How do intestinal probiotics restore the intestinal barrier?

1The intestinal barrier is an important line of defense to maintain the homeostasis of the intestinal microenvironment and is divided into mechanical, chemical, immune, and microbial barriers, which interact to maintain intestinal homeostasis. Intestinal microbes are an essential part of the intestinal barrier and help maintain normal intestinal barrier function. These microbes are abundant in the host intestine, which they colonize in a symbiotic manner. Some intestinal microbes act as probiotics, that is, they are beneficial to the host’s health. The Food and Agriculture Organization of the United Nations and the WHO define probiotics as “live microorganisms, which when administered in adequate amounts confer a health benefit on the host”. proposed the minimum standards required to correctly use the term “probiotics.” Probiotics must have “strain characteristics, intended use safety, clinical trials, and the ability to survive at effective doses within the product’s shelf life.” They have numerous functions, including participating in food digestion, promoting intestinal motility, synthesizing vitamins, decomposing harmful substances, and enhancing the intestinal barrier. With the development of molecular biology, genetic engineering, fermentation culture, microfluidics, and experimental models in recent years, it has been possible to use single or compound probiotics to restore the intestinal barrier, providing a new clinical treatment option for intestinal barrier damage-related diseases.

2The intestinal mechanical barrier comprises various intestinal epithelial cells and intercellular junction complexes differentiated from intestinal stem cells (ISCs) localized at the bottom of crypts. Probiotics can increase the expression of genes and proteins involved in tight junction (TJ) signaling, regulate the apoptosis of intestinal epithelial cells (IECs), and induce the proliferation of IECs to restore the intestinal mechanical barrier.

3The TJs between IECs can selectively transport substances, prevent pathogenic bacteria and harmful substances from entering the intestinal lumen, and maintain normal intestinal barrier function. The TJ-related complex proteins of IECs are intracellular and membrane proteins. Some of the intracellular proteins are zonula occludens (ZO)1, ZO2, ZO3, and cingulin. Membrane proteins include a variety of transmembrane proteins in TJs, such as the occlusive protein (occludin), synaptic connexin (claudin), and junction adhesion molecules (JAMS;; ). Probiotics affect the intestinal barrier function by regulating the expression of genes and proteins involved in TJ signaling in IECs (Table 1).

4found that when Escherichia coli Nissle1917 (EcN 1917) colonized the intestinal tract of germ-free mice, the gene and protein expression of the TJ molecule ZO1 increased, thereby increasing the TJ structure between IECs and decreasing intestinal permeability, which could improve intestinal epithelial barrier function. ZO1 expression also increased after transplanting EcN 1917 into colitis mice. However, this does not suggest that EcN 1917 has the same effect in the human intestinal environment, and the establishment and maintenance of the intestinal defense system may be a requirement for bacterial colonization and adhesion.

5Lactobacillus reuteri maintains the integrity of the intestinal barrier by increasing the expression of TJ proteins in IECs. revealed that L. reuteri LR1 could mitigate enterotoxigenic E. coli (ETEC)-induced membrane barrier damage by maintaining the correct localization of ZO1 and inhibiting the destruction of ZO1 protein. However, discovered that L. reuteri LR1 may increase the gene and protein expression of the TJ molecules ZO1 and occludin through the myosin light chain kinase signaling pathway, thereby alleviating the damage of intestinal epithelial barrier integrity caused by ETEC K88 infection. found that L. reuteri can also increase the expression of the TJ proteins, occludin and ZO1, strengthen the intestinal barrier, and ameliorate dextran sulfate sodium (DSS)-induced colitis. revealed that L. reuteri could prevent the decrease of TJ proteins expression in IECs caused by ovalbumin sensitization and substantially increase the protein expression of ZO-1; occludin; and claudin1, 3, 5, 7, 8, 9, and 15 in rat IECs, thereby effectively ameliorating the intestinal mucosal barrier function.

