The Leaky Gut and Human Diseases: “Can’t Fill the Cup if You Don’t Plug the Holes First”

The intestine is the largest interface between the internal body and the external environment, primarily aiding the absorption of nutrients, electrolytes, and water. Simultaneously, being the intestinal mucosa exposed to various external antigens, including food antigens, toxins, and commensal or pathogen microbes residing in the gut, another crucial function, commonly referred to as the barrier function, is to prevent these substances from entering the bloodstream.

Scientists have been interested in studying the structure of the intestinal barrier since the second half of the twentieth century. In 1968, Trier reviewed the morphology of the intestinal lining, but the limitations of the light microscope made it difficult to understand the nature of the surface of the cells. However, a hypothetical model of a limiting membrane was proposed [1]. It was not until 2004 that Cummings introduced the term “mucosal barrier” to describe the complex structure that separates the internal milieu from the luminal content [2].

Various intrinsic and extrinsic factors, including genetic predisposition, diet, drugs, alcohol, circadian rhythm disruption, psychological stress, and aging can weaken the barrier integrity [3]. The loss of barrier integrity, referred to as “leaky gut,” results in increased intestinal permeability, which allows the translocation of several compounds into the body, triggering systemic, low-grade inflammation [4].

In recent years, there has been increasing interest in the concept of leaky gut, both within the scientific community and among the general public. This interest has been emphasized by the media and internet communities, leading to a mix of accurate information, false myths, and bizarre theories, and consequently, generating a considerable economic burden. Indeed, a recent Australian study surveyed nearly 600 individuals with diagnosed or suspected leaky gut. The authors found that over the past 12 months, participants spent almost AUD 700 on consultation fees, AUD 287 on diagnostic tests, and over AUD 2,000 on dietary supplements for managing leaky gut. Additionally, many participants experienced a significant delay between first suspecting a leaky gut and receiving a diagnosis [5].

This uncertainty largely stems from the fact that the leaky gut has been merely deemed an extension of the concept of gut dysbiosis. It is a fact, however, that the leaky gut seems to be the prerequisite for numerous pathological conditions that are not limited to the gastrointestinal tract.

On account of its great potential pathogenetic impact, the measurement of intestinal permeability, reported in the scientific literature since the 1970s, has been extensively studied, but without achieving consensus about the best testing methods and their accuracy [6]. Moreover, although the research on the modulation of gut permeability is just starting, and no medical guidelines for the treatment of leaky gut syndrome are available, several treatment strategies are under investigation with promising results [7]. The objectives of this review are to discuss the composition of the intestinal barrier, the pathophysiology of the leaky gut and its implications on human health, the measurement of intestinal permeability, and the available therapeutic strategies to restore gut barrier integrity.

The gut barrier is a sophisticated and dynamic system that forms the frontline defense between the external environment and the body’s internal milieu and includes various structural and functional components engaged not only in digestion and nutrient absorption but also in immune regulation and overall health maintenance [8].

The gut barrier is comprised of four major lines of defense: the biological or microbiome barrier, the mucus barrier, the immune barrier, and the mechanical barrier [9]. A schematic representation of the gut barrier is reported in Figure 1.

The biological barrier is predominantly inhabited by the gut microbiome, an ecosystem of trillions of microorganisms residing in the gastrointestinal tract. These symbiotic bacteria contribute to nutrient metabolism, fermentation of dietary fibers, and synthesis of vitamins, while also exerting protective effects against pathogenic invaders through competitive exclusion and production of antimicrobial compounds [10].

The mucus layer, crucial for safeguarding against harmful substances and pathogens, comprises two distinct layers: an inner mucus layer and an outer mucus layer. While the inner mucus layer remains relatively sterile, serving for nutrient absorption and immune surveillance, the outer mucus layer hosts the community of microbes that bind to mucus through lectin-like molecules [11, 12]. Fundamental to the composition of mucus are mucins, synthesized and packaged into secretory granules within the cytoplasm of goblet cells scattered throughout the mucosa. Upon stimulation, goblet cells secrete mucins at the apical membrane, replenishing the mucus layer and fortifying the gut barrier. A steady production of mucus is maintained under physiological conditions, but various genetic and environmental factors can modulate mucus secretion, potentially compromising the integrity of the gut barrier [13]. Moreover, antimicrobial proteins and IgA molecules are secreted in the mucus gel in a gradient extending from the epithelium to the lumen, thereby enhancing the defense against the luminal microbes [6].

The immune barrier comprises an intricate network of immune cells strategically positioned throughout the intestinal mucosa. Gut-associated lymphoid tissue, including Peyer’s patches and isolated lymphoid follicles, serves as the primary site for antigen sampling and immune surveillance. T cells, B cells, macrophages, dendritic cells, and other immune mediators work in concert to detect, neutralize, and clear invading pathogens while maintaining immune tolerance to harmless antigens and commensal microorganisms [14].

The mechanical barrier, formed by a monolayer of epithelial cells lining the intestinal mucosa, is a selectively permeable barrier that allows for the absorption of nutrients and water while preventing the translocation of pathogens, toxins, and other harmful molecules from the gut lumen into the bloodstream [15]. The intestinal epithelial layer, the major barrier that separates our body from the external environment, is highly dynamic and characterized by a remarkable turnover rate; intestinal epithelial cells are rapidly renewed and replaced every couple of days. It contains different types of epithelial cells, such as enterocytes, Paneth cells, goblet cells, endocytes, and microfold cells, each with a distinct function [16]. The epithelial cells come into the closest possible contact in the most apical part of the lateral cell membranes by specific structures named “tight junctions” (TJs), which interconnect the cells and restrict the passage of ions, molecules, and cells through the paracellular space [17].

The dynamic concept of the intestinal barrier has further evolved with the identification of the gut vascular barrier (GVB). Indeed, in 2015, Rescigno and colleagues [18] uncovered in both mice and humans a new element in the gut barrier structure, the GVB, that controls the type of antigens that are translocated across blood endothelial cells to reach the portal vein. Following these preliminary findings, the GVB is now considered an integral component of the intestinal barrier, and its dysfunction has emerged as a concurrent cause of intestinal and extraintestinal diseases [19, 20].

