Anti-Atherogenic Effect of 10% Supplementation of Anchovy (Engraulis encrasicolus) Waste Protein Hydrolysates in ApoE-Deficient Mice

Abstract

Fish protein consumption exerts beneficial metabolic effects on human health, also correlating with a decreased risk for cardiovascular disease. Fish waste contains high amount of proteins and utilization may offer the opportunity for generating compounds advantageous for human health. Especially, fish waste protein hydrolysates beneficially influence pathways involved in body composition, exerting anti-inflammatory and antioxidant activities, making their potential supplementation in human disorders of increased interest. This study assessed the effect of a 10% (w/w) anchovy waste protein hydrolysate (APH) diet for 12 weeks in reducing atherosclerosis in ApoE^-/- mice, through histological and immunohistochemical methods. In addition, monitoring of plaque development was performed, using high-frequency ultrasound and magnetic resonance imaging. Overall, the APH diet attenuated atherosclerotic plaque development, producing a regression of arterial lesions over time (p < 0.05). Twelve weeks on an APH diet had an anti-obesity effect, improving lipid metabolism and reducing hepatic enzyme activity. A significant reduction in plaque size and lipid content was observed in the aortic sinus of APH-fed mice, compared to the control (p < 0.001), whereas no differences in the extracellular matrix and macrophage recruitment were observed. Supplementation of APH significantly attenuates atherosclerosis in ApoE^-/- mice, exerting a lipid-lowering activity. The opportunity to use fish waste protein hydrolysates as a nutraceutical in atherosclerosis is worthy of future investigations, representing a low cost, sustainable, and nutritional strategy with minimal environmental impact.

Keywords: ApoE-knockout mice; anchovy by-products; atherosclerosis; fish protein hydrolysates; fish proteins.

Figure 1

B-mode imaging of the abdominal aorta in longitudinal view. Atherosclerotic plaque (echolucent) protruding into the lumen of the aorta in ApoE^−/− HFD+APH at 8 (A) and 12 weeks (B). Atherosclerotic plaque into the lumen of the abdominal aorta in ApoE^−/− HFD at 8 (C) and 12 weeks (D).

Figure 2

In vivo magnetic resonance cross sectional images (T1 rare) of abdominal aorta. Atherosclerotic plaque protruding into the lumen of the aorta in ApoE^−/− HFD+APH at 8 (A) and 12 weeks (B). Atherosclerotic plaque in the abdominal aorta in ApoE^−/− HFD at 8 (C) and 12 weeks (D).

Figure 3

Histological characterization of atherosclerotic plaques in aortic sinus of ApoE^−/− HFD (control) (A,B) and ApoE^−/− HFD +APH mice (C,D) (HE). Quantification of plaque area, with a significant reduction in plaque area in ApoE^−/− HFD+APH compared to the ApoE^−/− HFD control mice (E) (p < 0.001). Significant reduction in percentage of aortic sinus covered by plaque in ApoE^−/− HFD + APH compared to the ApoE^−/− HFD mice (p < 0.001) (F). Quantification is expressed as mean ± SD for n = 6 animals per group. (Magnification 10×, (A,C)); (magnification 20×, (B,D).

Figure 4

Histochemical characterization of atherosclerotic plaques in aortic sinus of ApoE^−/− HFD + APH and ApoE^−/− HFD (control) mice. Representative photomicrographs and quantification of lipid deposition in plaque area (A–C). Representative photomicrographs and quantification of extracellular matrix deposition (D–F). Quantification of lipids and extracellular matrix deposition was expressed as percentage of positively stained area/total plaque area. Quantification is presented as mean ± SD for n = 6 animals per group. (Magnification 5×).

Figure 5

Immunohistochemical characterization of atherosclerotic plaques in aortic sinus of ApoE^−/− HFD+APH and ApoE^−/− HFD (control) mice. Representative photomicrographs of F4/80 expression (A,B). Representative photomicrographs of BDNF (C,D), TrkB (E,F), and FNDC5 (G,H) expression in atherosclerotic plaques (magnification 10×).