PMMB 2023, 6, 1; a0000339. doi: 10.36877/pmmb.0000339 http://journals.hh-publisher.com/index.php/pmmb 

Review Article 

The Ameliorative Role of Probiotics in 5-fluorouracil 

Induced Intestinal Mucositis 

Alexandra Arcilla Xin Hui Sim1†, Jia Ying Cheam1†, Jodi Woan-Fei Law1,2, Vengadesh 

Letchumanan1,3, Yatinesh Kumari4, Satoshi Ogawa5, Sunny Hei Wong6, Kok-Gan 

Chan1,7, Loh Teng-Hern Tan1,8* 

 

Article History 
1Novel Bacteria and Drug Discovery Research Group (NBDD), Microbiome 

and Bioresource Research Strength (MBRS), Jeffrey Cheah School of 

Medicine and Health Sciences, Monash University Malaysia, 47500, Selangor 

Darul Ehsan, Malaysia. asim0007@student.monash.edu (AAXHS); 

jche0331@student.monash.edu (JYC); jodi.law1@monash.edu (JW-FL) 

vengadesh.letchumanan1@monash.edu (VL); kokgan@um.edu.my (K-GC) 

2Next-Generation Precision Medicine and Therapeutics Research Group 

(NMeT), Jeffrey Cheah School of Medicine and Health Sciences, Monash 

University Malaysia, Bandar Sunway 47500, Selangor Darul Ehsan, Malaysia 

3Pathogen Resistome Virulome and Diagnostic Research Group (PathRiD), 

Jeffrey Cheah School of Medicine and Health Sciences, Monash University 

Malaysia, Bandar Sunway 47500, Selangor Darul Ehsan, Malaysia 

4Neurological Disorder and Aging Research Group (NDA), Microbiome and 

Bioresource Research Strength (MBRS), Jeffrey Cheah School of Medicine 

and Health Sciences, Monash University Malaysia, 47500, Selangor, Malaysia. 

yatinesh.kumari@monash.edu (YK) 

5Jeffrey Cheah School of Medicine and Health Sciences, Monash University 

Malaysia, 47500, Selangor Darul Ehsan, Malaysia. 

satoshi.ogawa@monash.edu (SO) 

6Lee Kong Chian School of Medicine, Nanyang Technological University, 

Singapore 308232, Singapore; sunny.wong@ntu.edu.sg (SHW) 

7Institute of Biological Sciences, Faculty of Science, University of Malaya, 

Kuala Lumpur 50603, Malaysia  

8Innovative Bioprospection Development Research Group (InBioD), Clinical 

School Johor Bahru, Jeffrey Cheah School of Medicine and Health Sciences, 

Monash University Malaysia, Johor Bahru 80100, Malaysia 

† These authors contributed equally to this work. 

*Corresponding author: Loh Teng Hern Tan, Innovative Bioprospection 

Development Research Group (InBioD), Clinical School Johor Bahru, Jeffrey 

Cheah School of Medicine and Health Sciences, Monash University Malaysia, 

Johor Bahru 80100, Malaysia; loh.teng.hern@monash.edu 

Received: 11 June 2023; 

Received in Revised Form: 

11 July 2023; 

Accepted: 14 July 2022;  

Available Online: 16 July 

2023 

Abstract: Colorectal cancer (CRC) remains one of the top cancers in the world. Although 

early detection improves the survival rate to around 90%, late detection would mean the need 

to use chemotherapy or radiotherapy, especially if surgery is not feasible. 5-fluorouracil (5-

FU) is one of the common anti-cancer drugs used in treating CRC. It is the drug that has the 



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greatest efficacy on CRC. Although it improves the survival rate, it has many detrimental 

side effects. One of these side effects is intestinal mucositis. It is described as having reduced 

villus height, villus atrophy, crypt fissure, abdominal discomfort, diarrhoea, and weight loss. 

Clinically, there is no conclusive treatment therapy for mucositis. This is possibly due to the 

complex mechanism of the pathobiology of intestinal mucositis that includes the production 

of pro-inflammatory cytokines and increased epithelial cell apoptosis. 5-FU itself is known 

to cause gut dysbiosis. Current studies revealed probiotics play a role in attenuating this 

inflammatory process of intestinal mucositis by reversing gut dysbiosis, reducing the 

expression of pro-inflammatory cytokines, and reducing intestinal damage. This review 

outlines the latest evidence supporting probiotic use in ameliorating 5-FU induced intestinal 

mucositis, thereby promoting good health and well-being in colorectal cancer patients 

receiving 5-FU chemotherapy. 

Keywords: Colorectal cancer; 5-fluorouracil; Intestinal mucositis; Gut dysbiosis; Probiotic; 

SDG 3 Good health and well-being 

 

1. Introduction 

Colorectal cancer (CRC) is the second leading cause of death due to cancer, with 

935,000 deaths worldwide[1–3]. The survival rate of CRC depends on the stages of cancer and 

when it was diagnosed[4]. Generally, 63% of CRC patients will live at least five years post-

diagnosis. Early stages can have survival rates up to 90%, while for later stages, especially 

those diagnosed with metastatic CRC, the survival rates are only around or less than 20%[5,6]. 

When treating CRC, the goal is to achieve as much total removal of tumours as possible while 

preserving patients' quality of life and survival. Typically, like most cancers, conventional 

surgery or chemotherapy is the most common treatment option depending on the size, 

location, and extent of metastasis. Typically, most early stages CRC patients would undergo 

tumour resection, while those who cannot undergo surgery would undergo chemotherapy to 

hinder or kill cancer cells. 5-Fluorouracil (5-FU) is the most common CRC chemotherapy 

treatment. Currently, 5-FU is either administered as a single drug or in combination with 

other drugs[7]. Combination regimens available now are the FOLFOX regimen (5-FU and 

oxaliplatin), FOLFIRI regimen (5-FU, leucovorin, and irinotecan), FOLFOXIRI (5-Fu, 

oxaliplatin, and irinotecan) and others[7]. 

Chemotherapy prolongs survival, reduces cancer-related symptoms, and preserves 

general well-being in advanced CRC patients[8]. Survival rates increase by almost 20% in 

high-risk CRC patients that undergo chemotherapy[9,10]. Being the backbone of CRC 

chemotherapy, 5-FU improves overall survival and disease-free interval in stage 3 CRC[10]. 

5-FU does this by inhibiting the normal function of DNA and RNA. 5-FU interferes with 

thymidylate synthesis and inhibits DNA synthesis during DNA replication and repair[11]. 

Eventually, 5-FU arrests the cell cycle and induces cell death[12]. Although 5-FU is a key drug 

to treat CRC, it comes with a cost. Since chemotherapy targets tissues with a high cell 

division rate, other normally dividing cells with the same rate of division are also affected[13]. 



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Repeated treatment with 5-FU, as frequently happens with chemotherapy, 5-FU causes 

changes to body weight, bowel movement and gut architecture. Weight loss starts around day 

five post-5-FU, along with diarrhoea two days before. There is around a 10% risk of 

developing grade three or four diarrhoea (with severe diarrhoea until requiring hospital 

admission or severe diarrhoea with life-threatening results) in patients that undergo the 

FOLFOX regimen, and it is doubled in patients receiving FOLFOXIRI[14].  

Zooming into the gut's architecture, 5-FU causes villi shortening and reduced number 

of crypts and crypts cells. Inflammation occurs, with neutrophil infiltration (measured by 

myeloperoxidase activity), an increase in pro-inflammatory cytokines (PIC) and apoptosis. 

All these events can be categorised as intestinal mucositis (IM). A common finding is the 

increased crypt apoptosis and villus atrophy leading to poor nutrition, malnutrition and 

bacteraemia[15,16]. Having IM is correlated with the application of total parenteral nutrition, 

analgesics and antibiotics, which are used to decrease the side effects of the inflammation 

and inflammation. However, in the long run, these are coupled with side effects that impact 

patients' quality of life, leading to a longer hospital stay, hospital fees and increases in the 

risk of nosocomial infections[17]. Apart from that, IM often results in dose reduction of 

chemotherapeutic agents or deferral of treatment, which further increases the mortality 

rate[18]. All these affect patients' ability to tolerate chemotherapy, affecting their chances of 

survival[17].  

Recognising these challenges, researchers are actively exploring natural resources to 

offer effective and less toxic alternatives to traditional chemotherapy[19–25], such as 5-FU in 

this case. The exploration of natural products, particularly those derived from microbes[26–

35], animals[36] and plants[37–40], holds promise in improving CRC treatment. While these 

natural products offer the potential for reduced toxicity and enhanced therapeutic effects, 5-

Fu remains the mainstay of chemotherapy used when CRC is at the advanced stage or has a 

higher risk of recurrence[41]. Among the many therapeutic alternatives, probiotics emerge as 

the most promising adjuvant in complementing and enhancing the effects of chemotherapy 

drugs. Probiotics offer several mechanisms through the modulation of the gut microbiome, 

which plays a crucial role in overall health and immune function[42]. By promoting a healthy 

gut microbiome, probiotics may improve the body's response to chemotherapy and enhance 

treatment efficacy[43]. Additionally, probiotics can support gastrointestinal health, alleviate 

chemotherapy-induced side effects such as diarrhoea, and enhance nutrient absorption[44–46]. 

