Research paperL-citrulline enriched fermented milk with Lactobacillus helveticus attenuates dextran sulfate sodium (DSS) induced colitis in mice
Graphical abstract
Introduction
Inflammatory bowel disease (IBD) refers to a range of chronic inflammations and disorders of the gastrointestinal tract, the most common forms of IBD being Crohn's disease (CD) and ulcerative colitis (UC) [1]. The clinical symptoms of CD and UC include abdominal pain, diarrhoea and rectal bleeding [2]. The pathogenesis of IBD is still not well understood, however, it is seemingly associated with genetic, environmental and microbial factors [3]. The traditional method of treating IBD is with anti-inflammatory agents, but recently some researchers have investigated the possibility of using alternative therapies to treat it, such as probiotics [4], herbal medicines [5] and omega 3 fatty acids [6].
Gut mucosal healing is an important goal to relieve and treat the pathogenesis of IBD, as the mucosa is the defence line for preventing the translocation of bacteria [7]. Therefore, different dietary supplementations have been extensively tested as coadjutants and alternative therapies for enhancing gastrointestinal barrier function. Especially, probiotics and amino acids might act as immunomodulators to promote gastrointestinal health [8]. L. helveticus, one of the lactic acid bacteria, is able to prevent the adhesion of uropathogenic E. coli to intestinal epithelium [9] and suppress the secretion of pro-inflammatory cytokine, IL-17 and up-regulate the anti-inflammatory cytokine, IL-10, in dextran sodium sulfate (DSS) injury model [10]. L. helveticus is commonly used for manufacturing various types of fermented products, which have been used as health-promoting functional foods due to the versatile benefits of lowering blood pressure, inhibiting infection and improving immune response [11]. Notably, the beneficial effects of L. helveticus fermented milk on gastrointestinal tract have been described. Oral administration of L. helveticus fermented milk modulates the gut mucosa immune response via escalating the number of IgA+ cells in the lamina propria of the small intestine [12,13], strengthens the gut immune system via activating the expression of calcineurin [14] and attenuates infection by inhibiting the pathogenic invasion [15].
Citrulline is a non-essential amino acid [16] and has been used for oral supplementation. It shows multiple positive effects on gastrointestinal functions, such as ameliorating the inflammatory response via reducing the level of pro-inflammatory cytokines (TNF-α, IL-6 and IFN-γ) in intestinal ischemia/reperfusion model [17], increasing mucosal protein content after massive intestinal resection [18] and preventing muscle atrophy in malnourished rats [19]. Citrulline is a precursor of arginine, which is used for generating nitric oxide (NO) via nitric oxide synthases (NOS). Citrulline is produced simultaneously by NOS during NO formation from arginine, and then arginine is re-generated from citrulline by argininosuccinate synthetase (ASS) and argininosccinate lyase (ASL) [16]. Because of the arginine-regenerating ability, citrulline is able to execute the same actions as arginine [20].
Intriguingly, some Lactobacillus strains are able to utilize arginine by the arginine deiminase (ADI) pathway. The degradation of arginine (citrulline) via ADI pathway plays an important role in improving the growth and survival of Lactobacillus [21]. The potential enhancement effect of combination of Lactobacillus strains with arginine in vivo has been investigated, and a synergistic effect was shown to increase protection against liver injury induced by D-galactosamine [22], high-cholesterol diet [23] and endotoxin [24], as well as to reduce bacterial translocation [22].
Recently, our research group determined that citrulline exerts a superior effect to arginine in improving intestinal epithelial barrier functions [25] and found an positive effect of citrulline enriched fermented milk with live L. helveticus ASCC 511 in mitigating the infection of pathogenic E. coli in an in vitro model by inhibiting the pathogenic adhesion, reinforcing tight junction (TJ) protein expression and distribution and modulation of the excessive inflammatory response via Toll-like receptors (TLRs) signalling pathway [26]. Moreover, previous studies reported the positive effect of probiotic fermented beverage [27], L. helveticus strain [28] and arginine [29] on dextran sulfate sodium (DSS)-induced colitis. However, there is no reported research related to the effect of the combination of citrulline and LAB fermented milk in protecting against gastrointestinal tract damage in vivo.
Thus, we hypothesized that citrulline enriched L. helveticus ASCC 511 fermented milk (Cit_LHFM) would show a synergistic effect in maintaining gastrointestinal tract integrity and had a superior effect to supplementation of citrulline or LAB fermented product alone. Hence, our aim was to investigate and compare the effect of supplementation of citrulline, L. helveticus ASCC 511 fermented milk (LHFM) and Cit_LHFM and examinate their anti-inflammatory effects against DSS-induced colitis in a mouse model.
Section snippets
Ethics statement
The protocol of this study was approved by the Committee on the Use of Live Animals in Teaching & Research (CULATR) of the University of Hong Kong (CULATR reference number 4729–18). This study was approved by the Department of Health of the Hong Kong SAR Government.
Animals
5-week-old male C57BL/6J mice were purchased from the Laboratory Animal Unit, The University of Hong Kong. Mice were acclimated for 1 week prior to the start of the experiment and were monitored every day during the experimental
Survival Study
Mice were fed with 4% (w/v) of DSS water for 5 days and fed with different treatments to determine the effect on survival rate for 12 days (Fig. 1). We found that DSS had a significantly lower survival rate dropping to 10% at day 12. The fermented milk samples (LHFM and Cit_LHFM) had a strong and significant preventative effect on DSS-induced injury, increasing the survival rate to 50% and 70% at day 12, respectively.