The intricate interplay among all the components of the gut barrier serves as the cornerstone of intestinal homeostasis, ensuring a delicate balance within the gut ecosystem. In recent years, significant attention has been directed towards understanding the role of intestinal biofilms in maintaining gut health. Biofilms are organized microbial communities that live within a self-produced matrix of extracellular polymeric substances (EPSs), adhering to biological surfaces within the intestinal tract [21]. The earliest observations of multispecies biofilms date back over 300 years when Antonie van Leeuwenhoek first noted them while examining his teeth and those of another individual with poor oral hygiene [22]. It is now well-established that both commensal and pathogenic bacteria can form biofilms as they colonize and establish niches within the gut environment. In healthy individuals, biofilms typically reside over the mucus layer, where they contribute to gut homeostasis and host-microbe interactions. However, in disease states, biofilms may breach the mucus barrier, allowing direct interaction with epithelial cells and potentially leading to pathological consequences [22]. Understanding the dynamics of intestinal biofilms and their relationship with the mucosal barrier holds promise for elucidating mechanisms of gut health and disease, offering potential avenues for therapeutic intervention and maintenance of intestinal homeostasis.

When one or more components of the intestinal barrier lose their structure and escape their function, this may result in a leaky gut. In 1955, Jacob Fine proposed an early version of the “leaky gut hypothesis,” suggesting that irreversibility in various forms of shock results from the systemic absorption of gut-derived bacterial endotoxins when host resistance to lipopolysaccharide (LPS) is impaired due to the adverse effects of shock on the reticuloendothelial system [1]. Although Fine’s thesis was supported by numerous experiments demonstrating improved survival with prior enteral administration of nonabsorbable antibiotics, elevated circulating concentrations of LPS in shocked animals, and increased lethality due to exogenously administered endotoxin in hemorrhage, it was not widely accepted by his contemporaries [23].

It was not until 1979 that Berg and Garlington [24] introduced the term “bacterial translocation” (BT) to describe the passage of viable bacteria from the gastrointestinal tract through the epithelial mucosa into the lamina propria and then into the mesenteric lymph nodes and possibly other organs. This initial definition was later expanded to include the translocation of nonviable bacteria or their products, namely pathogen-associated molecular patterns, with the main representative being the intestinal endotoxin.

BT occurs in healthy individuals at a low rate of 5–10%, serving physiological roles in preparing the gut immune system for effective responses to extensive pathogen invasion and developing immune tolerance to microbial antigens. However, compromised intestinal barriers in certain disease states lead to increased BT, causing infectious complications and a systemic inflammatory response that worsens underlying diseases [25].

Intestinal infections can play a role in disrupting the intestinal barrier. While it is expected that common intestinal pathogens can damage the intestinal barrier, it is interesting to note that other bacteria can compromise its integrity. For instance, Helicobacter pylori which typically infects the human stomach, is known to increase intestinal permeability by causing the ZO-1 protein to redistribute from the TJs [26]. Pathological intestinal barrier failure is also observed in situations like major abdominal surgery, critical illness, severe injury, sepsis, and intensive care unit hospitalization. However, gut barrier failure can also be found in stable patients with chronic conditions, where it promotes immune activation associated with disease progression and/or complications [25].

While compromised intestinal barrier integrity is observed in both intestinal and systemic diseases, it remains unclear whether the loss of barrier integrity is the cause or consequence of these diseases. Pathophysiological or environmental factors may play a role in disrupting normal physiology and increasing barrier permeability, highlighting the importance of understanding factors contributing to barrier integrity loss under pathological conditions [3].

To gain a comprehensive understanding of the impact of heightened intestinal permeability in diseases, it is essential to dissect fundamental concepts regarding how various molecules pass across the intestinal epithelium. Gut barrier function involves regulating the passage of luminal contents such as antigens and bacteria across the epithelial cell layer, either between the cells (paracellular route) or through the cells themselves (transcellular route), into the underlying mucosa [8].

The paracellular route denotes the passage between cells, facilitated by TJs and intercellular spaces [27]. This pathway typically permits the movement of medium-sized molecules (≤600 Da in vivo; ≤10 kDa in vitro in cell lines) that are hydrophilic [28]. Under normal conditions, the paracellular route is impermeable to molecules of protein size, thus serving as an effective barrier against antigenic macromolecules. Two distinct paracellular epithelial permeability pathways exist, the “leak” and “pore” pathways, both regulated by TJs [29]. Conversely, an “unrestricted” pathway, associated with pathological states (erosions/ulcers), operates independently of TJs, allowing luminal antigens to gain entry to the lamina propria [30].

The transcellular route involves passive diffusion through cells, primarily utilized by lipid-soluble and small hydrophilic compounds [31]. Additionally, active and energy-dependent uptake occurs through the cell membrane. Large particles and molecules, such as proteins, which cannot traverse the cell membrane or the paracellular space, can be taken up via endocytosis. This process involves the cell engulfing substances through the invagination of the plasma membrane, followed by the formation of vesicles [6]. The transcellular route is utilized not only for the uptake of large peptides and proteins but also for the entry of whole commensal bacteria into the lamina propria, as part of the BT process [32].

To evaluate intestinal permeability, researchers monitor the passage of permeability markers across the epithelium via these routes. While the paracellular route is commonly studied, the other routes of transepithelial transport remain poorly investigated. A deeper comprehension of intestinal permeability holds the potential for breakthroughs in the diagnosis and treatment of various gastrointestinal disorders.

Alterations in the microbial composition, referred to as dysbiosis, have been associated with dysfunction of the mucosal barrier [3]. The importance of gut microbes in maintaining the intestinal barrier is historically highlighted by studies conducted on animals bred under germ-free conditions showing that germ-free animals compared to conventionally raised mice exhibit morphological, structural, and functional abnormalities in the intestine. Interestingly, the reconstitution of the gut microbiome of germ-free mice with Bacteroides thetaiotaomicron is sufficient to restore intestinal health [33].