6Lactobacillus rhamnosus affects TJ protein expression to restore the intestinal mechanical barrier. found that E. coli O157:H7 caused abnormal distribution of ZO1 and claudin1 in polarized epithelial cells, as well as a decrease in ZO1 expression, resulting in increased permeability and decreased barrier function in an in vitro cell model. They cultured L. rhamnosus GG with polarized epithelial cells to induce the redistribution of ZO1 and claudin1 and increased expression of ZO1, improving barrier function. The postbiotic HM0539 from L. rhamnosus GG can enhance the resistance of mice to E. coli O157:H7 infection by attenuating the destruction of TJ proteins.

7Lactobacillus plantarum MB452 increases the gene and protein expression of ZO1, ZO2, occludin, and cingulin and regulates the expression of TJ protein-degrading genes (such as itchy E3 ubiquitin protein ligase and snail family transcriptional regressor 1), which stabilizes TJs and improves intestinal barrier function. Furthermore, the reduction in proteasome gene expression induced by L. plantarum MB452 may be an additional mechanism to enhance TJ integrity. found that L. plantarum WCFS1 increases the expression of occluding- and ZO1-coding genes. They injected L. plantarum WCFS1 strain into the duodenum of healthy subjects and found that TLR2 pathway activation affected the expression and distribution of TJ proteins. Another study revealed that L. plantarum regulates protein levels and distribution of claudin1, occludin, JAM1, and ZO1 in an in vitro model, protecting Caco-2 cells from enteroinvasive E. coli and improving intestinal barrier function.

8Bifidobacterium infantis and Lactobacillus acidophilus normalize the expression of the TJ proteins, occludin and claudin1, in an in vitro Caco-2 intestinal epithelial cell model, preventing barrier damage due to IL-1 stimulation. Furthermore, In a neonatal mouse necrotizing enterocolitis (NEC) model, B. infantis modulates the proper localization of claudin 4 and occludin in TJs, attenuates intestinal permeability, protects intestinal barrier function, and reduces the incidence of NEC. revealed that Bacillus subtilis CW14 could mitigate the damage of intestinal epithelial cell microvilli and the TJ proteins, ZO1 and claudin1, caused by ochratoxin A, and maintain genome stability. L. acidophilus LA1 considerably enhances the intestinal TJ barrier mediated by a TLR2 heterodimeric complex. LA1 increases the expression of the TJ protein occludin, but also prevents DSS-induced downregulation of occludin expression in mouse intestinal tissues and enhances TJ structure.

9In addition to a single strain that can restore the intestinal barrier and improve the intestinal barrier function, a mixture of probiotics can also restore the intestinal barrier. confirmed that the probiotic mixture (B. infantis, L. acidophilus, Enterococcus, and Bacillus cereus) could enhance the expression of claudin-1 and occludin, by regulating the pregnane X receptor-c-Jun N-terminal kinase signaling pathway, and ameliorate intestinal barrier damage in neonatal necrotizing enterocolitis. Furthermore, co-culture of Saccharomyces boulardii CNCM I-745 with T84 cells in vitro revealed that S. boulardii altered the distribution of ZO1 and maintained the barrier function.

10Intestinal probiotics can regulate the apoptosis of IECs, promote the proliferation of IECs, mitigate intestinal damage, and restore the intestinal mechanical barrier. L. rhamnosus GG promotes the proliferation of IECs by secreting the protein p40. p40 upregulates disintegrin and metalloproteinase domain-containing protein 17 catalytic activity to phosphorylate epidermal growth factor receptor and activate the phosphatidylinositol 3-kinase (PI3K)/protein kinase B signaling pathway, reduces apoptosis, and preserves barrier function. demonstrated that L. reuteri D8 stimulates lamina propria lymphocytes to secrete IL-22 via aryl hydrocarbon receptor, and then activates the phosphorylation of signal transducer and activator of transcription 3 (STAT3) to promote the proliferation of IECs and increase the growth of intestinal organs, thus recovering the intestinal mucosal barrier, which restores the structural damage to the intestinal epithelium caused by TNF treatment. In a mouse model of inflammatory bowel disease (IBD), continuous administration of B. subtilis during the remission period can maintain the integrity of the intestinal barrier by regulating the proliferation of IECs and alleviate IBD. Lactobacillus casei and Clostridium butyricum can also promote the proliferation of IECs and restore the intestinal barrier.

