The Antioxidant Effects of Whey Protein Peptide on Learning and Memory Improvement in Aging Mice Models

Abstract

This study investigated the antioxidant effects of whey protein peptide on learning and memory in aging C57BL/6N mice. A total of 72 SPF male C57BL/6N mice were used. Twelve mice were randomly selected as the control group, and the other mice were intraperitoneally injected with D-galactose (100 mg/kg body weight for 6 weeks), during which, the mice in the control group were intraperitoneally injected with the same amount of normal saline. After 6 weeks, the blood was taken from the epicanthus and the serum MDA level was measured, according to which, the mice were randomly divided into the model control group, the whey protein group (1.5 g/kg body weight), and three Whey protein peptide (WHP) intervention groups (0.3 g/kg body weight, 1.5 g/kg body weight, 3.0 g/kg body weight). The water solution of the test sample was administered by oral gavage every day. The intervention period was 30 days, during which, the model control group, the whey protein group, and the whey protein peptide group continued receiving intraperitoneal injections of D-galactose, while the control group continued receiving intraperitoneal injections of normal saline. After the intervention, behavioral experiments were conducted in the following order: open field test, water maze test, and new object recognition test. After the behavioral experiment, the morphology of hippocampal formation was observed by HE staining and TUNEL labeling. Oxidative stress-related indexes in the serum, liver, and brain were detected. Expression levels of the cholinergic system-related enzymes and proinflammatory cytokines in brain tissue were detected. Western blot was used to detect the expression of synaptic plasticity-related proteins in the mouse brain. The results showed that WHP could significantly improve the accumulation of MDA and PC, increase the activities of SOD and GSH-Px, resist oxidative stress injury, and enhance the potential of endogenous antioxidant defense mechanisms. WHP can significantly improve the decline of aging-related spatial exploration, body movement, and spatial and non-spatial learning/memory ability. Its specific mechanism may be related to reducing the degeneration of hippocampal nerve cells, reducing the apoptosis of nerve cells, improving the activity of AChE, reducing the expression of inflammatory factors (TNF-α and IL-1β) in brain tissue, reducing oxidative stress injury, and improving the expression of p-CaMKⅡ and BDNF synaptic plasticity protein. These results indicate that WHP can improve aging-related oxidative stress, as well as learning and memory impairment.

Keywords: D-galactose; antioxidation; learning and memory; whey protein peptide.

Figure 1

Typical roadmap of mice in the spatial probe test. C, control group; M, model control group; W, whey protein group at a dose of 1.5 g/kg; WL, whey protein peptide low dose group at a dose of 0.3 g/kg; WM whey protein peptide medium dose group at a dose of 1.5 g/kg; WH, whey protein peptide high dose group at a dose of 3.0 g/kg.

Figure 2

Effects of WHP on the learning and memory capability of the novel object recognition. Data are expressed as means ± SD (n = 8). ^a p < 0.05 indicates significant difference versus the control group; ^b p < 0.05 indicates significant difference versus the model group. Whey protein group at a dose of 1.5 g/kg; WHP-LG, whey protein peptide low dose group at a dose of 0.3 g/kg; WHP-MG, whey protein peptide medium dose group at a dose of 1.5 g/kg; WHP-HG, whey protein peptide high dose group at a dose of 3.0 g/kg.

Figure 3

HE staining of the coronal section of the hippocampus in each group. C, control group; M, model control group; W, whey protein group at a dose of 1.5 g/kg; WL, WHP-LG, whey protein peptide low dose group at a dose of 0.3 g/kg; WM, WHP-MG, whey protein peptide medium dose group at a dose of 1.5 g/kg; WH, WHP-HG, whey protein peptide high dose group at a dose of 3.0 g/kg.

Figure 4

HE staining of the hippocampal CA1 region in each group. C, control group; M, model control group; W, whey protein group at a dose of 1.5 g/kg; WL, WHP-LG, whey protein peptide low dose group at a dose of 0.3 g/kg; WM, WHP-MG, whey protein peptide medium dose group at a dose of 1.5 g/kg; WH, WHP-HG, whey protein peptide high dose group at a dose of 3.0 g/kg.

Figure 5

TUNEL labeling of the hippocampal CA1 region in each group. C, control group; M, model control group; W, whey protein group at a dose of 1.5 g/kg; WL, WHP-LG, whey protein peptide low dose group at a dose of 0.3 g/kg; WM, WHP-MG, whey protein peptide medium dose group at a dose of 1.5 g/kg; WH, WHP-HG, whey protein peptide high dose group at a dose of 3.0 g/kg.