By leveraging the symbiotic relationship between the gut microbiome and the body's 

response to cancer treatment, these studies aim to enhance the effectiveness of chemotherapy 

while reducing its associated toxicity, ultimately improving patient outcomes and quality of 

life[44,47]. 

Gut microbiota influences the responses of a host to chemotherapeutic agents[48,49]. 

For instance, certain groups of gut microorganisms can enhance drug efficacy, while some 

promote chemoresistance and mediate chemotherapy-induced side effects[13]. One of the 

explanations is that chemotherapy causes ROS-mediated DNA and non-DNA damage, which 

then leads to bacterial translocation through the gut epithelium, hence causing an 



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inflammatory reaction that can lead to systemic infections and other complications[13]. 

Unsurprisingly, reports have shown the involvement of gut microbiota in the 

pathophysiology of IM. Specifically, the crucial role of GM in 5-Fu induced IM has recently 

been highlighted with the recent study on probiotics, showing that probiotics lessen side 

effects such as diarrhoea and weight loss, possibly because of restoration of altered gut 

microbiome composition caused by 5-FU[15]. Therefore, this review discusses the 

pathophysiology of IM induced by 5-FU and how GM can mediate the inflammatory process. 

Although most of the studies are performed in animal models, overlapping features are shown 

in the studies and current understanding of the human body. Key findings in these animal 

studies would serve as a good fundamental for future human studies. In short, GM can present 

as a target therapeutic approach in ameliorating chemotherapy-related side effects, especially 

in patients undergoing the 5-FU regimen. 

2. The Role of Gut Microbiome and Their Involvement in The Pathophysiology of 5-

FU Induced Intestinal Mucositis 

The microbiome is a huge and intricate microbial community comprising bacteria, 

viruses, archaea, fungi and parasites that inhabit the human body[50]. Among them, 

Actinobacteria, Firmicutes and Bacteroidetes are the most prevalent phyla of bacteria. There 

are more than 100 trillion microorganisms in the gastrointestinal tract, which serves as the 

site of primary communication between the microbiota, the host cells and the host immune 

system[51,52]. Thus, the gut microbiome (GM) is associated with many functions in the human 

immune, metabolic, and structural functions. However, the occurrence of gut dysbiosis, 

specifically the disruption of the balance of beneficial bacteria in the gut microbiome, has 

been consistently linked to a range of human diseases[53–62]. Recent evidence suggests that 

certain pathogenic gut microbiota play a role in carcinogenesis[63,64]. For example, 

sulfidogenic bacteria produce hydrogen sulphide which is capable of damaging DNA. It also 

inhibits DNA repair mechanisms, leading to extensive DNA damage[65]. These sulfidogenic 

bacteria include Fusobacterium, Desulfovibrio and Bilophila wadsworthia. Besides that, 

Gram-positive organisms such as Streptococcus bovis or currently named S. gallolyticus are 

positively correlated with the development of adenomas and carcinomas. Various studies 

show an estimated likelihood of a 6–71% chance of developing CRC in patients that had S. 

gallolyticus bacteremia. Infection with S. gallolyticus can occur via infectious endocarditis 

or the presence of S. gallolyticus in the blood[66,67]. 

IM is a complex process that involves many different cells, tissues and signalling 

molecules. The pathophysiology of IM is yet fully unravelled. Sonis[68] proposed a 5- step 

theory consisting of the i) initiation phase, ii) upregulation and message generating phase, 

iii) amplification and signalling phase, iv) ulcerative phase, and v) the healing phase. It is 

worth mentioning that the activators for every phase would be a good target for intervention. 

5-FU induces dysbiosis in the gut, where it disturbs the delicate balance of homeostasis. Gut 

dysbiosis is worsened when given antibiotics. Antibiotics kill beneficial bacteria, such as 

those that secrete metabolites that protect the mucosa and those that influence the metabolism 

of 5-FU but spare pathogenic bacteria that contribute to decreasing the anti-tumour efficacy 



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of 5-FU[69]. With this being said, it could be thought that GM is important to humans and 

may play a role in preventing or treating IM. We will discuss Sonis[68] theory (summarised 

in Figure 1) with a focus on how the gut microbiome plays a role in this inflammatory 

process. GM also influences intestinal permeability (IP) and mucus layer, which are the gut's 

defence barrier. 

2.1. Initiation Phase 

Based on clinical evidence, the onset of mucositis begins within 24 hours to 48 hours 

upon the initiation of the cancer treatment[18]. The first stage, which occurs seconds after 

exposure to chemotherapy, is marked by the production of reactive oxygen species (ROS), 

direct DNA and non-DNA damage, and immune system activation[13]. It turns out that the 

human gut is constantly in a steady state of low-grade inflammation. This condition is due to 

the Toll-like receptors (TLR) on epithelial cells and intestinal-associated immune cells. The 

conserved molecular motifs on bacteria are recognised by the TLRs outside the cell, 

activating several intracellular signalling cascades, such as the NF-κB signalling pathway[70]. 

TLR also maintains homeostasis by preserving the mucosal layer and preventing pathogenic 

bacteria from disrupting this homeostasis[70]. 

NF-κB is a transcription factor that upregulates PIC, chemokines and adhesion 

molecules such as TNF-α, IL-1β and IL-6[68,70]. NF-κB is activated by chemotherapy or 

radiotherapy and leads to the generation of reactive oxygen species (ROS). ROS are capable 

of causing tissue damage in the gut epithelium, connective tissue and even blood vessels, 

eventually resulting in DNA damage and cell death[68]. TLRs trigger a signalling cascade. 

The importance of TLR can be seen in TLR4 knocked-out mice exhibiting altered neutrophil 

recruitment and epithelial proliferation. It is thought that TLR4 is able to limit bacterial 

translocation and modulate intestinal response to injury[71]. This shows that intact TLR 

signalling might be crucial for the anti-inflammatory response. Whether TLR agonism 

confers direct protection to either the gut itself or the intestinal microbiome is still unknown. 

It serves as a potential target site for treating IM as TLRs are frequently exposed to bacteria 

parts, such as bacterial cell wall. 

Due to the constant exposure between TLRs and bacteria, there is always a basal 

activation of pro-inflammatory transcription factor NF-κB. However, commensals do not 

trigger an inflammatory cascade[72]. It is postulated that the epithelium sequesters the 

commensals, preventing TLR activation, while pathogenic bacteria, equipped with virulence 

factors, are able to bypass the epithelial barrier to be detected by TLR ligands on immune 

cells[72]. It is also shown that commensals can suppress NF-κB, such as in the case of 

Bacteroides thetaiotaomicron that targets active NF-κB subunit RelA complex and decrease 

its activation. B. thetaiotaomicron also removes NF-κB complexes formed when epithelial 

cells were induced with virulent Salmonella strains resulting in decreased PIC production[73]. 

The full mechanism of how TLRs are able to differentiate pathogenic and commensal 

bacteria serves as a gap in present evidence, prompting further research.  



PMMB 2023, 6, 1; a0000339 6 of 34 

 

 

Figure 1. Pathogenic bacteria bind to Toll-like receptors (TLRs). TLRs activate IKK kinase. IKK 

phosphorylates NF-κB associated proteins, p50 and RelA. The phosphorylated complex is targeted for 

polyubiquitination, allowing degradation by proteasome, leaving only p50 and RelA. These free proteins 

translocate into DNA and activate pro-inflammatory gene expression. Commensal bacteria such as nonvirulent 

Salmonella can prevent IKK complex from being ubiquitinated, preventing degradation and incorporation into 

DNA. Other commensals act at the nuclear level, acting on RelA protein via transcription factor PPAR-γ. These 

two bind together and dampen NF-κB pro-inflammatory cascade. 

Besides B. thetaiotaomicron, NF-κB signalling can be suppressed directly by 

influencing other signalling pathways, as shown by Bifidobacterium. IL-10 is an anti-

inflammatory, having NF-κB inhibitory properties. IL-10 can also block the transcription of 

IL-1β, IL-6, IL-8, and tumour necrosis factor (TNF-α)[74]. IL-10 maintains gut immune 

homeostasis by decreasing PIC concentrations at the tissue damage site[75]. In IL-10 deficient 

rats, Bifidobacterium infantis managed to prevent proinflammatory-associated 

rearrangement of tight junction (TJ) proteins and instead increase TER. The study also 

showed that long-term exposure to B. infantis reduces inflammation, maintains colon 

permeability to normal levels and reduces IFN-γ secretion[76]. IFN-γ is an important recruiter 

for macrophages, leading to immune cell infiltration[77]. Moreover, Faecalibacterium 

prausnitzii secretes an unknown molecule that promotes IL-10 production[78]. Short-chain 

fatty acids (SCFA) such as butyrates by F. prausnitzii can also inhibit NF-κB and IL-2[79,80]. 