The gut microbiome, referred to as an “organ” due to the vast density and richness of microbes, plays a pivotal role in regulating epithelial barrier function through various mechanisms [34]. In addition to its role in safeguarding against pathogens through the competitive exclusion of pathogens and the synthesis of antimicrobial compounds, the gut microbiome also directly influences the maintenance of gut barrier integrity, by directly interacting with enterocytes or producing various metabolites [35].

The composition and quantity of certain bacteria play a pivotal role in determining the glycan composition of mucus and are closely linked to various glycosyltransferases, whose levels are heightened in the presence of gut microbes. For instance, certain bacteria have been found to stimulate the activity of host fucosyltransferases, responsible for adding L-fucose at the α−1,2 position, as well as sialyltransferases. Additionally, the bacterial communities residing in the host can influence the glycosylation patterns of both MUC2 and transmembrane mucins [11].

Several microbial components, including LPSs, peptidoglycans, and lipoteichoic acids, can activate various members of the toll-like receptor (TLR) family, belonging to pattern recognition receptors, some of which are implicated in the regulation of intestinal epithelial permeability [36]. Stimulation of TLR2 leads to activation of protein kinase C isoforms, which in turn induces ZO-1 redistribution an increase in barrier electrical resistance in vitro [37].

The commensal microbiota synthesizes various fermentation products, including short-chain fatty acids (SCFAs), indoles, and hydroxy fatty acids. These microbial metabolites act as external regulators for TJs, which are pivotal in maintaining intestinal integrity [38]. Butyrate, an SCFA derived from dietary fibers and mucin glycans, is typically produced by certain Clostridium clusters (IV and XIVa) from the Firmicutes phylum [39]. It enhances mucin 2 (MUC2) levels and stabilizes hypoxia-inducible factor-1 (HIF-1), thereby promoting the expression of genes encoding crucial TJ proteins like claudin-1 and occludin [40]. Similarly, indole-3-propionic acid suppresses the pro-inflammatory cytokine TNF-α and upregulates TJ-related proteins by activating the pregnane X receptor [41].

Conjugated fatty acids, such as conjugated linoleic acid (CLA), are produced by intestinal bacteria such as Bifidobacterium, Butyrivibrio, Enterobacter, Lactobacillus, Clostridium, Citrobacter, Roseburia, Klebsiella, and Megasphaera from diet fats. CLA promotes gut barrier function by upregulating zonulin-1, occludin, claudin-3, and E-cadherin in colonic tissues [42].

Primary bile acids, namely cholic acid and chenodeoxycholic acid, are initially produced in the liver and then transported via the enterohepatic circulation into the gut. Here, they undergo further metabolism by gut microbiota through processes such as dehydroxylation, dehydrogenation, and epimerization to generate secondary bile acids [43]. Secondary bile acids interact with the nuclear membrane farnesoid X receptor and the G protein-coupled receptor in the intestine, exerting both negative and positive effects on gut barrier TJ functions [44].

Polyamines such as spermine, spermidine, and putrescine are generated from polyamine-rich foods by gut microbes such as E. coli, Bacteroides spp., and Fusobacterium spp in the colon, where they favor gut barrier integrity through E-cadherin upregulation in an intracellular Ca2+-dependent manner [45]. Polyphenols (e.g., tannins, flavanols, flavanones, flavones, isoflavones, flavan-3-ols, lignans, chlorogenic acids, anthocyanidin) are metabolized by the gut microbiome and converted to active phenolic derivatives that promote gut barrier integrity through activation of aryl hydrocarbon receptor-nuclear factor erythroid 2-related factor 2-dependent pathways to upregulate epithelial TJ proteins [46].

The endocannabinoid system has emerged as a potential path by which the gut microbiota controls gut permeability and plasma LPS levels. Endocannabinoids have been shown to enhance the expression of occludin-1, while also decreasing the expression of claudin-1 [47]. Evidence on the role of gut microbiota in modulating the endocannabinoid system has been obtained by measuring the abundance of cannabinoids following the manipulation of the gut microbiota by various means, including antibiotic treatment, probiotic treatment, high-fat diet, and mutations in the Myd88 gene which disrupt TLR-mediated bacteria-host interaction [48]. In mice models metabolic endotoxemia arising from impaired gut barrier function was reduced by treatment with the cannabinoid-1 antagonist SR121716A, which resulted in the partial recovery of the efficacy of the TJ proteins, zonula occludens-1, and occludin [49]. The evidence discussed emphasizes the crucial role of the interplay between the intestinal microbiome and the epithelium in preserving the integrity of the gut barrier and provides insights into the pathophysiological implications of conditions related to leaky gut in humans.

The intestinal barrier plays a fundamental role in the pathogenesis of intestinal disorders as well as diseases in organs other than the intestine, gaining growing attention from the scientific community. Compromised intestinal barrier integrity, indeed, is evident in various diseases, including inflammatory bowel disease (IBD) [50], autoimmune disorders [51], neurological conditions [52], allergies [53], and cardiovascular [54] and metabolic diseases [55]. However, it remains unclear whether the loss of barrier integrity is the cause or consequence of these diseases. Pathophysiological or environmental factors may disrupt normal physiology and increase barrier permeability, thus a comprehensive understanding of their contributory role is needed.

IBDs and their related extraintestinal manifestations (EIMs) are paradigmatic of the link between the gut barrier and human diseases [56]. IBDs, classified into ulcerative colitis (UC) and Crohn’s disease (CD), are showing a rapidly growing incidence worldwide [57]. Environmental and autoimmune factors along with dysregulated mucosal immune responses are believed to play significant roles in the complex IBD pathogenesis, which remains largely undefined.