11The intestinal chemical barrier comprises mucin (MUC), antimicrobial peptides (AMPs), digestive fluids, lysozymes, mucopolysaccharides, other chemicals, and antimicrobial substances. Mucins are divided into transmembrane mucins and gel-forming mucins. The predominant membrane mucins in the intestine of humans and mice are MUC13 and MUC17, while MUC2 is the major secretory mucin in the gastrointestinal tract and is a major component of intestinal mucus. MUC2 is the main component of the intestinal chemical barrier, covering IECs to form an intestinal mucus layer, which improves food absorption, provides attachment sites for intestinal symbiotic bacteria, and limits the combination of pathogens and IECs. AMPs are a type of polypeptide secreted by Paneth cells and have bactericidal, anti-inflammatory, immunity-improving, and tissue restoration-promoting effects. Several studies have shown that probiotics can regulate the expression of mucin, affect the formation of the mucus layer, and maintain the intestinal barrier function.

12The increase in MUC2 expression by L. acidophilus A4 and its cell extracts significantly inhibited the attachment of E. coli O157:H7 to HT-29 IECs. Similar results were obtained when treating colorectal adenocarcinoma (Caco-2) cells with L. acidophilus LA1. In an in vitro model, L. casei GG increased the expression of MUC2 gene and protein to inhibit the translocation of specific pathogenic bacteria. revealed that the probiotic mixture VSL#3 (Lactobacillus, Bifidobacterium, and Streptococcus) increased the gene expression level of MUC1, MUC2, and MUC3.

13found that the oral administration of L. casei CRL 43 and L. paracasei collection Nationale de cultures de microorganisms (CNCM) I-1518 to mice could increase the number of Paneth cells in the small intestine, and the AMPs released by Paneth cells could kill microorganisms, reduce the translocation of pathogens in the mucosa, and induce a barrier against intestinal infection. found that lactic acid bacteria and L. casei can also secrete acetic and lactic acids to reduce intestinal pH, inhibit the growth of pathogens, promote the balance of intestinal flora, and maintain the intestinal barrier function.

14The intestinal immune barrier comprises gut-associated lymphoid tissue (GALT) and immune cells in the intestine. In the intestinal epithelial mucosa, propria contains Peyer’s node and immune cells from the innate and adaptive immune system, such as thymus (T) and bone marrow-or bursa-derived (B) cells, dendritic cells, and macrophages, together with the antibacterial peptides secreted by Paneth cells and secretory immunoglobulin A (IgA) from plasma cells. These participate in the immune defense mechanism of the intestinal barrier.

15Probiotics can directly or indirectly regulate immune and anti-inflammatory functions. In vivo experiments in mice demonstrated that Lactobacillus can upregulate the expression of major histocompatibility complex II and the costimulatory cluster of differentiation (CD) 86, CD80, and CD40 and promote the maturation of dendritic cells. B. infantis promotes the maturation of dendritic cells and accumulation of tolerogenic CD103 dendritic cells in GALT, which further regulates T-cell differentiation, induces anti-inflammatory factor expression, and improves intestinal mucosal immune response. EcN 1917 regulates T cell activation by affecting the T cell cycle and apoptosis to maintain intestinal immune homeostasis. found that L. reuteri induced macrophage activation in mice and enhanced macrophage phagocytosis to improve the immune function of the intestinal mucosa. L. plantarum 8,826 from the National Collection of Industrial Food and Marine Bacteria enhances the activity of T cell subsets and natural killer cells and increases the activity of the cytokine IL-10 in vitro. demonstrated that L. acidophilus and B. bifidum increased the phagocytic potential of aged mouse macrophages to improve immunity. A recent probiotic study showed that L. rhamnosus can reduce intestinal ischemia–reperfusion injury in mice by activating IL-10 release from macrophages through the TLR2 receptor signaling pathway.

