Intestinal mucositis is a commonly reported side effect in oncology patients undergoing chemoradiotherapy (Sonis, 2004; Kim et al., 2018) due to non-selective anticancer drugs (Miura et al., 2010; Crombie and Longo, 2016). 5-Fluorouracil (5-FU), an antimetabolite analog to uracil, is one of the most frequently used antitumor drugs that cause mucositis, with an incidence higher than 40% of cancer cases even if treated with the standard dose (10-15 mg/kg for 3-4 days intravenously). 5-FU exerts cytotoxic effects by inhibiting thymidylate synthase (TS) and incorporating its metabolites into DNA/RNA, leading to cell death and apoptosis (Longley et al., 2003; Miura et al., 2010). However, due to its non-selectivity, besides affecting neoplastic cells, this drug also promotes damage to healthy cells (Miura et al., 2010; Crombie and Longo, 2016).

Mucositis caused by 5-FU affects the small bowel mucosa, characterized by intense inflammation, dysbiosis, alteration in intestinal epithelium architecture such as villus atrophy, and loss of epithelial barrier integrity due to tight junction protein disruption and goblet cell degeneration. Additionally, it subsequently increases intestinal permeability and reduces mucin secretion (Sonis, 2004; van Vliet et al., 2010; Song et al., 2013). Except for palliative therapy, there is no known treatment option to prevent chemotherapy-induced intestinal mucositis (Batista et al., 2020).

Probiotic strains, such as Lactobacillus spp., mainly belong to the Lactic Acid Bacteria group (LAB), and they have been investigated as an alternative therapeutic approach against intestinal mucositis (Batista et al., 2020). Several studies have shown the efficacy of various LABs in preventing intestinal mucositis by regulating the microbiota in dysbiosis (Chang et al., 2018), improving the inflammatory process, modulating oxidative stress (Justino et al., 2015; Hu et al., 2020; Quaresma et al., 2020), and protecting the epithelial barrier by maintaining the integrity of tight junction proteins and goblet cells (Yeung et al., 2015; Barroso et al., 2021). Despite these beneficial effects, the use of these inactivated microorganisms has also been highlighted (Jin et al., 2020; Nakai et al., 2021; Trindade et al., 2021), to minimize possible risks of bacteria translocation producing systemic infections, especially in premature infants or immune-compromised patients (Doron and Snydman, 2015; Zawistowska-Rojek and Tyski, 2018). In addition, it has been demonstrated that many of the effects (such as immunomodulation and microbiota regulation) of inactivated probiotics are similar to their metabolically active form, suggesting that these effects can be attributed to cell wall proteins of these microorganisms that interact with host cells during their passage through the gastrointestinal tract (GIT) (Hsieh et al., 2016; Aiba et al., 2017; Jin et al., 2020; Nobre et al., 2022).

Among the Lactobacillus genus, Lactobacillus delbrueckii subsp. lactis CIDCA 133, a strain isolated from raw cow’s milk, has been related to probiotic properties. This includes its ability to inhibit the growth of spoilage bacteria, such as Escherichia coli and Pseudomonas aeruginosa (Kociubinski et al., 1999; Hugo et al., 2008), and to stimulate dendritic cells and murine macrophages infected with pathogenic Bacillus cereus or Citrobacter rodentium (Rolny et al., 2016; Hugo et al., 2017). One of our pioneering studies showed that a fermented milk formulation containing L. delbrueckii CIDCA 133 could attenuate antineoplastic 5-FU-induced intestinal epithelial tissue disruption by inhibiting degeneration of goblet cells, intestinal permeability, and inflammatory cell infiltration (de Jesus et al., 2019). In our other studies, we reported that no adverse effect was observed in healthy BALB/c mice after CIDCA 133 consumption, but its consumption stimulated the mucosal immune system and epithelial barrier by upregulating the gene expression of tight junction protein occludin (Ocln) (de Jesus et al., 2021a), anti-inflammatory cytokines (Il10 and Tgfb1) and mucin 2 (Muc2), and inhibiting the proinflammatory transcription factor Nfkb1 (p105) (de Jesus et al., 2021b). In this context, this study investigated whether the modulation of inflammatory and epithelial barrier biomarkers by Lactobacillus delbrueckii CIDCA 133 would be associated with its beneficial effects against 5-FU chemotherapy-induced intestinal mucositis.