Loss of barrier integrity is a hallmark of IBD, potentially serving as an initial event triggering inflammatory bowel disorders, to such an extent that some authors think of IBD as an impaired barrier disease [58, 59]. While debate persists regarding the precise contribution of leaky gut to disease pathology, recent research suggests that functional abnormalities in TJ proteins may contribute to disease development, independent of underlying immunity [60]. Notably, increased epithelial permeability precedes disease relapse in CD patients, emphasizing the importance of TJs in IBD pathogenesis [61, 62]. Moreover, it has been reported that first-degree relatives of patients with IBD have abnormal intestinal permeability [63‒65].

Patients with IBD also show gut microbiome dysbiosis characterized by a reduced bacterial diversity, with a decreased relative abundance of Firmicutes and an increase in pro-inflammatory bacteria, such as proteobacteria, or adherent-invasive Escherichia coli or mucolytic bacteria such as Ruminococcus gnavus and Ruminococcus torques [66‒68].

Genetic predisposition, environmental factors, and increased inflammation-induced apoptosis in intestinal epithelial cells may contribute to compromised barrier integrity in IBD. Regardless of genetic or environmental determinants, impaired permeability disrupts the balance between the mucosal barrier and luminal challenge, exacerbating immune dysregulation in IBD patients, and allowing bacteria and bacteria-derived molecules to be translocated into the mucosa and flaring up uncontrollable inflammatory signal cascades.

IBD often presents with EIMs, contributing significantly to morbidity and reduced quality of life. EIMs are classified based on their association with IBD disease activity but can occur before the onset of intestinal symptoms, throughout the natural course of the disease, and even after a colectomy in the case of UC [56]. Two different types of EIMs are generally associated the first are immune-related manifestations reactive to inflammatory intestinal activity and include arthritis, erythema nodosum, pyoderma gangrenosum, aphthous stomatitis, and iritis/uveitis. The latter are autoimmune disorders independent of intestinal activity but related to the increased susceptibility to autoimmunity and comprise primary biliary cirrhosis, alopecia areata, thyroid autoimmune disease, and others. The BT across the leaky gut barrier can trigger adaptive immune responses unable to distinguish between bacterial epitopes and epitopes of joints or the skin or an autoimmune response linked to genetic susceptibilities associated with human leukocyte antigen [69]. On the other hand, intestinal inflammation, dysbiosis, impaired intestinal permeability, and BT have been identified in patients with autoimmune joint diseases but the time at which they appear and their contribution to the pathogenesis of the disease are still a matter of debate [70]. Recently, in a rat model of reactive arthritis, the authors showed that transient dysbiosis, intestinal inflammation, and an increase in serum markers of a leaky gut precede the clinical onset of arthritis [71].

Based on this evidence, permeability-oriented therapies to manage these uncurable diseases aroused growing interest in recent years [50]. Agents commonly used in IBD treatment can promote and sustain mucosal remission not only through immunomodulation but also by restoring epithelial integrity and permeability, as shown by the effects of anti-TNF-α drugs in CD [72, 73]. Similar outcomes have been observed with elemental diets in CD [74], generating interest in dietary strategies involving immunomodulatory nutrients and probiotics. Supplementation with specific fatty acids, amino acids, and oligoelements has been shown to reduce inflammation and improve mucosal permeability in experimental models of IBD [75]. Different probiotic strains have been investigated for their effect in strengthening mucosal barrier in IBD, with conflicting results [76]. Nonetheless, the therapeutic effectiveness of these supplements in IBD is still debated, with the most substantial evidence supporting butyrate, zinc, and probiotics [50]. Further research aimed at elucidating mechanisms underlying gut barrier integrity and function, as well as related host responses, holds promise for preventing and managing diseases associated with a leaky gut and will pave the way for novel therapeutic approaches.

The increased interest in the leaky gut and its correlation with various gastrointestinal disorders has gained attention among researchers from diverse backgrounds to investigate the intestinal barrier’s functionality under a variety of conditions. As attention in this area grows, there is a rapid expansion in methodological tools for studying intestinal barrier function, with the ongoing development of new approaches, even if, selecting the most appropriate technique is still a challenge [77].

Techniques to assess intestinal barrier function include in vivo, in vitro, and ex vivo approaches. Table 1 summarizes the main methods available for the assessment of intestinal permeability and their advantages and disadvantages.

The most common method for evaluating intestinal permeability in vivo involves administering oral markers and measuring their recovery in urine. These markers, including Chromium-labelled EDTA (⁵¹Cr-EDTA), polyethylene glycol (PEG), and non-metabolizable sugars like lactulose, sucrose, and rhamnose are absorbed through the intestinal mucosa into the bloodstream, excreted by the kidneys into the urine, in which they can be measured [78]. Higher concentrations of these markers in urine samples indicate increased intestinal permeability, suggesting a greater passage through the intestinal barrier.

Using two different probes concurrently is considered the most effective approach for studying epithelial permeability. This method allows for the calculation of an excretion ratio, which helps adjust for variables such as gastric emptying, intestinal transit time, and renal function [9].

The gold standard for assessing small intestinal permeability evaluates the differential urinary excretion test of lactulose and mannitol [79]. Lactulose, being a disaccharide, crosses the barrier via the paracellular pathway in a highly regulated manner, while mannitol, a smaller monosaccharide, pass the intestinal epithelium freely through both transcellular and paracellular routes. The timing of urine collection is critical to assess the relative contributions of the small intestine and colon to measurements of permeability. Typically, the lactulose/mannitol ratio measured in urine collected within 0–2 h after ingestion serves as an indicator of small intestinal permeability. Indeed, the use of radioisotopes demonstrated that a significant portion of the administered probe remains within the small intestine during this time frame. Between 2 and 8 h, a combination of small intestinal and colonic presence is observed, while the 8–24 h urine collection is regarded as most indicative of colonic permeability [32]. However, when evaluating colonic permeability, lactulose/rhamnose or mannitol should not be used due to degradation by colonic bacteria. Hence, alternative probes, namely ⁵¹Cr-EDTA, PEG molecules, or sucralose which remain stable throughout the gastrointestinal tract, are needed [78]. Additionally, gastric permeability assessment can be achieved using sucrose, a sugar that undergoes degradation by sucrase in the duodenum [80]. Recent studies have also confirmed the feasibility and accuracy of multi-sugar tests with fractioned urine collections to simultaneously assess intestinal permeability at different locations [81]. Still, a major confounder is that probe molecules may be constituents identified at baseline, typically from inadvertent intake in the diet or cosmetic products. ¹³C-mannitol may be reliably measured in urine at specified time points to determine small intestinal and colonic permeability but is distinguished from mannitol that is inadvertently consumed by subjects outside the study. Recently, Camilleri et al. [77], by using a high-performance liquid chromatography-tandem mass spectrometry analysis, developed and validated a ¹³C-mannitol-based test to assess intestinal permeability in healthy adults [82].

