Anti-Aging and Tissue Regeneration Ability of Policosanol Along with Lipid-Lowering Effect in Hyperlipidemic Zebrafish via Enhancement of High-Density Lipoprotein Functionality

Abstract

We investigated the tissue regeneration and lipid-lowering effects of policosanol (PCO) by employing a hyperlipidemic zebrafish model. A reconstituted high-density lipoprotein containing policosanol (PCO-rHDL) facilitated greater cell growth and replication with less apoptosis and reactive oxygen species (ROS) production in BV-2 microglial cell lines. From in vivo study, injection of rHDL containing apolipoprotein A-I (ApoA-I) caused 76 ± 4% (p = 0.01) greater tissue regeneration activity than the phosphate-buffered saline (PBS) control, whereas PCO-rHDL caused 94 ± 7% (p = 0.002) increased regeneration. PCO in ethanol (EtOH) showed lower cholesteryl ester transfer protein (CETP) inhibitory ability than did anacetrapib, whereas PCO-rHDL showed higher inhibitory ability than anacetrapib, suggesting a synergistic effect between PCO and rHDL. Following 9 weeks of PCO consumption, the PCO group (0.003% PCO in Tetrabit) showed the highest survivability (80%), whereas normal diet (ND) and high-cholesterol diet (HCD) control groups showed 67% and 70% survival rates, respectively. Supplementation with a HCD resulted in two-fold elevation of CETP activity along with 3- and 2.5-fold increases in serum total cholesterol (TC) and triglycerides (TGs) levels, respectively. Consumption of PCO for 9 weeks resulted in 40 ± 5% (p = 0.01 vs. HCD) and 33 ± 4% (p = 0.02 vs. HCD) reduction of TC and TGs levels, respectively. Serum high-density lipoprotein cholesterol (HDL-C) level increased up to 37 ± 2 mg/dL (p = 0.004), whereas the percentage of HDL-C/TC increased up to 20 ± 2% from 5 ± 1% compared to the HCD control. The serum glucose level was reduced to 47 ± 2% (p = 0.002) compared to the HCD control. Fatty liver change and hepatic inflammation levels were remarkably increased upon HCD consumption and were two-fold higher than that under ND. However, the PCO group showed 58 ± 5% (p = 0.001) and 50 ± 3% (p = 0.006) reduction of inflammation enzyme levels and lipid content in hepatic tissue under HCD. In conclusion, PCO supplementation showed lipid-lowering and HDL-C-elevating effects with ameliorating fatty liver change. These in vivo anti-atherosclerotic and anti-diabetic effects of PCO are well associated with in vitro anti-apoptotic activities.

FIG. 1.

Cytoprotective effect of policosanol (PCO) in BV microglial cells. The extent of apoptosis and reactive oxygen species (ROS) production in BV cells in the presence of PCO either in organic solvent or reconstituted high-density lipoprotein (rHDL). (A) Cellular apoptosis and ROS production were determined by Acridine Orange (Ex = 502 nm, Em = 525 nm) staining and dihydroethidium (DHE) staining (Ex = 588 nm, Em = 605 nm), respectively. The graph (B) shows quantification of areas stained with Acridine Orange and DHE. (A) Photo a, phosphate-buffered saline (PBS); photo b, organic solvent (CHCl₃:Me-OH, 2:1); photo c, PCO (final 9 μM in CHCl₃:Me-OH); photo d, PCO (final 46 μM in CHCl₃:Me-OH); photo e, rHDL (1:0); photo f, PCO-rHDL (1:1), final 9 μM ; photo g, PCO-rHDL (1:5), final 46 μM. (B) Extent of apoptosis and ROS production in BV cells in the presence of PCO either in organic solvent or rHDL. Acridine Orange. Cellular apoptosis and ROS production were determined by Acridine Orange (Ex = 502 nm, Em = 525 nm) staining and DHE staining (Ex = 588 nm, Em = 605 nm), respectively. Graph shows quantification of areas stained with Acridine Orange and DHE. Color images available online at www.liebertpub.com/rej

FIG. 2.

Tissue regeneration activity of policosanol (PCO) hyperlipidemic zebrafish. Enhancement of zebrafish fin regeneration by injection of reconstituted high-density lipoprotein (rHDL) containing PCO and rHDL (2 μg of apolipoprotein A-I [Apo-A-I]). (A) Representative image of caudal fin regeneration upon injection of rHDL containing PCO with administration of a high-cholesterol diet (HCD). (B) Fin regeneration area and reactive oxygen species (ROS) production at 144 hr after protein injection. Data shown are the mean ± standard deviation (SD) of three independent experiments (n = 7). Color images available online at www.liebertpub.com/rej

FIG. 3.

Cholesteryl ester transfer protein (CETP) inhibitory activity of reconsituted high-density lipoprotein (rHDL) containing policosanol (PCO). Data shown are the mean ± standard deviation (SD) of three independent experiments performed in duplicate. Cholesteryl ester (CE) transfer from [³H]HDL (50 μg of apolipoprotein A-I [ApoA-I], 30,000 cpm) to human low-density lipoprotein (LDL) (50 μg of protein) by human HDL₃ (25 μg of protein) was inhibited by rHDL containing PCO.

FIG. 4.

Histological analysis of hepatic tissue and lipid quantification. (A) Representative photos for comparison of fatty liver changes in zebrafish as visualized by Oil Red O staining and reactive oxygen species (ROS) production as visualized by dihydroethidium (DHE) staining. Scale bar, 100 μm. ND, normal diet; HCD, high-cholesterol diet; HCD + PCO (policosanol). Hemaotxylin & Eosin staining to detect infiltrated inflammatory cells. (B) Quantification of Oil Red O- and DHE-stained area (red fluorescence, Ex = 588 nm, Em = 615 nm). (C) Quantification of total cholesterol and triglycerides in hepatic tissue as described in text. Color images available online at www.liebertpub.com/rej

FIG. 5.

Anti-oxidant ability of homogenate of hepatic tissue from each group. (A) Ferric ion reduction ability of hepatic tissue (50 μg in phosphate-buffered saline [PBS]). (*) p < 0.05 versus HCD; (**) p < 0.01 versus high-cholesterol diet (HCD). (B) Quantification of oxidized species (malondialdehyde) in hepatic tissue (50 μg in PBS). ND, normal diet; HCD, high-cholesterol diet; HCD + PCO (policosanol). Color images available online at www.liebertpub.com/rej