The gastric mucosa is lined by a single layer of epithelial cells that is supported by delicate elements of loose connective tissue underlaid by a thin layer of smooth muscle fibers. In many individuals, the gastric epithelium is exposed not only to its own acidic and enzymatic secretions, but also to duodenal bile, highly prevalent Helicobacter pylori (H. pylori), frequently used non-steroidal anti-inflammatory drugs (NSAIDs) and alcohol intake (1). Therefore, gastric mucosal damage is very common and may evolve into gastric ulcers in many patients. If not treated adequately, a gastric ulcer may lead to serious complications, such as perforation and bleeding, or may progress toward gastric cancer with substantial morbidity and mortality rates (2–4). Inhibition of acid secretion using proton pump inhibitors and eradication of H. pylori by treatment with clarithromycin, amoxicillin and metronidazole, are currently the most widely used therapeutic regimens for gastric ulcer (5). However, with the side effects of these therapeutic agents (6,7), the emerging resistance of H. pylori to antibiotics (8,9), and the high recurrence rate of gastric ulcer (10–12), efforts are being directed toward the identification of new therapeutic modalities.

With the increase of their popularity of use in the prevention and treatment of a number of systemic and gastrointestinal diseases (Fig. 1), probiotics have attracted the attention of numerous cell biologists and clinicians who are interested in exploring their effects on gastric ulcers and H. pylori. Even though the number of clinical studies investigating the impact of probiotics on gastric ulcer is relatively low, a number of experimental studies have generated promising results. The present review aims to summarize the available data concerning the potential role of probiotics in the prevention and healing of gastric ulcer.

Gastric ulcer is one of the most common and serious chronic diseases of the upper gastrointestinal tract. The prevalence of gastric ulcer is 2.4% in the Western population (13) and may be up to 6.1% in Asia (14). Despite advancements in anti-ulcer therapy, the recurrence rate remains high (10–12,15). A gastric ulcer is a localized deep necrotic lesion involving the entire mucosal thickness and the muscularis mucosa (16). It is generally considered that these ulcers develop from an imbalance between mucosal defensive mechanisms and damaging factors at the luminal surface of the stomach (1). In developing countries, the high prevalence of H. pylori, long-term frequent use of NSAIDs, and cigarette smoking represent the major risk factors involved in ulcer development (17,18).

Ulcerogenesis starts by disruption of the protective mucous layer formed by the epithelial cells. Enhanced secretion of acid and pepsin by parietal and zymogenic cells may contribute to damage of the mucous layer (1). Smoking contributes to ulcer formation by upregulating the production of the proton pump and, therefore, acid secretion (19). Damage to the mucous layer may lead to peeling of the surface epithelium and exposure of the endothelial cells of capillaries in the underlying connective tissue. Once capillaries are damaged, oxygen and nutrients will be deficient. As a consequence, hypoxic necrosis will occur in deep glandular cells, namely stem/progenitor cells, mucous neck cells, zymogenic cells, enteroendocrine cells and parietal cells. Moreover, damaged macrophages, mast cells and endothelial cells release vasoactive agents and pro-inflammatory mediators that worsen the mucosal microcirculation. Epithelia and connective tissue necrosis eventually lead to the formation of ulcers (1,20).

Healing of gastric ulcer involves an orchestrated array of different mechanisms that work together to correct the imbalance between damaging and defensive factors in the stomach (Fig. 2). Healing occurs by repairing the mucosal defect with epithelial cells and connective tissue elements, which involves the production of extracellular matrix, cell proliferation, migration, differentiation and gland reconstruction. These events are controlled by many factors, including epidermal growth factor, hepatocyte growth factor, insulin-like growth factor 1, trefoil factors, cyclooxygenase 2-generated prostaglandin, and several cytokines in a spatially and temporally coordinated manner (21). Healing also requires angiogenesis, which is triggered by hypoxia and involves vascular endothelial growth factor, fibroblast growth factor and angiopoietins (22). In addition to local mucosal cells from viable tissue at the ulcer edge, a study demonstrated that bone marrow-derived stem and progenitor cells are attracted to the site of injury and contribute to the regeneration of epithelial and connective tissue components (23). It has been proposed that the proliferation of these stem cells is followed by their commitment to different pathways and differentiation into parietal, surface mucous, mucous neck and zymogenic cells (24). Mucous neck cells are thought to be also involved in the healing of gastric ulcer (25,26). They synthesize and secrete trefoil factor 2, which downregulates acid secretion by parietal cells and, therefore, promotes mucosal healing (26).

