant interactions between colitis and creatine on day 37 in inflammatory cell infiltration scores, extension and depth (p=0.003 and p<0.0001, respectively).

DSS-treated male and female rats exhibited significant structural alterations in the colon, characterized by epithelial destruction, irregular crypt architecture, goblet cell depletion, and inflammatory cell infiltration. Representative images illustrating these histopathological changes are shown in Figure 2A. In previous research, using a total injury score, we demonstrated that chronic colitis induced by DSS resulted in similar colon damage in both sexes during active and remission phases [8]. In this work, first we used a radar chart to visualize the six key parameters included in the total score: destruction of epithelium, dilatation of crypts, loss of goblet cells, inflammatory cell infiltrate, oedema, and crypt abscesses. This representation suggested sex differences in colon damage and the effects of creatine in the active and remission phases (Figure 2B). Among all assessed parameters, crypt abscesses and oedema exhibited the lowest values in both sexes. In contrast, epithelial destruction, crypt dilation, goblet cell loss, and inflammatory cell infiltrate were the most prevalent histopathological alterations. Notably, goblet cell loss was the lesion most effectively restored spontaneously on day 44 in the colon of both sexes, whereas creatine supplementation resulted in a more pronounced improvement in epithelial destruction and crypt dilation. Post-hoc comparisons showed that the colon injury present in the active phase (day 37) decreased compared with the remission phase (day 44), being significant only in males. Although this damage was attenuated on day 44 but remained increased as evidenced by comparison with control groups (Figure 2C). Creatine significantly decreased the total score on days 37 and 44 in the rats with colitis in both sexes, with lower values observed in females; however, the score did not return to basal values in either sex, indicating that the colon damage did not completely disappear (Figure 2C).

Analysis of inflammatory cell infiltration, distinguishing between extension (spatial distribution) and depth, whether it spreads to other layers of the colon wall, revealed that on day 37, DSS-treated rats exhibited increased cellular infiltration, extension and depth, and both scores decreased by day 44 but remained elevated relative to baseline conditions. Creatine supplementation reduced these inflammatory scores in both sexes, but only in the active phase (day 37) (Figure 2D). Additionally, to confirm whether creatine had a sex-dependent effect we analyzed the differences between the DSS and DSS-creatine groups and found that its impact was greater in males for inflammatory cell infiltration scores in the active phase since they reached higher differences than females, whereas in females, creatine had a greater effect on overall colon injury in the remission phase (Table 1).

In summary, colon injury and inflammatory cell infiltration persist in the remission phase in males and females with chronic colitis. Creatine supplementation reduces colonic damage during both the active and remission phases of the disease, with a more pronounced effect in females only during the latter. Furthermore, creatine ameliorates the infiltration of inflammatory cells during the active phase with a stronger effect in males.

2.3. Creatine Prevents the Mucin-2 Depletion in Male and Female Rats with Chronic Colitis

The reduced mucus layer characteristic of UC is primarily attributed to goblet cell depletion and defective MUC2 production and secretion [16]. We wondered whether creatine supplementation could reduce or prevent this alteration. We performed immunofluorescence assays on distal colon sections from male and female rats and quantified MUC2 fluorescence signal in all experimental groups. Representative images are summarized in Figure 3A and showed the expected signal provided by the MUC2 antibody, localized within the goblet cells of the crypts.

Three-way ANOVA (Table S2) showed a significant interaction between colitis and creatine (p=0.001). Multiple comparisons revealed a significant reduction in MUC2 signal only in male DSS-treated rats during the active period of the chronic colitis (day 37), which was similar to control levels by day 44 of treatment (Figure 3B). Creatine supplementation avoided the decrease in MUC2 in the active phase. The effects of creatine on MUC2 did not differ significantly between male and female rats (Table 1).

2.4. Creatine Ameliorates Alterations in the Localization and Expression of Tight and Adherens Junctions’ Proteins in Colonic Epithelium of Male and Female Rats with Chronic Colitis

The colonic epithelial layer, in which adjacent cells are sealed by the TJs and AJs, constitutes a protective physical barrier. UC is characterized by alterations of these junctional complexes, including the dysregulated expression and mislocalization of proteins that make them up, such as ZO-1 and claudin-5, for TJs and E-cadherin and β-catenin for AJs. We investigated whether creatine supplementation exerts its effects by modifying the expression and/or localization of these structural proteins, thereby strengthening epithelial barrier integrity. To explore this, we performed immunofluorescence assays on distal colon sections from DSS-treated rats of both sexes, with and without creatine supplementation and analyzed surface and crypt epithelium of the mucosa. Confocal microscopy was used to examine the ZO-1 fluorescence signal, enabling enhanced visualization and accurate quantification, and a standard optical microscope for the other three proteins.