5-Fluorouracil (5-FU) is a commonly used antineoplastic drug that shows intestinal mucositis as one of its main side effects. This gastrointestinal disorder is characterized by inflammation and ulceration of the intestinal epithelium (Sonis, 2004) and is a limiting factor in oncology therapy success.

Probiotic consumption has been considered a promising therapeutic option to ameliorate the inflammatory and epithelial damage induced by 5-FU administration due to their reported anti-inflammatory effects (Batista et al., 2020; Cristofori et al., 2021). The protective effect of L. delbrueckii CIDCA 133 on 5-FU-induced epithelial damage was previously demonstrated using a fermented milk formulation (de Jesus et al., 2019). In this study, we investigated whether the beneficial effect of L. delbrueckii CIDCA 133 on damage caused by 5-FU-induced intestinal mucosal would be associated with the ability of this probiotic strain to regulate the gene expression of inflammatory and epithelial barrier markers.

Due to 5-FU cytotoxicity, severe epithelial damage has been reported as one of the most critical features of mucositis pathobiology. This damage includes epithelial architecture, mucosal barrier integrity alteration, goblet cell depletion, villus shortening, intestinal permeability, and increased polymorphonuclear cell infiltration. All these parameters were evaluated in this study to assess the ameliorative and anti-inflammatory activity of the CIDCA 133 strain. Although CIDCA 133 treatment did not improve the histological score, the strain was able to reduce the tissue damage and preserve villus height induced by 5-FU, corroborating with previous studies that had demonstrated the protective effect of other probiotics in the intestinal architecture of inflamed mice with 5-FU (Yeung et al., 2015; Kato et al., 2017; Oh et al., 2018).

It is essential to highlight that in addition to the damage reported at the ileal level, we also observed that 5-FU affected the whole digestive tract and other organs’ functions. This finding can be demonstrated by shortening the small intestine length after administering the antineoplastic agent. 5-FU promoted intestinal shortening, and mucosal destruction led to reduced nutrient absorption, which might correlate with body weight loss observed during mucositis (Vieira et al., 2012; Maioli et al., 2014; Kato et al., 2017; de Barros et al., 2018).

Our study shows that consumption of CIDCA 133 reduced the shortening of the small intestine but did not improve food and hydric intake, and body weight loss. Similar results were reported by Maioli et al. (2014) and Antunes et al. (2016) in animals with mucositis induced by 5-FU. The researchers observed that their experimental treatment did not influence food intake and weight loss. These results reinforce how intestinal mucositis, as a side effect of 5-FU administration, may become a severe problem in chemotherapy. In addition, unlike what was observed, the beneficial effect of milk fermented by CIDCA 133 on body weight loss and histopathological score in inflamed mice with 5-FU has been previously reported by us (de Jesus et al., 2019), demonstrating that dairy formulation with this probiotic seems to be more promising in the clinical manifestations of 5-FU administration-derived mucositis. One possible explanation for this outcome is that dairy formulations possess nutraceutical compounds (e.g., amino acid, fibers) as well as probiotic-derived bioactive metabolites (e.g., SCFA) (Galdino et al., 2018; Trindade et al., 2018), making them more nutritious and beneficial than MRS culture broth. Furthermore, despite improving the probiotic’s inflammatory parameters, the positive effects on liquid and food intake, and body weight may be more prominent with the intestinal healing progresses, ameliorating the gastrointestinal dysmotility (Soares et al., 2008), increasing the area of nutrient absorption, and consequently improving the mice’s clinical status (de Jesus et al., 2019). It is also interesting to point out that although probiotics demonstrate multiple beneficial effects, a single strain does not seem to be clinically sufficient, so it is proposed that the combination of several probiotic strains may be most effective (Prisciandaro et al., 2011a; Yeung et al., 2015).