Cell therapy may have some potential applications in gastric ulcer treatment. When bone marrow mesenchymal stem cells were injected (locally or intravenously) in rat models of gastric ulcer, they were found to promote ulcer healing (27,28). However, the involved mechanisms are not known and this stem cell injection method remains to be evaluated. Other studies have demonstrated the possibility of gastric tissue engineering with the formation of all cell lineages or only mucous cells using freshly isolated gastric organoids, isolated gastric stem cells or gastric stem cell line (29–31). These promising studies require further evaluation and testing in animal models of gastric ulcers.

Numerous studies have indicated that probiotics can be used for the treatment of gastric ulcers. The idea of using probiotics arose from the study conducted by Elliott et al in 1998 (32). In a rat model of acetic acid-induced gastric ulcer, colonization of gram-negative bacteria occurred rapidly at the site of the ulcer and significantly impaired ulcer healing. However, colonization by gram-positive bacteria promoted ulcer healing. Notably, administration of the exogenous probiotic strain Lactobacillus accelerated ulcer healing (32).

Historically, the concept of probiotics began around 1900 by the Nobel laureate Elie Metchnikoff who discovered that the consumption of live bacteria (Lactobacillus bulgaricus) in yogurt or fermented milk improves the biological features of the gastrointestinal tract (33,34). The Food and Agriculture Organization and the International Scientific Association for Probiotics and Prebiotics define probiotics as live microorganisms which, when administered in adequate amounts, confer a health benefit on the host (35).

The gut microbiota includes ~30 species of Bifidobacterium, 52 species of Lactobacillus, and others, such as Streptococcus and Enterococcus (36). The most extensively studied probiotics for treating and/or preventing gastrointestinal diseases are lactic acid bacteria, namely Lactobacillus and Bifidobacterium species. While these species are non-pathogenic, they can resist the harsh luminal environment of the gastrointestinal tract (37).

Several studies have revealed a number of beneficial effects of certain lactobacilli, such as the suppression of pathogenic bacteria in the gut and inhibition of allergic, inflammatory and neoplastic changes (38–41). Furthermore, it has been shown that lactobacilli are particularly useful in promoting gastric ulcer healing in rats, when administered as an individual probiotic strain, such as Lactobacillus rhamnosus GG (42), Lactobacillus gasseri OLL2716 (43,44), or Lactobacillus acidophilus (45,46) or as a probiotic mixture, VSL#3 (47). Lactobacillus rhamnosus GG increases the cellular proliferation to apoptosis ratio and therefore promotes regeneration of epithelial cells, particularly at the ulcer margins (42,48). In clinical studies, a probiotic mixture was demonstrated to be better than a single strain for improving the characteristics of indigenous microflora (47,49). In addition to bacteria, certain yeasts, such as Saccharomyces boulardii, have been investigated and have shown potential therapeutic effects in a rat model of ibuprofen-induced gastric ulcer (50,51). This yeast has neuraminidase activity, which removes sialic acid residues from the apical membranes of gastric epithelial cells. The loss of sialic acid prevents the adhesin-mediated binding of H. pylori to the epithelial cells (52).

To date, >13,438 research articles on probiotics have appeared in PubMed and ~1,422 articles were published during 2015 alone. Many of these articles report invaluable results demonstrating the effects of probiotics on the gastrointestinal tract using in vitro studies, animal models and healthy/unhealthy volunteers. The main gastrointestinal disorder targeted by probiotic research is irritable bowel syndrome (53–55). However, studies assessing the effects of probiotics on gastric ulcers are relatively limited. This could be due to the adverse physiological conditions of the host, such as an acidic environment, digestive enzymes, bile acids and mechanical stress that attenuate the survival and growth of certain probiotics. To overcome these conditions, a high dose of multiple probiotics has been administered (47,56,57), and probiotics packaged into a suitable delivery system have been developed (45,46).