Three-way ANOVA (Table S2) showed only a sex-dependent colitis effect on ZO-1 expression in the active phase (day 37) of the disease (p=0.007). We also found significant interactions between colitis and creatine on day 37 for ZO-1, E-cadherin, and β-catenin, and for E-cadherin on day 44 (p<0.001), and between sex, colitis, and creatine on day 44 for β-catenin (p=0.03).

First, we observed that, in the colonic mucosal epithelium of control rats, ZO-1 signal was continuous and belt-like, whereas in the rats with colitis (day 37) it showed discontinuous, fragmented and ruffled pattern. Multiple comparisons revealed that the ZO-1 signal intensity was significantly reduced in these rats, with a more pronounced effect in males (Figure 4A,B). Creatine supplementation prevented the ZO-1 downregulation in males and females, although the normal appearance of ZO-1 signal was only partially preserved in DSS-creatine groups (Figure 4A,B). On day 44, during the remission phase of the colitis, ZO-1 intensity was similar to controls, but the appearance remained somewhat irregular across DSS groups. Claudin-5 fluorescence signal presented a discontinuous pattern at cell junctions and the protein levels diminished in the epithelial cells on day 37 of DSS treatment in both sexes (Figure 5A,B). This claudin-5 disruption was subsequently restored to the same levels as controls on day 44 in females, but it remained in males. Creatine supplementation preserved normal claudin-5 localization at cell junctions in the rats with colitis in both sexes. Additionally, creatine avoided the expression decrease of this protein on day 37 in both sexes, and in males on day 44 because females had already reached the normal levels (Figure 5A,B). Regarding E-cadherin protein, in both males and females with colitis (days 37 and 44), mislocalization was observed with loss of the lateral belt and reduced expression in the colonic epithelium (Figure 6A,B). This effect was not observed in the DSS-creatine groups where the epithelium presented localization and E-cadherin intensity similar to controls. Finally, β-catenin analysis showed that, in both sexes, DSS-treated rats (days 37 and 44) exhibited a signal wavy and micro-tortuosity pattern and localized not only at lateral membrane but also away from the borders in the cytoplasm of epithelial cells. Quantification of β-catenin signal revealed significantly increased intensity in the DSS groups in the active phase of colitis which normalized together with a proper localization on day 44 in both sexes. Creatine supplementation prevented this increase and abnormal localization (Figure 7A,B).

Analysis of the differences between DSS and DSS-creatine groups comparing males with females only showed significant sex differences on creatine supplementation in claudin-5 on day 44, with stronger effect in males (Table 1).

These findings reveal that mainly during the active phase of the chronic colitis in male and female rats, TJs and AJs lose their uniform structural appearance and become irregular and discontinuous due to alterations in the expression levels and localization of junctional proteins. In the remission phase there is tissue self-repair, except for claudin-5 in males and E-cadherin in both sexes where these impairments remained. Furthermore, creatine supplementation prevented the mislocalization and dysregulated expression of these proteins during both phases of colitis, thereby preserving epithelial barrier integrity.

2.5. Creatine Preserves the Scutoid Geometry of Colonic Epithelial Cells in Male and Female Rats with Chronic Colitis

Colonic epithelium is a curved tissue which cells undergo constant junctional remodeling; thus, they could adopt the novel geometrical shape, the scutoid, to facilitate cellular packing, minimize cell surface tension and lead to a balanced energetic state. We wanted to identify this geometry in the colon and whether it is impaired by colitis and, if so, whether creatine supplementation could reduce or prevent this effect. For that, we used the immunofluorescence of β-catenin performed on distal colon sections from DSS-treated rats, with and without creatine supplementation, and analyzed the epithelium by confocal microscopy, which allowed us to observe the three-dimensional structure and cross-section of the scutoids.

