modulazione del processo infiammatorio ed aterogenico Cristian - PowerPoint PPT Presentation

Studio in vitro per valutare il ruolo di antociani e metaboliti nella modulazione del processo infiammatorio ed aterogenico Cristian Del Bo’, Mirko Marino, Patrizia Riso, Dorothy Klimis-Zacas, Marisa Porrini Cristian Del Bo’, PhD Dipartimento di Scienze per gli Alimenti la Nutrizione e l’Ambiente DeFENS – Divisione di Nutrizione Email: cristian.delbo@unimi.it

BACKGROUND • Inflammation plays an important role in the aetiology and pathogenesis of atherosclerosis and plaque formation . • Tumour necrosis factor-alpha (TNF- α) is a mediator of systemic inflammation and implicated in the pathogenesis of atherogenesis/atherosclerosis. • TNF- α induces the transcription factor NF- κ B leading to enhanced expression of intercellular adhesion molecules, vascular endothelial and fibroblast growth factors, chemokines that promote the recuitment and the adhesion of monocytes to inflamed luminal endothelium triggering the atherogenic process. Steyers et al., 2014; Int. J. Mol. Sci. 15:11324-49.

• Anthocyanins (ACNs) are a group of abundant and widely consumed flavonoids providing the red, blue, violet colors at many fruit- and vegetable-based food products. • ACNs may prevent endothelial cells dysfunction thanks to their capacity to modulate the expression and activity of several enzymes involved in nitric oxide (NO) metabolism by influencing NO levels. • Furthermore, ACNs can down- regulate the expression of adhesion molecules and prevent the adhesion of monocytes to endothelial cells challenged by pro-inflammatory agents. Speciale et al., 2014 Genes Nutr. 9:404

• The link between atherogenesis and health promoting effect of ACNs has not been deeply clarified and some studies reported a different effect depending on the compound tested. • Most of ACNs are rapidly transformed by human gut in metabolic products, reaching a plasmatic concentration much higher than that of parental ACNs, and their contribution in the biological activity observed should be considered. Karga et al., 2016 Arch. Biochem. Biophys. 599:51-9.

AIM OF THE STUDY To investigate the capacity of anthocyanins and related metabolites to counteract inflammation and atherogenic process through the evaluation of monocytes (THP-1) adhesion to stimulated endothelial cells (HUVECs), and the levels of E-selectin and VCAM-1 as potential molecules involved in such modulation.

ACNs METABOLITES Protocatechuic acid Cyanidin 3-glucoside (Cy 3-glc) (PrA) Delphinidin 3-glucoside (Dp 3-glc) Gallic acid (Ga) Peonidin 3-glucoside (Peo 3-glc) Vanillic acid (VA) Malvidin 3-glucoside (Mv 3-glc) Syringic acid (SA) 3-Methyl Gallic acid (3-MetGa A) Petunidin 3-glucoside (Pet 3-glc) Ferulic acid (FA) Benzoic acid (BA)

METHODS: PROTOCOL TO STUDY THE ANTI-INFLAMMATORY AND ANTIATHEROGENIC ACTIVITY OF ACNs AND METABOLITES • Day 1-Preparation of 96 wells plate (2x10 4 HUVEC per well); • Day 2-Labelling of THP-1 cells with CellTrackerTM Green CMFDA, addition of THP-1 (2x10 5 cells/well) and TNF-  (100 ng mL -1 ) to HUVEC, and incubation for 24h; • Day 3-Incubation with ACNs or meabolites at different concentrations (from 0.01 till 10 μ g mL -1 ); • Day 4- Reading of the fluorescence (excitation: 485 nm, emission: 538 nm, mod. F200 Infinite, TECAN Milan, Italy) Supernatants were collected for the evaluation of markers of inflammation and vascular function (e.g. E-selectin, VCAM-1, VEGF) by ELISA kits

RESULTS: Effect of Mv-3-glc and Syringic acid on THP-1 adhesion to HUVECs Syringic acid Malvidin-3-glucoside 2,5 Fold increase THP-1 adhesion to HUVEC Fold increase THP-1 adhesion to HUVEC 2,5 b 2 c 2 c c c 1,5 1,5 a 1 1 0,5 0,5 0 0 TNF- α TNF- α SA SA SA SA Mv-3-glc Mv-3-glc Mv-3-glc Mv-3-glc NO TNF- α NO TNF- α 0.01 µg mL -1 0.1 µg mL -1 1 µg mL -1 10 µg mL -1 0.01 µg mL -1 0.1 µg mL -1 1 µg mL -1 10 µg mL -1 100ng mL -1 100ng mL -1 Significant reduction of THP-1 adhesion to HUVEC at No signficant effect after SA supplementation all concentrations tested with respect to the control treatment with TNF- α . Maximum reduction at 10 µg/mL -1 (-33.9%; p<0.001).

