Kampo formulas alleviate aging-related emotional disturbances and neuroinflammation in male senescence-accelerated mouse prone 8 mice

Abstract

Aging-induced neuroinflammation, also known as neuroinflammaging, plays a pivotal role in emotional disturbances, including depression and anxiety, in older individuals, thereby leading to cognitive dysfunction. Although numerous studies have focused on therapeutic strategies for cognitive impairment in older individuals, little research has been performed on treating its preceding emotional disturbances. Here, we examined whether Kampo formulas (kososan [KS], nobiletin-rich kososan [NKS], and hachimijiogan [HJG]) can ameliorate aging-induced emotional disturbances and neuroinflammation in mice. The depression-like behaviors observed in SAMP8 mice, relative to normally aging SAMR1 mice, were significantly prevented by treatment with Kampo formulas for 13 weeks. Western blot analysis revealed that hippocampal neuroinflammation was significantly abrogated by Kampo formulas. KS and NKS also significantly attenuated the hippocampal neuroinflammatory priming induced by lipopolysaccharide (LPS, 0.33 mg/kg, i.p.) challenge in SAMP8 mice. Hippocampal IL-1β, IL-6, and MCP-1 levels were significantly decreased in NKS-treated SAMP8 mice. KS and NKS showed significantly reduced tau accumulation in the brains of SAMP8 mice. RNA-sequencing revealed that each Kampo formula led to unique dynamics of hippocampal gene expression and appeared to abrogate hippocampal inflammatory responses. HJG significantly blocked the LPS-induced increase in serum IL-6 and MCP-1. These results suggest that Kampo formulas would be useful for treating aging-induced depression, in part by regulating neuroinflammatory pathways. This finding may pave the way for the development of therapeutic strategies for aging-related emotional disturbances, which may contribute to the prevention of cognitive dysfunction in older individuals.

Keywords: SAMP8 mice; aging; emotional disturbances; kososan; neuroinflammation.

Figure 1

Oral administration of Kampo formulas ameliorates depression-like behaviors but not anxiety-like behaviors in SAMP8 mice. The tail suspension test (TST, A), sucrose preference test (SPT, B), and open field test (OFT, C) were performed following treatment with the indicated Kampo formulas or with water for 13 weeks. Data are shown as the mean ± SEM (n = 10−12). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, and ^****P < 0.0001 using Bonferroni’s post-hoc test. Abbreviations: KS: kososan; NKS: nobiletin-rich kososan; HJG: hachimijiogan.

Figure 2

Oral administration of Kampo formulas does not improve circadian rhythm disruption in SAMP8 mice. Time course of locomotor activity of mice aged 7 weeks (A) and 19 weeks (B) in their home cages. The time between 8:00 and 20:00 and between 20:00 and 8:00 represents the diurnal and nocturnal phases, respectively. The cumulative activity during the diurnal and nocturnal phases of mice aged 7 weeks (C) and 19 weeks (D) is shown. Data are shown as the mean ± SEM (n = 4−6). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, and ^****P < 0.0001 using paired t-test. Abbreviations: KS: kososan; NKS: nobiletin-rich kososan; HJG: hachimijiogan; W: weeks of age.

Figure 3

Distinct efficacy of different Kampo formulas for blocking neuroinflammatory priming in the hippocampus of SAMP8 mice at 19 weeks of age. (A) Representative western blotting images of NLRP3, Arg1, and GAPDH expression. Expression of NLRP3 (B) and Arg1 (C) were normalized based on GAPDH expression. (D) Pro-inflammatory/anti-inflammatory balance. Data are shown as the mean ± SEM (n = 5 or 6). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, and ^****P < 0.0001 using Bonferroni’s post-hoc test. Abbreviations: KS: kososan; NKS: nobiletin-rich kososan; HJG: hachimijiogan; LPS: lipopolysaccharide.

Figure 4

Oral administration of Kampo formulas results in characteristic gene expression patterns in the hippocampus of 19-week-old SAMP8 mice. (A) Genes that were significantly down-regulated in the water-administered SAMP8 mice as compared with water-administered SAMR1 mice and that were significantly up-regulated by treatment with Kampo formulas, i.e., the green line, are referred to as V-shaped recovered genes. Genes that were significantly up-regulated in the water-administered SAMP8 mice as compared with water-administered SAMR1 mice and that were significantly down-regulated by treatment with Kampo formulas, i.e., the orange line, are referred to as reverse V-shaped recovered genes. (B) Venn diagrams showing differences and similarities in gene expression among Kampo formula–treated groups. Numbers in circles indicate the number of genes with statistically significant changes in expression (P < 0.05). The full list of genes depicted using lower-case letters is shown in Supplementary Table 1. (C) Heatmap of differential gene expression related to the inflammatory response in the hippocampus of SAMR1 and SAMP8 mice. ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001 (n = 5) vs. water-administered SAMP8 mice, using the likelihood ratio test. Abbreviations: KS: kososan; NKS: nobiletin-rich kososan; HJG: hachimijiogan; LPS: lipopolysaccharide.

Figure 5

Oral administration of KS and NKS blocks the increase in the level of tau observed in the hippocampus of SAMP8 mice. (A) Representative western blot images of tau and GAPDH expression. (B) Expression of tau was normalized based on GAPDH expression. (C) Expression of Mapt was normalized based on Gapdh expression. Heatmap of differential gene expression related to phosphorylation and splicing of tau (D) and autophagy (E) in the hippocampus of mice. Data are shown as the mean ± SEM (n = 5 or 6). ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001 using Bonferroni’s post-hoc test. Abbreviations: KS: kososan; NKS: nobiletin-rich kososan; HJG: hachimijiogan.

Figure 6

Oral administration of NKS modulates pro- and anti-inflammatory cytokine and chemokine levels in the hippocampus of mice after saline or LPS injection. Data are shown as the mean ± SEM (n = 5 or 6). ^*P < 0.05 and ^**P < 0.01 using Bonferroni’s post-hoc test. Abbreviations: KS: kososan; NKS: nobiletin-rich kososan; HJG: hachimijiogan; LPS: lipopolysaccharide.

Figure 7

Serum levels of a pro-inflammatory cytokine (IL-6) and chemokine (MCP-1) are suppressed by oral administration of HJG after LPS injection. Data are shown as the mean ± SEM (n = 5 or 6). ^*P < 0.05 using Dunnett’s post-hoc test. Abbreviations: KS: kososan; NKS: nobiletin-rich kososan; HJG: hachimijiogan; LPS: lipopolysaccharide.

Figure 8

Schematic representation of the experimental schedule. Abbreviations: OFT: open field test; SPT: sucrose preference test; TST: tail suspension test; Decap: decapitation; LPS: lipopolysaccharide.