In Figure 8A, a schematic illustrates a group of cells forming the three-dimensional structure and cross-section of the scutoids. We observed that control rats exhibited a colonic epithelium with scutoid architecture, with hexagonal and pentagonal shapes alternating in a mosaic-like pattern in a curved surface (Figure 8B). Rats with active colitis (day 37) displayed an epithelium with a disruption of this hexagon–pentagon alternation, and in areas with more severe lesions, cells appeared rounded, with irregular shapes and a loss of that mosaic pattern (Figure 8C). During the remission phase (day 44), scutoid geometry was to some extent restored (Figure 8D). Creatine supplementation partially prevented alterations in scutoid geometry in rats in the active phase of colitis (Figure 8F) and nearly preserved scutoid architecture in rats in the remission phase (Figure 8E). These findings indicate that creatine contributes to preserving the three-dimensional architecture and normal cellular packing in the colonic epithelium under chronic inflammatory conditions.

2.6. Creatine Prevents Impairments in the Intestinal Vascular Barrier Integrity in Male and Female Rats with Chronic Colitis

Finally, we investigated the more recently described additional layer of the intestinal barrier formed by endothelial cells located beneath the epithelial layer. The importance of vascular integrity for intestinal barrier function is highlighted by its disruption in UC and its potential contribution to disease pathogenesis. We evaluated the effect of creatine on this vascular barrier by performing immunofluorescence assays on distal colon sections from male and female rats with chronic colitis and analyzing the localization and abundance of plasmalemma vesicle-associated protein-1 (PV-1) and caveolin-1 (Cav-1) as indicators of vascular barrier impairments and increased permeability. Figure 9A and 10A show representative images of immunofluorescence assays of PV-1 and Cav-1, respectively, where the antibody signal was primarily localized in the endothelial layer within the lamina propria, just beneath the colonocytes and surrounding the crypts. This localization remained consistent across all experimental groups, while the protein abundance varied.

Three-way ANOVA analysis (Table S2) showed a significant interaction between colitis and creatine on day 37 on PV-1 signal intensity (p=0.0007). In the case of Cav-1 significant interactions were observed between sex and colitis on day 37 (p=0.001), between sex and creatine (p=0.032), and between colitis and creatine on days 37 (p<0.0001) and 44 (p=0.001). As shown in Figure 9B and 10B, post-hoc comparisons revealed that DSS-treated rats of both sexes on day 37 exhibited significant increased levels of both proteins PV-1 and Cav-1 in the endothelial layer, as compared with controls. The colitis-induced Cav-1 increase was significantly lower in females (Figure 10B). During the remission phase (day 44), compared with the active phase (day 37), PV-1 intensity decreased in both sexes, although it remained elevated compared to control rats; however, that of Cav-1 decreased only in males, remaining similar between days 37 and 44 in females. Creatine prevented the colitis-induced increase of both proteins PV-1 and Cav-1 in both sexes and at both time points, with levels similar to controls (Figure 9B and 10B). Analysis of the differences (Table 1) showed that the effect of creatine on Cav-1 was significantly more pronounced in males on day 37, while that on PV-1 had no significant sex effect.

These results reveal a compromised colonic vascular barrier in chronic colitis and suggest that creatine contributes to maintaining the integrity of this barrier during active and remission phases of the disease in both sexes.

3. Discussion

Despite the availability of multiple therapeutic options for UC, patients frequently continue to experience alternating active and remission phases of throughout the course of the illness. This relapsing–remitting pattern highlights the limitations of current treatments in achieving sustained disease control and emphasizes the need for therapeutic strategies that not only suppressing inflammation but also promoting the preservation of intestinal barrier integrity for maintaining long-term disease remission in UC. In the present study, we provide evidence that creatine, under chronic colitis conditions, reduced the severity of symptoms and colon inflammation, and preserved the intestinal barrier integrity contributing to its recovery by attenuating the impairments of scutoid shape and expression and localization of critical proteins from epithelial junctions and vascular. These results were observed either during both the active and remission phases of the disease, and, in addition, revealed some sex-specific differences and suggested the use of creatine as a therapeutic strategy to sustain remission of UC.

We used a DSS-induced chronic colitis model in male and female rats, which exhibited relapsing-remitting periods like the phases of UC in humans, as previously described [8,35]. Our previous study, using this model, revealed sex-dependent alterations, including clinical symptoms and colonic surface epithelial injury, and creatine supplementation mitigated these impairments, also with sex differences [35]. While in our previous study we reported creatine effects on the severity of chronic colitis symptoms in an active phase (day 37 of DSS treatment) [35], in this work, we incorporated an additional time point, day 44 of DSS treatment, that mimics a remission phase, during which the rats underwent a 9-day water period to allow a recovery, and compared these two critical phases of chronic colitis progression, while also considering sex-specific differences. Moreover, we conducted a more in-depth analysis of additional colitis-associated parameters and innovative aspects to explore the potential mechanisms underlying creatine protective effects in the gut.

