Research paper
L-citrulline enriched fermented milk with Lactobacillus helveticus attenuates dextran sulfate sodium (DSS) induced colitis in mice

https://doi.org/10.1016/j.jnutbio.2021.108858Get rights and content

Abstract

Inflammatory bowel disease (IBD) is a group of chronic inflammatory gastrointestinal diseases that causes worldwide suffering. L. helveticus is a probiotic that can enhance intestinal barrier function via alleviation of excessive inflammatory response. Citrulline, a functional amino acid, has been reported to stimulate muscle synthesis and to function with a prebiotic-like action with certain Lactobacillus strains. The aim of this study was to investigate the potential synergistic effect of combining L. helveticus and citrulline on protection against damage induced by dextran sulfate sodium (DSS) in a mouse model. 6-week-old male C57BL/6J mice were fed with DSS water and randomly divided for administering with different milk treatments: 1) plain milk (control or DSS control), 2) 1% (w/v) citrulline enriched milk (Cit_milk), 3) milk fermented with L. helveticus (LHFM) and 4) DSS+milk fermented with L. helveticus with 1% (w/v) citrulline (Cit_LHFM). The treatment effects on the survival and macroscopic and microscopic signs were examined. All treatments presented different degrees of protective effects on attenuating the damages induced by DSS. All treatments reduced the body weight loss, disease activity index (DAI), histological scores, pro-inflammatory cytokine expression (IL-6, TNF-α and IFN-γ) and production (IL-4) (all P <0.05) and the tight junction (TJ) protein (zonula occluden-1 (ZO-1) expression. LHFM and Cit_LHFM improved survival rate (both at P<0.05). Particularly, Cit_LHFM showed greater effects on protecting the damages induced by DSS, especially in ameliorating colonic permeability, TJ protein (ZO-1, occludin and claudin-1) expression and distribution as well as in reducing IL-4 and IL-17 expression (all P <0.05). Our findings suggested that the combination of and citrulline had significant synergistic effect on protecting against injury from DSS-induced colitis. Therefore, citrulline enriched L. helveticus fermented milk is suggested to be a potential therapy for treating IBD.

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.

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