To evaluate the expression of the transmembrane protein TF, HUVEC were cultured with 10 [micro]g/mL of DHZ or DHZ dimer and, after an overnight incubation, were stimulated with TNF-[alpha] (Tebu-bio) for 6 hours.
5] cells/mL) were preincubated with DHZ or DHZ dimer overnight and then treated with 10 ng/mL TNF-[alpha] (Tebu-bio) for 1 hour.
Preliminary dose-response experiments conducted to evaluate a possible toxic effect of DHZ dimer and monomer on ECs showed a reduction of cell viability after a treatment with 50 [micro]g/mL of DHZ dimer and with 100 [micro]g/mL of monomer (Figure 1).
To evaluate the antioxidant activity of DHZ and DHZ dimer on ECs we assessed the ability of these compounds to counteract ROS production induced by [H.
Cytofluorimetric analysis of adhesion molecule surface expression showed a significant reduction of TNF-[alpha]-induced ICAM-1 and VCAM-1 surface expression evaluated as MFI in response to both DHZ and DHZ dimer (Figures 3(a) and 3(b)).
Analysis of ICAM-1 and VCAM-1 concentrations in culture supernatants from TNF-[alpha]-stimulated HUVEC showed lower levels of these molecules in samples pretreated with DHZ and DHZ dimer compared to those from non-pretreated samples (Figures 5(a) and 5(b)).
In all cases, preincubation with DHZ and DHZ dimer significantly reduced such expression (Figures 6(a) and 6(b)), confirming their potential anti-inflammatory activity.
To assess the possible antithrombotic activity of DHZ and DHZ dimer, we evaluated TF expression on cell membrane.
When HUVEC were pretreated with DHZ or DHZ dimer a significant reduction of active p50 and p65 levels was observed (Figure 7(b), (i) and (ii)).
Our data here demonstrate that DHZ and its dimer exert antioxidant and anti-inflammatory activities on human endothelial cells, thus suggesting a possible usefulness of these compounds in the prevention and therapy of atherosclerosis.
Our data showing that pretreatment with DHZ and DHZ dimer reduces ROS levels in ECs exposed to oxidative stress are in accordance with previous results demonstrating that CUR improves cell viability and reduces ROS production in HUVEC treated with [H.
The potent inhibitory effects of DHZ dimer and at less extent of its monomer on adhesion molecule expression and secretion by ECs strongly suggest that these compounds could be useful in hampering leukocyte recruitment and inflammation, thus contrasting atherosclerotic disease progression.