First, we found that, as expected, in both sexes, colitis-associated symptoms were more severe in the active phase (day 37) than in the remission phase (day 44) and with greater effect observed in males, which agrees with our previous study and those of others [6,7,8]. During the active phase of chronic colitis, creatine supplementation alleviated the symptoms and prevented the increase of pro-inflammatory cytokine expression, and the colon weight-length ratio, with some of these effects more pronounced in males. Furthermore, creatine maintained these values close to control levels on day 44. These results lead to two main conclusions: first, creatine was more effective in males because their colitis was more severe, although females also showed improvement. Second, in general terms creatine exerted its effects during the active phase of the disease, as in the remission phase, the colon is already nearly recovered on its own. To further investigate, we analyzed the overall colon damage along with specific scores related to the extension and depth of inflammatory infiltration in both sexes at the two previously mentioned time points. Consistent with our previous findings, we observed severe colon damage on day 37 of DSS treatment [8]. Here, during the remission phase, the damage was reduced but remained significantly greater than in controls, as did inflammatory infiltration scores, indicating that the tissue did not recover spontaneously. Creatine attenuated the colon damage at both time points, but its effect was more pronounced in the active phase of the disease and similar in both sexes. Intriguingly, the effect of creatine was greater in females during the remission phase. Despite these effects, creatine did not normalize the score in either phase or sex suggesting that although it had a protective effect and contributed to recovery, the colon damage did not completely disappear. In addition, creatine decreased inflammatory cell infiltration only in the active phase of colitis with more impact in males. In the remission phase, both inflammatory scores remained elevated compared to baseline conditions, in all DSS groups without creatine effect.

Our results suggest that creatine, in both sexes, may play a role in managing and recovering from colitis by reducing damage and preventing colonic inflammation. In line with our findings, others author and we have previously demonstrated this protective role of creatine during active colitis in murine models of acute colitis in females [32,33], chronic colitis in males [38], and chronic colitis in males and females [8], as well as in a pilot clinical trial involving patients with mild to moderate UC [37]. However, this work is the first to evaluate creatine protective effects by directly comparing two critical phases of chronic colitis progression, the active and remission phases, while also considering sex-specific differences.

After establishing that creatine alleviates chronic colitis, we sought to investigate whether creatine could modulate intestinal barrier integrity as a plausible underlying mechanism. The intestinal barrier plays a crucial role in maintaining gut homeostasis, protecting not only the intestine itself but also the entire organism by preventing the entry of pathogens into the systemic bloodstream, which could otherwise spread throughout the body [39]. Furthermore, our research and that of others have shown that sustained inflammation in the colon can lead to neuroinflammation, potentially contributing to the development of neurodegenerative disorders and psychiatric disorders [8,40,41]. Recent studies from our group demonstrated that creatine supplementation prevents motor deficits, anxiety- and depression-like behaviors, as well as neuroinflammation, neuronal damage and electrophysiological impairments found in cortex and hippocampus of rats with chronic colitis [35,42]. Given these findings, we considered it relevant to explore whether these beneficial effects of creatine could be, at least in part, mediated by improvements in intestinal barrier function during colitis. Our results demonstrate for the first time that creatine may enhance intestinal barrier function at four levels: (1) sustaining MUC2 production, (2) maintaining the correct localization and expression of tight and adherens junction proteins at colonic epithelial cells, (3) conserving the epithelial scutoid geometry, and (4) preserving vascular barrier integrity.

The secreted mucus in the intestine is predominantly composed of MUC2. In patients with active UC structural weakening of the mucus barrier occurs with a reduction of MUC2, whereas increased MUC2 is associated with a protective response [15,16]. The importance of MUC2 for colon protection is shown in studies of mice with deficiency of MUC2 that spontaneously develop severe colitis [43,44,45]. We found MUC2 depletion in colonic mucosa of male rats during the active phase of chronic colitis. In the remission phase the MUC2 levels were normal in both sexes, suggesting a natural recovery. Creatine supplementation prevented this MUC2 decrease in the active phase in males. However, as shown in the radar chart, creatine only attenuated the loss of goblet cell during this phase remaining their abundance below normal levels. These findings suggest that creatine maintains normal MUC2 levels despite persistent goblet cell loss, indicating that its protective effect may involve a direct regulation of MUC2 production. To our knowledge, no previous study showed this protective effect that linked creatine supplementation to MUC2 production in the colonic mucosa.

