Long-Term Effect of Porcine Brain Enzyme Hydrolysate Intake on Scopolamine-Induced Memory Impairment in Rats

Abstract

No study has revealed the effect of porcine brain enzyme hydrolysate (PBEH) on memory impairment. We aimed to examine the hypothesis that PBEH intake modulates memory deficits and cognitive behavior in scopolamine (SC)-induced amnesia rats, and its mechanism, including gut microbiota changes, was determined. Sprague-Dawley male rats had intraperitoneal injections of SC (2 mg/kg body weight/day) at 30 min after daily feeding of casein (MD-control), PBEH (7 mg total nitrogen/mL) at 0.053 mL (Low-PBEH), 0.159 mL (Medium-PBEH), 0.478 mL (High-PBEH), or 10 mg donepezil (Positive-control) per kilogram body weight per day through a feeding needle for six weeks. The Normal-control rats had casein feeding without SC injection. PBEH dose-dependently protected against memory deficits determined by passive avoidance test, Y-maze, water-maze, and novel object recognition test in SC-induced rats compared to the MD-control. The High-PBEH group had a similar memory function to the Positive-control group. Systemic insulin resistance determined by HOMA-IR was lower in the PBEH groups than in the Normal-control but not the Positive-control. In parallel with systemic insulin resistance, decreased cholesterol and increased glycogen contents in the hippocampus in the Medium-PBEH and High-PBEH represented reduced brain insulin resistance. PBEH intake prevented the increment of serum TNF-α and IL-1β concentrations in the SC-injected rats. Hippocampal lipid peroxide and TNF-α contents and mRNA TNF-α and IL-1β expression were dose-dependently reduced in PBEH and Positive-control. PBEH decreased the hippocampal acetylcholinesterase activity compared to the MD-control, but not as much as the Positive-control. PBEH intake increased the α-diversity of the gut microbiota compared to the MD-control, and the gut microbiota community was separated from MD-control. In metagenome function analysis, PBEH increased the energy metabolism-related pathways of the gut microbiota, including citric acid cycle, oxidative phosphorylation, glycolysis, and amino acid metabolism, which were lower in the MD-control than the Normal-control. In conclusion, alleviated memory deficit by PBEH was associated potentially with not only reducing acetylcholinesterase activity but also improving brain insulin resistance and neuroinflammation potentially through modulating gut microbiota. PBEH intake (1.5-4.5 mL of 7 mg total nitrogen/mL for human equivalent) can be a potential therapeutic agent for improving memory impairment.

Keywords: acetylcholinesterase; insulin resistance; memory deficit; porcine brain enzyme hydrolysates; scopolamine.

Figure 1

Memory deficits in rats after scopolamine injection. Male rats had intraperitoneal injections of scopolamine (2 mg/kg body weight/day) at 30 min after daily feeding of casein (MD-control), porcine brain enzyme hydrolysates (PBEH; 7 mg total nitrogen/mL) at 0.053 mL (Low-PBEH), 0.159 mL (Medium-PBEH), 0.478 mL (High-PBEH), or 10 mg donepezil (Positive-control) per kilogram body weight per day for six weeks. (A) Latency time to enter the dark box (s) during the first, second, and third trials; (B) novel object recognition index during novel objective recognition test and the number of the right turns among the total turns in the Y-maze test; (C) latency time to the first visit, frequencies to visit zone 5, and duration in zone 5. The bars and error bars represent the means ± standard deviations (n = 10). ^a–c Different superscript letters on the bars indicate a significant difference among the groups according to a Tukey test at p < 0.05.

Figure 2

Depression measurement by forced swimming test. Male rats had intraperitoneal injections of scopolamine (2 mg/kg body weight/day) at 30 min after daily feeding of casein (MD-control), porcine brain enzyme hydrolysates (PBEH; 7 mg total nitrogen/mL) at 0.053 mL (Low-PBEH), 0.159 mL (Medium-PBEH), 0.478 mL (High-PBEH), or 10 mg donepezil (Positive-control) per kilogram body weight per day for six weeks. Bars and error bars represent the means ± standard deviations (n = 10). ^a–c Different superscript letters on the bars indicate a significant difference among the groups by Tukey test at p < 0.05.

Figure 3

Changes in serum glucose and insulin concentrations and areas under the curve during oral glucose tolerance testing (OGTT) and intraperitoneal insulin tolerance testing (IPITT). Male rats had intraperitoneal injections of scopolamine (2 mg/kg body weight/day) at 30 min after daily feeding of casein (MD-control), porcine brain enzyme hydrolysates (PBEH; 7 mg total nitrogen/mL) at 0.053 mL (Low-PBEH), 0.159 mL (Medium-PBEH), 0.478 mL (High-PBEH), or 10 mg donepezil (Positive-control) per kilogram body weight per day for six weeks. (A) Changes in serum glucose concentration during oral glucose tolerance test after oral consumption of 2 g glucose/kg body weight; (B) changes of serum insulin concentrations during OGTT; dots and error bars represent the means ± standard deviations (n = 10). * Significantly different among the groups at each time point at p < 0.05.