Butyrates are the main products excreted by microbes during the fermentation of dietary fibre 

(also known as prebiotics). It is found that butyrate suppresses inflammation by reducing 

TNF-α, IFN-γ, and IL-12 and prevents carcinogenesis by strengthening the mucosal barrier 

and modulating oxidative stress[81,82]. Probiotic supernatants have immune-modulating 

properties as well. Supernatant of Bacillus coagulans directly increases IL-10 production, 

which leads to reduced ROS production[11]. Supernatant of Escherichia coli Nissle 1917 

(EcN) was anti-inflammatory in rats with colitis, reducing TNF-α and increasing IL-10[83].  

In short, probiotic is capable of downregulating tissue-damaging ROS by targeting upstream 



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factors, particularly NF-κB. The exact target to pinpoint in this pathway could be key in 

reducing IM. 

2.2. Upregulation and Message-Generating Phase 

After the initiation phase, begins the upregulation and message-generating phase. 

DNA damage and ROS production activate NF-κB similar to the initiation phase. TNF-α is 

also activated, leading to epithelial cell apoptosis. NF-κB can transcribe up to 200 genes, 

most of which are toxic to the gut mucosa. For example, pro-apoptotic proteins are produced 

by the upregulation of Bcl2 genes and BAX genes. The overall upregulation of genes such 

as Bcl2 and BAX genes hastens mucosal damage. NF-κB also can increase PIC production 

in the mucosa. These cytokines damage the connective tissues and endothelium by initiating 

mesenchymal epithelial signaling and reducing oxygen concentrations in the epithelium 

resulting in epithelial cell death and injury[68]. This process causes thinning of the mucosa, 

making it erythematous. This condition can be ameliorated by supplementing with lactic acid 

bacteria (LAB) such as Bifidobacterium breve. LAB protects against inflammation by 

releasing antimicrobial peptides, IgA. Its presence itself competes with pathogens to adhere 

to the gut epithelium. LAB have a stronger adherence to epithelial cells by increasing the 

expression of binding proteins. Besides that, LABs are able to secrete antioxidant enzymes 

as well[84]. Another study showed Streptococcus thermophilus metabolites can cross the 

mucosa and suppress TNF-α[85]. 

2.3. Amplification and Signalling Phase 

The third phase is the amplification and signalling phase. There is positive feedback 

by the mediators released in previous phases that magnify other biological factors. 

Chemotherapy such as 5-Fu can activate NF-κB (a transcription factor), such as found in the 

gut's epithelial, endothelial, and mesenchymal cells. NF-κB in macrophages can be activated 

as well. Activation of NF-κB leads to the production of PIC such as TNF-α and interleukin-

1β (IL-1β). They participate in positive feedback, further activating NF-κB. NF-κB 

upregulates transcription of mitogen-activated protein kinase (MAPK), cyclooxygenase 2 

(COX2) and tyrosine-kinase signalling molecules.  

These biological factors gather in the mucosa and target the tissue. For example, 

increased TNF-α expression has positive feedback on NF-κB activity. TNF-α can activate 

caspase 3 (pro-apoptotic protein), which causes cell apoptosis. Upregulation of MAPK also 

can lead to activation of pro-apoptotic protein caspase 3. Additionally, upregulation of 

MAPK, COX2 and tyrosine-kinase leads to the activation of matrix metalloproteinases 

(MMPs) 1 and 3 present in epithelial cells and the lamina propria. MMPs ultimately cause 

damage to the gut mucosa[86]. Clinically, the gut mucosa may seem erythematous, and 

patients can manifest very few signs and symptoms while still in the early phases of 

inflammation[68]. 

 



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2.4. Ulcerative Phase 

The gut barrier consists of the mucus layer, TJ and epithelial cells. Disruption of any 

of these can impair the barrier. The mucus layer, secreted by goblet cells, provides a physical 

barrier around the mucosa lining. TJ acts as a seal between epithelial cells comprising a bunch 

of proteins such as occludin and junctional adhesion molecules. TJ interspersed between 

epithelial cells is a physical barrier to prevent unwanted substances such as pathogenic 

bacteria, toxins, or byproducts from penetrating the mucosa. Zonula occludins (ZO-1) tighten 

the TJ seal at the cell junction[87]. 5-FU decreases both TJ and ZO-1 expression[88]. Upstream 

inflammatory factors like TNF-α (through NF-κB activation) and IL-1β  exacerbate this 

cycle by down-regulating ZO-1 proteins and altering the localisation of ZO-1 proteins[89,90]. 

Instead of the normal cohesive binding of ZO-1, it becomes gaplike and zig-zag in 

appearance. Disruption in both TJ and ZO-1 leads to a penetrable mucosal layer, leading to 

a more permeable intestine[89].  

Besides the connection between epithelial cells, epithelial cells themselves are 

damaged. Cell apoptosis and villous atrophy occur, forming crypts. Crypts serve as a harbour 

for pathogenic bacteria to grow on. Crypts also leave nerve endings in the lamina propria 

exposed, making this phase painful for patients. Besides losing the mucus layer, the epithelial 

barrier itself is damaged. The gut mucosa is very permeable in this phase, allowing bacteria 

to translocate across this barrier. The 'leaky' gut mucosa puts patients at risk of developing 

bacteraemia and sepsis. Direct exposure of the gut to pathogenic bacteria causes the human 

body to recognise foreign bacteria and start another cascade of inflammatory processes. 

Mononuclear infiltrates by chemotaxis lead to more inflammation, possibly promoting pro-

apoptotic gene expression and PIC, further potentiating further tissue injury (Figure 2)[68]. 

 

Figure 2. The gut mucosa is protected by one layer of epithelial cells with tight junction interspersed in between 

cells. This barrier is also reinforced with a mucus layer. Bacteria bind to Toll-like receptors (TLRs). The binding 

activates NF-κB. NF-κB causes an upregulation of PIC. Bacteria are also phagocytosed by dendritic cells. 

Dendritic cells present bacteria parts to immunoglobins, subsequently setting off an inflammatory response as 

well. 



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Regulating TJ and ZO-1 expression is vital to the progression of intestinal 

permeability. With the administration of Bifidobacteria, there was increased expression TJ 

forming proteins and decreased intestinal permeability[76]. Lactobacilli also has been shown 

to elevate TJ protein expression and restore intestinal permeability[91–94]. Probiotic strains 

such as E. coli Nissle 1917 and L. rhamnosus GG upregulates ZO-1, protecting the gut from 

being more permeable[95,96]. Upregulation of ZO-1 is important as it protects the epithelial 

cells from damage such as those caused by pathogenic bacteria like Escherichia 

coli O157:H7[96]. SCFA produced by bacteria is able to reduce IP and increase cell 

viability[81].  It also helps modulate the intracellular environment, maintaining the intestinal 

structure, function and integrity and promoting gene expression and proliferation of the 

epithelial cells[97]. Butyrate, propionate and acetate are usually produced due to the 

fermentation of undigested carbohydrates or prebiotics by B. breve and Bacteroides 

ovatus[98]. 

2.5. Healing Phase 

The last phase is the healing phase. Gut mucosa self-heals itself once the causative 

factor is removed. In this phase, the extracellular matrix (ECM) releases molecules which 

promote the healing of the ulcer. First, the molecules migrate from ECM to the ulcer border. 

Then, migration of the cells occurs to cover the ulcer. Then these cells begin to differentiate 

into epithelial cells. Normal architecture is then restored. Other molecules that promote 

hyperplasia are downregulated. Once the ulcer has healed, symptoms of IM begin to 

resolve[68]. 

It is known that gut dysbiosis with increased numbers of pathogenic bacteria on an 

exposed intestinal surface can activate more TLRs. Meanwhile, chemotherapy activates 

TLRs via NF-κB[70]. It turns out the steady-state inflammation by TLRs and commensal 

bacteria is proven to allow the repair of the epithelium. Commensal bacteria help to induce 

the recovery of the gut mucosa. For instance, in a study on chicks, migration of the cells is 

faster by more than half the time of those in germ-free chicks[99,100]. This allows faster healing 

of the gut epithelium. In this case, TLR is also significant because it can sense injured or dead 

cells as TLR recognises and binds to heat shock protein (HSP) produced by dying epithelial 

cells[70,71]. Cytoprotective effects of HSP are suppressed in TLR knockout mice. For example, 

a finding indicates that mice deficient in TLR (specifically TLR4) could not repair damaged 

cells. They experienced more severe damage in the epithelium, with decreased epithelial cell 

proliferation. TLR4 knockout mice show impaired TLR4 and HSP signalling, delaying 

response to dead or dying epithelial cells and leading to delayed epithelial healing[71]. As seen 

before, butyrate is a key player. Butyrate promotes the migration of epithelial cells, thus 

enhancing mucosal healing[81,101]. Other bacterial products, such as the peptides (p75 and 

p40) secreted by L. rhamnosus GG, prevent epithelial cell apoptosis and stimulate cell 

growth, hence stimulating the repair of the mucosal layer[102]. L. lactis NZ900 producing 

pancreatitis-associated protein I (PAP) also stimulates epithelial cell growth, including 

Paneth cells. PAP alters the expression of growth factors and proliferation-related molecules 



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along with antioxidant enzymes[103]. Figure 3 summarises the 5 phases of mucositis induced 

by 5-FU.  