To gain further insight into the mechanisms by which creatine preserves intestinal barrier integrity, we examined the expression and localization of key structural proteins forming TJs and AJs, specifically, the junctional proteins ZO-1, claudin-5, E-cadherin, and β-catenin. Previous studies have shown that expression or localization changes of these proteins lead to structural alterations in the junctions, resulting in increased intestinal permeability and a compromised barrier function, which promotes the development of IBD [2,18,19,46]. During intestinal inflammation, one of the most notable defects in molecular composition of TJs is the loss of ZO-1 from the cytosolic plaque of TJs [2,19]. Downregulation of epithelial claudin-5 has been reported in the colonic mucosa of mice with acute colitis [18,46] and patients with Crohn’s disease [47]. Strikingly, a study using intestinal epithelial cell cultures and an experimental colitis model identified claudin-5 as a key mediator linking inflammatory signaling to TJ disruption, intestinal barrier dysfunction, and UC progression [18]. In addition, this same study reported that restoration of claudin-5 was associated with improved epithelial barrier function, supporting its value as a marker of barrier integrity and a potential therapeutic target in UC [18]. Inflammation and injury of the intestinal mucosa also downregulate E-cadherin expression leading to dissociation of the E-cadherin/β-catenin complex and loss of membranous β-catenin [19].

Our findings regarding colitis-associated alterations in junctional proteins are consistent with previous reports. As expected, we observed a compromised colonic epithelial barrier in active phase of the chronic colitis, in both sexes, evidenced by the altered localization and reduced expression of epithelial ZO-1, claudin-5 and E-cadherin. During the remission phase the localization and expression of ZO-1 was restored, but E-cadherin expression remained reduced in both sexes, whereas claudin-5 returned to baseline exclusively in female rats. Additionally, at the active phase of disease there was an increase in the epithelial β-catenin signal together with a cytoplasmic redistribution. A plausible explanation for this is that our quantification included both junctional and cytoplasmic β-catenin, and the loss of E-cadherin may result in β-catenin redistribution from AJs to the cytoplasmic compartment following complex dissociation. Since no nuclear β-catenin staining was detected, our findings likely represent an intermediate stage preceding canonical Wnt activation, characterized by the cytoplasmic β-catenin accumulation before nuclear translocation. This may constitute a response to mucosal injury promoting epithelial proliferation and an early molecular event linking UC to an increased risk of development of colorectal cancer [20]. Notably, creatine supplementation reduced β-catenin accumulation, suggesting that preservation of AJ integrity limits cytoplasmic β-catenin availability and may consequently restrain aberrant Wnt/β-catenin signaling. These findings support the concept that targeting early alterations in β-catenin localization, before overt activation of the Wnt pathway, could constitute an effective strategy to prevent inflammation-driven colorectal cancer, as previously proposed [20].

Furthermore, creatine, in both sexes, also effectively prevented the alterations in the localization and expression of proteins ZO-1, claudin-5 and E-cadherin during the active phase of colitis, and when the changes occurred during the remission phase, maintaining normal levels and localization. The action of creatine on intercellular junctions could be direct or indirect. It is known that proinflammatory cytokines, including IL-1β and TNF, alter expression of junctional proteins through different mechanisms [19,48]. In this regard, creatine may exert its barrier-protective effect by reducing those proinflammatory cytokines. On the other hand, creatine could also act by maintaining epithelial energy requirements, which increase during damage, such as colitis, to sustain the barrier. Maintaining strong and stable intercellular junctions requires significant energy expenditure, as the reorganization of junctional proteins involves cytoskeletal movements, actin-myosin polymerization and depolymerization, and endocytic vesicle trafficking [49,50]. This rearrangement is energetically costly, which may explain the beneficial effects of creatine supplementation. This hypothesis is supported by studies that have demonstrated the relevance of the Cr/PCr/CK system in the maintenance of correct localization and expression of intercellular junction proteins contributing to preserving the intestinal barrier integrity [31,32,33,34]. In this context, it has been shown that the brain-type creatine kinase (CKB) colocalized with E-cadherin in mice colonic epithelial cells, where this colocalization and the E-cadherin expression decreased in acute colitis, and creatine supplementation prevented these changes [32]. In intestinal epithelial culture cells, junctional assembly of ZO-1 and E-cadherin proteins was impaired by inhibition of CKs [32], and CrT deletion resulted in mislocalization of TJ proteins such as ZO-1 [31].

