HelixComplex snail mucus exhibits pro-survival, proliferative and pro-migration effects on mammalian fibroblasts

Abstract

Snail mucus is a mixture of active substances commonly thought to have healthy properties for the treatment of skin disorders. Although snail mucus is an ingredient of several cosmetic and para-pharmaceutic products, a comprehensive characterization of chemical composition and biological effects is still missing. Crude purified extracts from Helix aspersa muller mucus (HelixComplex) were prepared and, after chemical characterization, tested on in vitro experimental models. Differently from what expected, HelixComplex was characterized by the presence of small amounts of glycolic acid and allantoin. By using different in vitro assays on fibroblast cultures, we found that HelixComplex lacked of cytotoxicity, protected cells from apoptosis (p < 0.05) and, importantly, was able to significantly induce cell proliferation and migration through direct and indirect mechanisms. These effects were associated to morphological changes, cytoskeleton re-organization and release of cytokines. In conclusion, our findings suggest that snail mucus biological effects are attributable to cell proliferation and migration, and pave the way for further investigating snail mucus potential as therapeutic agent.

Figure 1

Chemical characterization HelixComplex. In (A) infrared spectrum from a representative a HelixComplex sample. In (B) HPLC chromatogram of allantoin and glycolic acid from a representative HelixComplex sample.

Figure 2

Lack of cell cytotoxicity by HelixComplex treatment. In (A) fibroblasts were exposed to increasing doses of HelixComplex for analysis of cell viability (from 4 µg/ml to 400 µg/ml). Left panel: cell viability, examined by MTT colorimetric assays, was calculated at 24 hours as percentage with respect to the untreated cultures (set to 100%). Right panel: cell number was monitored over time for up to 72 hours, starting from a high density cell culture. DMSO (10%) was used as positive control of cell death. In (B) apoptosis was evaluated on fibroblasts treated for 48 hours with a high dose of HelixComplex (400 µg/ml) and calculated as percentage of Annexin V/PI double positive cells on the total population for each treatment. Representative plots of apoptotic cells analyzed by flow-cytometry are shown. In (C) fibroblasts were cultured for 48 hours in medium with 2% serum with or without 400 µg/ml HelixComplex. Medium with 10% serum was used in the untreated control (Untreated). Left panel: representative immunofluorescence images of actin organization (red staining). Nuclei were colored with DAPI (blue staining). Right panel: cell surface was measured and reported in arbitrary units (AU). In (A–C) data are reported as the mean ± SD of results from at least three independent experiments. The asterisk indicates p < 0.05 respect to untreated cultures. HC: HelixComplex.

Figure 3

HelixComplex promotes cellular proliferation. Cultures of fibroblasts were exposed to high dose of HelixComplex (400 µg/ml) for up to 48 hours. In (A) representative images taken by light microscopy of monolayers of fibroblasts untreated or treated with HelixComplex or glycolic acid (0.1 mM) at 48 hours. Magnification 100x. In (B) cell distribution in the different phases of the cell cycle was calculated from the flow cytometric dot plots after BrdU/PI staining of cultures after 36 hours of treatment and was expressed as percentage of the total population. Representative flow cytometric dot plots of cell-cycle profiles are shown: the rectangles represent the cells in the different (G0/G1, S, G2/M) phases of the cell cycle and the percentage of cells in S-phase is indicated for each treatment. In (C) fibroblasts were treated with HelixComplex or glycolic acid and monitored for proliferation with the xCELLigence system. Upper panel: representative plot of Cell index of fibroblasts treated with HelixComplex or glycolic acid. Fibroblasts grown in starvation medium (0.1% serum) were used as negative control of proliferation. Dashed arrow indicates the time in which treatments were added to cell cultures. Arrow indicates the time in which Cell index was quantified. Lower panel: Cell index quantification at 16 hours after treatment (normalized relative to 0 hour) and expressed as fold of modulation with respect to untreated cultures set at 1. In (B,C) data are reported as the mean ± SD of results from at least three independent experiments. The asterisk indicates p < 0.05 respect to untreated cultures. HC: HelixComplex.

Figure 4

HelixComplex exhibits protection from apoptosis. Fibroblasts were grown in starvation medium (0.1% serum) and treated with HelixComplex (400 µg/ml). Fibroblasts grown with standard medium (10% serum) were used as negative control of apoptosis induction. The induction of apoptosis was calculated after 48 hours of treatment as percentage of Annexin V/PI double positive cells on the total population for each treatment. Data are reported as the mean ± SD of results from at least three independent experiments. The asterisk indicates p < 0.05 respect to untreated cultures. Representative plots of apoptotic cells analyzed by flow-cytometry are shown. HC: HelixComplex.

Figure 5

HelixComplex promotes cell migration and wound repair. Dynamic monitoring of fibroblasts migration in response to HelixComplex treatment (400 µg/ml) by xCELLigence and scratch assays. In (A) fibroblasts were seeded into the upper chamber and monitored for migration through the lower chamber in response to HelixComplex with the xCELLigence system. Left panel: a representative plot of Cell index (normalized to the first reading of the plate) of migrated fibroblasts is shown. Right panel: Cell index quantification at different time points after the beginning of migration (normalized to the first reading). In (B) scratch-wound healing assay. Left panel: representative images taken at the indicated time points post wounding are displayed. Right panel: quantification of wound repair at the indicated time points expressed as percentage of wound repair in comparison with the 0-hour time point. In (C) a schematic representation of the experiments performed with supernatants of HelixComplex shortly-exposed fibroblasts. In (D) scratched fibroblasts were treated with supernatants from fibroblasts cultures shortly exposed to HelixComplex (HC-Sup) and observed 24 hours after wounding. Upper panel: immunofluorescence images of actively migrating fibroblasts stained for endogenous actin (red staining). Lines show the extent of the wound closure. Scale bars: 20 μm. Bottom panel: quantification of wound repair expressed as percentage of repair in comparison with the 0-hour time point. In (E) histograms represent cytokines detectable in supernatants of fibroblast 24 hours after short-exposure to HelixComplex. In (F) fibroblasts were seeded into the upper chamber and monitored for migration through the lower chamber in response to recombinant human IL-8 (rIL-8) with the xCELLigence system; a representative plot of Cell index (normalized to the first reading of the plate) of migrated fibroblasts is shown. Data are reported as the mean ± SD of results from at least three independent experiments. The asterisk indicates p < 0.05 respect to untreated cultures. HC: HelixComplex.