16Probiotics stimulate plasma cells to secrete IgA, but probiotic-induced IgA secretion may be strain-specific. reported that C. butyricum and Enterococcus faecalis increase the level of IgA in pig serum and improve immunity. Another study on probiotics found that after gavage of mice with E. faecalis CECT7121 increased intestinal mucosal IgA levels and enhanced local mucosal immune responses. showed that different concentrations of B. bifidum significantly stimulated the production of immune IgA in the intestinal mucosa of mice. Long-term consumption of fermented milk containing L. casei DN-114001 increased the amount of IgA in the large intestine and proved to be beneficial to the immune system of the intestinal mucosa. S. boulardii was able to increase intestinal secretory IgA levels and modulate intestinal immunity in mice.

17The metabolites of probiotics, such as short-chain fatty acids (SCFAs), can act on immune cells, such as mononuclear phagocytes and lymphocytes, affect the release of inflammatory factors and immune chemotaxis, and inhibit the proliferation of immune effector cells. Moreover, they can participate in immune regulation in the intestine.

18SCFAs can reduce the activity of the nuclear transcription factor NF-κB by inhibiting histone deacetylase; inhibit neutrophils and macrophages from releasing IL-8, tumor necrosis factor-α, and other inflammatory factors; and prevent chemotaxis of neutrophils to inflammation sites, thereby reducing intestinal inflammation. found that SCFAs activate mitogen kinase signaling through G protein-coupled receptors on intestinal epithelial cells, promote the production of immune factors, improve intestinal immune function in mice, and enhance intestinal barrier function.

19The intestinal microbial barrier is a bacterial membrane barrier formed by the commensal gut microbiota tightly adhering to the surface of the intestinal epithelial mucosa. Intestinal probiotics are an important part of the intestinal microbial barrier, which helps regulate the balance of the number and structure of intestinal microflora. Probiotics can compete with pathogens for nutrients, and through space barriers, they competitively inhibit the attachment sites of targeted cells or the spread of microcolonies to resist the invasion of pathogens.

20L. rhamnosus and L. acidophilus can competitively attach to the adhesion sites of HEp-2 cells and T84 cells, reducing the adhesion sites on the cell surface and preventing the invasion of pathogenic microorganisms, such as EPEC. EcN can secrete a non-bacteriocin component to act on pathogenic microorganisms or host cells to weaken the adhesion of pathogenic microorganisms. Bacillus mesentericus, C. butyricum and E. faecalis increase the diversity and abundance of intestinal microbes and maintain the balance of intestinal flora.

21L. plantarum increased the abundance of Bifidobacterium and Lactobacillus in the cecum of mice treated with cyclophosphamide and decreased the abundance of E. coli and Enterococcus. Additionally, the mixture of Lactobacillus fermentum GOS57 and L. plantarum GOS42 was able to reduce the number of Enterobacteriaceae, increase the abundance of Lactobacillus, adjust the balance of intestinal flora, and enhance the intestinal barrier function. Lactobacillus rhamnosus and L. bulgaricus can increase the abundance of beneficial bacteria in the intestines, inhibit the growth of harmful bacteria, and help restore the imbalance of intestinal flora caused by antibiotics. However, revealed that the intestinal flora of children with repeated respiratory infections is imbalanced, where the number of Bifidobacterium and Lactobacillus significantly decreases, and the number of E. coli increases. The administration of Bifidobacterium quadruple live bacterial tablets can effectively increase the number of Bifidobacterium and Lactobacillus in infected children, thereby maintaining the balance of intestinal flora and reducing the incidence of infection.