In addition to the findings described above, our immunofluorescence of β-catenin was also used to reveal that the colonic epithelium exhibits structures highly reminiscent of scutoids. This scutoid geometry was partially lost during chronic colitis but preserved with creatine supplementation. No previous studies have reported the presence of scutoids in vivo beyond mouse embryos, let alone in the intestinal epithelium, or their alteration in a pathological context such as chronic colitis. Our findings align with the proposed role of scutoids in epithelial organization. Scutoids are thought to provide curvature to the lateral membranes of epithelial cells, allowing proliferating cells to integrate into the epithelial layer without compromising the integrity of the epithelial seal [25]. The observed loss of scutoid geometry during chronic colitis is consistent with the impairment of intestinal barrier function in this condition, which plays a critical role in disease progression. Conversely, the preservation of this epithelial geometry in creatine-supplemented animals further supports our hypothesis that creatine strengthens the epithelial barrier, in part by conserving the three-dimensional architecture necessary for its maintenance.

To further investigate the effects of creatine on the intestinal barrier, we also examined the vascular barrier, consisting of a fenestrated endothelium connected by intercellular junctions and localized below the epithelial layer. We evaluated two proteins involved in vascular barrier integrity, PV-1, which increases in response to vascular damage and indicates augmented permeability [22] and Cav-1 considered vital for maintaining endothelial cell integrity and its increased expression is linked to enhanced transcytosis or vesicular traffic [23]. We observed that endothelial PV-1 increased in the colonic mucosa of rats with colitis, agreeing with the only study to date in patients with UC and a mouse model of acute colitis which demonstrated compromised gut vascular barrier integrity, resulting in increased vascular permeability [51]. Additionally, we showed that Cav-1 staining was primarily detected in endothelial cells of colonic mucosa, whereas its presence in epithelial cells was very limited, and that this endothelial expression increased in the inflamed mucosa. Previous results showed that the accumulation of Cav-1 contributes to intestinal barrier disruption [52,53]. In our colitis model, the upregulation of these proteins reflects a compromised colonic vascular barrier and suggests an increased permeability during both phases of disease and in both sexes. During the active phase, the increases were higher than in the remission phase. PV-1 did not exhibit sex differences in either phase, but the Cav-1 increase was significantly lower in females in the active phase and similar to that in the remission phase. During the remission period, the expression of both proteins remained elevated compared to control rats. Creatine supplementation effectively prevented the colitis-induced increase of both proteins PV-1 and Cav-1, in both sexes and at both time points, being more pronounced this effect on Cav-1 in the active phase in males. Importantly, these results suggest that creatine by targeting both components, contributes to maintaining and recovering the integrity of this barrier with sex- and time-dependent nuances, and reinforce its potential as a therapeutic strategy for preserving intestinal barrier integrity. According to our observations, several studies performed in different tissues and models, but not in the intestine, showed various mechanisms by which creatine could benefit vascular health and function, such as its impact on inflammation and oxidative stress [54].

4. Materials and Methods

4.1. Chemicals

Dextran sulfate sodium (DSS) (MW = 40 kDa) was purchased from PanReac AppliChem (Spain). Creatine monohydrate was obtained from Best Medical Diet S. L. (Sevilla, Spain). Paraformaldehyde (PFA), salts for the preparation of phosphate-buffered saline (PBS), sodium citrate, Triton X-100, bovine serum albumin (BSA), and paraffin were purchased from PanReac AppliChem (Spain). Normal goat serum was purchased from Chemicon International, Inc. (California, USA), MUC2 antibody was obtained from Santa Cruz Biotechnology, Inc. (Texas, USA), ZO-1, Claudin-5 and PV-1 antibodies from Thermo Fisher Scientific (Massachusetts, USA), E-cadherin and β-catenin antibodies from BD Biosciences (California, USA), Caveolin-1 antibody from AbCam (Cambridge, UK), and Hoechst 33258, and Alexa Fluor-546 and -488 secondary antibodies from Thermo Fisher Scientific (Massachusetts, USA).