Circulating biomarkers have also been used for the assessment of intestinal permeability in vivo in human subjects. These biomarkers include, among others, LPS and LPS-binding protein, zonulin, intestinal fatty acid-binding protein (I-FABP), citrulline, and glucagon-like peptide (GLP)-2, which serve as indicators of epithelial damage and provide indirect evaluations of the intestinal barrier [83].

Serum levels of the endotoxin LPS, have been suggested as a potential marker of increased intestinal permeability and BT, and elevated serum-LPS levels originating from the gut microbiome, have been associated with various diseases [84]. However, the methodologies employed for LPS detection in blood have met criticism for their inaccuracy, leading to conflicting results that are difficult to interpret [85]. Moreover, it remains unclear whether the LPS identified through these assays is bioactive and capable of causing systemic inflammation, and whether it may originate from bacteria in body parts other than the gastrointestinal tract [86]. Therefore, the interpretation of serum LPS levels as an indicator of a disrupted intestinal barrier should be approached with caution [8].

Due to the challenges associated with measuring and interpreting LPS levels, LBP has gained significant interest as a marker of the immune reaction to LPS and, consequently, as an indirect indicator of endotoxemia [87]. LBP is an acute-phase protein synthesized by hepatocytes, which binds to bacterial LPS. The LPS-LBP complex interacts with CD14, leading to the initiation of an inflammatory response [88]. However, circulating LBP levels exhibit variability during acute and chronic conditions and are influenced by factors such as diet and infection. Therefore, repeated measurements of plasma LBP are necessary to ensure acceptable reliability. Furthermore, elevated levels of LBP merely indicate the occurrence of an immune response to LPS in the bloodstream, but it remains challenging to ascertain the precise source of LPS within the body [8].

Zonulins, which are 47 kDa paracrine proteins released by diverse cell lines, including those in the small intestine epithelium, play a substantial role in regulating intercellular TJs. Particularly, they are significant in conditions like celiac disease, diabetes mellitus, IBD, and obesity where changes in TJs influence antigen trafficking and mucosal immune response [89]. However, it should be considered that the most widely used enzyme-linked immunosorbent assays do not detect solely zonulin but also haptoglobin, complement C3, and possibly properdin, providing unspecific results [90, 91]. Moreover, increased serum zonulin levels do not correlate with colonic paracellular permeability assessed in Ussing chambers [92].

I-FABP, derived from enterocytes and released in the blood, functions as a biomarker for enterocyte damage [93]. Thus, its circulating levels, present in low amounts upon normal conditions, may be an indirect marker of gut barrier failure [32]. Increased I-FABP levels have been demonstrated in intestinal ischemia, necrotizing enterocolitis, celiac disease, CD, and irritable bowel syndrome [94]. Finally, I-FABP levels correlated to an increased small intestinal permeability as assessed by the lactulose/mannitol ratio in urine [95]. Hence, I-FABP has emerged as a potential biomarker of intestinal barrier dysfunction in routine clinical practice.

Citrulline, a nonprotein amino acid mainly produced by enterocytes, is suggested as a marker of reduced enterocyte mass [96]. Citrulline measurement demonstrates higher specificity and sensitivity compared to the in vivo multi-sugar test in detecting intestinal permeability [97]. However, in autoimmune disorders like rheumatoid arthritis, citrulline exists as citrullinated peptides due to post-translationally modified arginine residues [98]. Thus, interpreting its levels as a marker of intestinal permeability in autoimmune conditions would need caution.

GLP-2, a cleavage product of glucagon released by intestinal enteroendocrine cells, plays a crucial role in maintaining the growth and absorptive function of the intestinal epithelium [99]. Therefore, a reduction in GLP-2 levels may indicate disrupted intestinal barrier function [8]. Additionally, increased populations of Bifidobacterium and Lactobacillus species in the gut microbiota appear to be dependent on GLP-2 in mice [100]. Finally, GLP-2 secretion is stimulated by common food components such as glucose, fatty acids, and dietary fibers [101]. Thus, it is essential to closely monitor food intake and gut microbiota composition, when dealing with GLP-2 as a marker of gut permeability.

Although no single circulating biomarker is specific and sensitive enough to detect a dysfunctional barrier or increased permeability independently, serological biomarkers, alone or in combination, may complement other methods such as the in vivo multisugar test in evaluating intestinal health. Together with circulating biomarkers, fecal and urinary biomarkers have also been evaluated for the assessment of intestinal permeability [9].

Calprotectin is a well-studied marker, found in leukocyte cytoplasm, and released into the gut lumen upon neutrophilic infiltration of the gut mucosa during inflammation [102]. This protein remains stable at room temperature for several days and can be extracted from feces using commercially available kits [78]. Quantification is commonly performed using enzyme-linked immunosorbent assay techniques, which offer good diagnostic precision. However, despite its effectiveness as an inflammation biomarker, calprotectin could not fully represent the gut mucosal function and could miss the identification of gut barrier defects that occur independently of intestinal inflammation [9].

Alpha 1-antitrypsin (AAT) is primarily synthesized in the liver but is also secreted by various cell types including macrophages, enterocytes, and Paneth cells to protect tissues from the proteolytic activity of immune cells [103]. Fecal AAT clearance serves as a marker of clinical disease severity in IBD and gastrointestinal graft-versus-host disease [104, 105]. In conditions with increased intestinal permeability due to mucosal barrier disruption, AAT leaks from serum into the intestine, making it a useful marker of intestinal permeability.