The beneficial effects of probiotics depend mainly on their ability to survive the acidic conditions and the hydrolytic enzymes and bile content in the stomach and duodenum (37). Several studies have shown that the strength of acidity, length of exposure and strain of probiotic are major factors affecting their survival (58–60). Among probiotic strains, lactic acid bacteria such as Lactobacillus and Bifidobacterium exhibit a great ability to survive gastric transit and, therefore, are extensively used in many pharmaceutical and dairy probiotic products.

Screening of different probiotics has revealed that Lactobacillus acidophilus and Bifidobacterium longum can survive and adhere better to the gastric mucosa than Streptococcus thermophilus and Bifidobacterium infantis/adolescentis/bifidum (61,62). Studies have shown that Lactobacillus acidophilus survive at pH ≥3 after a 3-h incubation (60) and Lactobacillus rhamnosus survive a 4-h incubation at pH 2.5 (63). Also, the viability of several strains of Lactobacillus acidophilus and Bifidobacterium was maintained for ~3 h in the pH range of 1.5–3.0 (60). While the viability of a Bifidobacterium strain remains unchanged at a pH of 3 for 3 h, which even declines slowly to pH of 2 or 1 after 1 h (59), Lactobacillus delbrueckii and Streptococcus thermophilus do not readily survive stomach acidity (64).

The reason underlying the survival of some probiotic strains in the stomach has been attributed to F-type ATPase. This bacterial membrane-bound ATP synthase is responsible for generating a constant gradient between extra- and intracellular pH for protection against acidic conditions (65). So, in an acidic environment, the F₀F₁-ATPase is upregulated and generates a proton motive force via proton expulsion and, therefore, increases the intracellular pH (66). It has been reported that Lactobacillus acidophilus has a high cytoplasmic buffering capacity, which allows changes in cytoplasmic pH and stability in acidic conditions (67). Glucose enhances the survival of lactobacilli in acidic conditions because glycolysis provides ATP to F₀F₁-ATPase, and thereby enables proton exclusion (68,69).

To overcome the inability of some probiotics to survive, microencapsulated or coated probiotic strains have been developed (70–72). Recently, Villena and coworkers designed gastro-resistant tablets containing Lactobacillus fermentum CECT5716 using sodium alginate (73). Calcium alginate beads have also been proposed to protect the delivery of viable probiotic strains in the gastrointestinal tract (74,75) and have even been used to treat cold restraint-induced gastric ulcers (46).

In addition to microencapsulation, coating and food supplements, the use of non-living probiotic strains could also contribute to overcoming the problem of acid-sensitive probiotic strains not surviving in the stomach. Even though some viable probiotic strains do not survive gastric transit, their dead forms remain beneficial (76). Substantial evidence from in vitro and animal studies has shown that both live and dead probiotics can act as biological response modifiers (76–78). Nonviable probiotics are now known as ‘paraprobiotics’ or ‘ghost probiotics’ (79).

Studies have shown that heat-killed Enterococcus faecalis fraction stimulates the gastrointestinal immune system against vancomycin-resistant enterococci (80) while heat-killed bifidobacteria induce significant increases in tumor necrosis factor (TNF)-α and interleukin (IL)-6 production (81). Using fractions of heat-killed Lactobacillus acidophilus and Lactobacillus casei, it is possible to protect immunodeficient mice against Candida albicans (82). Further studies have shown that even non-viable gamma ray-irradiated probiotic mixtures or their DNA can ameliorate the anti-inflammatory response in rats with experimental colitis (83). Also, it has been shown that viable and nonviable probiotic Lactobacillus paracasei IMPC2.1 and Lactobacillus rhamnosus GG can exert the same antiproliferative and proapoptotic effects on cancer cells in vitro (84).

The prophylactic and therapeutic effects of probiotics in some gastrointestinal and non-gastrointestinal diseases are summarized in Fig. 1. In addition to their conventional benefits for gastrointestinal functions, probiotics have shown potential therapeutic effects in some metabolic diseases, such as hyperlipidemia or hypercholesterolemia (85–89), obesity (90,91) and diabetes (92,93). Therefore, the use of probiotics may contribute to a reduced risk of atherosclerosis (94) and hypertension (95,96).

