Comparison of Donepezil, Memantine, Melatonin, and Liuwei Dihuang Decoction on Behavioral and Immune Endocrine Responses of Aged Senescence-Accelerated Mouse Resistant 1 Mice

Abstract

Aging is a natural biological process associated with cognitive decline and neuroendocrine-immune system changes; the neuroendocrine-immune system plays crucial role in brain aging and neurodegeneration, and it is essential to discern beneficial attempts to delay the aging progress based on immunological aging. In this study, we have investigated the effects of Traditional Chinese Medicine (TCM)-Liuwei Dihuang decoction (LW)-and donepezil, memantine, and melatonin on cognitive decline in aging mice. The aged SAMR1 mice received oral administration of donepezil (1mg/kg), memantine (10 mg/kg), melatonin (10 mg/kg), and LW (10 g/kg) for 3 months. A shuttle box, Morris water maze, and elevated-zero maze were performed to assess cognitive function, and flowcytometry, Luminex, and radioimmunoassay were performed to measure the lymphocyte subsets, inflammatory factors, and hormones. We observed that survival days of mice was increased with melatonin and LW, the anxiety behavior was significantly improved by memantine, melatonin, and LW treatment, active avoidance responses significantly improved by LW, donepezil, and memantine, the spatial learning ability was significantly improved by donepezil, and LW and melatonin were beneficial to the spatial memory of old mice. For immune function, LW increased CD4⁺ and CD4⁺CD28⁺ cells and reduced TNF-α, IL-1β, and G-CSF in plasma, and it also promoted the secretion of anti-inflammatory factors IL-4, IL-5, and IL-10 by regulating the active of Th2 cells in spleen. Donepezil and memantine exerted protective effects against CD4⁺CD28⁺ cell decrease caused by aging and reduced the pro-inflammatory factors TNF-α, IL-1β, and G-CSF in plasma. Melatonin could reverse CD8⁺CD28⁺ cell imbalances and increased B cells. For endocrine factors, LW increased TSH levels in the pituitary, and melatonin increased the GH level in blood. Our findings indicated that LW improved the cognitive decline in aging mice, and this might be associated with modulation of the active T cells and HPG axis hormones as well as increasing anti-inflammatory factors. Meanwhile, donepezil and memantine have advantages in regulating adaptive immunity, melatonin has advantages in the regulation of B cells and pituitary hormones, and LW exhibits a better effect on neuroendocrine immune function compared with the others from a holistic point of view. LW might be a potential therapeutic strategy for anti-aging-related syndromes, and it can also provide a value on medication guidance about drug combinations or treatment in clinic.

Keywords: Liuwei Dihuang decoction; aging; cognition; immune response; inflammation.

Figure 1

The experimental timeline and the effects of donepezil, memantine, melatonin, and LW on the aging performance of old SAMR1 mice. (A) The weight of mice during drug administration. (B) The locomotor activity of mice during drug administration. (C) The score of mouse nest building after 3 months of treatment. (D) The percentage survival of the mice. Log-rank test of survival curve, P=0.1. (E) Survival days of mice. (F) The aging degree score of mice during drug administration. One-way ANOVA and Dunnett test, mean ± S.D., n=13~16 (male: n = 4~6, female: n = 7~9). (G) The nest building scores. (H) Performance of nest building and fur of mice. (I) The peak of 1, 2, 3, 4, 5, and 7 are the main compounds α-morroniside, β-morroniside, oxypaeoniflorin, loganin, paeoniflorin, and 5-hydroxymethylfurfural of LW, respectively.

Figure 2

Effects of donepezil, memantine, melatonin, and LW on anxiety behavior in old SAMR1 mice. (A) Time spent in the open quadrants, (B) number of the open quadrants entries, and (C) total distance traveled in the elevated zero maze test. *P < 0.05, **P < 0.01 vs control group. Kruskal-Wallis test, mean ± S.D., n=11~13(male: n = 4~5, female: n = 7~8).

