The 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 (Camilleri et al., 2012). 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” (Hill et al., 2014; Araya et al., 2015). Binda et al. (2020) 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 (Vitetta et al., 2018; Rose et al., 2021; Tang et al., 2021). 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.

The intestinal mechanical barrier comprises various intestinal epithelial cells and intercellular junction complexes differentiated from intestinal stem cells (ISCs) localized at the bottom of crypts (Capaldo et al., 2017; Ge et al., 2020). 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 (Ohland and Macnaughton, 2010; Sharma et al., 2010; Ashida et al., 2011; La Fata et al., 2018).

The intestinal chemical barrier comprises mucin (MUC), antimicrobial peptides (AMPs), digestive fluids, lysozymes, mucopolysaccharides, other chemicals, and antimicrobial substances (Ren et al., 2019; Ge et al., 2020). Mucins are divided into transmembrane mucins and gel-forming mucins (Johansson and Hansson, 2016). The predominant membrane mucins in the intestine of humans and mice are MUC13 and MUC17 (Layunta et al., 2021), while MUC2 is the major secretory mucin in the gastrointestinal tract and is a major component of intestinal mucus (Tailford et al., 2015; Johansson and Hansson, 2016). 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 (Schoenborn et al., 2019) and have bactericidal, anti-inflammatory, immunity-improving, and tissue restoration-promoting effects (Bakshani et al., 2018). 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.

The 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 (Kim et al., 2008). Similar results were obtained when treating colorectal adenocarcinoma (Caco-2) cells with L. acidophilus LA1 (Bernet et al., 1994). In an in vitro model, L. casei GG increased the expression of MUC2 gene and protein to inhibit the translocation of specific pathogenic bacteria (Mattar et al., 2002). Caballero-Franco et al. (2007) revealed that the probiotic mixture VSL#3 (Lactobacillus, Bifidobacterium, and Streptococcus) increased the gene expression level of MUC1, MUC2, and MUC3.

Cazorla et al. (2018) found 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. Ogawa et al. (2001) 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.

The 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 (Wells et al., 2017).

The intestinal microbial barrier is a bacterial membrane barrier formed by the commensal gut microbiota tightly adhering to the surface of the intestinal epithelial mucosa (Kayama et al., 2020). 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 (Corr et al., 2009; Hynönen and Palva, 2013; Ge et al., 2020).

L. 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 (Sherman et al., 2005). EcN can secrete a non-bacteriocin component to act on pathogenic microorganisms or host cells to weaken the adhesion of pathogenic microorganisms (Smajs et al., 2012). Bacillus mesentericus, C. butyricum and E. faecalis increase the diversity and abundance of intestinal microbes and maintain the balance of intestinal flora (Chen et al., 2010).

L. 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 (Meng et al., 2019). 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 (Linninge et al., 2019). 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 (Li et al., 2020). However, Li et al. (2019) 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.

Research 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 (Hill et al., 2014; Abid and Koh, 2019; Suez et al., 2019). Common experimental models for probiotic studies and their advantages and limitations are listed in Table 2.

Although 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 (Bron et al., 2017; Rose et al., 2021), 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.

After 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 (Castillo et al., 2011; Ryu et al., 2016; Paveljšek et al., 2021). 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 (Patel and Lin, 2010; Thomas and Versalovic, 2010; Yousefi et al., 2019), and the effect of probiotics may result from the coordination of multiple signaling pathways.

For 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 (Good et al., 2014; Gao et al., 2017; Geng et al., 2021). Yang et al. (2021) 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 (Barroso et al., 2022). Bacillus enhances intestinal immune function and is associated with the TLR2/4/Myd88/NF-κB signaling pathway (Du et al., 2018). However, the specific immune mechanism requires further research.

Peroxisome 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 (Dubuquoy et al., 2006; Marion-Letellier et al., 2009). Eun et al. (2007) 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 (Ewaschuk et al., 2007). However, there is still a paucity of studies on the relationship between probiotics and PPAR-γ.

Probiotics 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 (Paone and Cani, 2020). Reportedly, L. reuteri improves the intestinal barrier by increasing mucus thickness in a mouse model of colitis (Ahl et al., 2016). In addition, probiotics increase the expression and localization of TJ proteins and mucin-related genes.

Host 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 (O'Callaghan and Corr, 2019). 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 (Malago, 2015). Host and microbial interactions in the intestinal lumen maintain the homeostasis of the intestinal microenvironment.

Supplementation 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, Sugahara et al. (2015) 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.

Probiotics 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 (Bermudez-Brito et al., 2012; Stavropoulou and Bezirtzoglou, 2020). Furthermore, the metabolites of probiotics, such as SCFAs, can regulate intestinal pH, increase mucin gene expression (Burger-van Paassen et al., 2009), increase mucus production, and change the nature of mucus to prevent the adhesion of pathogenic bacteria. Fermentation products can also regulate intestinal immunity (Halloran and Underwood, 2019; Ratajczak et al., 2019), 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.

In 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).

Presently, 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.

All 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.

This 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).

The 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.

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