Fucoidan protege contra a disfunção endotelial e aterosclerose em camundongos apoe-/- através da redução da inflamação e estresse oxidativo

Abstract

Introduction: Atherosclerosis is characterized by chronic oxidative stress and inflammatory changes in the vascular tissue. Endothelial injuries caused by oxidized low-density lipoprotein (oxLDL) play a key role in this disease. Previous studies have shown that fucoidan, a sulfated polysaccharide extracted from brown seaweed, is associated to beneficial effects on atherosclerosis prevention. However, the role of fucoidan in regulating vascular endothelial function and mechanisms associated to atherogenesis is not well understood. Objective: Investigate the effect of fucoidan in the atherosclerosis development, assessing the molecular mechanisms involved in protection against vascular cell damage: inflammation and oxidative stress. Methods: To test the anti-atherosclerotic effect of fucoidan, eight to ten-week-old male apolipoprotein E-deficient (ApoE-/-) mice were fed during ten weeks with either a high-cholesterol diet (HCD) or standard diet (SD), with or without 1% of fucoidan. At the end of this period, blood sample was collected for biochemical analysis, and aorta for en face quantification of atherosclerotic lesions (Sudan IV staining). We also evaluated the absorption and tissue distribution of fucoidan, as well as the leukocyte-endothelial interactions in ApoE-/- after a single-dose oral administration. To investigate the effect of fucoidan on endothelial dysfunction induced by oxLDL, thoracic aortic rings from wild type mice and human endothelial cells (EA.hy926) were pre-incubated with fucoidan (1, 5 or 10 μg/mL), by 30 minutes before 4 hours of coincubation with oxLDL (50 μg/mL). After incubation, the aortic rings were used to perform vascular reactivity studies. The endothelial cells were used to perform cell viability and adhesion assay, and to analyze the cytokines production, adhesion molecules expression and intracellular reactive oxygen species (ROS) generation. Further, the correlation between fucoidan and ROS/NF-κB signaling pathway was explored. All animal experiments were approved by the Federal University of Minas Gerais Animals Care and Use Committee according to the Protocol n° 284/2016. Statistical analyses: One-way ANOVA followed by Tukey post-test was performed to compare groups, using the GraphPad Prism 7 software. All data were presented as mean ± SD. Differences were considered statistically relevant at p<0.05. Results: Fucoidan treatment significantly reduced the lesion area in aorta of ApoE-/- mice fed on a high-cholesterol diet (HCD 7.61 ± 1.48 vs HCD + Fuc 1% 4.39 ± 1.95), without changes between SD groups. This result was associated with a decrease in serum levels of total cholesterol (22%), triglycerides (35%) and non-HDL cholesterol fractions (21%). We observed that oxLDL reduced acetylcholine (ACh)-induced vasodilation in mouse aorta, but the treatment with fucoidan reversed oxLDL-induced endothelial dysfunction. In in vitro studies, pretreatment with fucoidan reduced the inflammatory responses. Fucoidan reduced the oxLDL-induced monocyte adherence to endothelial cells (>50%), production of proinflammatory mediators (MCP-1, IL-6 e TNF), and the expression of adhesion molecules (P-Selectin and VCAM). In addition, fucoidan attenuated ROS generation, and reduced the NF-κB signaling pathway (decreasing NF-κB phosphorylation). Conclusions: Our study demonstrated that fucoidan reduced atherosclerosis in ApoE-/- mice suppressing oxLDL-induced vascular inflammation and oxidative stress. Thus, fucoidan has a vasoprotective property, suggesting possible therapeutic effects in atherosclerotic diseases.