Additional markers of intestinal inflammation that could complement the assessment of in vivo permeability include fecal lipocalin 2 (LCN2) and albumin. LCN2 is produced by intestinal epithelial cells and other cell types in response to pro-inflammatory stimuli such as cytokines or TLR activation [106]. Albumin, when the intestinal barrier is damaged, can escape from blood vessels into the interstitial space, and eventually, into the intestinal lumen [107].

In urine samples, claudin protein levels have been proposed as suitable candidates for assessing intestinal TJ loss. Claudins are key components of TJs, crucial for regulating the paracellular barrier pathway. Studies in various conditions including hemorrhagic shock, major surgery, IBD, and necrotizing enterocolitis have shown the release of the sealing protein claudin-3 in urine [108]. Whether other TJ proteins could serve as urinary biomarkers remains to be investigated. Alternative urinary biomarkers include I-FABP and glutathione s-transferases (GSTs), which correlate with intestinal epithelial cell damage and are easily detectable using enzyme-linked immunosorbent assays [9].

Developed in 2004, confocal laser endomicroscopy (CLE) has recently emerged as a novel method for directly assessing intestinal barrier function [109]. Intravenous fluorescein serves as a contrasting agent, enabling real-time visualization of cellular and subcellular alterations in the epithelial barrier. This method aids in the visualization of defects in vascular permeability, enlarged intervillous spaces, epithelial leaks or gaps, cell shedding, and intraepithelial lymphocytes [110]. CLE is evolving rapidly, with new options being explored, such as using labeled antibodies to characterize lesions on a molecular level or marked drugs to estimate drug affinity to target organs. The classical CLE method with fluorescein was useful and quick in determining cell shedding and loss of intestinal barrier function in humans, particularly in IBD [59]. However, it is essential to note that CLE visualizes the basolateral (subepithelial) to the apical (luminal) flux of a small molecule (fluorescein) while the correlation between this flux and the passage of luminal compounds from the lumen to the subepithelial space remains unclear. Furthermore, conducting CLE necessitates specialized training, and the validation of its relevance and potential applications requires larger scale studies [110].

Endoscopic assessments of intestinal barriers in humans incorporate mucosal impedance measurement [111]. This involves the insertion of a 2 mm diameter catheter through an endoscope, positioning it directly onto the duodenal mucosa under direct visual guidance. Two circumferential sensors placed 2 mm apart are deployed on the mucosa for 0.10 s within the four quadrants of the duodenum. The procedure is conducted with a decompressed lumen, and any fluid present is aspirated to ensure accurate readings [112]. It is worth noting that while significant data exist for impedance measurement in the esophageal mucosa, further advancements are needed for its application in assessing intestinal permeability. Nonetheless, these techniques, although needing further validation, hold promise as an important option for real-time evaluation of intestinal barrier function in a wide range of diseases.

The Ussing chamber technique was first described in 1951 by the Danish physiologists Ussing and Zerhan [113] who used frog skin to reliably measure ion and molecule transport across fresh epithelial tissue specimens. In 1988, the complex original methodology was modified and simplified by Grass and Sweetana [114]. The basic principle involves mounting a flat mucosal sheet between two half-chambers filled with continuously oxygenated buffer. One pair of electrodes supplies current, while another pair monitors electrophysiological parameters. The electrical resistance across the tissue can then be determined, providing insight into the integrity of the tissue concerning paracellular permeability. Experimental probes can be added to the apical side of the chamber and collected from the basolateral side for analysis. The Ussing chamber technique offers advantages such as good viability with oxygenation, effective fluid circulation, and the ability to monitor membrane electrophysiological parameters, making it superior to other in vitro systems for intestinal tissue experiments [115].

The first ex vivo study using Ussing chambers was conducted in 1999. It evaluated the epithelial barrier function in colonic tissue from patients with mild to moderate UC and found a 50% decrease in total electrical wall resistance compared to healthy subjects. This reduction was accompanied by increased paracellular permeability of mannitol and decreased numbers of TJs [116].

Since then, the Ussing chamber has been crucial in supplying mechanistic insight into permeability pathways and has been used in numerous human and animal studies, providing the proof of concept to develop newer in vivo approaches, such as the sugar test [8]. However, while the Ussing chamber is currently the most advanced method for assessing intestinal barrier function, it is important to note that the tissue is removed from its natural context, lacking contact with nerves and luminal contents. Ultimately, the need for fresh intestinal tissue makes this assay quite invasive.

Cell culture-based models employing immortalized intestinal cell lines provide a valuable tool for assessing electrical resistance and intestinal flux parameters in scenarios where biological samples are inaccessible. Numerous cellular models have been employed to evaluate the functionality of the intestinal barrier, including enterocyte cell lines derived from human cancer cells (e.g., Caco-2, HT-29) and from normal human cells (e.g., HIEC-6, H4), colonocyte cell lines from human cancer cells (e.g., T84, DLD1, SK-CO15) and from normal human cells (e.g., NCM460, CCD18-co, FHC), as well as secretory cells (such as Goblet, Paneth, Enteroendocrine, and Tuft cells) [117]. These cell culture techniques have proven particularly beneficial for drug screening and toxicity studies, facilitating the assessment of compound transport across the intestinal barrier [118]. In these models, cells are grown on permeable filter supports to form polarized monolayers that closely mimic in vivo conditions. The culture plates generally provide access to both apical and basolateral compartments [117].

To create more realistic in vitro models simulating the intestinal environment, various co-culture models and even triple-cultures have been developed. These models, representative of the small intestine, aim to establish a more accurate representation of the intestinal milieu. However, it is important to recognize that the correlation between in vitro and in vivo permeability data can vary significantly due to factors such as the molecular characteristics of the cells used, the compounds being studied, the transport route, and potential interaction effects [9].

