Safety evaluation of the antimicrobial peptide bovicin HC5 orally administered to a murine model

Abstract

Background: Bovicin HC5 is an antimicrobial peptide that shows a broad spectrum of activity and potential for biotechnological and therapeutic applications. To gain insight about the safety of bovicin HC5 application, the histological and immunostimulatory effects of orally administrated bovicin HC5 to BALB/c mice were evaluated. BALB/c mice were divided into three groups: negative control (NC group); mice given purified bovicin HC5 (Bov group); mice given ovalbumin (positive control, PC group; a murine model of enteropathy). The mice were initially pre-sensitized, and PBS, bovicin HC5 or ovalbumin were administered for 30 days by daily gavages. Histological and morphometric analysis were performed and the relative expression of cytokines was analyzed by real-time RT-PCR.

Results: The oral administration of bovicin HC5 to BALB/c mice reduced weight gain and caused alterations in the small intestine, although absorptive changes have not been detected. The number of total goblet cells and the mucopolysaccharides production were not affected by bovicin HC5 administration. A hypertrophy of Paneth cells and an increase in the number of mitotic cells were observed in Bov group, while the number of mast cells remained unaltered. Increased expression of TNF-α, INF-γ and IL-12 was observed in the small intestine upon bovicin HC5 administration.

Conclusion: Bovicin HC5 has only minor effects on intestinal permeability and did not elicit an allergenic response upon oral administration to animal models. Considering the low in vivo toxicity of bovicin HC5, it might be a good candidate for enteral applications.

Figure 1

Gain or loss of body weight in BALB/c mice during the experimental period. The gain/loss of weight is shown as percentage of the animals’ weight, which was calculated comparing the weight at the end of the experiment (day 58) to the weight at the day of the first immunization (day 0). Each bar represents the percentage of weight gain obtained from two independent experiments, with the standard deviation (SD) (N = 8 animals per group). Statistically significant differences among treatments by the Dunn’s multiple comparison test (p < 0.05) were indicated by different lowercase letters (“a” or “b”) above the error bars. (NC) negative control group; (Bov) mice treated with bovicin HC5; (PC) positive control group.

Figure 2

Concentration of β-lactoglobulin in animal sera from treatment groups. Upon an intragastrically dose of β-LG, blood was collected at the indicated time points and the levels of β-LG in mice sera were determined by FPLC. The results are shown as the average of β-LG concentration detected in a pool of animal’s sera from each experimental group (N = 8 mice per group), in two independent experiments. (NC) negative control group; (Bov) mice treated with bovicin HC5; (PC) positive control group.

Figure 3

Comparison of the total number of splenocytes among experimental groups. Data are shown as average ± SD, from two independent experiments (N = 8 mice per group). Statistically significant differences among treatments by the Dunn’s multiple comparison test (p < 0.05) were indicated by different lowercase letters (“a” or “b”) above the error bars. (NC) negative control group; (Bov) mice treated with bovicin HC5; (PC) positive control group.

Figure 4

Photomicrographs of longitudinal sections of small intestine of the experimental groups. Jejunum segments were collected and processed for optical microscopy analysis at the end of the experiment (day 58) (N = 8 mice per group). (NC), negative control group, figures A and D; (Bov) mice treated with bovicin HC5, figures B and E; (PC) positive control group, figures C and F. The sections were stained with hematoxylin and eosin (HE; left panel) or PAS/Alcian Blue (right panel). Abbreviations: L: lumen; EP: simple cuboidal epithelium; BB: brush border; V: villum; LP: lamina propria; LC: Lieberkühn crypt; Sm: submucosa; IC: inner circular muscle layer; OL: outer longitudinal muscle layer. The asterisks indicate intraepithelial lymphocytes; simple arrow indicates Paneth cells. Black arrow head indicates goblet cells PAS/AB⁺; red arrow head indicates PAS⁺ cells. Right panel – Scale bar: 100 μm; Left panel – Scale bar: 50 μm.