22Research on probiotics regarding the restoration of intestinal barrier function should be verified in human trials, which would lead to the development of treatments to improve the intestinal barrier, alleviate symptoms, and allow patients to recover quickly from disease. The safety of probiotics in humans needs to be prioritized, and although experiments in other animals and in vitro tests can further our understanding of the mechanisms of action, the human gut is rich in microbial species, the crosstalk between host and microbes is complex, and the physiology of non-human animals differs from that of humans. Therefore, animal and in vitro experiments are not sufficient for predicting the function of probiotic microorganisms in humans, and inferences from animal models and in vitro experiments cannot be extrapolated to humans. Common experimental models for probiotic studies and their advantages and limitations are listed in Table 2.

23Although these experimental models cannot be extrapolated to humans, they can be used to study the mechanism of action of probiotic strains. The results of such experiments do have therapeutic value for disease models related to intestinal damage. Probiotics from such experiments are safe to use without side effects before clinical trials if they meet the expected therapeutic goals and are administered under clinic conditions. Recent studies have reported probiotics that positively affect intestinal barrier function, but the specific mechanisms still need to be elucidated. In the future, these models can be used to study the specific mechanisms of action of probiotics and the effect of single or mixed strains on certain intestinal immune cells, identify more strains that can improve intestinal barrier function, and reveal the biological properties of the strains.

24After entering the intestinal tract, the effect of probiotics on intestinal barrier function is intricate, and the specific mechanism of action may vary depending on the strain. Furthermore, the beneficial effect may be a result of a combination of actions, which may be related to the enzymes or metabolites produced by specific strains. Previous research on the mechanism of action of probiotics has been relatively superficial, with much of the research limited to in vitro or animal experiments. Regarding intestinal barrier function, increasing evidence indicates that probiotics improve intestinal barrier function through TLR-like receptors (especially TLR2) and that probiotics can enhance host intestinal immunity through TLR-like receptor-related cytokines and signaling pathways. In addition, myeloid differentiation factor (MyD88); nuclear factor-kappa-B (NF-κB); mitogen-activated protein kinase (MAPK); protein kinase C(PKC); protein PI3K; and the STAT, p38, and ERK1/2 signaling pathways may be related to intestinal barrier function, and the effect of probiotics may result from the coordination of multiple signaling pathways.

25For example, L. rhamnosus can stimulate GALT to induce an immune response, produce immune factors, and resist invasion by pathogenic microorganisms. Furthermore, L. rhamnosus and its effective components (surface-layer protein and exopolysaccharides) pass through TOLL-like receptors to mediate the regulation of NF-κB, MAPK, and extracellular signal-regulated kinase (ERK) signaling pathways to regulate intestinal cytokines. found that L. plantarum may inhibit the activation of the p38 and ERK1/2 MAPK signaling pathways mediated by TLR-4 to combat the excessive activation of the innate immune response caused by ETEC K88. Lactobacillus delbrueckii CIDCA 133 improves 5-fluorouracil chemotherapy-induced mucositis by regulating inflammatory pathways through the TLR2/4/Myd88/NF-κB signaling pathway. Bacillus enhances intestinal immune function and is associated with the TLR2/4/Myd88/NF-κB signaling pathway. However, the specific immune mechanism requires further research.

26Peroxisome proliferator activated receptor-gamma (PPAR-γ), a nuclear hormone receptor that can regulate intestinal inflammation, is mainly expressed in the colon and may be another target of probiotics to regulate the intestinal barrier. reported that the inhibition of intestinal inflammatory mediator expression by L. casei may be associated with PPAR-γ activation. Another study found that the protective effect of probiotics on epithelial barrier function is dependent on PPAR-γ activation. However, there is still a paucity of studies on the relationship between probiotics and PPAR-γ.

