Administration of Protein Hydrolysates from Anchovy (Engraulis Encrasicolus) Waste for Twelve Weeks Decreases Metabolic Dysfunction-Associated Fatty Liver Disease Severity in ApoE-/-Mice

Abstract

Metabolic dysfunction-associated fatty liver disease (MAFLD) includes several diseases, ranging from simple steatosis to steatohepatitis, fibrosis and cirrhosis. Fish-rich diets are considered helpful in the prevention of MAFLD, and the enzymatic hydrolysis of fish waste has been explored as a means of obtaining high-value protein hydrolysates, which have been proven to exert beneficial bioactivities including anti-obesity and hypocholesterol effects. This study aimed to assess the effect of the administration of protein hydrolysates from anchovy waste (APH) for 12 weeks on attenuated high-fat diet-induced MAFLD in apolipoprotein E-knockout mice (ApoE^-/-). Thirty ApoE^-/- mice were divided into two groups (n = 15/group) and fed a high-fat diet (HFD), with and without the addition of 10% (w/w) APH. After 12 weeks, serum and hepatic lipid profiles, hepatic enzyme activities, liver histology and immunohistochemistry were analyzed to assess hepatic steatosis, inflammation and fibrosis. Twelve-weeks on a 10% (w/w) APH diet reduces total cholesterol and triglyceride serum levels, hepatic enzyme activity and hepatic triacylglycerol content (p < 0.0001), and results in a reduction in hepatic fat accumulation and macrophage recruitment (p < 0.0001). The results suggest that a 10% APH diet has an anti-obesity effect, with an improvement in lipid metabolism, hepatic steatosis and liver injury as a result of a high-fat diet. Protein hydrolysates from fish waste may represent an efficient nutritional strategy in several diseases, and their use as nutraceuticals is worthy of future investigation.

Keywords: ApoE-deficient mice; MAFLD; anchovy; hydrolysates.

Figure 1

Body weight trends in mice throughout study period. An increasing trend in body weight was observed in ApoE^–/– mice of both groups (p < 0.05), with a percentage increase of 50.47% in ApoE^–/– mice fed HFD and 40.94% in ApoE^–/– fed HFD + APH. Body weight gain was significantly lower in ApoE^–/– fed HFD + APH from T14 to T84 compared to the control group (p < 0.001). Data are presented as mean ± SD for n = 6 mice per group. Abbreviations: HFD high-fat diet, APH anchovy protein hydrolysates.

Figure 2

Liver-to-body weight ratio (LBW) (A) and levels of hepatic free cholesterol (B) cholesteryl ester (C) and triacylglycerol (D). LBW in ApoE^–/– mice fed with HFD was significantly increased (i.e., 2.47%) compared to ApoE^–/– mice fed with HFD + APH (i.e., 2.01%) (p < 0.0001). No statistical difference was found in either hepatic free cholesterol (B) or cholesteryl ester (C), whereas hepatic triacylglycerol (D) content was significantly reduced in ApoE^–/– mice fed HFD+APH (214.39 ± 29.67 nmol/mg) compared to values obtained from ApoE^–/– control mice fed HFD (365.97 ± 46.20 nmol/mg). Data are presented as mean ± SD for n = 6 mice per group. Abbreviations: HFD, high-fat diet, APH, anchovy protein hydrolysates.

Figure 3

Serum lipid levels and hepatic enzyme activity. Administration of APH for 12 weeks produced significant serum total cholesterol and triglycerides-lowering effects (A,B). Serum ALT (alanine aminotransferase) (C) and AST (aspartate amino transferase) (D) levels were statistically lower in ApoE^–/– HFD + APH compared to the control group (p < 0.0001), suggesting minor damage of the hepatocytes. Data are presented as mean ± SD for n = 6 mice per group. Abbreviations: HFD high-fat diet, APH anchovy protein hydrolysates.

Figure 4

Hepatic histology. Representative liver sections of H&E (A,D) Oil Red O (B,E) and Mallory’s Trichrome (C,F) histological staining of ApoE^–/–mice included in both groups (Scale bar, 100 µm). Abbreviations: HFD high-fat diet, APH anchovy protein hydrolysates.

Figure 5

Representative liver sections (A,B) and quantification (% of positive stained area) of Oil-Red O histological staining (C). ApoE^–/–mice fed a HFD showed a statistically twofold increase in hepatic fat accumulation (39.06 ± 3.67%) compared to ApoE^–/–mice fed with a APH diet (19.26 ± 4.32%). Data are presented as mean ± SD for n = 6 mice per group (Scale bar, 50 µm). Abbreviations: HFD high-fat diet, APH anchovy protein hydrolysates.

Figure 6

Representative liver sections (A,B) and quantification (% of positive stained area) of F4/80 immunohistochemistry (C). ApoE^–/–mice fed a HFD showed a statistically significant threefold increase in F4/80 expression levels (24.14 ± 2.04%) compared to ApoE^–/– mice fed with a APH diet (9.81 ± 1.03%) (p < 0.001). Data are presented as mean ± SD for n = 6 mice per group (Scale bar, 50 µm). Abbreviations: HFD high-fat diet, APH anchovy protein hydrolysates.