 

Figure 3. The 5 phases of mucositis. The initiation phase begins with the exposure of gut mucosa to 

chemotherapy or radiotherapy. Here, we show exposure of the gut to 5-fluorouracil. Reactive oxygen species 

(ROS) are then formed, promoting DNA damage. ROS activates NF-κB, a transcription factor that subsequently 

activates PIC, such as interleukin 6 (IL-6), interleukin 8 (IL-8), IL-1β, TNF-α, and cyclooxygenase 2 (COX-2). 

DNA damage occurs and leads to apoptosis. In the third phase, there is a positive feedback loop in which PIC 

further reinforce stimulation to produce more PIC, causing further oxidative stress and worsening the injury. 

Eventually, a vicious cycle is created, and ulcers form due to intestinal injury. After stopping stimuli, the mucosa 

begins to heal by itself in a few days until the mucosa is restored. 

2.6. Effects of 5-FU and Its Impact on Gut Microbiota Composition 

5-FU causes a shift in gut microbiota profile by decreasing the abundance of intestine 

Firmicutes while increasing the abundance of Bacteroidetes and Verrucomicrobia[104].   

Specific strains were shown to decrease in abundance, such as Clostridium spp., 

Lactobacillus spp. and Streptococcus spp., while some increased in abundance, like 

Escherichia spp.[105]. The gut microbiome is in a dynamic state, allowing external factors to 

influence it, such as antibiotics[106]. Studies have found antibiotics do not offer any clear 

protection against IM. This was demonstrated by mice that were treated with both 5-FU and 

a cocktail of antibiotics; pathogenic bacteria such as Escherichia, Shigella, and Enterobacter 

significantly increased in abundance while other commensals such as Firmicutes decreased 

in abundance. The combination of antibiotics and 5-FU downregulates genes involved 

in amino acid metabolism, replication and repair translation and nucleotide metabolism, 

which contributed to overall reduced antitumor efficacy. The effect of GM on 5-FU anti-



PMMB 2023, 6, 1; a0000339 11 of 34 

 

tumour efficacy is evident with the administration of antibiotics. Antibiotics use instead 

worsens patients' body weight in clinical trials. They also showed a higher tumour/body 

weight ratio in comparison to the 5-FU treatment alone[69]. Therefore, since antibiotics are 

not working in ameliorating IM, other strategies suitable for use along with 5-FU regimens 

to ameliorate IM need to be investigated. 

3. Gut Microbiota Modulation-Based Strategies to Ameliorate 5-Fu-Induced Intestinal 

Mucositis 

The commensals residing in the human gut are crucial for sustaining a healthy human 

body. Healthy individuals usually contain bacterial species belonging to four phyla 

Firmicutes, Bacteroidetes, Actinobacteria and Proteobacteria[107,108]. GM helps hosts 

maintain immunity, protecting hosts from bacterial invasion and nutrient metabolism[47,109]. 

Shifts in this dynamic structure are present in inflammatory diseases such as inflammatory 

bowel disease (IBD), psoriasis and multiple sclerosis[107,110,111]. Neurological and psychiatric 

conditions, such as depression, obsessive compulsive disorder and myasthenia gravis, are 

also linked to gut microbiome changes[112,113]. Although the full mechanism of all of these 

interactions has yet to be unravelled, associations between GM and many common illnesses 

have been evidenced in numerous studies. For instance, gut dysbiosis is also evident in CRC 

patients. As mentioned earlier, 5-FU and antibiotics are also able to cause shifts in the gut 

microbial composition. Various strategies such as probiotics, prebiotics, dietary 

modifications, postbiotics, and fecal microbiota transplantation are utilized for gut 

microbiota modulation to promote health and manage diseases[114–118]. Among all of these 

strategies, probiotic use is one of the most common strategies, and it has been shown to 

prevent or ameliorate dysbiosis of the normal microbiota by maintaining a balanced gut 

microbiome by reducing potential pathogenic bacteria while increasing the number of 

beneficial bacteria[119–125]. With that, restoring the disturbed gut microbiota by supplementing 

probiotics can lessen the side effects of 5-FU, especially IM[126]. 

3.1. Use of Probiotics in Intestinal Mucositis 

Probiotics are "live microorganisms when administered in adequate amounts that 

confer a health benefit on the host"[97]. They are non-invasive and safe when compared to 

pathogenic bacteria. Only viable bacteria can be considered probiotics, not including bacteria 

components that are not viable[18]. Some requirements that must be met to consider a bacterial 

strain as a probiotic include the ability to adhere to and colonise the gut mucosa to interact 

with the host to modulate the antagonism against pathogens and for immune system 

responses[44,127,128]. They colonise the gut temporarily and without releasing toxins or 

metabolites detrimental to the host. To ensure the viability of the bacteria for colonisation of 

the intestinal tract, they must be able to resist the environment of low pH and bile salt in the 

gastrointestinal tract[18]. They help to restore gut dysbiosis by cultivating good commensal 

bacteria. Probiotics currently are used as adjuvants for many gastrointestinal disorders such 

as diarrhoea due to infections (Rotavirus infection) or iatrogenic (antibiotic use) or due to 

other inflammatory diseases such as IBD[12,129]. It is thought that probiotics decrease 



PMMB 2023, 6, 1; a0000339 12 of 34 

 

pathogenic bacteria that activate the immune system[107]. PIC and ROS have clearly been 

shown to be the main factors driving the inflammatory process of IM. As such, probiotics 

can come into play by being an anti-inflammatory agent.  

Since it has been shown that commensal organisms play a role in the inflammatory 

cascade, it would be a good target site to help ameliorate the manifestation of IM. Having 

said that, not only can probiotics suppress the inflammatory process in IM, but they also 

promote healing. Probiotics can improve host defence, such as mucus layer, TJ proteins and 

immune defence, subsequently ameliorating the side effects of 5-FU by reducing apoptosis 

and improving mucin production. Clinically, weight loss and diarrhoea were also attenuated 

with the administration of probiotics. Together, probiotics are capable of ameliorating 5-FU 

induced side effects via different mechanisms[105,130,131]. 

3.1.1. Gut dysbiosis 

The composition of the gut microbiota is altered and modified through a process 

called dysbiosis due to undergoing chemotherapy, which is often related to the 

gastrointestinal tract's biochemistry and immunologic disorders[46]. Gut dysbiosis is shown 

to be common among CRC patients, indicating the possible relationship between the gut 

microbiome and the carcinogenesis of colorectal cancer[13,132]. A primary increase in Gram-

negative bacteria was found following 5-Fu administration in rats, as shown in a preclinical 

study[13]. At the jejunal level, 5-FU decreases Clostridium sp., Lactobacillus sp. and 

Streptococcus sp., but increases Escherichia sp. In the colon, 5-FU caused decreases in 

Enterococcus sp., Lactobacillus sp. and Streptococcus sp. Faecal samples showed decreasing 

trends in Lactobacillus sp. and Bacteroides sp., and an increasing trend in E. coli and 

Staphylococcus sp.[133]. This was also validated in another study of mice administered with 

5-Fu showing a shift away from good bacteria such as Actinobacteria and towards 

Bacteroidetes and Verrucomicrobia phyla with pro-inflammatory traits[13]. Hence, gut 

microbiota modulation-based strategies which can shift dysbiosis towards eubiosis or achieve 

gut microbiota homeostasis should be developed[46]. Probiotics have evidently been able to 

change this shift in organisms. 