Figure 5

Comparison of the mucopolysaccharides production and number of total goblet cells among experimental groups. (A) PAS⁺ cells; (B) PAS/AB⁺ cells; (C) Total number of goblet cells. Data are shown as average ± SD, from two independent experiments (N = 8 mice per group). Statistically significant differences among treatments by the Dunn’s multiple comparison test (p < 0.05) were indicated by different lowercase letters (“a” or “b”) above the error bars. (NC) negative control group; (Bov) mice treated with bovicin HC5; (PC) positive control group.

Figure 6

Analysis of the Lieberkuhn glands. Size of Paneth cells (A) and number of cells in mitosis (B) at the small intestinal crypts of the experimental groups. Data are shown as average ± SD, from two independent experiments (N = 8 mice per group). Statistically significant differences among treatments by the Dunn’s multiple comparison test (p < 0.05) were indicated by different lowercase letters (“a” or “b”) above the error bars. (NC) negative control group; (Bov) mice treated with bovicin HC5; (PC) positive control group.

Figure 7

Number of mast cells in small intestine of the experimental groups. Longitudinal sections from jejunum segments were stained with toluidine blue/sodium borate (1%), and the mast cells were counted in the mucosa and submucosa. Data are shown as average ± SD, from two independent experiments (N = 8 mice per group). Statistically significant differences among treatments by the Dunn’s multiple comparison test (p < 0.05) were indicated by different lowercase letters (“a” or “b”) above the error bars. (NC) negative control group; (Bov) mice treated with bovicin HC5; (PC) positive control group.

Figure 8

Morphometric analysis of the small intestinal villi. Panel (A) and panel (B) show the height and diameter of the small intestinal villi, respectively. Data were shown as average ± SD, from two independent experiments (N = 8 mice per group). Statistically significant differences among treatments by the Dunn’s multiple comparison test (p < 0.05) were indicated by different lowercase letters (“a”, “b” or “c”) above the error bars. (NC) negative control group; (Bov) mice treated with bovicin HC5; (PC) positive control group.

Figure 9

Photomicrographs of longitudinal sections of large intestine of the experimental groups. Large intestine segments were collected and processed for optical microscopy analysis at the end of the experiment (day 58) (N = 8 mice per group). (NC), negative control group, figures A and B; (Bov) mice treated with bovicin HC5, figure C; (PC) positive control group, figure D. The sections were stained with hematoxylin and eosin (HE; figure A) or PAS/Alcian Blue (figures B-D). Abbreviations: EP: simple cuboidal epithelium; LP: lamina propria; MT: mucosal thickness; E: edema; ML: muscle layer. Red arrow head indicates goblet cells. Scale bar = 200 (figure A) or 100 μm (figures B, C and D).

Figure 10

Comparison of the mucosal thickness of the large intestine among the experimental groups. Data are shown as average ± SD, from two independent experiments (N = 8 mice per group). Statistically significant differences among treatments by the Dunn’s multiple comparison test (p < 0.05) were indicated by different lowercase letters (“a” or “b”) above the error bars. (NC) negative control group; (Bov) mice treated with bovicin HC5; (PC) positive control group.

Figure 11

Cytokine production in small intestine of five-week old female BALB/c mice treated with bovicin HC5 or ovalbumin. The relative expression of IL-12p40 (A), IFN-γ (B), IL-5 (C), IL-13 (D), TNF-α (E), TGF-β (F), IL-10 (G), IL-4 (H) and IL-17 (I) mRNA was determined by real time-PCR and calculated by reference to the β-actin in each sample, using the threshold cycle (Ct) method. Results are shown as the mean value ± SD of duplicate samples from three independent mice within the NC, Bov and PC groups. Differences among treatments were indicated by different lowercase letters and were considered statistically significant by the Bonferroni multiple comparison test (p < 0.05). (NC) negative control group; (Bov) mice treated with bovicin HC5; (PC) positive control group.