Nano-Encapsulated Phytosterols Ameliorate Hypercholesterolemia in Mice via Dual Modulation of Cholesterol Metabolism Pathways

Abstract

Background: The limited bioavailability of free phytosterols restricts their clinical application in managing hypercholesterolemia. This study aimed to develop phytosterol nanoparticles (PNs) to enhance bioactivity and investigate their cholesterol-lowering efficacy and underlying mechanisms in vivo. Methods: Phytosterol nanoparticles (PNs) (93.35 nm) were engineered using soy protein isolate and administered orally at concentrations of 4.00-12.50 mg/mL to high-fat-diet-induced hypercholesterolemic mice (n = 60) over a 4-week period. Serum and hepatic lipid profiles, histopathology, gene/protein expression related to cholesterol metabolism, and fecal sterol content were evaluated. Results: PNs dose-dependently reduced serum total cholesterol (TC: 28.6-36.8%), triglycerides (TG: 22.4-30.1%), and LDL-C (31.2-39.5%), while increasing HDL-C by 18.7-23.4% compared to hyperlipidemic controls (p < 0.01). Hepatic TC and TG accumulation decreased by 34.2% and 41.7%, respectively, at the highest dose, with histopathology confirming attenuated fatty degeneration. Mechanistically, PNs simultaneously suppressed cholesterol synthesis through downregulating HMGCR (3.2-fold) and SREBP2 (2.8-fold), while enhancing cholesterol catabolism via CYP7A1 upregulation (2.1-fold) at protein level. Although less potent than simvastatin (p < 0.05), the nanoparticles exhibited unique dual-pathway modulation absent in conventional phytosterol formulations. Fecal analysis revealed dose-responsive cholesterol excretion (36.01 vs. 11.79 mg/g in controls), indicating enhanced enteric elimination. While slightly less potent than simvastatin (p < 0.05), PNs offered unique dual-pathway modulation absent in conventional phytosterol formulations. Conclusions: Nano-encapsulation significantly improves the bioavailability and hypocholesterolemic efficacy of phytosterols. PNs represent a promising nutraceutical strategy for cholesterol management by concurrently regulating cholesterol synthesis and catabolism, with potential application in both preventive and therapeutic contexts.

Keywords: CYP7A1; cholesterol regulation; high-fat diet; mouse; phytosterol nanoparticles.

Figure 1

Average body weight (A) and average daily feed intake (B) of mice. Nor: fed basic feed + 0.5 mL saline gavage daily; HCM: fed high-fat feed + 0.5 mL saline gavage daily; L-PN: fed high-fat feed + 0.5 mL low concentration (4.00 mg/mL) PNs by gavage daily; M-PN: fed high-fat feed + 0.5 mL medium concentration (8.25 mg/mL) PNs by gavage daily; H-PN: fed high-fat diet + 0.5 mL high concentration (12.50 mg/mL) PNs by daily gavage; PM: fed high-fat diet + 0.5 mL 0.4 mg/mL simvastatin by daily gavage. * (p < 0.05) vs. Nor. n = 10.

Figure 2

Serum TC (A), TG (B), HLD-C (C), LDL-C (D) and liver lipid content (E) concentrations. Nor: fed basic feed + 0.5 mL saline gavage daily; HCM: fed high-fat feed + 0.5 mL saline gavage daily; L-PN: fed high-fat feed + 0.5 mL low concentration (4.00 mg/mL) PNs by gavage daily; M-PN: fed high-fat feed + 0.5 mL medium concentration (8.25 mg/mL) PNs by gavage daily; H-PN: fed high-fat diet + 0.5 mL high concentration (12.50 mg/mL) PNs by daily gavage; PM: fed high-fat diet + 0.5 mL 0.4 mg/mL simvastatin by daily gavage. Different capital letters and lowercase letters indicate highly significant (p < 0.01) and significant (p < 0.05) differences between groups. n = 10.

Figure 3

Liver histomorphology (×200). Nor: fed basic feed + 0.5 mL saline gavage daily (A); HCM: fed high-fat feed + 0.5 mL saline gavage daily (B); L-PN: fed high-fat feed + 0.5 mL low concentration (4.00 mg/mL) PNs by gavage daily (C); M-PN: fed high-fat feed + 0.5 mL medium concentration (8.25 mg/mL) PNs by gavage daily (D); H-PN: fed high-fat diet + 0.5 mL high concentration (12.50 mg/mL) PNs by daily gavage (E); PM: fed high-fat diet + 0.5 mL 0.4 mg/mL simvastatin by daily gavage (F). Red arrows indicate representative hepatocytes exhibiting cytoplasmic vacuolization or lipid droplets, characteristic of hepatic steatosis.

Figure 4

The mRNA expression of HMGCR (A), CYP7A1 (B), LDLR (C), LXR-α (D) and SREBP 2 (E). Different capital letters and lowercase letters indicate highly significant (p < 0.01) and significant (p < 0.05) differences between groups. HMGCR, 3-Hydroxy-3-Methylglutaryl-CoA Reductase; SREBP2, Sterol Regulatory Element-Binding Protein 2; CYP7A1, Cholesterol 7α-Hydroxylase; LDLR, Low-Density Lipoprotein Receptor; LXR-α, Liver X Receptor Alpha; Nor: fed basic feed + 0.5 mL saline gavage daily; HCM: fed high-fat feed + 0.5 mL saline gavage daily; L-PN: fed high-fat feed + 0.5 mL low concentration (4.00 mg/mL) PNs by gavage daily; M-PN: fed high-fat feed + 0.5 mL medium concentration (8.25 mg/mL) PNs by gavage daily; H-PN: fed high-fat diet + 0.5 mL high concentration (12.50 mg/mL) PNs by daily gavage; PM: fed high-fat diet + 0.5 mL 0.4 mg/mL simvastatin by daily gavage. n = 10.

Figure 5

The protein expression of HMGCR (A), CYP7A1 (B), LDLR (C), LXR-α (D) and SREBP 2 (E). Different capital letters and lowercase letters indicate highly significant (p < 0.01) and significant (p < 0.05) differences between groups. HMGCR, 3-Hydroxy-3-Methylglutaryl-CoA Reductase; SREBP2, Sterol Regulatory Element-Binding Protein 2; CYP7A1, Cholesterol 7α-Hydroxylase; LDLR, Low-Density Lipoprotein Receptor; LXR-α, Liver X Receptor Alpha; Nor: fed basic feed + 0.5 mL saline gavage daily; HCM: fed high-fat feed + 0.5 mL saline gavage daily; L-PN: fed high-fat feed + 0.5 mL low concentration (4.00 mg/mL) PNs by gavage daily; M-PN: fed high-fat feed + 0.5 mL medium concentration (8.25 mg/mL) PNs by gavage daily; H-PN: fed high-fat diet + 0.5 mL high concentration (12.50 mg/mL) PNs by daily gavage; PM: fed high-fat diet + 0.5 mL 0.4 mg/mL simvastatin by daily gavage. n = 3. Numbers 1 to 6 correspond to the following groups: 1 = Nor, 2 = HCM, 3 = L-PN, 4 = M-PN, 5 = H-PN, 6 = PM.