Antioxidant and Anti-Inflammaging Ability of Prune (Prunus Spinosa L.) Extract Result in Improved Wound Healing Efficacy

Abstract

Prunus spinosa L. fruit (PSF) ethanol extract, showing a peculiar content of biologically active molecules (polyphenols), was investigated for its wound healing capacity, a typical feature that declines during aging and is negatively affected by the persistence of inflammation and oxidative stress. To this aim, first, PSF anti-inflammatory properties were tested on young and senescent LPS-treated human umbilical vein endothelial cells (HUVECs). As a result, PSF treatment increased miR-146a and decreased IRAK-1 and IL-6 expression levels. In addition, the PSF antioxidant effect was validated in vitro with DPPH assay and confirmed by in vivo treatments in C. elegans. Our findings showed beneficial effects on worms' lifespan and healthspan with positive outcomes on longevity markers (i.e., miR-124 upregulation and miR-39 downregulation) as well. The PSF effect on wound healing was tested using the same cells and experimental conditions employed to investigate PSF antioxidant and anti-inflammaging ability. PSF treatment resulted in a significant improvement of wound healing closure (ca. 70%), through cell migration, both in young and older cells, associated to a downregulation of inflammation markers. In conclusion, PSF extract antioxidant and anti-inflammaging abilities result in improved wound healing capacity, thus suggesting that PSF might be helpful to improve the quality of life for its beneficial health effects.

Keywords: C. elegans; HUVEC; MicroRNA; aging phenotype; biological aging; cell migration; lifespan; polyphenols; tissue regeneration.

Figure 1

DPPH analysis of P. spinosa extract antioxidant activity was carried out at different P. spinosa extract (PSF) concentrations (from 12.5 to 100 µg/mL). DPPH inhibition (I, expressed in %) was used to evaluate PSF antioxidant activity. Linear regression analysis (R² = 0.982) shows an excellent concentration-dependent effect. P. spinosa exerts an antioxidant activity with an EC50 = 64.2 µg/mL (calculated (given y = 50) as: 50 = 0.502x + 17.906).

Figure 2

Anti-inflammatory effect of P. spinosa extract on IL-6, IRAK-1, and miR-146a expression levels in LPS (lipopolysaccharide, 1 µg/mL) stimulated young (P3) and older (P15) human umbilical vein endothelial cells (HUVECs) after 6 h of treatment. LPS stimulus downregulated miR-146a (LPS). When treated with P. spinosa extract during LPS stimulation (20/40/80 µg/mL P. spinosa + LPS), cells showed miR-146a upregulation with a decreased expression of both IRAK-1 and IL-6. Results are reported as fold change related to CTRL. Two-tailed paired Student’s t-test: * p < 0.05 LPS vs. P. spinosa + LPS; ** p < 0.01 LPS vs. P. spinosa + LPS; ° p < 0.05 LPS vs. CTRL; °° p < 0.05 LPS vs. CTRL.

Figure 3

High concentrations (400 µg/mL) of Prunus extract extend lifespan and healthspan in C. elegans. (A) Kaplan–Meier survival curves of C. elegans treated with the indicated concentrations of Prunus extract showing extended lifespan at the highest concentration. (B) Kaplan–Meier survival curves of C. elegans treated with the indicated concentrations of Prunus extract showing extended healthspan at the highest concentration. For 3A and B panels, pooled data of 180 worms/condition in three independent replicates are shown. Bonferroni p-value ETOH vs. 400 µg/mL 1.2 × 10⁻⁷ in panel A. Bonferroni p-value ETOH vs. 400 µg/mL 0.0007 in panel B. Statistical test: Log Rank test.