Probiotic bacteria that can colonise the intestinal tract have been shown to restore 

eubiosis by releasing antimicrobial compounds such as bacteriocins and decreasing the pH 

to limit the development of other harmful bacteria[36]. Gut microbiota is brought back into 

balance when the amount of pathogenic bacteria reduces due to their inability to survive in 

the acidic environment while the amount of good bacteria which flourish in the acidic 

environment increases[18]. This was apparent when researchers used a probiotic mixture in 

mice and showed a decrease in opportunistic pathogens[69]. In male mice that received both 

5-FU and Lactobacillus reuteri DSM 17938 (BG), it was found Bacteroides and 

Bacteroidaceae were most bountiful in 5-FU treated rats supplemented with the mixture, 

while those that received only saline solution showed that Muribaculaceae (S24_7) was most 

abundant[134]. Similarly, Lactobacillus plantarum 299v reduced 5-FU stimulated the growth 

of facultative anaerobes in the intestine[135]. Both probiotic strains,  Lactobacillus casei 



PMMB 2023, 6, 1; a0000339 13 of 34 

 

variety rhamnosus (Lcr35) and Bifidobacterium bifidum G9-1 (BBG9-1), also can 

significantly reverse the perturbed microbiota composition of 

Firmicutes and Bacteroidetes induced by FOLFOX. However, supplementation of Lcr35 

itself did not affect faecal GM composition[12,131]. This shows that probiotics are strain-

dependent. Hence, further research must be carried out in the future to find out more 

functional probiotic strains. Another strain, Rhodotorula mucilaginosa UFMGCB 18377, 

was also found to be able to reduce intestinal enterobacteria levels in 5-Fu treated mice with 

induced mucositis[136]. In vitro studies, non-recombinant L. lactis NZ9000 showed 

antagonistic activity against the invasive gut pathogen Listeria monocytogenes and its 

secreted compound called PAP had an inhibitory effect against opportunistic Enterococcus 

faecalis[137,138]. PAP is not the only property possessed by L. lactis NZ9000; it is thought that 

its antagonistic actions against pathogens could also be due to the secretion of lactic acid, 

bacteriocins or metabolites[137].  

3.1.2. Pro-inflammatory cytokines  

In the pathobiology of IM, TNF-α is a key PIC that causes IM. Other cytokines 

involved include interleukin (IL)-4, IL-6, IL-10 and IL-17. IL-17 is an inflammatory 

mediator. It acts together with IFN-y to synergistically enhance IL-8 secretion by human fetal 

intestinal epithelial cells[139].  When supplemented with probiotics Lcr35 and 

Bifidobacterium bifidum (LaBi, Infloran®),  levels of PIC decreased[12,140,141]. The study 

shows Lcr35 has the ability to suppress NF-κB activity, also leading to suppression of 

inflammation and subsequently reduced IM in the intestine[12].  

Other Lactobacillus strains, such as Lactobacillus delbrueckii CIDCA 133 

(pExu:hsp65), a recombinant strain of L. delbrueckii is able to increase serum IL-10 in ileum 

tissue while reducing inflammatory infiltrate and cytokines such as TNF-α, IL-6, IL-12, and 

IL-1β[142]. Other strains S. thermophilus CRL 808 decreases IL-6 but increases IL-10. This 

vitamin-overproducing strain can reverse the rise of the anti-/proinflammatory cytokines 

ratio. It increases concentrations of IL-10 in the serum along with decreased IFN-γ levels[143]. 

In another experiment on a mouse model, it was found that the levels of TNF-α, IL-1β and 

IL-6 were higher in the 5-Fu induced mouse group than the control group. However, those 

concentrations markedly decrease in the use of S. thermophilus ST4 along with 5-Fu 

treatment[97]. 

B. infantis also successfully reduced high levels of PIC caused by 5-FU in CRC rats. 

As mentioned, IFN-γ recruits macrophages[77]. IL-2 is important for IFN-γ stimulation while  

IL-12 stimulates production of IFN-γ by naïve T cells[144] . In this case, it is found that Th1 

cells and their cytokines, IL-2, IL-12 and IFN-γ, which are upregulated during chemotherapy, 

can be reversed by B. infantis[145].  

 

 



PMMB 2023, 6, 1; a0000339 14 of 34 

 

3.1.3. Intestinal damage 

As mentioned before, changes in the architecture of the intestinal mucosa are side 

effects of 5-Fu, including a decrease in villus high, an increase in crypt depth and intestinal 

permeability and a shortening of the intestinal length. Villus blunting and crypts fissure in 

the small intestine are mainly caused by an increase in apoptosis and a decrease in cell 

proliferation as a result of chemotherapy[97]. Restoration of the intestinal structure is very 

important as it influences the ability of nutrients to be absorbed by the body. A taller villus 

has more surface area for the absorption of nutrients, and it also lessens water loss and 

electrolyte loss[146]. Inefficient nutrient absorption can lead to loss of weight (LOW) in cancer 

patients[142]. Hence, probiotics have been proven to play a role in restoring intestinal damage.  

In an in vivo experiment, S. thermophilus ST4 was proven to be able to ameliorate the 

shortening of colon length of mice treated with 5-Fu. This is shown by a shorter average 

colon length in the mice treated only with 5-Fu when compared to the 5-Fu + S. thermophilus 

ST4 group. It was also found that the administration of S. thermophilus ST4 had also 

mitigated the decrease in the villus heights and crypt depths[97]. Another probiotic strain, R. 

mucilaginosa UFMGCB 18377, was also demonstrated to be able to decrease intestinal 

shortening of mice induced with mucositis by 5-Fu from around 17% shortening compared 

to the control group to 7.5% only. It is also found that this probiotic strain is also efficient in 

ameliorating the structure of the damaged intestinal mucosa, restoring its architecture and 

villus to crypt ratio, and reducing the increasing permeability of the intestine as a result of 5-

Fu treatment[136]. 

Mucosal damage, such as crypt depths, has been shown to be significantly restored, 

even to levels similar to those only injected with saline, with Lcr35 and 

LaBi supplementation. Villus height to crypt depth ratio managed to return to normal[140], 

although another study by Huang et al.[141] has shown similar results without it being 

statistically significant. Other strains also showed similar results, with Saccharomyces 

boulardii and Lactobacillus fermentum BR11[130,147]. S. boulardii also induced the recovery 

of intestinal permeability by normalising the lactulose: mannitol ratio[130]. 

Effects of supernatant and live S. thermophilus TH-4 reduced ileal crypt fissure by at 

least half of the controls, although it unsuccessfully ameliorated 5-FU induced mucositis[148]. 

Lactobacillus plantarum ATCC 8014 showed a potential beneficial effect on ameliorating 5-

Fu induced microscopic changes in the intestinal mucosa[149]. Lactobacillus 

delbrueckii (pExu:hsp65), L. delbrueckii subsp. Lactis CIDCA 133 strain, L. acidophilus or 

milk fermented with L. rhamnosus FLRH93 all were able to stop the shortening of the gut 

due to 5-FU. These probiotic strains also reduced 5-FU's adverse effects on IP, crypt depth, 

villus height, as well as villus/crypt ratio and goblet cells[142,146,150–152]. L. rhamnosus 

FLRH93 has also managed to prevent small intestine shortening, with an almost 5% 

difference compared to mice with 5-FU alone. This is consistent with the finding from the 

same study that the mice had less LOW[150].  



PMMB 2023, 6, 1; a0000339 15 of 34 

 

Two tested probiotic mixtures, such as  Lactobacillus acidophilus and B. lactis, or a 

mix of four strains (Lactobacillus acidophilus, Lactobacillus paracasei, L. rhamnosus, 

and B. lactis) partially reversed histopathological changes induced by 5-FU[153].  BBG9-1 as 

mentioned before, prevented the increase in crypts quantity and reduction of small intestine 

length (although not statistically significant) but could not stop the induction of crypt cell 

apoptosis, as shown with B. infantis supplementation[145,154]. 

Supernatants from E. coli Nissle 1917 and F. prausnitzii enhanced barrier integrity 

by normalising crypt depth and prevented the change in barrier by 5-FU. S. 

thermophilus CRL 808 and S. thermophilus CRL 415 are strains that can produce folate, 

which was shown to induce less inflammation in the jejunum and preserved villus 

height/crypt depth ratio[143]. Improvement in the histopathological score was also apparent 

with P. freudenreichii WT[155]. Treatment with Saccharomyces cerevisiae UFMG A-905 

successfully re-established a normal state in the intestine. Intestinal permeability returned to 

normal. S. cerevisiae UFMG A-905 has antioxidant and anti-inflammatory properties. 

Inflammatory markers were further reduced after giving selenium-enriched yeast[156].  

3.1.4. Immune cells infiltration 

The infiltration of inflammatory cells is broadly related to mucosal damage[136]. 

Neutrophil infiltration, associated with intestinal damage and its recruitment, can be 

measured quantitatively by myeloperoxidase activity (MPO). In a recent study, 5-FU can 

increase this by almost 500 times with an 83% decrease in sucrase[157]. On the other hand, 

eosinophil peroxidase (EPO) activity can be used to quantify the accumulation of another 

inflammatory cell called eosinophils. Eosinophils play an important role in inducing 

inflammation in the intestine by acting as antibacterials through the production of PIC and 

EPO activity. 5-Fu induced mucositis often results in the recruitment of neutrophil and 

eosinophil infiltrates or causing an increase in MPO and EPO levels, hence triggering 

inflammatory reactions in the tissues[136].  In a mucositis mouse model, MPO level has been 

shown to decrease with L. delbrueckii subsp. Lactis CIDCA 133 strain, S. boulardii, 

Lactococcus lactis NZ9000, Lactobacillus acidophilus A4 (A4), BBG9-1, R. mucilaginosa 

UFMGCB 18377 and probiotic mixtures[130,131,136,137,151,153,158,159]. R. mucilaginosa 

UFMGCB 18377 can also reduce EPO levels in the ileum and jejunum of mice with induced 

mucositis due to 5-Fu[136]. Lower MPO and EPO levels also indicate that there is lesser 

immune cell infiltration, which will reduce inflammatory responses. Besides, fewer 

polymorphonuclear cells in the lamina propria lead to less inflammatory activity by these 

cells, following fewer lesions occurring[137]. L. delbrueckii subsp. Lactis CIDCA 133 has also 

been shown to minimise 5-FU induced leukopenia[151]. 

