Extracts from Myrtle Liqueur Processing Waste Modulate Stem Cells Pluripotency under Stressing Conditions

Abstract

Nutraceuticals present in food are molecules able to exert biological activity for the prevention and treatment of various diseases, in form of pharmaceutical preparations, such as capsules, cream, or pills. Myrtus communis L. is a spontaneous Mediterranean evergreen shrub, widely known for the liqueur obtained from its berries rich in phytochemicals such as tannins and flavonoids. In the present study, we aimed to evaluate the properties of myrtle byproducts, residual of the industrial liqueur processing, in Adipose-derived stem cells (ADSCs) induced at oxidative stress by in vitro H₂O₂ treatment. Cells were exposed for 12-24 and 48h at treatment with extracts and then senescence-induced. ROS production was then determined. The real-time PCR was performed to evaluate the expression of inflammatory cytokines and sirtuin-dependent epigenetic changes, as well the modifications in terms of stem cell pluripotency. The β-galactosidase assay was conducted to analyze stem cell senescence after treatment. Our results show that industrial myrtle byproducts retain a high antioxidant and antisenescence activity, protecting cells from oxidative stress damages. The results obtained suggest that residues from myrtle liqueur production could be used as resource in formulation of food supplements or pharmaceutical preparations with antioxidant, antiaging, and anti-inflammatory activity.

Figure 1

Expression of proinflammatory cytokines Il-6 and TNF-α. The expression of Interleukin 6 (IL-6) (a) and Tumor necrosis factor alpha (TNF-α) (b) was evaluated in H₂O₂-senescent ADSCs exposed for 12, 24, or 48h to ascorbic acid (CTRL+, blue bar) or to Lab by-P (yellow bar) or Ind by-P (red bar). The mRNA levels for each gene were expressed as fold of change (2−∆∆Ct) of mRNA levels observed in untreated ADSCs (CTRL-, black bar) defined as 1 (mean ±SD; n=6) and normalized to Glyceraldehyde-3-Phosphate-Dehidrogenase (GAPDH). Data are represented as mean± SD referring to the control (∗  p ≤ 0.05).

Figure 2

Measuring nitric oxide production after oxidative stress induction. The NO concentration was evaluated in ADSCs exposed for 12, 24, or 48h to ascorbic acid (CTRL+, blue bar), at Lab by-P (yellow bar), or at Ind by-P (red bar) and then induced to oxidative stress, compared to untreated H₂O₂-senescent cells (CTRL-, black bar). The nitrite concentrations were read as the absorbance at 548 nm for each sample and were expressed as mean± SD referring to the control (∗  p ≤ 0.05).

Figure 3

Expression of pluripotency related genes. The expression of octamer-binding transcription factor 4 (Oct-4) (a). Sex determining region Y-box 2 (Sox2) (b) and Homeobox protein Nanog (NANOG) (c) were evaluated in H₂O₂-senescent ADSCs exposed for 12, 24, or 48h to ascorbic acid (CTRL+, blue bar), at Lab by-P (yellow bar), or at Ind by-P (red bar). The mRNA levels for each gene were expressed as fold of change (2−∆∆Ct) of mRNA levels observed in untreated ADSCs (CTRL-, black bar) defined as 1 (mean ±SD; n=6) and normalized to Glyceraldehyde-3-Phosphate-Dehidrogenase (GAPDH). Data are represented as mean± SD referring to the control (∗  p ≤ 0.05).

Figure 4

Expression of Sirtuins and Heat Shock Proteins in ADSCs induced to oxidative stress. The expression of NAD-dependent deacetylase sirtuin-1 (SIRT1) (a) and Heat Shock Protein 90b (Hsp90B) (b) was evaluated in H₂O₂-senescent ADSCs exposed for 12, 24, or 48h to ascorbic acid (CTRL+, blue bar), to Lab by-P (yellow bar), or to Ind by-P (red bar). The mRNA levels for each gene were expressed as fold of change (2−∆∆Ct) of mRNA levels observed in untreated ADSCs (CTRL-, black bar) defined as 1 (mean ±SD; n=6) and normalized to Glyceraldehyde-3-Phosphate-Dehidrogenase (GAPDH). Data are represented as mean± SD referring to the control (∗  p ≤ 0.05).

Figure 5

Senescence-associated β-galactosidase activity. (a) β-galactosidase was evaluated in H₂O₂-senescent ADSCs treated with ascorbic acid (CTRL+), with Lab by-P, or with Ind by-P for 12, 24, or 48h, compared to untreated ADSCs (CTRL-). Scale bar=100 μm. (b) The numbers of positive (blue) and negative cells were counted under the light microscope and the percentage of SA-β-Gal-positive cells for each treatment was calculated as the number of positive cells divided by the total number of cells counted using an image software analysis (ImageJ). Data are expressed as mean± SD referring to the control (∗  p ≤ 0.05).

Figure 6

Mtt assay of the ADSCs treated with Myrtus extracts related to the untreated cells (grey bar). Cell Viability = {OD570 of treated cells} × 100%/{OD570 of control cells, considered as 100}. The data were entered using SPSS Version 2.0 (IBM SPSS, 2013). Data are expressed as mean± SD referring to the control (∗  p ≤ 0.05).