To address the need for more biologically relevant answers, more complex models have been developed. Three-dimensional (3D) gut organoids, either derived from biopsy or generated from pluripotent stem cells via directed differentiation, have emerged as promising tools [119]. These organoids retain regional identities of the small intestine or colon but consist solely of epithelial cells. Real-time measurement of barrier permeability in intestinal organoids can be obtained by microinjection of fluorescently labeled dextran and imaging using an inverted microscope equipped with epifluorescent filters [120].

Despite their potential, recent studies have highlighted the limitations of intestinal organoids [121]. Organoids established from inflamed tissue lose some inflammatory characteristics after 1 week of culturing and can be distinguished from those established from non-inflamed tissue after 4 weeks [122]. These findings underscore the importance of continued research to refine and improve these models for accurate representation of intestinal barrier function.

Although valid measurements of intestinal permeability are now accessible, and the concept of “leaky gut” is recognized in clinical practice, currently, there are no approved medications demonstrated to specifically target and restore normal barrier function. However, various treatments and interventions have been proposed to ameliorate gut barrier dysfunction, such as dietary interventions, probiotics, and dietary supplements.

Dietary habits can profoundly influence the integrity of the intestinal barrier, both directly and indirectly through alterations in gut microbiota composition [123]. Carbohydrates and lipids, in particular, have been implicated in inducing cellular inflammation through intestinal dysbiosis, ultimately affecting the metabolism of the gastrointestinal tract and the immune homeostasis, thereby damaging the gut barrier [123]. Studies have shown that certain carbohydrates, such as fructose, glucose, and sucrose, along with long-chain fatty acids, can contribute to the dysfunction of TJs, which are critical for maintaining the integrity of the intestinal barrier [124, 125]. Conversely, other carbohydrates, like galacto-oligosaccharides, can promote the growth of beneficial bacteria, while dietary fiber serves as a substrate for fermentation by gut microorganisms, producing SCFAs like butyrate and propionate, which play key roles in protecting the intestine [123, 126].

Ultra-processed foods and food additives, particularly emulsifiers, organic solvents, and nanoparticles have also been linked to increased intestinal permeability by interfering with TJs [127, 128]. A low-FODMAP (fermentable oligosaccharides, disaccharides, monosaccharides, polyols) diet is often recommended for symptom relief in patients with irritable bowel syndrome [129]. This diet restricts certain carbohydrates that are poorly absorbed in the small intestine and can ferment in the colon. Research suggests that a low-FODMAP diet increases fecal bolus volume, improves calcium absorption, enhances SCFAs production, and positively influences gut microbiome composition and function, all of which may contribute to improved gut barrier integrity [130, 131]. In summary, consuming a balanced diet rich in whole foods and low in processed foods, sugar, and artificial additives, along with considering dietary strategies like the low-FODMAP diet when appropriate, can help support intestinal barrier function, reduce inflammation, and improve overall gut health.

Probiotics are “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host” [132]. Probiotics may potentially restore the intestinal barrier and treat intestinal injury-related diseases by enhancing TJs, increasing the expression of mucin, regulating the immune system, and inhibiting the adhesion of pathogenic bacteria [133]. Different probiotic strains have been evaluated for their effects on intestinal permeability, including Lactobacillus rhamnosus GG, Lactobacillus acidophilus, Lactobacillus Plantarum, Bifidobacterium infantis, Bifidobacterium animalis lactis BB-12, and Escherichia coli Nissle 1917 [7].

A recent meta-analysis, including 26 RCTs on a total of 1,891 patients, indicated that probiotics significantly improved gut barrier function (p < 0.00001), serum zonulin (p = 0.0007), endotoxin (p = 0.005), and LPS (p = 0.02). Furthermore, probiotic groups demonstrated better efficacy over control groups in reducing inflammatory factors, including CRP, TNF-a, and IL-6. Finally, probiotics modulated the gut microbiota structure by boosting the enrichment of Bifidobacterium and Lactobacillus [134]. However, the authors acknowledge that the heterogeneity was high, the probiotics information in some studies was unclear, and the assessment of gut permeability involved a wide range of indicators [134].

The mechanisms underlying the restoration of the intestinal barrier by probiotics have not been fully studied, and thus, more in-depth research is needed. Probiotics encompass a wide range of microorganisms, each with its characteristics and potential benefits that may have varying effects on the intestinal barrier. Compound probiotics, which consist of multiple strains or other bioactive components, present additional challenges. Understanding the interactions between these components and their combined effects on the intestinal barrier is complex and requires sophisticated research methods.

Moreover, supplementing probiotic formulations with prebiotics has the potential to enhance the beneficial impacts of probiotics in improving gut barrier function [135]. Despite the potential of probiotics for restoring the intestinal barrier, practical challenges persist related to dosage, delivery methods, and patient variability. Addressing these challenges is warranted before recommending probiotics use for restoring the intestinal barrier in disease states.

The term “prebiotics” was introduced in 1995 to describe “non-digestible food ingredients that beneficially affect the host by selectively stimulating the growth and/or activity of one or a limited number of bacterial species already resident in the colon” [136]. In 2017, the definition of prebiotics was updated to include substrates that are “utilized by host microorganisms conferring a health benefit” [137]. Given the positive effects of probiotics on the intestinal barrier, it is not surprising that prebiotic supplementation also supports intestinal barrier function and repair.

In vitro and in vivo supplementation with inulin, fructooligosaccharides, and galactooligosaccharide significantly upregulates TJ genes such as occludin, claudin-3, and zonulin-1 or triggers the redistribution of proteins like zonulin-1 and occludin to the TJs [138]. A randomized, placebo-controlled trial in children aged 8 to 17 with type 1 diabetes found that those given prebiotic oligofructose-enriched inulin for 12 weeks had decreased intestinal permeability, whereas the placebo group experienced an increase [139]. Despite these promising results, clinical studies evaluating the effect of probiotics on the gut barrier are limited, and the few available studies show conflicting and more often negative outcomes.