Intestinal microbiota plays a part in the adaptive immune system by regulating IgA 

secretion. IgA is secreted by immune cells scattered across the mucosa[142]. IgA prevents 

bacteria colonisation and invasion into the mucosa. Toxic materials are blocked by IgA from 

penetrating the mucosa. IgA modulates the inflammatory response by inhibiting 

inflammatory response by activating regulatory T-cells[160]. Clearly, serum IgA has a dual 



PMMB 2023, 6, 1; a0000339 16 of 34 

 

function in modulating microorganisms present in the gut. It protects the gut from pathogens 

but also regulates good bacteria. It seems that IgA has a different affinity for different 

microorganisms. There is a high affinity for pathogenic microorganisms but a low affinity 

for commensal bacteria[161]. IgA and commensals also regulate each other. Bifidobacteria is 

capable of modulating the expression of secretory IgA. Serum IgA was increased after 3 days 

of Bifidobacteria supplementation in rats that suffered burn injuries in the gut, but these 

levels were returned to normal by day 5. This self-modulating expression reduced incidences 

of bacteria translocated and reduced counts of E. coli and fungi[162]. Clearly, strains that 

increase IgA is a good probiotic to consider as well. L. delbrueckii CIDCA 133 (pExu:hsp65), 

a recombinant strain can also increase serum IgA[142].  

3.1.5. Apoptosis 

5-FU is capable of inducing cell apoptosis by inducing the expression of IL-1β and 

TNF-α[15]. This is shown by its antagonist, interleukin-1 receptor (IL-1R) antagonist, in 

successfully reducing apoptosis after chemotherapy. Up-regulation of Bax and caspase 3 

(pro-apoptotic proteins) along with downregulation of Bcl-2 and Bcl-xL (antiapoptotic 

proteins) occur during 5-FU induced intestinal apoptosis[163]. Caspases and Bcl-2 proteins are 

important in early apoptosis[164]. Bcl-2 can prevent cells from harsh environments such as 

radiation, heat and chemotherapy. Bcl-2 prevent these cells from undergoing apoptosis[163]. 

In vitro study shows that IL-1R antagonist inhibits apoptosis without affecting its anti-tumour 

efficacy. This shows that antagonising IL-1β activation can protect the gut from 

chemotherapy-induced IM[163]. 

Lcr35 has been shown to suppress apoptosis caused by FOLFOX administration. The 

reduction of the increased BAX/BCL-2 ratio induced by FOLFOX and a shift toward anti-

apoptosis when Lcr35 is given[12]. Besides that, S. boulardii, a probiotic yeast, has been 

shown to reduce 5-FU induced intestinal cell apoptosis. 5-FU modifies the TLR response to 

activate PIC. When this cascade is activated, there is phosphorylation of ERK1/2, p38 and 

JNK. In particular, TNF-α and IL-1β activate these protein kinases in response to oxidative 

stress. The IL-1β induced MyD88 pathway and phosphorylation of protein kinases are 

inhibited by S. boulardii[165]. In another case, the probiotic Propionibacterium freudenreichii 

requires SlpB protein to alleviate mucositis induced by chemotherapy[130].   

3.1.6. Mucin production 

The human gut mucosa is protected by a mucus layer. This mucus layer comprises 

water, glycoproteins, trefoil factors, defensins and mucins. Mucin is an important component, 

it being the first-line defence in the gut. Mucins are produced by goblet cells that reside all 

over the small and large intestines and produce mucin[166]. Bacteria are able to adhere to 

mucin oligosaccharides. Commensals can compete with pathogens to bind and colonise the 

mucin, preventing pathogens from attaching to the mucosal surface. For example, the parasite 

Entamoeba histolytica requires contact with epithelial cells in order to invade it. Having a 

thick mucus layer prevents it from having contact with the epithelium. Once this mucus layer 



PMMB 2023, 6, 1; a0000339 17 of 34 

 

is disrupted, E. histolytica can easily penetrate through the gut barrier leading to phagocytosis 

of epithelial cells[167]. Our gut epithelium is endlessly exposed to various noxious chemicals 

and physical insults such as digestive enzymes, faecal material, resident bacteria, and 

intestinal pathogens and their products. Mucin blocks the mucosa from bacterial enzymes by 

acting as a substrate for enzymes such as α-galactosidase, β-N-acetylgalactosaminidase, 

sialidase, β-glucuronidase, blood group degrading enzymes, and proteases[166]. Having a 

mucosal shield as a barrier between underlying epithelium and these substances is essential 

in preventing IM. 

The mucus layer is also thickened in response to the presence of pathogens. This quick 

response is key to eliminating pathogens. In other words, pathogens and their toxins have a 

positive stimulation on mucin production. Thick mucus allows easier excretion of pathogens 

but also carries a risk of diminished mucin[167,168]. Vibrio cholerae enterotoxin stimulated 

mucin production[169]. However, Helicobacter pylori downregulate mucin-producing genes 

MUC1 and MUC5AC resulting in reduced mucin synthesis[170]. Another important gene is 

MUC2. MUC2 is a key player in the formation of the mucus layer. It is stored in goblet cells 

and is key in determining goblet cell shape[171]. In MUC2 knockout mice, defective or 

depleted mucus defence layer showed bacteria colonisation and inflammation (with multiple 

crypts), subsequently leading to carcinogenesis[168,171]. For example, Lactobacillus 

plantarum 299v, L. rhamnosus strain GG and L acidophilus strain DDS-1 are shown to 

increase gene expression of MUC2 and MUC3 at the mRNA level[172,173]. This managed to 

prevent adherence of pathogenic E. coli onto gut epithelium[172]. Besides, non-pathogenic 

commensal E. coli Nissle 1917 regulates multiple genes. Besides MUC2 and MUC3, it also 

upregulates MUC5AC and MUC5A[174]. Butyrate-producing organisms also have shown 

abilities to increase MUC2 gene expression in in vitro studies. Human colon biopsies in ex 

vivo studies showed increased mucous secretion in the presence of butyrate-producing 

organisms. Trefoil factors, a contributor to the viscosity and elasticity of mucus, can reduce 

the chemotaxis of inflammatory cells and are thought to repair damaged mucosa. Like mucin, 

it is only produced by GC. Trefoil factors are reduced in rats with colitis, but with butyrate 

administration, these levels increased[175]. 

5-FU treatment has been shown to influence the mucus layer and decrease goblet cells 

while increasing the number of crypts in the jejunum[176]. This is possibly due to the pro-

inflammatory state 5-FU induces. It is found that Rhodotorula mucilaginosa UFMGCB 

18377 decreases the loss of goblet cells in mice induced with mucositis by 5-Fu[136]. Other 

than that, treatment with L. delbrueckii rCIDCA 133:Hsp65 also increases goblet cells and 

expression of the MUC2 gene. A higher quantity of goblet cells and increased MUC2 gene 

expression can reinforce each other's positive effect in protecting the mucus layer[142]. 

3.1.7. Weight loss 

Regarding weight loss, probiotic strains such as Lcr35 and LaBi have been shown to 

reduce body weight loss by 20%. Mice in the Lcr35 probiotic group showed a lesser degree 

of LOW compared to 5-FU and saline group[177]. Other Lactobacillus strains, such as L. 



PMMB 2023, 6, 1; a0000339 18 of 34 

 

plantarum 299v, have been shown to improve food intake, resulting in less LOW, while 

others, such as L. delbrueckii subsp. Lactis CIDCA 133 strain and L. acidophilus could 

increase food and milk intake but still reduce LOW[135,142,151]. Riboflavin overproducing L. 

plantarum CRL2130 also showed similar results[152,178]. 

Besides these, L. fermentum BR11 and L. rhamnosus GG increase colon weight in 

conjunction with 5-FU treatment[157]. Lactobacillus plantarum 299v and BBG9‐1, at a 

concentration of 1 × 109 CFU/mL, improved both food consumption and body weight post 

5-FU administration[131,147]. Interestingly, high doses resulted in LOW, while low doses 

resulted in body weight gain after one week of 5-FU treatment. On day 8 after the treatment, 

low doses increased body weight compared to high doses of the probiotic, which decreased 

food intake[147]. B. infantis also has shown weight-gaining properties[145,154]. 

