Protective effect of snail secretion filtrate against ethanol-induced gastric ulcer in mice

Abstract

Gastric ulcer or peptic ulcer is a common disease worldwide. Basically, it develops when there is an imbalance between the protective and aggressive factors, especially at the luminal surface of epithelial cells. Thus, there is a constant interest in research new drugs for treatment of gastric ulcer. The snail secretion is a dense mucous, that covers the external surface of the snails, with important functions for the survival of snails. The biological proprieties of snail Helix Aspersa Muller mucus it has been known for centuries to treat human disorders in particular for skin disease. Recently the use of snail mucus has seen a worldwide increase, as a component in cosmetic product and it has been used in particular for the management of wound and skin disorders. In this study we use a murine model of ethanol intragastric administration which has been widely used to test the drugs efficacies and to explore the underlying mechanism for gastric ulcer development. The intragastric ethanol administration causes several mucosal damages and an induction of a severe inflammatory response. Our results show a significant protective effect of snail secretion filtrate in reducing macroscopic and histological lesions, as well the protective effect on mucus content, oxidative stress and inflammatory response. In conclusion this study demonstrate the protective effect of intragastrical snail secretion filtrate, in a model of ethanol-induced gastric ulcer in mice, suggesting its possible useful use in the treatment or prevention of gastric ulcer.

Figure 1

Qualitative and quantitative analysis of crude SSF. Full chemical characterization are shown in supplementary figure S1-S7.

Figure 2

The macroscopic picture of stomach from different experimental groups. stomachs from EtOH group (the negative control group) showed a significant presence in mucosa hyperemia and mucosal damage with large ulcer formation, instead the treatment with omeprazole at dose of 20 mg/kg significant prevent the ulceration and mucosal damage induced by EtOH. The treatment with SSF showed a dose dependent protective effect. Data are presented as means ± SEM, or median with interquartile range for non-parametric data of 10 mice for each group .***p < 0.001 versus control; ◦p < 0.05 versus EtOH; ◦◦◦p < 0.001 versus EtOH; ###p < 0.001 versus omeprazole.

Figure 3

Stomach tissue section from: Control group healthy mice; EtOH group negative control; omeprazole 20 mg/kg positive control group; Histological section was assessed for: a) epithelial cell loss; b) edema in the upper mucosa; c) hemorrhagic damage; d) presence of inflammatory cells. The treatment with SSF showed a dose dependent protective effect SSF 3 ml/kg by os; SSF 7.5 ml/kg by os; SSF 15 ml/kg by os. Panel b showed the resuts of MPO assay. Data are presented as means ± SEM, or median with interquartile range for non-parametric data of 10 mice for each group.***p < 0.001 versus control; ◦p < 0.05 versus EtOH; ◦◦p < 0.01 versus EtOH; ◦◦◦p < 0.001 versus EtOH.

Figure 4

PAS staining from control group showed the normal contents of mucosa glycoprotein(magenta color); EtOH group showed a significantly reduction in PAS staining, significantly inhibited by treatment wit omeprazole 20 mg/Kg; SSF treatment showed a dose dependent protective effect as showed by the increase of PAS staining. Image J software (1.49v, https://imagej.nih.gov/ij/, 1997–2018. Schneider, C.A., Rasband, W.S., Eliceiri, K.W.) was used for the quantification of glycoprotein as the positively stained area (pixel/field). Data are presented as means ± SEM of 10 mice for each group.***p < 0.001 versus control; ◦◦◦p < 0.001 versus EtOH.

Figure 5

Representative image of collagen within mucosa layer using Masson’s trichrome staining. Control group showed the staining in healthy gastric tissue. EtOH group stain indicated fragmented and disorganized collagen fibers, while omeprazole group showed a significative protective effect. For the SSF treatment only the dose of 15 ml/Kg showed a significant protective effect.

Figure 6

(a) The graph showed the mucus contents evaluation performed by alcian blue binding assay. (b) MDA assay levels. (c, d) CAT and SOD determination. Data are presented as means ± SEM of 10 mice for each group; .***p < 0.001 versus control; ◦◦p < 0.01 versus EtOH; ◦◦◦p < 0.001 versus EtOH;

Figure 7

ELISA assays for (a) PEG₂; (b) IL-6; (c) IL-1β; (d) TNF-α . Data are presented as means ± SEM of 10 mice for each group; ***p < 0.001 versus control; ◦◦p < 0.01 versus EtOH; ◦◦◦p < 0.001 versus EtOH;