Docosahexaenoic acid inhibits Helicobacter pylori growth in vitro and mice gastric mucosa colonization

Abstract

H. pylori drug-resistant strains and non-compliance to therapy are the major causes of H. pylori eradication failure. For some bacterial species it has been demonstrated that fatty acids have a growth inhibitory effect. Our main aim was to assess the ability of docosahexaenoic acid (DHA) to inhibit H. pylori growth both in vitro and in a mouse model. The effectiveness of standard therapy (ST) in combination with DHA on H. pylori eradication and recurrence prevention success was also investigated. The effects of DHA on H. pylori growth were analyzed in an in vitro dose-response study and n in vivo model. We analized the ability of H. pylori to colonize mice gastric mucosa following DHA, ST or a combination of both treatments. Our data demonstrate that DHA decreases H. pylori growth in vitro in a dose-dependent manner. Furthermore, DHA inhibits H. pylori gastric colonization in vivo as well as decreases mouse gastric mucosa inflammation. Addition of DHA to ST was also associated with lower H. pylori infection recurrence in the mouse model. In conclusion, DHA is an inhibitor of H. pylori growth and its ability to colonize mouse stomach. DHA treatment is also associated with a lower recurrence of H. pylori infection in combination with ST. These observations pave the way to consider DHA as an adjunct agent in H. pylori eradication treatment.

Figure 1. DHA effect on H. pylori growth.

Growth of H. pylori strains A) 26695, B) SS1 and C) B128 during 48 hours in the presence of increasing concentrations of DHA from 50 to 1000 µM. Ethanol 0.06% v/v was used as a vehicle in DHA original stock solution, and therefore the presence of ethanol at the same concentration was also analyzed on H. pylori control culture with no effect on bacterial growth for the three strains. Data are expressed as the mean ± Standard Deviation and are representative of three independent experiments. * Refers to significant differences in H. pylori growth between controls and DHA-treated conditions (50 µM to 1000 µM of DHA).

Figure 2. Alteration of H. pylori morphology upon DHA treatment.

H. pylori strains A) 26695 and B) SS1 cultured for 12 hours incubated with 100 µM of DHA show changes in cell shape as observed by scanning electron microscopy. The presence of 100 µM of DHA leads to spherical/coccoids bacterial forms for both strains.

Figure 3. Inhibition of H. pylori SS1 gastric colonization by DHA treatment and a combination of standard antibiotherapy (ST) and DHA in C57BL/6 mice.

A) Analysis of H. pylori SS1 gastric colonization inhibition by DHA treatment in C57BL/6 mouse over time. Four groups each encompassing six mice: one group of non-treated, non-infected controls; one group of animals infected by H. pylori strain SS1; one group of mice supplemented with 50 µM of DHA in the drinking water; and one group infected by H. pylori strain SS1 and supplemented with 50 µM of DHA in the drinking water as described in materials and methods. The non-infected control group and DHA treated mice are not colonized and are not reported in the figure. In each condition mice were sacrificed after one, three, six and nine months. Median values of gastric colonization between H. pylori infected mice non-DHA treated and DHA treated were 18.36×10⁵ vs 0.0049×10⁵, 8.24×10⁵ vs 0.069×10⁵, 15.71×10⁵ vs 0.0096×10⁵ and 27.45×10⁵ vs 0.034×10⁵ cfu/g of gastric tissue after 1, 3, 6 and 9 months, respectively. * P<0.05; **P<0.005. B) Analysis of the effect of the Standard antibiotics Therapy (ST) on the level of gastric mucosa colonization by H. pylori infection compared with DHA treatment and DHA in combination with ST in mice. Four groups of mice, each encompassing six (n = 6) were infected by H. pylori strain SS1. Control group (n = 4) was given peptone trypsin broth alone. As described in materials and methods, three therapeutic options were given to mice: ST p.o. for 7 days, DHA addition to mice drinking day water for 15 days, or a combination of both ST for 7 days, and at the same time DHA for 15 days. All treatments were administered at 4 weeks post H. pylori infection and gastric colonization measured at 6 and 14 weeks post infection. * P<0.05; **P<0.01. C) Number of mice colonized and not colonized by H. pylori under the different conditions of treatment described above. Open bars represent the number of mice infected by H. pylori (Hp⁺), whereas grey bars represent the number of non-infected mice (Hp⁻). The Kruskal-Wallis test was used to assess the consequences on H. pylori mouse gastric colonization of DHA treatment. Our results show a significant (P = 0.0036) reduced rate of recurrence of H. pylori infection in animals supplemented with DHA.

Figure 4. Anti-inflammatory effects of DHA in the infected gastric mucosa of mice.

A) Histological analysis of inflammatory lesions of the gastric mucosa of H. pylori infected or non-infected mice treated or not with DHA 50 µM after 6 and 9 months. No differences were observed between non-infected mice DHA-treated or non-treated. In infected mice, the DHA treatment in infected mice leads to a decrease of the gastric mucosa thickness compared to H. pylori infected mice but non-DHA treated at both time-points. Infiltrates of polymorphonuclear (PMN) cells and plasmocytes as well as number of lymphoid aggregates (arrows) were lower in H. pylori infected mice treated with DHA compared to infected mice non-treated. The infection leads to the formation of lymphoid aggregate (arrows) not observed in mice treated with DHA. Scale bars correspond to 100 µm. B) Semi-quantification of inflammation score grading in the gastric mucosa. At each time-point, 6 and 9 months, mean score grading for each group of mice are higher in the antrum than fundus part. The presence of DHA leads to a significant decrease of the antrum inflammation of the H. pylori infected gastric mucosa. PMN: Polymorphonuclear cells; Linf: Lymphocytes aggregates; Submuc: Submucosa. Comparison was made between infected mice non-DHA supplemented and infected mice DHA supplemented. C) Measurement of PGE2 in the mice serum. Lower PGE2 values were observed in infected mice that received DHA 50 µM compared to infected mice non-DHA treated (adjusted P values 0.016 at 1 month; 0.008 at 3 months; 0.054 at 6 months; 0.012 at 9 months). PGE2 levels were lower at 6 months in DHA-treated mice compared to untreated mice, even though the difference cannot be considered statistically significant. Control group corresponds to non-infected and non-DHA treated mice. Bars represent means with standard deviation.