In the last few decades, several studies have suggested a potential role for probiotics in cancer prevention and therapy (97). Data have shown specific alterations of the gut microbial composition (dysbiosis) in patients with colon cancer (98). Induction of colon cancer in rats using 1,2-dimethylhydrazine is associated with significant dysbiosis, which could be inhibited by the oral administration of Lactobacillus salivarius Ren, leading to effective suppression of colon carcinogenesis (99). In mice, treatment with the probiotics Clostridium butyricum and Bacillus subtilis has been found to inhibit the development of 1,2-dimethylhydrazine-induced colorectal cancer (100). As for gastric cancer, little is known about the possible association between probiotics and carcinogenesis. However, some in vitro studies have demonstrated very promising anti-proliferative and pro-apoptotic effects of probiotics on gastric cancer cells (84,101–104). Moreover, clinical studies have provided evidence for the possible effects of probiotics in preventing the toxic effects of chemotherapy and radiation therapy in cancer patients (105,106).

The possible use of probiotics as supplements or even alternatives to oral antibiotic therapy has been suggested, especially with increasing cases of resistance to antibiotics. When frequently and unspecifically used, antibiotics may not only induce resistance, but also harm the gastrointestinal microflora. In these cases, the administration of probiotics may restore the normal microflora, compete with the pathogenic resistant bacteria and, therefore, help patients to recover (107,108).

Novel approaches have been used to design some genetically modified probiotic strains with specific capabilities for the delivery of anti-inflammatory cytokines, vaccines and anti-pathogenic molecules (109–111). Engineered Lactococcus lactis strains were produced as live mucosal vaccines for a large number of antigens derived from bacteria, viruses and parasites (112). In addition, recombinant strains of Lactococcus lactis were used to produce the rotavirus spike-protein subunit VP8 that can prevent rotavirus infection (113). The future use of probiotics as vectors targeted to gastrointestinal mucosal lesions is promising. This new targeted drug delivery approach using probiotics is known as ‘pharmabiotics’ (35).

There are data suggesting that probiotics could be useful for gastrointestinal colic, acute infectious diarrhea, inflammatory bowel syndrome, antibiotic-associated diarrhea, travelers' diarrhea, lactose malabsorption and inflammatory bowel diseases (85,86). However, the data available regarding the possible association between probiotic administration and gastric ulcer healing and prevention are limited.

Over the last two decades, the use of probiotics in the management of gastric ulcer has been investigated in a number of studies. Promising results for studies exploring both prophylactic (Table I) and therapeutic (Table II) effects of probiotics have been obtained. The studies concerning the roles of probiotics in gastric ulcer healing reported in the literature were mainly conducted in rats. These studies were based on the use of either individual probiotic strains, such as Lactobacillus rhamnosus GG (42,48), Lactobacillus gasseri OLL2716 (44), Lactobacillus acidophilus (45,46), Escherichia coli Nissle 1917 (114), Bifidobacterium animalis VKL/VKB (115), Bifidobacterium bifidum/brevis (116) and Saccharomyces boulardii (51), or a mixture of probiotic strains, such as VSL#3 (47). A number of studies have reported that probiotics not only inhibit the development of acute gastric mucosal lesions, but also accelerate the process of healing of induced gastric ulcers (42,44,47). The effects of probiotics on gastric ulcer are attributed to several cellular and molecular mechanisms (Fig. 3).

For a long time, gastric ulcers were considered to be a result of stress, improper diet and NSAID usage. However, the discovery of H. pylori and its association with gastric ulcers has changed the gastroenterological practice worldwide (165). H. pylori can uniquely survive and colonize in the harsh acidic environment of the stomach for decades, leading to progressive inflammatory, ulcerative and neoplastic changes (166). Among patients infected by H. pylori, 10–20% may ultimately develop ulcers (16). Recent regression of ulcer incidence is highly dependent on the worldwide eradication of H. pylori (167).

Eradication of H. pylori and associated gastric mucosal changes remain a challenge for gastroenterologists. This could be due to the fact that H. pylori infection may start early during childhood where developing gastric glands are characterized by prominent dividing stem cells (Karam and Bharwani, unpublished data). No antibiotic is effective enough to eliminate H. pylori when given as a monotherapy. The gold standard triple regimen (clarithromycin and amoxicillin/metronidazole combined with a proton pump inhibitor) represents the worldwide accepted protocol used in the eradication of H. pylori. Studies using this triple therapy have demonstrated an eradication rate of 90% (168). However, none of the studies reported 100% eradication of H. pylori.

