Enhancement of memory and emotional functions by long-term ingestion of protease-treated porcine liver extract in mice

Abstract

The relationship between diet and brain functions has garnered attention. Previous studies have shown that ingesting a protease-treated porcine liver decomposition product (PLDP) improves cognitive function in humans. In this study, we investigated the effects of PLDP ingestion on cognitive and emotional functions in mice. Mice were fed a PLDP-enhanced diet for 16 weeks and subjected to various behavioral assessments. PLDP ingestion enhanced long-term memory in Barnes maze test. Moreover, mice fed the PLDP diet exhibited reduced anxiety- and depression-like behaviors as evidenced by their performance in open-field, elevated plus maze, marble-burying, and forced swim tests. They also increased locomotor activity. RNA sequencing analysis of the brain tissue revealed substantial changes in gene expression, particularly in pathways associated with learning, memory, and anxiety regulation. Collectively, these results suggest that PLDP induces changes in gene expression associated with brain function, potentially contributing to the enhancement of cognitive function and psychological health. Furthermore, our findings not only enhance our understanding of the relationship between nutrition and brain function but also indicate the potential of interventions utilizing dietary components, such as PLDP, to support cognitive function and psychological health.

Keywords: Anxiety; Locomotor activity; Memory; Nutrition; Porcine liver.

Fig. 1

Schematic of the experimental design for evaluating the effects of PLDP ingestion in mice. (A) Sixteen-week-old male C57BL/6N mice were fed a PLDP-containing diet (PLDP mice) or a control diet (control mice) for 16 weeks, and behavioral analyses were conducted. One day after completing behavioral analyses, total RNA was extracted from their brains. The schema presents the timeline of PLDP diet feeding, behavioral analyses, and subsequent RNA extraction. EPM, elevated plus maze; FS, forced swimming; MB, marble burying, OF, Open field; YM, Y-maze. The graphic was created with Biorender.com. (B) Weekly changes in the body weights of the PLDP and control mice. Body weight was measured weekly. Two-way repeated measures ANOVA revealed no significant effect of diet on body weight (F(1, 24) = 0.9485, p = 0.3398, n = 13 each). (C) Food intake of the PLDP and control mice. The daily food intake of the PLDP and control mice was measured weekly. Two-way repeated measures ANOVA revealed no significant effect of diet on food intake (F(1, 24) = 3.293, p = 0.0821, n = 13 each). Data are represented as mean ± SEM.

Fig. 2

Ingestion of PLDP enhances memory and locomotor activity. (A–E) Barnes maze test. (A) Time taken to reach the target hole during the training session and probe tests. There was no significant difference in escape latency during the session between control and PLDP mice (two-way repeated measures ANOVA, n = 13 each, F(1,24) = 2.465, p = 0.1295). PLDP mice showed significantly shorter escape latency than control mice during the probe session (two-way repeated measures ANOVA, n = 13 each, F(1,24) = 7.950, p = 0.0095). (B and D) Representative trajectories of the PLDP and control mice in the 1st (B) and 2nd (D) probe tests. (C and E) Percentage of time spent in each quadrant during the 1st (C) and 2nd (E) probe tests. (F–I) Y-maze test. (F) Representative trajectories of the PLDP and control mice. (G) Total number of entries into the arms. (H) Percentage of correct alternations. (I) Distance traveled during the test. Data are represented as mean ± SEM in (A) and boxplots in (C, E, and G–I). The horizontal line in each box shows the median; the box shows the interquartile range (IQR); and the whiskers are 1.5 × IQR. n = 13 in each group. **p < 0.01, *p < 0.05; Mann–Whitney’s U–test in (C, E, and G–I).

Fig. 3

Ingestion of PLDP enhances locomotor activity and reduces anxiety-like behaviors. (A) Representative trajectories of the PLDP and control mice in the open field test. The trajectories at 10-min intervals are shown. (B) Distance traveled, (C) vertical activity, (D) immobility time, (E) time spent in the central zone, (F) number of entries into the central zone, and (G) number of grooming bouts at 10-min intervals. Data are represented as mean ± SEM. n = 13 in each group. The effects of the PLDP diet were analyzed using two-way repeated measures ANOVA; F(1,24) = 5.401; p = 0.0289 in (B), F(1,24) = 14.85; p = 0.0008 in (C); F(1,24) = 4.596, p = 0.0424 in (D); F(1,24) = 13.97, p = 0.001 in (E); F(1,24) = 8.072, p = 0.009 in (F) ; and F(1,24) = 0.6812, p = 0.4173 in (G). Statistical significance was analyzed by two-way repeated measures ANOVA.

Fig. 4

Ingestion of PLDP reduces anxiety-like behaviors. (A–F) Elevated plus maze test. (A) Representative trajectories of the PLDP and control mice. (B) Distance traveled, (C) number of entries into open arms, (D) percentage of open arm entries, and (E) percentage of time spent in open arms within 10 min. (F and G) Marble burying test. (F) Representative images captured before and after the test of the PLDP and control mice. (G) Number of marbles buried after a 10-min test. The data are represented as boxplots. The horizontal line in each box shows the median, the box shows the IQR and the whiskers are 1.5 × IQR. n = 13 in each group. **p < 0.01, *p < 0.05; Mann–Whitney’s U–test.

Fig. 5

Ingestion of PLDP exerts antidepression effect. (A) Representative photos of the PLDP and control mice in the forced swim test. (B) Latency to immobility. (C) Immobility time during a 10-min period. The data are presented as boxplots. The horizontal line in each box shows the median; the box shows the IQR; and the whiskers are 1.5 × IQR. n = 13 in each group. *p < 0.05; Mann–Whitney’s U–test.

Fig. 6

Long-term ingestion of PLDP alters gene expression in the brain. (A) Volcano plot showing differentially expressed genes in the brains of PLDP and control mice. Genes in PLDP mice with expression levels that significantly increased by more than twofold or decreased to less than half are shown in red. Genes in PLDP mice with expression changes within the range of 0.5-fold to 2.0-fold, showing significant decreases or increases, are shown in green. (B) IPA canonical pathway analysis. The canonical pathways related to cognitive and emotional functions with the lowest p-values are shown. Blue and orange bars indicate negative (inhibition) and positive (activation) z-scores, respectively. AChR, acetylcholine receptor; CREB, cyclic AMP response element-binding protein; CRH, corticotropin-releasing hormone; ERK, extracellular signal-regulated kinase; IL-10, interleukin-10; MAPK, mitogen-activated protein kinase. (C) IPA diseases and functional analysis. The relationship between gene expression and functional changes in the brain are shown. Molecules in dataset; A list of molecules associated with each function in IPA. Red characters represent genes significantly increased in PLDP mice, while blue characters represent genes significantly decreased.