4.2. Animals

Wistar rats, both male and female, aged 45 days, were utilized in this study. Each experimental group consisted of 10 to 30 rats, with an equal distribution of 5 to 15 males and 5 to 15 females. The animals were handled with care, adhering to ethical standards to reduce both the number of subjects used and any potential distress. All procedures complied with European Union Council regulations (Directive 2010/63/EU) and the Spanish Royal Decree (BOE 34/11370, 2013) regarding the welfare of experimental animals. The study protocol received approval from the Animal Ethics Committee of the University of Seville and the “Junta de Andalucía” (Approval number: CEEA-US2021-14, 21/03/2022/051). The rats were supplied by the “Centro de experimentación animal de la Universidad de Sevilla.” Anesthesia was induced via intraperitoneal administration of ketamine (50 mg/kg) combined with xylazine (10 mg/kg) before euthanasia.

4.3. Experimental Design: Induction and Assessment of Experimental Chronic Colitis and Creatine Supplementation

The induction of colon inflammation was conducted as previously described [8,35]. Briefly, rats with comparable body weight values were randomized into untreated or control groups and dextran sulfate sodium (DSS)-treated groups. Control rats consumed normal drinking water. Chronic colitis was induced by administering three cycles of DSS in drinking water for seven days, with three seven-day intervals between the DSS cycles with normal water (Figure 1A). Rats were euthanized on day 37 (active phase) and day 44 (remission phase) of the experimental protocol (red). The daily DSS dose was 5.5 g per kilogram of body weight, which corresponded to approximately a 4% DSS solution and was given based on body weight rather than ad libitum consumption to ensure that all rats in the DSS groups received the same dose. Before DSS administration, water intake and body weight were monitored to determine the average daily consumption, allowing for appropriate dosage adjustments throughout the treatment. This protocol simulates the relapsing-remitting inflammation periods observed in human UC.

The induction and progression of colon inflammation were examined by determining clinical manifestations using the disease activity index (DAI) and colon-specific parameters such as the weight-to-length ratio, histopathological scoring, and the relative mRNA expression of colonic pro-inflammatory cytokines, as previously described [8,35]. To evaluate the DAI, animals were monitored every 2–3 days throughout DSS treatment, assessing body weight changes, stool consistency, and fecal blood presence. DAI scoring involved assigning a score between 0 and 3 to each criterion, with the total score representing the overall severity of the condition.

The creatine monohydrate supplementation protocol was based on a previously described method [35]. Briefly, creatine was administered in the drinking water to rats of both sexes at a dose of 1 g per kilogram of body weight per day. Similar to DSS, the dose was adjusted according to daily water intake. Before starting DSS treatment, rats in the creatine-supplemented groups received a preload of the same dose for 10 days, after which supplementation continued uninterrupted until the end of the experimental protocol (Figure 1A).

4.4. Biological Preparations

After euthanasia on day 37 or day 44 of the experimental protocol, the colons were excised and rinsed with an ice-cold saline solution, and their lengths and weights were recorded. Distal colon segments were either fixed overnight in phosphate-buffered saline (PBS; in mM: 137 NaCl, 2.7 KCl, 10 Na₂HPO₄, and 1.8 KH₂PO₄; pH 7.4) containing 4% paraformaldehyde (PFA) and embedded in paraffin for histological examination and immunofluorescence assays or rapidly frozen in liquid nitrogen and stored at -80 °C for subsequent RNA extraction and PCR analysis. The paraffin-embedded distal colon segments were cut into sections (5-10 μm-thick) using a microtome and applied to adhesive-coated glass slides.

4.5. Histopathological Analysis

For histopathological scoring, on day 37 or day 44 of the experimental protocol, paraffin-embedded sections of the removed distal colon were stained with hematoxylin/eosin. Colon sections were then scored for analyzing the injury, according to the scoring protocol published by Erben (2014) [55] with some modifications. Total colon injury was graded (0-3 scale) by evaluating the following parameters: the destruction of epithelium, the dilatation of crypts, the loss of goblet cells, the inflammatory cell infiltrate, oedema, and crypt abscesses, and is expressed as the sum of each of these six parameters. Inflammatory cell infiltration was graded by evaluating separately the extension and the depth scoring. The score for the extension (0-3 scale) was based on whether infiltration was absent, focal (restricted to a single or very few isolated spots scattered areas), zonal or extensive (widespread and continuous), and for the depth (0-3 scale) whether infiltration was absent, reached subepithelial area and crypt basis, the muscularis mucosae, submucosa, and muscular layers.