Figure 4

High concentrations of Prunus extract protect from H₂O₂-induced oxidative stress in C. elegans. (A) The pharyngeal pumping rate has been measured by counting the number of times the terminal bulb of the pharynx contracted over a 1-min interval (pump/min). Data are shown as mean pumps/min ± SE (n = 50 worms/assay, 3 assays). *** p < 0.001 and **** p < 0.0001, according to two-way ANOVA and Bonferroni’s post hoc test. °°°° p < 0.0001 vs. H₂O₂-fed worms and grown on ETOH plates, according to two-way ANOVA and Bonferroni’s post hoc test. Interaction < 0.0001. (B) The locomotor activity has been evaluated by measuring the number of body bends in liquid over a 1-min interval (body bends/min). Data are shown as mean pumps/min ± SE (n = 40 worms/assay, 3 assays). * p < 0.05, ****p < 0.0001, according to two-way ANOVA and Bonferroni’s post hoc test. °°°° p < 0.0001 vs. H₂O₂-fed worms and grown on ETOH plates, according to two-way ANOVA and Bonferroni’s post hoc test. Interaction < 0.0001. (C) MicroRNAs (miR-124 and miR-39) expression levels in P. spinosa treated C. elegans worms. Treatment of C. elegans from embryos with 400 µg/mL of PSF lead to miR-124 upregulation and miR-39 downregulation in the adults. Two-tailed paired Student’s t-test: * p < 0.05 vs. CTRL; ** p < 0.01 vs. CTRL.

Figure 5

Wound healing (in % wound closure) in P. spinosa fruit extract (PSF) treated cells and expression levels of inflammation markers assessed in the same cells. Experimental conditions were the same as those employed for anti-inflammaging assays. (A) Percentage of wound healing closure related to CTRL in young (P3) and senescent (P15) HUVECs after 48 h of P. spinosa treatment. Our data showed, both in young and older cells, a significant improvement (up to 70%) of wound healing closure (expressed in percentage related to untreated cells) through cell migration. The same cells were detached and used for the evaluation of expression levels of anti-inflammatory markers, IL-6 (B), IRAK-1 (C), and miR-146a (D). MiR-146a upregulation was observed during PSF extract treatment (20/40/80 µg/mL P. spinosa). PSF extract treatment caused a decreased expression of both IRAK-1 and IL-6, thus revealing a downregulation of the inflammatory response. Results are reported as fold change related to CTRL. Two-tailed paired Student’s t-test: *p < 0.05 vs. CTRL; **p < 0.01 vs. CTRL.

Figure 6

Senescence of HUVECs treated with P. spinosa at different concentrations (20, 40, and 80 µg/mL) during 160 days (P2-P15). (A) Growth curve showing the cumulative population doubling level (CPDL) during P. spinosa treatment (P2-P15). (B) Percentage of senescent HUVECs (P15) as a fraction of cells expressing SA-β-Gal. (C), Cellular morphology of P15 senescent HUVECs after 160-day P. spinosa treatment (Optical microscopy, 10x). The samples CTRL, ETOH and 20 µg/mL treated cells showed large cobblestone shaped cells, while cells treated with 40 µg/mL and 80 µg/mL P. spinosa extract showed long cells (spindle-shaped) with multiple protrusions (connecting neighboring cell clusters).

Figure 7

Anti-inflammatory effect of P. spinosa during HUVEC senescence (observed at P2, P5, and P15). Untreated (i.e., without any additional triggers) CTRL old cells showed increased IL-6/IRAK-1 expressions and miR-146a downregulation. Notably, in older cells, P. spinosa treatments (20/40/80 µg/mL) decreased IL-6/IRAK-1 expressions and upregulated miR-146a, showing a modulation of miR-146a expression level and IL-6 and IRAK-1 amounts comparable to what was observed in younger cells. The relative expression has been determined using the 2^−ΔCt method and is expressed as fold change related to the TATA binding protein (TBP) endogenous control.

Figure 8

PSF extract delays biological age by acting as an antioxidant and anti-inflammatory agent. Beneficial effects promote in vitro endothelial cell functions (wound healing) and in vivo lifespan/healthspan of C. elegans.