Figure 3

The effects of donepezil, memantine, melatonin, and LW on the active avoidance responses, spatial learning and memory in old SAMR1 mice. The successful avoidance times in training phase (A) and testing phase (B) in shuttle box test. n=9~10(male: n=4, female: n=5~6).The escape latency of mice in learning task (C) and probe trial (E), number of crossing the platform (D), the time spent in the target quadrant (F), distance traveled in the target quadrant (G), and swimming speed (H) in the Morris water maze test; *P < 0.05, **P < 0.01, ***P < 0.001, vs control group. Kruskal-Wallis test. ^#P < 0.05, ^##P < 0.01, vs memantine group, Kruskal-Wallis test. mean ± S.D., n = 7~9 (male: n = 2~4, female: n=5).

Figure 4

Effects of donepezil, memantine, melatonin, and LW on lymphocyte subsets of splenocyte in old SAMR1 mice. 1× 10⁶ spleen cells isolated from mouse (n = 6) spleen were harvested, (A) CD19⁺ B cells, (B) CD3⁺ CD4⁺ T cells, (C) CD3⁺ CD8⁺ T cells, (D) CD4⁺ CD25⁺ T cells, (E) CD4⁺ CD28⁺ T cells, (F) CD8⁺ CD28⁺ T cells quantified by flow cytometry. *P < 0.05, **P < 0.01, ***P < 0.001 vs control, one-way ANOVA and Dunnett test. mean ± S.D., n=6~9(male: n = 2~4, female: n = 4~6). (G) The scatter plot of lymphocyte subsets.

Figure 5

Effects of donepezil, memantine, melatonin, and LW on cytokine levels in the splenocyte culture supernatant and plasma of old SAMR1 mice. After the behavior testes, the mice were sacrificed, and spleen cells isolated from the spleen were harvested, 5×10⁵ cell/mL was seeded in 96-well plates, cultured for 56 h, and then the supernatants were harvested for cytokine detection. (A, I) tumor necrosis factor α (TNF-α), (B, J) interleukin-1β (IL-1β), (C, D, K) interleukin-6 (IL-6), (E, L) granulocyte colony stimulating factor (G-CSF), (F) interleukin-4 (IL-4), (G, M) interleukin-5 (IL-5), (H, N) interleukin-10 (IL-10). *P < 0.05, **P < 0.01, ***P < 0.001, vs control, one-way ANOVA and Dunnett test, Kruskal-Wallis test. mean ± S.D., n = 6~9(male: n=2~4, female: n=4~6).

Figure 6

The neuroendocrine hormone secretion in old SAMR1 mice after donepezil, memantine, melatonin, and LW administration. (A) corticosterone (CORT), (B) growth hormone (GH), (C) dehydroepiandrosterone (DHEA) in the plasma, (D) thyrotropin (TSH), (E) adrenocorticotropic hormone (ACTH), (F) luteinizing hormone (LH) in the pituitary, (G) estradiol (E2), (H) testosterone (T), (I) the ratio of T/E2 in male or female mice. *P < 0.05, **P < 0.01, ***P < 0.001, vs control group, one-way ANOVA and Dunnett test, Kruskal-Wallis test. mean ± S.D., n=6~9(male: n=2~4, female: n=4~6).

Figure 7

Correlation analysis between neuroendocrine-immune factors and behavior tests in old SAMR1 mice. The pro-inflammatory factors (A1-3) TNFα, (B1-3) IL-1β, (C1-3) IL-6, and (D1-3) G-CSF as well as the anti-inflammatory factors (E1-3) IL-5 and (F1-3) IL-10, hormones (G1-3) TSH, (H1-3) GH and (I1-3) ACTH related with active avoidance response, spatial memory and anxiety behavior of old SAMR1 mice. Pearson correlation analysis, n = 24-33, P < 0.05 was considered to be a significant correlation.