In another in vivo experiment in mice, it was found that mice with 5-Fu induced IM 

showed a significant decrease of about 50% in food intake and about a 20% decrease in their 

body weight in relation to the initial weight on the 18th day. However, in the 5-Fu + S. 

thermophilus ST4 mice group, there was no big difference in body weight loss (only 1.85%) 

and food intake compared to the control group[97]. Another strain of probiotic, Rhodotorula 

mucilaginosa UFMGCB 18377 ameliorated the decrease in food consumption of mice 

induced with mucositis by 5-Fu hence reducing the weight loss in comparison to their initial 

weight from 20.3% to 12.4% in the probiotic-treated group as compared to the mucositis 

group[136]. A probiotic mixture (L. acidophilus, L. paracasei, L. rhamnosus, and B. lactis) 

reduced body weight loss[153]. However, the addition of Streptococcus genus, resulting in a 

mixture of B. breve DM8310 + L. acidophilus DM8302, L. casei DM8121 + S. 

thermophilus DM8309 showed no differences in faecal output, and food intake[158]. E. 

coli Nissle 1917 supernatant and F. prausnitzii supernatant prevented further weight loss 

from happening post 5-FU induction resulting in normal urine and faecal output[179]. 

These mixed results show that, clinically, some probiotic strains are better than others 

at preventing weight loss. None of the probiotics mentioned exacerbated weight loss, 

suggesting its importance in maintaining patients' weight. Further work should be done to 

understand the underlying mechanisms exhibited by some strains to preserve the body 

weight, potentially via improved feeding behaviour and the host's metabolism[180]. 

3.1.8. Diarrhoea 

Severe diarrhoea is presumed to be closely related to the shortened colon, one of the 

side effects of the administration of 5-Fu[97]. Diarrhoea occurs when the colon's absorption 

capacity is surpassed by the rising fluid volume out of the small intestine[44]. Rat studies show 

that probiotics can ameliorate diarrhoea. The research was carried out on a mice model by 

assessing the severity of diarrhoea using Bowen's score system, in which diarrhoea is 

categorised into 4 grades based on the consistency of stool (0 — normal stool; 1 — mild 

diarrhoea, the stool is slightly soft and wet;  2 — moderate diarrhoea, stool are wet and 

unformed; 3 — severe diarrhoea, stool are watery). It is found that the 5-Fu + S. thermophilus 



PMMB 2023, 6, 1; a0000339 19 of 34 

 

ST4 group shows a lower mean diarrhoea score that changed from 1.0 to 0.1 when compared 

to the group of mice induced with 5-Fu only that scored 2.5 and 2.5 respectively, on the most 

severe day 14th to 15th[97]. For example, Lcr35 group and LaBi group, L. delbrueckii CIDCA 

133 (pExu:hsp65), L. plantarum CRL2130, BBG9-1 and also folate-producing S. 

thermophilus CRL 808 were shown to successfully reduce the incidence of 

diarrhoea[140,143,178].  

Probiotics reduced the incidence of diarrhoea in human patients. In a systematic 

review, researchers found probiotics and fibre reduced the incidence of grade 3 or 4 

abdominal discomfort (flatulence, borborygmi and abdominal distension) and the need for 

chemotherapy dose reduction, compared with placebo[14,181]. L. rhamnosus GG decreased the 

incidence of diarrhoea in grades 3 or 4 by around 15%. In patients with severe diarrhoea, 

metabolic and nutritional imbalances may occur, which may further worsen their condition. 

It is evident that probiotics show improvement in diarrhoea in animal models and humans[45]. 

In another clinical trial on patients treated with 5-Fu based chemotherapy and administered 

with probiotic Lactobacillus kefiri LKF01, it was found that the incidence of diarrhoea was 

reduced. Only 4.7% and 8.7% of patients treated with 5-Fu and FOLFOXIRI respectively 

developed G3-4 severe diarrhoea [CTCAE grade [G] 3-4]. In contrast, no incidence of high-

grade diarrhoea was reported by patients treated with FOLFOX and FOLFIRI, respectively. 

Since their results also showed that the onset of diarrhoea mostly started at the early stages 

of chemotherapy, hence it is hypothesised that the intake of probiotics earlier before the 

treatment begins and not concomitantly as they did in their studies could possibly work as a 

preventive measure to reduce the rates of early-onset diarrhoea, but further studies are 

required to validate it[44]. 

Table 1. Probiotic genus, strains and mixtures that show beneficial effects toward IM. Different probiotics 

ameliorate IM in various aspects are summarised in this table. 

Probiotic Effects of probiotic on ameliorating intestinal mucositis Reference 

Lactobacillus acidophilus • Preserved villus and crypt length ratio 

• Reduced GSH levels 

• Reduced myeloperoxidase activity 

• Reduced nitrite levels 

• Reduced level of PIC, TNF-α, IL-1β 

• Reduced levels of chemokines CXCL-1 

• Increased levels of anti-inflammatory cytokines IL-10 

[152] 

Lactobacillus acidophilus 

A4 
• Prevented attachment of Escherichia coli O157:H7  

• Reduced level of PIC, IL-8, TNF-α, IL-1β 

• Induced the expression of MUC2  

[182] 

Lactobacillus brevis 47 • Partially restored expression of Ki-67 epithelial 
proliferation cell marker 

[183] 

Lactobacillus plantarum 

CRL2130 
• Reduced weight loss 

• Reduced diarrhoea scores 

• Maintained mucosal architecture 

• Reduced mucosal inflammation 

[178] 



PMMB 2023, 6, 1; a0000339 20 of 34 

 

Probiotic Effects of probiotic on ameliorating intestinal mucositis Reference 

• Preserved villus and crypt length 

• Reduced level of PIC, IL-10 

Lactobacillus plantarum 

supernatant  
• Increased sensitivity of colorectal cancer cells to 5-

fluorouracil 

• Inhibited CD44 gene expression 

• Inhibited CD133 gene expression 

• Inhibited CD166 gene expression 

• Inhibited ALDH1 gene expression 

• Increased caspase 3 activity  

• Inhibited signalling pathway Wnt/β-catenin 

[43] 

Lactobacillus 

casei variety rhamnosus (L

cr35) 

 

• Reduced diarrhoea scores 

• Reduced loss of weight  

• Normalised crypt depth 

• Preserved villus and crypt length ratio 

• Reduced villous inflammation 

• Diminished apoptosis 

• Reduced level of PIC, TNF-α and IL-6 

• Reduced levels of NF-κB-, and BAX-activated cells  

• Restored composition of fecal gut microbiota 

• Decreased Firmicutes and Bacteroidetes ratio  

• Reduced CD3+/CD8+ count  

• Increased CD3+CD4+/CD3+CD8+  

• Reduced CD44 the number of Ki67 proliferative cells 

[177] 

Lactobacillus delbrueckii 

CIDCA 133 (pExu:hsp65) 

 

• Reduced inflammatory infiltrate 

• Reduced intestinal permeability  

• Increased serum IgA in the intestinal fluid  

• Improved histological score  

• Prevented shortening of the intestine 

• Preserved villus and crypt length ratio 

• Reduced myeloperoxidase activity  

• Reduced level of PIC, TNF-α, IL-6, IL-12, IL-1β 

• Reduced level of Toll-like receptors  

• Increased level of anti-inflammatory cytokines IL-10 

• Increased gene expression of MUC2 

• Increased gene expression of claudin 1  

• Increased the number of goblet cells 

[142] 

Lactococcus lactis NZ9000 

 

• Prevented growth of Listeria monocytogenes  

• Pancreas-associated peptide secretion prevented growth 
of E. faecalis 

• Improved histological score  

• Reduced infiltration of neutrophils 

• Reduced infiltration of eosinophils  

• Reduced secretion of Immunoglobulin-A in the gut  

• Reduced gut inflammation 

[137] 

Lactobacillus sp.  • Induced expression of MUC3 [173] 

Lactobacillus rhamnosus 

FLRH93  
• Preserved villus and crypt length ratio 

• Lessen the decrease of goblet cells 

[150] 



PMMB 2023, 6, 1; a0000339 21 of 34 

 

Probiotic Effects of probiotic on ameliorating intestinal mucositis Reference 

 • Increased the expression Bcl-2 in the intestinal tract 

• Decreased expression of NLRP3 

• Reduced level of PIC, TNF-α, IL-1β 

• Increased survival rate of mice  

Lactobacillus rhamnosus 

GG 
• Reduced diarrhea scores 

• Reduced incidence of abdominal discomfort  

[45] 

Streptococcus 

thermophilus CRL 808 
• Enhanced chemotherapeutic effect of 5-FU 

• Reduced diarrhoea scores 

• Reduced jejunal inflammation 

• Increased histological score 

• Reduced level of PIC, IL-6 

• Increased level of anti-inflammatory cytokines IL-10 
 

[143] 

Streptococcus 

thermophilus ST4 
• Increased food intake of mice 

• Decreased body weight loss 

• Reduced mean diarrhoea score 

• Reduced shortening of colon length 

• Restored villus heights and crypts depths 

• Reduced level of PIC, TNF-α, IL-1β and IL-6 

[97] 

