Nutr Res Pract.  2017 Feb;11(1):11-16. 10.4162/nrp.2017.11.1.11.

Anti-bacterial effects of enzymatically-isolated sialic acid from glycomacropeptide in a Helicobacter pylori-infected murine model

• Noh HJ
• Koh HB
• Kim HK
• Cho HH
• Lee J

• KMID: 2395288
• DOI: http://doi.org/10.4162/nrp.2017.11.1.11

Abstract

BACKGROUND/OBJECTIVES
Helicobacter pylori (H. pylori) colonization of the stomach mucosa and duodenum is the major cause of acute and chronic gastroduodenal pathology in humans. Efforts to find effective anti-bacterial strategies against H. pylori for the non-antibiotic control of H. pylori infection are urgently required. In this study, we used whey to prepare glycomacropeptide (GMP), from which sialic acid (G-SA) was enzymatically isolated. We investigated the anti-bacterial effects of G-SA against H. pylori in vitro and in an H. pylori-infected murine model.
MATERIALS/METHODS
The anti-bacterial activity of G-SA was measured in vitro using the macrodilution method, and interleukin-8 (IL-8) production was measured in H. pylori and AGS cell co-cultures by ELISA. For in vivo study, G-SA 5 g/kg body weight (bw)/day and H. pylori were administered to mice three times over one week. After one week, G-SA 5 g/kg bw/day alone was administered every day for one week. Tumor necrosis factor-α (TNF-α), IL-1β, IL-6, and IL-10 levels were measured by ELISA to determine the anti-inflammatory effects of G-SA. In addition, real-time PCR was performed to measure the genetic expression of cytotoxin-associated gene A (cagA).
RESULTS
G-SA inhibited the growth of H. pylori and suppressed IL-8 production in H. pylori and in AGS cell co-cultures in vitro. In the in vivo assay, administration of G-SA reduced levels of IL-1β and IL-6 pro-inflammatory cytokines whereas IL-10 level increased. Also, G-SA suppressed the expression of cagA in the stomach of H. pylori-infected mice.
CONCLUSION
G-SA possesses anti-H. pylori activity as well as an anti-H. pylori-induced gastric inflammatory effect in an experimental H. pylori-infected murine model. G-SA has potential as an alternative to antibiotics for the prevention of H. pylori infection and H. pylori-induced gastric disease prevention.

Keyword

Sialic acid; Helicobacter pylori; cytotoxin-associated gene A

MeSH Terms

Animals
Anti-Bacterial Agents
Body Weight
Coculture Techniques
Colon
Cytokines
Duodenum
Enzyme-Linked Immunosorbent Assay
Helicobacter pylori
Helicobacter*
Humans
In Vitro Techniques
Interleukin-10
Interleukin-6
Interleukin-8
Methods
Mice
Mucous Membrane
N-Acetylneuraminic Acid*
Necrosis
Pathology
Real-Time Polymerase Chain Reaction
Stomach
Stomach Diseases
Whey
Anti-Bacterial Agents
Cytokines
Interleukin-10
Interleukin-6
Interleukin-8
N-Acetylneuraminic Acid

Figure

• 

  Fig. 1 Effects of G-SA on AGS cell viability. Cells were incubated with various concentrations of SA for 24 h, after which cell viability was measured using MTT assay. Data are presented as growth rate of cells cultured in the presence of SA compared to the percentage of untreated control cells, taken as 100%. Data are expressed as mean ± SD (n = 3). Significant differences were determined using Duncan's multiple range test at P < 0.05. S-SA, synthetic sialic acid; G-SA, enzymatically isolated sialic acid from GMP.

• 

  Fig. 2 The anti-bacterial activity of G-SA was evaluated by the macrodilution method, and the results are shown on a logarithmic scale. The experiments were carried out using three different SA concentrations (0.1%, 0.25%, 0.5%). Data are expressed as mean ± SD (n = 8). Significant differences were determined using Duncan's multiple range test at P < 0.05. S-SA, synthetic sialic acid; G-SA, enzymatically isolated sialic acid from GMP.

• 

  Fig. 3 Effect of G-SA on H. pylori adhesion to host human epithelial cells AGS cells were infected with H. pylori SS1 inocula exposed to SA for (A) 2 h, (B) 4 h, and (C) 6 h at a multiplicity of infection (MOI) of 100:1 (H. pylori:AGS). Supernatants from H. pylori infected-AGS cells were collected and used to determine IL-8 concentration as evaluated by ELISA. Data are expressed as mean ± SD (n = 3). Significant differences were determined using Duncan's multiple range test atP < 0.05. # P < 0.05 compared with unexposed cells. *P < 0.05 compared with H. pylori inoculation control. S-SA, synthetic sialic acid; G-SA, enzymatically isolated sialic acid from GMP.

• 

  Fig. 4 Effects of G-SA on pro-inflammatory cytokine levels in the serum of H. pylori-infected mice (A) TNF-α, (B) IL-1β, (C) IL-6, and (D) IL-10 concentrations were determined by enzyme-linked immunosorbent assay (ELISA). Normal control (NC), no infection; Sham, experimental H. pylori infection; S-SA, experimental H. pylori infection + synthetic sialic acid (5 g/kg bw/day); G-SA, experimentally H. pylori infection + enzymatically isolated sialic acid from GMP (5 g/kg bw/day). Data are expressed as mean ± SD (n = 8). Significant differences were determined using Duncan's multiple range test at P < 0.05. #P <0.05 compared with NC group. *P < 0.05 compared with sham group.

• 

  Fig. 5 Effect of G-SA on cagA expression in H. pylori-infected gastric tissue. The mRNA level of cagA was determined by real-time PCR. NC, normal control, no infection; Sham, experimental H. pylori infection; S-SA, experimental H. pylori infection + synthetic sialic acid (5 g/kg bw/day); G-SA, experimentally H. pylori infection + enzymatically isolated sialic acid from GMP (5 g/kg bw/day). Data are expressed as mean ± SD (n = 8). Significant differences were determined using Duncan's multiple range test at P < 0.05. # P < 0.05 compared with NC group. * P < 0.05 compared with sham group.

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