4.6. Relative Quantification of Gene Expression by Real-Time PCR

Real-time PCR was carried out as previously reported [56] with some modifications. Briefly, total RNA was isolated from the distal colon of both control rats (water and water-creatine) and treated rats (DSS and DSS-creatine) using the RNeasy^® kit (Qiagen, Germany). cDNA synthesis was performed using 1 µg of total RNA with the QuantiTect^® reverse transcription kit (Qiagen, Germany), following the manufacturer’s guidelines. The primers utilized are listed in Table S1. For real-time PCR, the NZYSupreme qPCR Green Master Mix^® (MB41903; Nzytec, Portugal) was used. PCR amplification was carried out in a MiniOpticon™ thermocycler (Bio-Rad^®) using the following cycling conditions: an initial denaturation step at 95 °C for 3 min, followed by 40 cycles of 94 °C for 5 s and 60 °C for 30 s. β-Actin served as the housekeeping gene for sample normalization. The relative mRNA expression levels were calculated using the comparative Ct method, with the lowest recorded value for each gene set to 1.

4.7. Immunofluorescence Assays

Immunofluorescence assays were conducted on distal colon sections from both male and female rats in the control groups (water and water+creatine) and treated groups (DSS and DSS-creatine). Briefly, 5-10 µm-thick paraffin-embedded sections mounted on adhesive-coated glass slides were washed with PBS, boiled in sodium citrate buffer (10 mM; pH 6) for 10 min and blocked with 5% bovine serum albumin (BSA), 5% normal goat serum, and 0.1% Triton X-100 in PBS for 1 h at room temperature. Next, the sections were incubated overnight at 4 °C with the appropriate primary antibody, diluted in the blocking solution. To visualize primary antibody binding, appropriate secondary antibodies Alexa Fluor-546 or -488 were used. Nuclei were counterstained with Hoechst 33258. Negative controls were processed simultaneously by omitting the primary antibody to confirm specificity. The colon slides were mounted (Vectashield, Vector, Burlingame, CA, USA), observed with a Zeiss Axioskop 40 fluorescence microscope or a Leica Stellaris 8 STED confocal microscope, and photographed using the ZEISS ZEN 3.5 software or LAS X Office software, respectively. Immunofluorescence images of confocal microscopy were obtained at the “Centro de Investigación Tecnología e Innovación de la Universidad de Sevilla” (CITIUS). We analyzed 3 immunolabeled sections per rat in 8–10 fields per section, covering a representative area of the distal colon mucosa. An average of all fluorescence intensity measurements for each animal was calculated, and the intensity level of the male control group was used as the reference and assigned a value of 1; the levels of all other experimental groups were normalized and expressed relative to it. Image analysis was performed using ImageJ software (ImageJ, National Institutes of Health, Bethesda, MD, USA).

4.8. Statistical Analysis

Data are presented as the mean ± the standard error of the mean (SEM). For comparisons involving two or more variables, two-way or three-way analysis of variance (ANOVA) was used. To correct for multiple comparisons between the experimental groups ANOVA was followed by the Bonferroni post hoc test using the GraphPad Prism software version 8.0. The differences were considered statistically significant at p<0.05.

5. Conclusions

To summarize, we demonstrate that creatine protects the colon against chronic colitis, through a preventive and restorative function, in both the active phase of the disease, with particularly more pronounced during the active phase, and in both males and females. In our study, we integrated classical readouts with innovative aspects of intestinal barrier assessment, such as epithelial scutoid architecture and vascular parameters, and identify the anti-inflammatory effects and the preservation of epithelial and vascular barrier integrity as key mechanisms underlying the protective effects of creatine in the gut. We propose that creatine exerts a multilevel protection at six interconnected levels: 1) ameliorating the symptoms and colonic damage, 2) avoiding colonic inflammation, 3) preventing the MUC2 depletion, 4) maintaining the normal expression and distribution of structural proteins involved in epithelial TJs and AJs, 5) conserving the epithelium scutoid geometry, and 6) preventing increases in proteins involved in vascular permeability. By stabilizing junctional complexes and preserving scutoid geometry, creatine not only prevents barrier disruption but also promotes the architectural configuration necessary for accommodating proliferating cells without compromising epithelial sealing. Additionally, our study incorporates a sex perspective. Our results indicate that, in general terms, creatine sustains key physiological parameters at normal levels and is particularly effective in the active phase; although it benefits both sexes, some of its effects were more pronounced in males, probably due to a higher susceptibility to colitis-associated damage. Creatine oral supplementation could be beneficial in preserving colonic homeostasis against the damage induced by colitis with a potential translational application as a safe and accessible adjuvant option for UC treatment in both the active and remission phases.