Bifidobacterium infantis  • Mice showed higher body weight  

• Mice had taller villus 

• Reduced level of PIC, IL-6, IL-1β and TNF-α 

• Down-regulated levels of T-bet (Th transcription factor) 

• Increased CD4+ levels 

• Increased CD25+ levels 

• Increased Foxp3+ levels 

• Increased Tregs levels 

• Reduced CD4+ IL17A+ cells  

[145] 

 

Saccharomyces boulardii 

 

• Changed expression of Toll-like receptors, TLR2, TLR4 

• Changed expression of MyD88 

• Changed expression of NF-κB 

• Changed expression of ERK1 

• Changed expression of phospho-p38 

• Changed expression of phospho-JNK 

• Reduced level of PIC, TNF-α, IL-1β  

• Reduced level of chemokine CXCL-1 

[165] 

Saccharomyces cerevisiae 

UFMG A-905 

 

• Partially reduced intestinal permeability  

• Reduced myeloperoxidase activity 

• Reduced level of chemokine CXCL-1 

• Reduced mucosal inflammation 

• Reduced oxidative stress 

[156] 

Propionibacterium 

freudenreichii 
• Reduced level of PIC, IL-12, IL-17a, IL-8, TNF- α 

• Changed expression of cld1 

[155] 



PMMB 2023, 6, 1; a0000339 22 of 34 

 

Probiotic Effects of probiotic on ameliorating intestinal mucositis Reference 

Wild strain of 

Propionibacterium 

freudenreichii 

• reduced weight loss 

• reduced gut inflammation 

• increased histopathological scores 

• induced production of Th17 cells  

• reduced level of IgA  

[155] 

Rhodotorula mucilaginosa 

UFMGCB 18377 
• Ameliorated the decrease in food consumption 

• Reduced weight loss 

• Reduced shortening of intestinal length 

• Restored villus to crypt ratio 

• Reduced intestinal permeability 

• Reduced goblet cells loss 

• Reduced enterobacteria in intestine 

• Reduced MPO and EPO levels / Reduced neutrophils 
and eosinophils infiltration 

[136] 

Probiotic mixture : 

• Bifidobacterium 
breve DM8310 

• Lactobacillus 
acidophilus DM8

302 

• Lactobacillus 
casei DM8121 

• Streptococcus 
thermophilus DM

8309 

• Reduced level of PIC   

• Reduced infiltration of neutrophils 

• Reduced intestinal permeability  

• Changes of Toll-like receptors, TLR2, TLR4 signalling 
pathway  

[158] 

4. Discussion and Future Direction 

Despite extensive evidence shown in this review, there is no solid guideline for 

treating intestinal mucositis due to chemotherapy. Current medications or other ways to 

combat chemotherapy-induced side effects are generally not completely effective, mostly fail 

to address possible long-term impacts, and may even cause additional side effects that merely 

exacerbate patients' sufferings[13]. Some of the interventions being used currently are limited 

to ice, analgesics, barrier protectors and topical antimicrobials. Hence, it is necessary to look 

for other possible alternatives to treat IM[136]. Probiotics have clearly shown to be a valuable 

adjuvant therapy in treating intestinal mucositis. Many strains successfully reduce levels of 

PIC, inhibit inflammatory pathways, and strengthen the gut's physical defence system. 

Probiotics also manage to prevent adherence of pathogenic bacteria and prevent gut cell 

apoptosis.  

When patients undergo cancer treatment, they are at risk of gut dysbiosis. They will 

be less protected from harmful external factors due to lack of commensal GM and also due 

to a weak immune system from cancer itself as well as the chemotherapy drug. 



PMMB 2023, 6, 1; a0000339 23 of 34 

 

Supplementing with probiotics before, during and after chemotherapy can increase one more 

level of protection for cancer patients. Further research is required to investigate the 

integration of probiotic interventions into the clinical management of IM, effectively halting 

mucositis in its track. With the current understanding that the gut microbiome plays a crucial 

role in all stages of intestinal mucositis, including initiation, upregulation, message 

generation, amplification, signaling, ulceration, and healing, it is anticipated that this 

integration will be realised in the near future. There is also a risk of bacterial translocation, 

causing invasive infections. Immunocompromised cancer patients would be particularly 

vulnerable, especially the risk of bacteraemia or sepsis, worsening patients' survival and 

mortality rate. However, with all of these studies mentioned, there were no events of 

bacteraemia or sepsis. Probiotics are beneficial. They inhibit the invasion of pathogens and 

strengthen the immune system. It is also important to note that probiotics do not compromise 

5-FU efficacy, supporting their further use[17].  

As shown in this review, there is a large shortage of studies conducted on humans. 

Although some of the preclinical studies show promising results, they might not translate 

similarly to humans. Most of the studies are also done in mice induced for mucositis or 

induced with carcinoma, which could be different from humans with chemotherapy-induced 

mucositis with underlying CRC. Perhaps this is why there is currently no definitive proof 

supporting the use of probiotics in treating IM. Therefore, this review calls for future study 

of ethical, large, randomised, double-blind human trials. This is to further assess the 

capability of probiotics in real-life clinical settings.  

Studies should include isolated strains that can ameliorate all side effects of IM rather 

than just ameliorating one specific side effect. Other than that, the most suitable doses along 

with the most effective probiotic strains or combination of different probiotic strains, should 

also be further evaluated to maximise the beneficial effects of probiotics on the host. Also, 

human factors such as genetics[184,185], race, and environmental factors could affect patients' 

response to probiotics supplementation. Thus, knowing the correlation between these 

external factors and response to probiotics should be studied as well. By doing this, we might 

be able to predict which patients are specifically at risk of developing certain side effects. 

Perhaps we can tailor the probiotic supplementation to patients' needs, thus developing a 

personalised probiotic regimen for each patient to reach optimal treatment outcomes. 

Individual variability is impossible to predict; however, more studies on the association of 

certain external factors or internal factors, such as gene polymorphism, can be a starting point 

for having proper guidelines for IM therapy.   



PMMB 2023, 6, 1; a0000339 24 of 34 

 

A challenge in using probiotics as a therapeutic method would be modulating its 

effects to target inflamed tissues while leaving normal tissues undisturbed and, most 

importantly, without disturbing the efficacy of 5-FU. Evidence showed probiotics do not 

affect the efficacy of 5-FU and its actions on tumour cells. However, similar to other studies, 

probiotics' influence on 5-FU's mode of action is only primarily known in rat studies. There 

has yet to be a large human trial to allow full justification. The challenge of maximising 

probiotic protection of mucosa while minimising the protection of tumour will need to be 

overcome. Perhaps, new techniques or technology should be used to overcome this challenge.  

Also, probiotics are required to remain in the body for a considerable amount of time. 

So far, it is uncertain how long the effects of probiotics will last and whether the effects will 

continue to last even after probiotics are no longer in the body[186]. This review also does not 

discuss the viability of probiotics after going through the digestive tract, such as stomach acid 

and digestive enzymes[146].  

5. Conclusion 

In conclusion, this review shows promising evidence that would encourage future 

studies on the use of probiotics in ameliorating the side effects of 5-FU or chemotherapy in 

general. More studies are important, as we have seen that IM is a complex process, and its 

association with gut microbes is even more complex. 5-FU continues to be used today, and 

yet there is only a superficial understanding of the relationship between GM and 5-FU. This 

review highlights that GM plays an important role in the host's response to 5-FU. Also, this 

review gives a framework on how probiotics may increase the efficacy and lessen the side 

effects of 5-FU; thus, future studies on this will be beneficial to many patients that use 5-FU. 

There has yet to be fully validated that probiotics could be the magic bullet, and for now, it 

serves better as an adjuvant targeted therapy. Nevertheless, this emerging field of research 

provides hope for finding safer and more effective treatment options for CRC and mitigating 

the adverse effects associated with traditional chemotherapy. These approaches not only 

combat cancer but also preserve patients' quality of life during treatment. Continued efforts 

in this direction may open new avenues for personalized and targeted approaches to CRC 

treatment, revolutionizing the way we combat this devastating disease. 

Author Contributions: AAX-HS and J-YC performed the literature search, critical data analysis as well as 

manuscript writing. J-YC, JW-FL, VL, YK, SO, SHW, and K-GC performed editing, revision and proofreading. 

JW-FL, VL, YK, SO, SHW, K-GC and LT-HT provided technical support and expertise. LT-TH conceptualised 

this review writing project.  

Acknowledgments: This work was inspired by the Jeffrey Cheah School of Medicine and Health Sciences 

(JCSMHS) "MED5101 Scholarly Intensive Placement (SIP)". The author also would like to acknowledge and 

sincerely thank the Young Scholar’s Program (YSP) conducted at JCSMHS, Monash University Malaysia. 



PMMB 2023, 6, 1; a0000339 25 of 34 

 

Conflicts of Interest: The authors declare no conflict of interest. 

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