It is evident that certain poorly absorbed antibiotics, such as rifaximin and vancomycin, can increase the relative abundance of Lactobacilli, Bifidobacteria, and Fecalibacterium prausnitzii, in addition to exhibiting anti-inflammatory properties [140, 141]. In rat models of visceral hyperalgesia, oral administration of rifaximin modified the bacterial community composition in the ileum (with Lactobacillus species becoming the most prevalent) and prevented mucosal inflammation, damage to the intestinal barrier, and visceral hyperalgesia resulting from chronic stress [142]. Furthermore, the administration of rifaximin to the proteoglycan-induced ankylosing spondylitis mice for 4 consecutive weeks resulted in downregulation of inflammatory factors, prevented ileum histological alterations, restored intestinal barrier function, and inhibited TLR-4/NF-κB signaling pathway activation [143]. Finally, recent experimental studies have indicated that rifaximin can enhance the intestinal barrier in conditions like ethanol-induced liver fibrosis and hepatic sinusoidal obstruction syndrome [144, 145].

In human studies, rifaximin has demonstrated high efficacy in eradicating small intestinal bacterial overgrowth (SIBO), and it has been observed that the increased intestinal permeability in SIBO-positive patients is reversed following rifaximin treatment [146, 147]. Additionally, the RIFSYS trial revealed that rifaximin reduces gut-derived inflammation and mucin degradation in cases of cirrhosis and hepatic encephalopathy [148]. Nevertheless, more comprehensive evidence is required to support the translation of these findings into practical clinical guidelines.

Polyphenols, anthocyanins, and ellagitannins are nonnutritive secondary plant compounds often with health-promoting and disease-preventive properties, mainly found in fruits, vegetables, grains, herbs, spices, and other plant foods [149]. They have been shown to enhance TJ expression in vitro [150]. In rats fed a high-fat diet, polyphenols reduced chronic low-grade inflammatory responses by modulating gut microbiota, and this was also associated with reduced circulating endotoxin [151].

SCFAs, mainly butyrate, serve as an essential energy source for colonocytes and influence both host and microbial activities [152]. Optimal levels of butyrate contribute to gastrointestinal health in animal models by enhancing colonocyte function, reducing inflammation, preserving the gut barrier, and fostering a healthy microbiome [152]. Additionally, butyrate exhibits protective effects in IBD, graft-versus-host disease, and colon cancer [153‒155]. Despite these benefits, clinical attempts to elevate butyrate levels in humans have yielded inconsistent outcomes.

Vitamins A and D play crucial roles in maintaining the integrity of the epithelium and gut microbiome, while also modulating immune responses at various levels. In vitro studies have shown that both vitamins modify the expression of TJ molecules and modulate innate and adaptive immunity [156]. Clinical trials have further substantiated the importance of these vitamins in impacting components of the mucosal barrier. Indeed, they have been found to influence epithelial integrity, immune system function, and the composition of gut microbiota [157].

Glutamine, the most abundant amino acid in both intracellular and extracellular compartments, is currently considered the most important nutrient for healing the leaky gut. A significant body of evidence, indeed, has shown that glutamine preserves the gut barrier function and prevents permeability to toxins and pathogens under various conditions of gastrointestinal mucosal injury [158]. Low serum concentrations of glutamine are associated with disruptions in the intestinal barrier and inflammation, particularly among children. Experimental research has further demonstrated the importance of glutamine supplementation in maintaining the function of the gastrointestinal mucosal barrier and preventing the translocation of bacteria and endotoxins in various stressful conditions such as parenteral nutrition, sepsis, infection, radiation, and other catabolic stress states [159].

Arginine is a semi-essential amino acid and a substrate for different enzymes such as arginases and nitric oxide synthases (NOS), among others [160]. It can affect the maturation and functions of immune cells of lamina propria and modulate serum IgM levels. In contrast to glutamine, only a few studies evaluated arginine supplementation in protecting intestinal epithelial integrity. In preclinical models, dietary treatment with arginine showed improvement in histological abnormalities and TJ protein expression [159]. Moreover, arginine increased the abundance of Bacteroides and reinforced the innate immune system by increasing the production of pro-inflammatory cytokines IL-1β, IFN-γ, and TNF-α, secretory immunoglobulin A, mucins, and Paneth antimicrobials in the small intestine of mice [161].

Overall, numerous in vitro, in vivo, and clinical studies suggest that various dietary supplements play a beneficial role in maintaining mucosal barrier integrity. However, to confidently recommend their use in clinical practice, larger randomized trials providing more robust evidence regarding the efficacy, safety, optimal dosages, and potential interactions with other treatments are necessary.

Our understanding of leaky gut physiology has expanded significantly in recent years, highlighting its intricate interplay with various factors including diet, gut microbiota, and immune responses. Evidence indicates an association between increased gut permeability and many intestinal and extraintestinal diseases, but the fundamental question of whether barrier dysfunction is a primary event in the pathophysiology or a consequence of the disease remains unresolved in many cases.

Assessment of intestinal permeability has evolved from invasive techniques to noninvasive biomarkers, providing valuable insights into gut health. However, challenges remain in standardizing assessment methods and establishing clinical thresholds. A combination of different techniques seems to be the best approach to give the most accurate picture of the intestinal barrier.

Regarding treatment, while certain dietary interventions, such as supplementation with glutamine, show promise in preserving intestinal integrity, further research is needed to elucidate their efficacy and mechanisms of action. Moreover, probiotics offer potential avenues for modulating gut microbiota and reducing gut permeability. Overall, a multifaceted approach incorporating dietary modifications, microbiota-targeted therapies, and personalized interventions holds promise for addressing leaky gut syndrome, but continued research efforts are essential to refine our understanding and therapeutic strategies. Identifying specific patient populations who may benefit most from a specific intervention would lead to personalized and effective management strategies for the conditions associated with mucosal barrier dysfunction.

The authors have no conflict of interests related to this publication.

The authors have no funding sources to declare.

Review conception and design and draft manuscript preparation: D.C.and G.N.; data collection: C.S., A.R., P.C., and C.A. All authors reviewed and approved the final version of the manuscript.