Anti-SASP and anti-inflammatory activity of resveratrol, curcumin and β-caryophyllene association on human endothelial and monocytic cells

Abstract

A challenging and promising new branch of aging-related research fields is the identification of natural compounds able to modulate the senescence-associated secretory phenotype (SASP), which characterizes senescent cells and can contribute to fuel the inflammaging. We investigated both the anti-SASP and anti-inflammatory activities of a nutritional supplement, namely Fenoxidol™, composed of turmeric extract bioCurcumin (bCUR), Polydatin (the natural glycosylated precursor of Resveratrol-RSV), and liposomal β-caryophyllene (BCP), in two human cellular models, such as the primary endothelial cell line, HUVECs and the monocytic cell line, THP-1. Replicative and Doxorubicin-induced senescent HUVECs, both chosen as cellular models of SASP, and lipopolysaccharides (LPS)-stimulated THP-1, selected as a model of the inflammatory response, were treated with the three single natural compounds or with a combination of them (MIX). In both senescent HUVEC models, MIX treatment significantly reduced IL-1β and IL-6 expression levels and p16^ink4a protein, and also increased SIRT1 protein level, as well as downregulated miR-146a and miR-21 expression, two of the so-called inflamma-miRNAs, more effectively than the single compounds. In THP-1 cells stimulated with LPS, the MIX showed a significant effect in decreasing IL-1β, IL-6, TNF-α, and miR-146a expression levels and Caspase-1 activation, in association with an up-regulation of SIRT1 protein, compared to the single compounds. Overall, our results suggest that the three analysed compounds can have a combined effect in restraining SASP in senescent HUVECs as well as the inflammatory response in LPS-stimulated THP-1 cells.

Keywords: Inflammaging; Nutraceuticals; Polyphenols; SASP; Senescence.

Fig. 1

a The main molecules of bioCurcumin b HPLC–DAD chromatogram with the peaks of ar-turmerone (i) at 240 nm, bisdemethoxycurcumin (ii), demethoxycurcumin (iii) and curcumin (iv) at 430 nm

Fig. 2

Effect of bCUR, RSV, BCP and MIX on yHUVECs (a–d) and THP-1 cells (e–h). Cells were treated with different concentrations of bioCurcumin (from 0.5 to 5 µg/ml), Resveratrol (from 0.5 to 10 µg/ml), β‐caryophyllene (from 1.5 to 40 µg/ml) and MIX for 3 h. The viability of cells was determined by MTT assay. The results are expressed as a percentage of cell viability normalized to the viability of DMSO treated cells (CTR) and presented as mean value ± SEM from three independent biological experiments. bCUR bioCurcumin, RSV resveratrol, BCP β‐caryophyllene

Fig. 3

Characterization of endothelial cells in RS- and IS-HUVEC models. a Growth curve of a pool of HUVECs—Y-axis: Cumulative Population Doubling (cPD); X-axis: cell passages from P1 to P22 (i); two representative pictures of SA-β-Gal staining of young (ii) and senescent (iii) HUVECs; % of SA- β-Gal (iv). Cells with SA-β-Gal < 10% were considered young cells (yHUVEC), while those with SA- β-Gal > 60% were identified as senescent cells; b representative picture of SA- β-Gal-positive cells (i), dose–response curve (ii) and % of SA- β-Gal (iii) after treatment with 5 nM (left image, 50 nM (central image) and 100 nM (right image) of Doxorubicin; c relative expression of p16^ink4a mRNA in yHUVEC (P4), RS-HUVECs (P21) and IS-HUVECs (Doxorubicin 50 nM); d densitometric analysis of p16^ink4a protein level in RS-HUVEC and IS-HUVEC. Histograms represent the mean of the protein level and the relative expression measured in three different experiments ± SD. Paired t test, *p < 0.05 versus yHUVEC. RS replicative senescence, IS induced senescence

Fig. 4

mRNA and miRNA expression, IL-6 and IL-1β concentration after 3 h of treatment with bCUR, RSV and BCP as single compound and in combination in RS- and IS-HUVECs. a and d mRNA expression of IL-1β and IL-6 in RS- and IS-HUVECs; b and e concentration (pg/ml) of IL-1β and IL-6 in the culture medium of RS- and IS-HUVECs; c and f miRNA expression of miR-146a, miR-126 and miR-21 in RS- and IS-HUVEC. Data are reported as fold change vs untreated senescence HUVECs according to 2-ΔCt method and using β -actin and RNU48 (respectively for mRNA and miRNA) as housekeeping; histograms represent the mean of the fold change detected in three different experiments ± SD. Paired t test, *p < 0.05 versus RS- and IS-HUVECs; **p < 0.01 versus RS- and IS-HUVECs. bCUR bioCurcumin, RSV resveratrol, BCP β‐caryophyllene, RS replicative senescence, IS induced senescence. RS-HUVECs data are depicted in the left panel (solid-coloured histograms); IS-HUVECs data are reported in the right panel (striped histograms)

Fig. 5

p16^ink4a and SIRT1 protein level after treatment with bCUR, RSV and BCP and their combination in RS- and IS-HUVECs. a and c p16^ink4a protein level and densitometric analysis; b and d SIRT1 protein level and densitometric analysis. Data are reported as fold change vs untreated senescent HUVECs. All data were normalized using α-tubulin as internal control. Bands were quantified by ImageJ; histograms represent the mean of the protein expression detected in three different experiments ± SD. Paired t test *p < 0.05 versus RS- and IS-HUVECs; **p < 0.01 versus RS- and IS-HUVECs. bCUR, bioCurcumin; RSV, resveratrol; BCP, β‐caryophyllene; RS, replicative senescence; IS, induced senescence. RS-HUVECs data are depicted in the left panel (solid-coloured histograms); IS-HUVECs data are reported in the right panel (striped histograms)

Fig. 6

Anti-inflammatory activity of bCUR, RSV and BCP as single compound and in combination in LPS-stimulated THP-1 cells a IL-1β, IL-6 and TNFα mRNA expression. Data were reported as fold change vs untreated LPS-stimulated THP1 cells according to 2-ΔCt method and using GAPDH as housekeeping b Concentration (pg/ml) of IL-1β in the culture medium of untreated and treated THP-1 cells; c miR-146a and miR-21 expression. Data were reported as fold change vs untreated LPS-stimulated THP-1 cells according to 2^−ΔCt method and using RNU48 as housekeeping. d and e SIRT1 and Caspase-1/pro-Caspase-1 protein level and densitometric analysis. Data were normalized using α-tubulin as internal control and reported as fold change versus LPS-stimulated THP-1 cells untreated with natural compounds. Bands were quantified by ImageJ; histograms represent the mean of the protein expression detected in three different experiments ± SD. Paired t test, *p < 0.05 versus THP-1 + LPS; **p < 0.01 versus THP-1 + LPS. LPS lipopolysaccharide, bCUR bioCurcumin, RSV resveratrol, BCP β‐caryophyllene