The production of several inflammatory markers, such as proinflammatory cytokines (IL-6, TNF-α, IL-1β, IFN-γ, CXCL1), via exacerbated activation of the TLR2/4/Myd88/NF-κB signaling pathway, is another feature caused by the administration of 5-FU (Chang et al., 2012; Li et al., 2017; Justino et al., 2020). Probiotics can modulate the host immune response through their interaction with intestinal epithelial cells via Toll-like receptors (TLRs) and regulate the balance between the T helper type 1 and 2 (Th1 and Th2) responses induced by NF-κB activation (Kawai and Akira, 2007; Mukherjee et al., 2016). Therefore, we also investigated the effects of CIDCA 133 consumption and its impact on the gene expression associated with immune system activation. We observed a decrease in the relative gene expression of Tlr2, Tlr4, Myd88, and Nfkb1 after CIDCA 133 strain consumption by inflamed mice, while the mRNA expression of proinflammatory cytokines (Il6 and Il1b) and regulatory cytokine Il10 were down- and upregulated, respectively. Regarding TNF cytokine, its gene expression was upregulated after 5-FU administration, and transcript levels were maintained after consumption of CIDCA 133. TNF is a key factor in inflammatory diseases. However, it has been demonstrated that epithelial-derived TNF-α can stimulate epithelial cell proliferation, a mechanism used by probiotics to promote intestinal epithelial barrier regeneration and improve the innate immune response (Pagnini et al., 2010; Giorgetti et al., 2015). This property may be a mechanism used by CIDCA 133 in its beneficial effect against 5-FU epithelial damage.

The balance of pro-and anti-inflammatory cytokines is fundamental to control the exacerbation of intestinal damage in 5-FU-induced mucositis. Upregulation of Il10 in mice inflamed with 5-FU and treated with CIDCA 133 evidenced the anti-inflammatory effects that this strain can provide. This finding corroborates de Jesus et al. (2021b), who showed that CIDCA 133 could modulate inflammatory responses possibly by controlling NF-κB signaling pathway activation through upregulation of immunoregulatory molecules such as Il10 and Tgfb1 to maintain intestinal homeostasis.

Our results are supported by other studies in which administration of probiotics, such as Saccharomyces boulardii CNCM I-745 (Justino et al., 2020), Lactobacillus acidophilus (Justino et al., 2015), or Lacticaseibacillus casei (Yeung et al., 2015), also improved the inflammatory and functional aspects of intestinal inflammation induced by 5-FU through the modulation of the expression of Tlr2, Tlr4, Myd88, Nf-κB, and cytokines Il6, Il1b, Il10, and Ifng. It should be noted that not all probiotics showed anti-inflammatory effects in 5-FU-induced epithelial damage (Prisciandaro et al., 2011b; Maioli et al., 2014). Despite sharing similar probiotic features (Plaza-Diaz et al., 2019), the mechanisms used in a 5-FU inflammation context may be different from one strain/specie to another, and this may be related to the probiotic dose used, the antineoplastic agents type and experimental protocols for inducing mucositis, as well as the biotic form as beneficial microorganisms are administrated (viable, inactivated or their secreted products) (Chang et al., 2012; Yeung et al., 2015; Batista et al., 2020; Wu et al., 2021).

The intestinal mucosa destruction and production of proinflammatory cytokines induced 5-FU cytotoxicity, resulting in the recruitment of eosinophils and neutrophils into the lamina propria. This study shows increased activities of MPO and EPO in 5-FU-inflamed mice, which was reduced after administration of CIDCA 133. The beneficial effect of this strain on the recruitment of eosinophils and neutrophils to the intestinal mucosa reduced the oxidative stress and production of proinflammatory cytokines induced by these cells. Consequently, it prevented the exacerbation of tissue damage and the development of ulcers. These findings are consistent with other studies that also observed beneficial effects of different probiotic microorganisms in decreasing inflammatory cell infiltrate induced by 5-FU administration, such as Bifidobacterium sp. (Kato et al., 2017; Quaresma et al., 2020), Lactobacillus acidophilus (Oh et al., 2018) Lacticaseibacillus rhamnosus (Trindade et al., 2021) and Rhodotorula mucilaginosa (Coutinho et al., 2021).