Figure 4

Gut microbiota α- and β-diversity and profiles. Male rats had intraperitoneal injections of scopolamine (2 mg/kg body weight/day) at 30 min after daily feeding of casein (MD-control), porcine brain enzyme hydrolysates (PBEH; 7 mg total nitrogen/mL) at 0.053 mL (Low-PBEH), 0.159 mL (Medium-PBEH), 0.478 mL (High-PBEH), or 10 mg donepezil (Positive-control) per kilogram body weight per day for six weeks. (A) Chao1 index of fecal bacteria; (B) Shannon index of fecal bacteria; (C) principal coordinate analysis (PCoA) of fecal bacteria; (D) relative amounts (%) of fecal samples at the family level; (E) relative amounts (%) of fecal samples at the genus level. Bars and error bars represent the means ± standard deviations (n = 10). ^a,b Different superscript letters on the bars indicated a significant difference among the groups by Tukey test at p < 0.05.

Figure 4

Gut microbiota α- and β-diversity and profiles. Male rats had intraperitoneal injections of scopolamine (2 mg/kg body weight/day) at 30 min after daily feeding of casein (MD-control), porcine brain enzyme hydrolysates (PBEH; 7 mg total nitrogen/mL) at 0.053 mL (Low-PBEH), 0.159 mL (Medium-PBEH), 0.478 mL (High-PBEH), or 10 mg donepezil (Positive-control) per kilogram body weight per day for six weeks. (A) Chao1 index of fecal bacteria; (B) Shannon index of fecal bacteria; (C) principal coordinate analysis (PCoA) of fecal bacteria; (D) relative amounts (%) of fecal samples at the family level; (E) relative amounts (%) of fecal samples at the genus level. Bars and error bars represent the means ± standard deviations (n = 10). ^a,b Different superscript letters on the bars indicated a significant difference among the groups by Tukey test at p < 0.05.

Figure 5

Gut microbiome functions and short-chain fatty acids in the portal vein. Male rats had intraperitoneal injections of scopolamine (2 mg/kg body weight/day) at 30 min after daily feeding of casein (MD-control), porcine brain enzyme hydrolysates (PBEH; 7 mg total nitrogen/mL) at 0.053 mL (Low-PBEH), 0.159 mL (Medium-PBEH), 0.478 mL (High-PBEH), or 10 mg donepezil (Positive-control) per kilogram body weight per day for six weeks. (A) Glycolysis (Embden–Meyerhof pathway), glucose → pyruvate; (B) pyruvate oxidation, pyruvate→ acetyl-CoA; (C) beta-oxidation, acetyl-Co A synthesis; (D) citrate cycle (TCA cycle); (E) leucine degradation, leucine → acetoacetate + acetyl-CoA; (F) fatty acid biosynthesis, initiation; (G) bile acid biosynthesis, cholesterol → cholate/chenodeoxycholate; (H) 3-Hydroxypropionate bi-cycle. (I) Bars and error bars represent the means ± standard deviations (n = 10). ^a–d Different superscript letters on the bars indicate a significant difference among the groups by Tukey test at p < 0.05.

Figure 5

Gut microbiome functions and short-chain fatty acids in the portal vein. Male rats had intraperitoneal injections of scopolamine (2 mg/kg body weight/day) at 30 min after daily feeding of casein (MD-control), porcine brain enzyme hydrolysates (PBEH; 7 mg total nitrogen/mL) at 0.053 mL (Low-PBEH), 0.159 mL (Medium-PBEH), 0.478 mL (High-PBEH), or 10 mg donepezil (Positive-control) per kilogram body weight per day for six weeks. (A) Glycolysis (Embden–Meyerhof pathway), glucose → pyruvate; (B) pyruvate oxidation, pyruvate→ acetyl-CoA; (C) beta-oxidation, acetyl-Co A synthesis; (D) citrate cycle (TCA cycle); (E) leucine degradation, leucine → acetoacetate + acetyl-CoA; (F) fatty acid biosynthesis, initiation; (G) bile acid biosynthesis, cholesterol → cholate/chenodeoxycholate; (H) 3-Hydroxypropionate bi-cycle. (I) Bars and error bars represent the means ± standard deviations (n = 10). ^a–d Different superscript letters on the bars indicate a significant difference among the groups by Tukey test at p < 0.05.

Figure 6

Experimental design.