Atheroprotective effects and safety evaluation of saffron extract in early and established atherosclerotic-induced rabbits

Cardiovascular disease (CVD) is the leading cause of death worldwide, with atherosclerosis being a major risk factor. Current therapies for managing atherosclerosis have limitations, including side effects. Saffron has demonstrated anti-atherogenic properties, but the mechanisms by which saffron extract reduces atherosclerotic lesions at the protein and gene expression levels remain unclear. Additionally, the plaque­ stabilizing effects of saffron and its safety in individuals with atherosclerosis are not well understood. This study aims to investigate the atheroprotective effects, underlying mechanisms, and safety of saffron extract in experimentally induced early and established atherosclerosis in high-cholesterol diet (HCD)-fed rabbits. Sixty-six male New Zealand White (NZW) rabbits were randomized into two groups: (i) a baseline group, which included subgroups euthanized without intervention (Al), fed 1% HCD for 4 weeks to induce early atherosclerosis (A2), and fed 1% HCD for 8 weeks to induce established atherosclerosis (A3); and (ii) four treatment groups, receiving 1 % HCD for 4 and 8 weeks and then force-fed with (i) saffron ethanolic extract (SEE) 50 mg/kg/day, (ii) SEE 100 mg/kg/day, (iii) distilled water (placebo), or (iv) simvastatin 2.5 mg/kg/day. Blood samples were collected for lipid, biochemical, and hematological profiles. After euthanasia, the a011a and vital organs were excised. Parts of the aorta were preserved in RNA later for qRT-PCR, while others were preserved in formalin for Sudan IV staining to analyze the percentage lesion area, and immunohistochemical analysis to assess biomarkers of endothelial activation and inflammation. Vital organs underwent histological examination. Plaque stability was assessed by calculating the ratio of matrix metalloproteinase-9 (MMP-9) to tissue inhibitor of metalloproteinases- 1 (TIMP-1 ). In the early atherosclerosis group, post-treatment with 50 and 100 mg/kg/day SEE resulted in significant reductions in low-density lipoprotein cholesterol (LDL-C) by 43.0% and 44.5%, respectively, compared to the placebo group (p<0.01), while total cholesterol (TC) levels decreased by 28.8% and 25.6%, respectively (p<0.01). In the established atherosclerosis group, SEE treatment reduced LDL-C by 46.2% and 49.6%, and TC by 60.1% and 64.5%, compared to placebo (p<0.01). The percentage of lesion area in the established atherosclerosis group decreased by 4.82% and 10.1 % after treatment with 50 and 100 mg/kg/day SEE, respectively (p<O.O 1 ). Tissue and gene markers of endothelial activation and inflammation were significantly reduced in SEE-treated groups compared to placebo in both early and established atherosclerosis groups (p<0.01). The plaque instability index (MMP-9/TIMP-l) increased by 137.5% in early atherosclerosis and decreased by 88.3% compared to placebo in established atherosclerosis after treatment with 100 mg/kg/day SEE (p<0.01). Oral administration of both doses of SEE in early and established atherosclerotic NZW rabbits significantly improved hematological and biochemical parameters. Histopathological analysis on the vital organs indicated that both doses of SEE ameliorated tissue damage in early and established atherosclerosis, potentially due to its hypolipidemic effects, which modulate lipid metabolism and its associated impact on tissues. There were no adverse effects and mortality at the evaluated doses. In conclusion, the reduction of endothelial dysfunction and inflammation through oral supplementation of SEE decreased lipid levels, atherosclerotic lesion areas, and plaque instability. This suggests that SEE may protect the a011a and reduce the risk of atherosclerotic complications in HCD-induced atherosclerotic rabbits.