Polymorphonuclear cell infiltration is also associated with an increase in intestinal permeability (IP), probably as a result of reactive oxygen species (ROS) production by these cells, villous atrophy, and reduced expression of tight junction proteins (van Vliet et al., 2010; Li et al., 2017). In this study, IP was studied by measuring blood radioactivity after oral intake of ^99mTc-DTPA, a hydrophilic macromolecule that rarely crosses the intestinal barrier under healthy conditions, making it a good radiotracer marker for measuring IP (Andrade et al., 2015). CIDCA 133 consumption was able to prevent intestinal permeability induced by 5-FU significantly. The permeability was decreased due to upregulation of tight junctions (Cldn1, F11r, and Hp) and Muc2 gene expression, reduction in mucus-producing goblet cell number loss, and villus preservation by the strain. These results agree with other studies that also showed beneficial effects of probiotics in attenuating 5-FU-induced intestinal permeability (Tang et al., 2017; Trindade et al., 2018; Porto et al., 2019) and goblet cell number loss (Yeung et al., 2015; Kato et al., 2017; de Jesus et al., 2019).

Reactive oxygen species production by inflammatory cells promotes intestinal cell apoptosis (Soares et al., 2008). On the basis of this information, we suggest CIDCA 133 can maintain the maturation of mucus-producing goblet cells and tight junctions gene expression by reducing apoptosis of epithelial cells resulting from inflammatory pathways activation, via direct contact of its bacterial cells components (e.g., surface or extracellular proteins, SCFA) with intestinal cells receptors (e.g., TLR2/TLR4; GPR43) (Karczewski et al., 2010; van Vliet et al., 2010; Wang et al., 2018; Liu et al., 2020; Rose et al., 2021). This process would reduce the inflammatory cells infiltrate and their oxidative stress generated on the intestinal mucosa, thus preserving the function and structure of intestinal epithelial components, such as tight-junction proteins, goblet cells, and enterocytes. Furthermore, our results showed that although there was no difference between the groups related to crypt depth, there was an improvement in the crypt-villus ratio after consumption of CIDCA 133, demonstrating that possibly villus height improvement may be associated with the migration of enterocytes, produced by the crypt stem cells, to repair the intestinal epithelium by replacing the dead cells (Yeung et al., 2015). This, therefore, reflects the increase in the number of goblet cells present in the villi, as observed in our study.

Stringer et al. (2009) suggested that rapid mucin secretion in the small intestine due to 5-FU administration occurs primarily in the crypts, mainly caused by enteric neurotransmitters acting on epithelial cells, including goblet cells (Stringer et al., 2009). Furthermore, studies demonstrate that the maturation of goblet cells results from epithelial activation of TLR2/TLR4 (Podolsky et al., 2009; Dheer et al., 2016). On the other hand, TLR activation is also attributed to dysbiotic commensal microbiota (Li et al., 2017). Based on this information, we suggest that CIDCA 133 can upregulated mucin 2 as a mechanism to control the dysbiotic microbiota and maintain intestinal homeostasis. Altogether, these above mechanisms may lead to translocation inhibition of pathogenic microorganisms and toxins into the lamina propria, thereby attenuating an exacerbated inflammatory response (Sonis, 2004; Krishna Rao and Samak, 2013; Maioli et al., 2014), and, thus, reinforcing the importance of this strain in maintaining the epithelial barrier, as previously demonstrated by de Jesus et al. (2019) and de Jesus et al. (2021a).

The ameliorative effect of CIDCA 133 has already been demonstrated by our research group using a dairy formulation, which so far has been shown to be the best matrix for the beneficial effects of CIDCA 133 when compared to some results of our work. However, our findings showed the main molecular pathways used by this probiotic strain to promote its beneficial effects, which have not been reported in previous studies.

Our results reinforce that Lactobacillus delbrueckii CIDCA 133 strain consumption improved mucosal damage in mice undergoing chemotherapy with 5-FU, demonstrating to be a promising adjuvant therapeutic strategy to attenuate 5-FU-induced intestinal mucositis. Furthermore, our work suggests that this strain can re-establish intestinal mucosa homeostasis involving different molecular mechanisms through modulation of NF-κB activation, proinflammatory cytokines, and tight junction proteins expression. Therefore, more studies will be performed to fully explain the mechanisms used for this strain on intestinal inflammation context, reinforcing its use as a probiotic.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The animal study was reviewed and approved by Local Animal Experimental Ethics Committee of the Federal University of Minas Gerais (CEUA-UFMG).

FB, PM-A, and LJ: conceptualization. FB, LJ, VB, MD, KV, NC-R, FM, SF, and VC: methodology. LJ, FB, and EF: formal analysis and investigation. FB, LJ, JL, and TS: writing-original draft preparation. AB, DB, and VA: writing-review and editing. All authors read and approved the final manuscript.

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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