Fig 1: Dihydroethidium (DHE), acridine orange (AO), and senescence-associated β-galactosidase (SA-β-gal) staining in the liver of zebrafish following 12 weeks of consumption of a high-cholesterol diet (HCD) supplemented with Raydel® and BOC Sciences policosanol. (A) DHE fluorescent staining for the detection of reactive oxygen species (ROS). (B) AO fluorescent staining to show the magnitude of apoptosis. (C) SA-β-gal staining to ascertain cellular senescence; the red arrow indicates the SA-β-gal-positive cells [100 μm, scale bar]. (D) and DHE fluorescence intensity quantification by Image J software, version 1.53r. (E) AO fluorescence intensity quantification by the Image J software. (F) Percentage quantification of SA-β-gal-stained area. Values depicted in the graphs are the mean ± SEM of three assorted studies. ND, normal diet; HCD, high-cholesterol diet (i.e., ND containing 4% cholesterol), while HCD + Raydel or BOC represent the HCD + 0.1% Raydel® policosanol or BOC Sciences policosanol, respectively. Significance levels of p < 0.01 and p < 0.001 are denoted by ** and *** with respect to the HCD group, while ††† depicts significance levels at p < 0.001 between Raydel® and BOC Sciences policosanol groups. “ns” denotes non-significant differences between the groups.
Fig 2: Body weight of zebrafish consuming a high-cholesterol diet (HCD) supplemented with Raydel® and BOC Sciences policosanol. (A) Zebrafish body weight at different time points (0–12 weeks). (B) Zebrafish body weight at 12 weeks. Values depicted in the graphs are the mean ± SEM of three assorted studies. ND, normal diet; HCD, high-cholesterol diet (i.e., ND containing 4% cholesterol), while HCD + Raydel or BOC represent the HCD + 0.1% Raydel® policosanol or BOC sciences policosanol, respectively. Significance level of p < 0.01 is indicated by ** with respect to the HCD group. ns, non-significant.
Fig 3: Hepatic histology of zebrafish consuming a high-cholesterol diet (HCD) supplemented with Raydel® and BOC Sciences policosanol for 12 weeks. (A,B) Hematoxylin and eosin (H&E) staining at 400× and 1000× magnification, respectively. Red arrow indicates infiltrated neutrophil. (C) Oil red O (ORO) staining. (D) Immunohistochemistry (IHC) for the detection of IL-6. (E) The brown color IL-6-stained area is interchanged with red color (to enhance visibility) at the brown color threshold values of 20–120 utilizing Image J software, version 1.53r). (F) Percentage of neutrophils quantified in the H&E section. (G) Image J-based assessment of ORO-stained areas. (H) Image J-based IL-6-stained areas. Values depicted in the graphs are the mean ± SEM of three assorted studies. ND, normal diet; HCD, high-cholesterol diet (i.e., ND containing 4% cholesterol), while HCD + Raydel or BOC represent the HCD + 0.1% Raydel® policosanol or BOC Sciences policosanol, respectively. Significance levels of p < 0.01 and p < 0.001 are denoted by ** and *** with respect to the HCD group, while †† and ††† depict significance levels at p < 0.01 and p < 0.001, respectively, between Raydel® and BOC Sciences policosanol groups. “ns” denotes non-significant differences between the groups.
Fig 4: Levels of the hepatic function biomarkers aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in zebrafish consuming a high-cholesterol diet (HCD) supplemented with Raydel® and BOC Sciences policosanol for 12 weeks. Values depicted in the graphs are the mean ± SEM of three assorted studies. ND, normal diet; HCD, high-cholesterol diet (i.e., ND containing 4% cholesterol), while HCD + Raydel or BOC represent the HCD + 0.1% Raydel® policosanol or BOC Sciences policosanol, respectively. Significance levels of p < 0.01 and p < 0.001 are denoted by ** and *** with respect to the HCD group. ns, non-significant.
Fig 5: Plasma lipid profile of zebrafish consuming a high-cholesterol diet (HCD) supplemented with Raydel® and BOC Sciences policosanol for 12 weeks. Values depicted in the graphs are the mean ± SEM of three assorted studies. ND, normal diet; HCD, high-cholesterol diet (i.e., ND containing 4% cholesterol), while HCD + Raydel or BOC represent the HCD + 0.1% Raydel® policosanol or BOC Sciences policosanol, respectively. Significance levels of p < 0.05, p < 0.01, and p < 0.001 are denoted by *, **, and *** with respect to the HCD group. †, ††, and ††† depict significance levels at p < 0.05, p < 0.01, and p < 0.001, respectively, between Raydel® and BOC Sciences policosanol groups. ns, non-significant.
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