27Probiotics improve intestinal barrier function by not only inhibiting host intestinal inflammatory response but also altering the thickness, nature, and secretion of intestinal mucus. The composition of intestinal microorganisms affects the nature of intestinal mucus, and a possible mechanism is the expression of glycosyltransferases, which varies according to the type and number of strains. The entry of probiotics into the organism changes the composition of intestinal microorganisms, which can change the nature and increase the secretion of mucus. Reportedly, L. reuteri improves the intestinal barrier by increasing mucus thickness in a mouse model of colitis. In addition, probiotics increase the expression and localization of TJ proteins and mucin-related genes.

28Host intestinal epithelial cells form a structural interface that separates the lamina propria from the intestinal lumen. Therefore, the intestinal microorganisms in the lumen are in close contact with epithelial cells. The intestinal epithelium can distinguish between commensal and pathogenic microbiota through pattern recognition receptors that activate inflammation-related signaling pathways to resist pathogen invasion. The interaction between the microbiota in the intestinal lumen and intestinal epithelial cells and immune cells affects intestinal immunity. Microbiota interferes with bacterial adhesion, colonization, and invasion by affecting the expression of mucin genes in host goblet cells and stimulate the expression of antimicrobial peptides secreted by Paneth cells to affect intestinal immunity. Host and microbial interactions in the intestinal lumen maintain the homeostasis of the intestinal microenvironment.

29Supplementation of probiotics may increase the number of certain microbes in the gut or metabolites of some specific strains that may improve intestinal barrier function by modulating intestinal immunity, TJ fraction, mucins, and changes in the intestinal microenvironment. These effects may be mediated by a crosstalk mechanism between probiotic and commensal bacteria. For example, supplemented human gut microbes in model mice with B. longum and found increased production of fecal pimelate, biotin, and butyrate, which may be caused by crosstalk between B. longum and the intestinal commensal microbiota.

30Probiotics can compete with potentially pathogenic bacteria for nutrients and adhesion sites and inhibit their growth. As microbes establish a balanced microecosystem in the intestine, pathogenic bacteria must compete for binding sites and nutrients to survive. This competition between probiotics and pathogenic bacteria may reduce the possibility of colonization by pathogenic bacteria, reducing the chance of developing infectious diseases in the intestine. Furthermore, the metabolites of probiotics, such as SCFAs, can regulate intestinal pH, increase mucin gene expression, increase mucus production, and change the nature of mucus to prevent the adhesion of pathogenic bacteria. Fermentation products can also regulate intestinal immunity, the specific mechanism of which may result from the combined action of the strains and their metabolites with intestinal immune cells or immune signaling pathways.

31In summary, the intestinal barrier can separate substances from the intestinal cavity, prevent the invasion of pathogens, maintain the stability of the internal environment of the body, and protect the life and health of the human host. Probiotics can restore the intestinal mechanical, chemical, immune, and microbial barriers through various ways and maintain normal intestinal barrier function (Figure 1).

32Presently, a myriad of studies has shown that probiotics can 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. However, the mechanisms underlying the restoration of the intestinal barrier by probiotics have not been fully studied, and thus, more in-depth research is needed. Moreover, owing to the diversity of probiotics, limitations of the technology for extracting single probiotics, and complexity of the mechanism of compound probiotics, applying probiotics to the treatment of intestinal-related diseases by restoring the intestinal barrier faces ongoing challenges. With the development of biomarkers, genetic engineering, fermentation, separation, extraction technologies, and experimental design, researchers can further clarify the mechanism of probiotics at the genetic and molecular levels, make further breakthroughs in the screening, processing, and clinical application of probiotics, and develop more probiotics for the treatment of intestinal injury-related diseases.

33All authors made significant contributions to the conception and design of the study, participated in the drafting of the article and in the critical revision of the intellectual content, and approved the final manuscript. All authors contributed to the article and approved the submitted version.

34This study was supported by grants from the National Natural Science Foundation of China (31960236), Natural Science Foundation of Gansu Province (21JR7RA369), and the Talent Innovation and Entrepreneurship Project of Lanzhou City (2019-RC-34).

35The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

36All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.