In some countries, the marked rise in resistance to clarithromycin has caused a steady decline in the efficiency of the standard triple therapy (169,170). To overcome this problem, new regimens including quadruple, sequential, concomitant, dual and rescue therapies have been introduced (168). However, the development of resistance to antibiotics and their side effects has caused poor patient compliance and, therefore, has limited their applications (171).

During the last decade, numerous studies have explored the possible use of probiotics to improve the protocol of H. pylori eradication and to prevent its side effects (172–176). The use of probiotics has also been tested in asymptomatic H. pylori-infected patients and found to lower the risk of gastric ulcer development (177). Kabir and co-workers were one of the first groups to report that probiotic strain Lactobacillus salivarus can prevent and eliminate H. pylori colonization in the stomach of gnotobiotic BALB/c mice (178).

On the basis of in vitro studies using gastric epithelial cells and different probiotic strains, several effects for probiotics against H. pylori infection have been identified (179). Probiotics can inhibit H. pylori infection by non-immunological and immunological mechanisms (179–181). The non-immunological effects of probiotics include: i) Production of antimicrobials and antioxidants that could inhibit either the growth or urease activity of H. pylori (182,183), ii) competing with H. pylori for binding to the surface of gastric epithelial cells and blocking their specific membrane receptors (125,184–186), and iii) stabilizing the gastric mucosal barrier by stimulating mucus production by surface epithelial cells (187).

The immunological effects of probiotics include: i) Maintaining the balance between pro- and anti-inflammatory cytokines, which leads to recovery from gastritis (188), ii) downregulating the production of IL-8 and TNF-α by producing conjugated linoleic acid that targets the nuclear factor κB pathway (189,190), iii) upregulating the anti-inflammatory suppressor of cytokine signaling through activation of signal transducer and activator of transcription (STAT)-1/STAT-3 transcription factors and inactivation of Janus kinase 2 (191), and iv) enhancing gastric mucosal barrier by stimulating IgA secretion and transport (181).

In animal models of H. pylori infection and also in humans, the use of different probiotic strains has demonstrated different favorable effects, specifically, prophylaxis against H. pylori, inhibition of H. pylori colonization, and alleviation of H. pylori-associated gastric inflammation (173,181,192,193). Therefore, some clinical and laboratory-based studies have demonstrated an improvement in H. pylori eradication by using probiotics (194,195).

The most frequently used probiotic strains for H. pylori infection are Lactobacillus johnsonii La1 (177,196,197). Lactobacilli, the predominant gut bacteria, inhibit adhesion of H. pylori to gastric epithelial cells in vitro. Thus, using lactobacilli exogenously can help in the eradication of H. pylori. Other probiotic strains such as Saccharomyces boulardii, Lactobacillus acidophilus and Bifidobacterium lactis, have been used either alone or combined with antibiotics specific to H. pylori. Meta-analytic studies have recommended the use of either Saccharomyces boulardii or Lactobacillus species supplementation in combination with the standard triple therapy (198,199).

Some in vitro studies demonstrating the inhibition or even killing of H. pylori by probiotics have been followed by preclinical and clinical applications (200,201). These studies indicated only a partial efficacy of probiotics against H. pylori when administered alone. Increase of efficacy and/or reduction of side effects was demonstrated when probiotics were administered in combination with the standard triple treatment of H. pylori (201). However, to date, there is no study that demonstrates complete eradication of H. pylori infection by probiotic treatment.

Gastric ulcers develop due to an imbalance between damaging factors and the defense mechanisms of the gastric mucosa (Fig. 2). The available studies in the literature indicate that probiotics can accelerate the healing of gastric ulcers via multiple mechanisms that involve both damaging and defensive factors (Fig. 2). Even though only limited in vivo studies have explored the impact of probiotics in gastric ulcer, some cellular and molecular findings have suggested their protective and therapeutic effects (Fig. 2). Several studies also identified probiotic strains effective in H. pylori eradication via immunological and non-immunological mechanisms. Therefore, the use of probiotics in the management of gastric ulcer appears promising and further studies are required. Taking in consideration the probiotic strains, dosage, commercial preparations and the heterogeneity of patients, a combined clinical and basic science experimental approach is likely to yield important strategies to optimize the use of probiotics in health and disease (202).