Colonic permeability
The colonic permeability (Fig. 2) was significantly increased with DSS
Discussion
In this study, all treatments showed multiple effects in protecting against injury and inflammatory response resulting from DSS-induced colitis.
We first used a higher dosage of DSS (4%) to investigate the protective effects of the treatments in increase of survival compared to DSS. We found that LHFM and Cit_LHFM increased the survival rate. Treatment of Cit_milk altered the survival rate from 10% to 40% at day 12 compared with DSS but not statistically significant (P = 0.09). In a previous
Conclusion
The present study indicates that oral supplementation of citrulline, LH511 fermented milk and citrulline enriched LH511 fermented milk had protective effects on reducing the damage caused by DSS administration. Interestingly, treatment with Cit_milk and LHFM showed similar effects on modulating several parameters. Notably, we found that the combination of these two components had synergistic effects, and significantly superior effects on reinforcing intestinal structure and reducing
Author contributions
The author's responsibilities were as follows: S.W.H. and N.P.S. designed the study. S.W.H conducted the study and wrote the manuscript. H.E-N assisted with the RNA extraction. H.C revised the manuscript. C.S.H. assisted with the histological imaging and the immunohistochemical analysis. N.P.S edited and revised the manuscript and approved the final manuscript.
Acknowledgments
This study was supported by the University of Hong Kong. The authors are grateful to Dr. Wai Hung Sit and Miss Iris Tse for their expert assistance with animal handling.
Declaration of Competing Interest
The authors declare no competing interests.
References (54)
- et al.
The role of immunomodulators on intestinal barrier homeostasis in experimental models
Clin Nutr
(2015) - et al.
Immunomodulating effects of peptidic fractions issued from milk fermented with Lactobacillus helveticus
J Dairy Sci
(2002) - et al.
Mucosal immunomodulation by the non-bacterial fraction of milk fermented by Lactobacillus helveticus R389
Int J Food Microbiol
(2007) - et al.
Milk fermented by Lactobacillus helveticus R389 and its non-bacterial fraction confer enhanced protection against Salmonella enteritidis serovar Typhimurium infection in mice
Immunobiology
(2007) - et al.
Mechanisms and kinetics of citrulline uptake in a model of human intestinal epithelial cells
Clin Nutr
(2008) - et al.
Arginine, citrulline and ornithine metabolism by lactic acid bacteria from wine
Int J Food Microbiol
(1999) - et al.
Effect of Lactobacillus plantarum and L-arginine against endotoxin-induced liver injury in a rat model
Life Sci
(2011) - et al.
Effects of supplementation of citrulline and Lactobacillus helveticus ASCC 511 on intestinal epithelial cell integrity
J Funct Foods
(2020) - et al.
A cheese-containing diet modulates immune responses and alleviates dextran sodium sulfate-induced colitis in mice
J Dairy Sci
(2012) - et al.
Induction of nitric-oxide metabolism in enterocytes alleviates colitis and inflammation-associated colon cancer
Cell Rep
(2018)
Oral administration of Lactobacillus plantarum K68 ameliorates DSS-induced ulcerative colitis in BALB/c mice via the anti-inflammatory and immunomodulatory activities
Int Immunopharmacol
Lactobacillus plantarum TWK10 supplementation improves exercise performance and increases muscle mass in mice
Nutrients
Evaluation of chemical mediators and cellular response during acute and chronic gut inflammatory response induced by dextran sodium sulfate in mice
Biochem Pharmacol
Citrulline: a potential immunomodulator in sepsis
Surgery
The tight junction in inflammatory disease: communication breakdown
Curr Opin Pharmacol
Effects of lactobacilli with different regulatory behaviours on tight junctions in mice with dextran sodium sulphate-induced colitis
J Funct Foods
Nitric oxide supplementation ameliorates dextran sulfate sodium-induced colitis in mice
Labo Invest; J Tech methods pathol
Increasing incidence and prevalence of the inflammatory bowel diseases with time, based on systematic review
Gastroenterology
Uridine ameliorates dextran sulfate sodium (DSS)-induced colitis in mice
Sci Rep
Understanding and preventing the global increase of inflammatory bowel disease
Gastroenterology
Systematic review of randomized controlled trials of probiotics, prebiotics, and synbiotics in inflammatory bowel disease
Clin Exp Gastroenterol
On the use of herbal medicines in management of inflammatory bowel diseases: a systematic review of animal and human studies
Dig Dis Sci
Maintenance of remission in inflammatory bowel disease using omega-3 fatty acids (fish oil): a systematic review and meta-analyses
Inflamm Bowel Dis
Mucosal healing in inflammatory bowel diseases: a systematic review
Gut
In vitro antibacterial activity of lactobacillus helveticus strain KS300 against diarrhoeagenic, uropathogenic and vaginosis-associated bacteria
J Appl Microbiol
Single probiotic supplement suppresses colitis-associated colorectal tumorigenesis by modulating inflammatory development and microbial homeostasis
J Gastroenterol Hepatol
Health-promoting properties of lactobacillus helveticus
Front Microbiol
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