RESULTS: Effect of Peo-3-glc and Vanillic acid on THP-1 adhesion to HUVECs Peonidin-3-glucoside Vanillic acid 2,5 b b Fold increase THP-1 adhesion to HUVEC 2,5 Fold increase THP-1 adhesion to HUVEC b b b 2 2 c c c 1,5 c 1,5 a a a 1 1 0,5 0,5 0 0 VA VA TNF- α TNF- α VA Peo-3-glc Peo-3-glc Peo-3-glc VA Peo-3-glc NO TNF- α NO TNF- α 1 µg mL -1 10 µg mL -1 0.1 µg mL -1 0.1 µg mL -1 1 µg mL -1 10 µg mL -1 0.01 µg mL -1 100ng mL -1 0.01 µg mL -1 100ng mL -1 Significant reduction of THP-1 adhesion to HUVEC at Significant reduction after VA only at the maximum all concentrations tested with respect to the control concentration (-20.8%; p<0.005). treatment with TNF- α . Maximum reduction at 10 µg/mL -1 (-46.8%; p<0.001).

RESULTS: Effect of ACNs and metabolites on E-selectin concentration Malvidin-3-glucoside Syringic acid b b 350 350 b b b b 300 300 -66% 250 250 -67% pg mL -1 pg mL -1 d 200 200 d 150 150 c c 100 100 a a 50 50 0 0 TNF- α TNF- α SA Mv-3-glc SA Mv-3-glc Mv-3-glc SA SA Mv-3-glc NO TNF- α NO TNF- α 10 µg mL -1 1 µg mL -1 10 µg mL -1 100ng mL -1 0.1 µg mL -1 1 µg mL -1 100ng mL -1 0.01 µg mL -1 0.1 µg mL -1 0.01 µg mL -1 Peonidin-3-glucoside Vanillic acid 350 350 b b b b b 300 300 250 250 -75% pg mL -1 -65% pg mL -1 200 200 c c c 150 c,a 150 c 100 100 a a 50 50 0 0 TNF- α Peo-3-glc 0.01 µg mL -1 Peo-3-glc Peo-3-glc Peo-3-glc TNF- α VA VA NO TNF- α VA VA NO TNF- α 100ng mL -1 0.1 µg mL -1 10 µg mL -1 1 µg mL -1 0.01 µg mL -1 10 µg mL -1 1 µg mL -1 100ng mL -1 0.1 µg mL -1

RESULTS: Effect of ACNs and metabolites on VCAM-1 concentration Malvidin-3-glucoside Syringic acid 18000 18000 b b 16000 16000 b b b b c b c b 14000 14000 pg mL -1 12000 pg mL -1 12000 10000 10000 a 8000 8000 a 6000 6000 4000 4000 2000 2000 0 0 TNF- α Mv-3-glc Mv-3-glc Mv-3-glc Mv-3-glc NO TNF- α TNF- α SA SA SA SA 0.01 µg mL -1 0.1 µg mL -1 1 µg mL -1 10 µg mL -1 NO TNF- α 100ng mL -1 1 µg mL -1 10 µg mL -1 100ng mL -1 0.01 µg mL -1 0.1 µg mL -1 Peonidin-3-glucoside Vanillic acid 18000 b b b 18000 b b b 16000 b c 16000 b b 14000 14000 12000 12000 pg mL -1 pg mL -1 10000 10000 8000 a 8000 a 6000 6000 4000 4000 2000 2000 0 0 TNF- α TNF- α VA VA VA Peo-3-glc VA 0.01 µg mL -1 Peo-3-glc Peo-3-glc Peo-3-glc NO TNF- α NO TNF- α 0.01 µg mL -1 0.1 µg mL -1 10 µg mL -1 100ng mL -1 1 µg mL -1 100ng mL -1 0.1 µg mL -1 10 µg mL -1 1 µg mL -1

CONCLUSIONS 1-Mv and Peo-3-glc showed to counteract THP-1 adhesion to HUVEC, while the effects of metabolites seem to be compound dependent and only at high doses. 2-ACNs and metabolites seem to reduce the production of E-selectin, but not VCAM-1. 3-Since the effects were observed at the low doses, these results could suggest that the protective effect may be reached also in vivo at physiological concentrations. 4-Ongoing experiments are attempting to confirm and clarify the mechanisms of action of each compound involved in the above observations.

THANK YOU FOR YOUR ATTENTION !