Figure 6

Effects of WHP on the TUNEL labeling of the hippocampus CA1 region. Data are expressed as means ± SD (n = 8). ^a p < 0.05 indicates significant difference versus the control group; ^b p < 0.05 indicates significant difference versus the model group; ^c p < 0.05 indicates significant difference versus the whey protein group. Whey protein group at a dose of 1.5 g/kg; WHP-LG, whey protein peptide low dose group at a dose of 0.3 g/kg; WHP-MG, whey protein peptide medium dose group at a dose of 1.5 g/kg; WHP-HG, whey protein peptide high dose group at a dose of 3.0 g/kg.

Figure 7

Effects of WHP on serum SOD (a), liver SOD (b), serum GSH-Px (c), liver GSH-Px (d). Data are expressed as means ± SD (n = 12). ^a p < 0.05 indicates significant difference versus the control group; ^b p < 0.05 indicates significant difference versus the model group. SOD, superoxide dismutase; GSH-Px, glutathione peroxidase. Whey protein group at a dose of 1.5 g/kg; WHP-LG, whey protein peptide low dose group at a dose of 0.3 g/kg; WHP-MG, whey protein peptide medium dose group at a dose of 1.5 g/kg; WHP-HG, whey protein peptide high dose group at a dose of 3.0 g/kg.

Figure 8

Effects of WHP on serum MDA (a), brain MDA (b), serum PC (c), brain PC (d). Data are expressed as means ± SD (n = 12). ^a p < 0.05 indicates significant difference versus the control group; ^b p < 0.05 indicates significant difference versus the model group. MDA, malondialdehyde; PC, protein carbonyl. Whey protein group at a dose of 1.5 g/kg; WHP-LG, whey protein peptide low dose group at a dose of 0.3 g/kg; WHP-MG, whey protein peptide medium dose group at a dose of 1.5 g/kg; WHP-HG, whey protein peptide high dose group at a dose of 3.0 g/kg.

Figure 9

Effects of WHP on brain AChE (a) and brain ChAT (b). Data are expressed as means ± SD (n = 8). ^a p < 0.05 indicates significant difference versus the control group; ^b p < 0.05 indicates significant difference versus the model group. AChE, acetyl cholinesterase; ChAT, choline acetyltransferase. Whey protein group at a dose of 1.5 g/kg; WHP-LG, whey protein peptide low dose group at a dose of 0.3 g/kg; WHP-MG, whey protein peptide medium dose group at a dose of 1.5 g/kg; WHP-HG, whey protein peptide high dose group at a dose of 3.0 g/kg.

Figure 10

Effects of WHP on brain TNF-α (a), brain IL-1β (b). Data are expressed as means ± SD (n = 8). ^a p < 0.05 indicates significant difference versus the control group; ^b p < 0.05 indicates significant difference versus the model group; ^c p < 0.05 indicates significant difference versus the whey protein group. TNF-α, tumor necrosis factor-α; IL-1β, interleukin-1β. Whey protein group at a dose of 1.5 g/kg; WHP-LG, whey protein peptide low dose group at a dose of 0.3 g/kg; WHP-MG, whey protein peptide medium dose group at a dose of 1.5 g/kg; WHP-HG, whey protein peptide high dose group at a dose of 3.0 g/kg.

Figure 11

Effects of WHP on the expression of synaptic plasticity related proteins in the brain tissue of mice. (a) Synaptic plasticity related proteins were analyzed by Western blot. C, control group; M, model control group; W, whey protein group at a dose of 1.5 g/kg; WL, WHP-LG, whey protein peptide low dose group at a dose of 0.3 g/kg; WM, WHP-MG, whey protein peptide medium dose group at a dose of 1.5 g/kg; WH, WHP-HG, whey protein peptide high dose group at a dose of 3.0 g/kg. (b) Effects of WHP on p-CREB in the brain tissue of mice. (c) Effects of WHP on CREB in the brain tissue of mice. (d) Effects of WHP on p-CaMKⅡ in the brain tissue of mice. (e) Effects of WHP on CaMKⅡ in the brain tissue of mice. (f) Effects of WHP on BDNF in the brain tissue of mice. Data are expressed as means ± SD (n = 8). ^a p < 0.05 indicates significant difference versus the control group; ^b p < 0.05 indicates significant difference versus the model group; ^c p < 0.05 indicates significant difference versus the whey protein group. CREB, cAMP-response element binding protein; CaMKⅡ, calcium/calmodulin-dependent protein kinases Ⅱ; BDNF, brain-derived neurotrophic factor. Whey protein group at a dose of 1.5 g/kg; WHP-LG, whey protein peptide low dose group at a dose of 0.3 g/kg; WHP-MG, whey protein peptide medium dose group at a dose of 1.5 g/kg; WHP-HG, whey protein peptide high dose group at a dose of 3.0 g/kg.