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

Results

Kampo formulas ameliorate aging-induced depression-like behaviors in SAMP8 mice

In the TST, the immobility time of SAMP8 mice was significantly increased relative to that of senescence-accelerated mouse resistant 1 (SAMR1) mice, as normal aging controls (Figure 1A; Bonferroni’s post-hoc test, P < 0.0001). This effect was significantly prevented by treatment with Kampo formulas (Bonferroni’s post-hoc test; KS, P < 0.0001; NKS, P < 0.0001; HJG, P < 0.001). Moreover, a significant difference in immobility was found between NKS and HJG treatment (Bonferroni’s post-hoc test, P < 0.001), suggesting that the preventive effect of NKS was superior to that of HJG.

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.

In the SPT, the sucrose preference was significantly lower in SAMP8 mice than in SAMR1 mice (Figure 1B; Bonferroni’s post-hoc test, P < 0.05). This decrease in sucrose preference was not affected by treatment with Kampo formulas. However, a significant negative correlation was also found between the immobility time in the TST and the sucrose preference in the SPT (Supplementary Figure 1; Pearson’s correlation coefficient test, r = −0.3048, P < 0.05), suggesting that Kampo formulas ameliorate aging-induced apathy-like behavior, along with anhedonia-like behavior, in SAMP8 mice. This behavioral recovery indicates the antidepressant-like effect of the Kampo formulas.

In the OFT, the time spent in the center and intermediate zones was significantly decreased and the time spent in the peripheral zone was significantly increased in SAMP8 mice relative to SAMR1 mice (Figure 1C; Bonferroni’s post-hoc test; center zone, P < 0.001; intermediate zone, P < 0.01; peripheral zone, P < 0.0001). These changes were retained after treatment with Kampo formulas. There was also no difference among the groups in the distance traveled during the OFT. Thus the Kampo formulas do not prevent aging-induced anxiety-like behaviors in SAMP8 mice.

At the beginning of the Kampo treatment period (7 weeks of age), mice in all groups had increased locomotor activity during the nocturnal phase relative to the diurnal phase (Figure 2A). Similarly, cumulative activity during the nocturnal phase was significantly higher than that during the diurnal phase for all groups (Figure 2C; paired t-test; SAMR1/water, P < 0.0001; SAMP8/water, P < 0.05; SAMP8/KS, P < 0.0001; SAMP8/NKS, P < 0.001; SAMP8/HJG, P < 0.001), indicating that both SAMR1 and SAMP8 mice showed normal circadian rhythms at 7 weeks of age. At 19 weeks of age, however, there was no difference in cumulative activity between diurnal and nocturnal phases in the water-administered SAMP8 mice (Figure 2B, 2D). There was still a significant difference between these phases in the water-administered SAMR1 mice (paired t-test, P < 0.05), although a nanotag datum of a water-administered SAMR1 mouse was not extracted because of the built-in battery exhaustion of nanotag. These results suggest that SAMP8 mice aged 19 weeks exhibit circadian rhythm disruption. This disruption was not alleviated by treatment with Kampo formulas, even when the SAMP8 mice were assessed at earlier ages (Supplementary Figure 2), implying that Kampo formulas are ineffective for treating circadian rhythm disruption in SAMP8 mice.

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.

With regard to body weight changes, the water-administered SAMP8 mice had significantly greater body weight gain from 7 to 19 weeks of age as compared with the water-administered SAMR1 mice (Bonferroni’s post-hoc test, P < 0.05). No differences in body weight were found between water-administered and Kampo formulas–administered SAMP8 mice (Supplementary Figure 3).

Hippocampal neuroinflammatory priming is attenuated in different ways by Kampo formulas in SAMP8 mice

To unravel the mechanisms underlying the antidepressant-like effect of Kampo formulas, we examined whether Kampo formulas regulate neuroinflammation in the hippocampus, which is involved in the depressive phenotype [53–55]. We used expression of NLRP3 and Arg1 as markers of pro- and anti-inflammatory phenotypes, respectively. Under physiological conditions, there was no difference in NLRP3 expression among the groups (Figure 3A and 3B, left), whereas Arg1 expression tended to decrease in the water-administered SAMP8 mice relative to the water-administered SAMR1 mice (Figure 3A and 3C, left; Bonferroni’s post-hoc test, P < 0.1). The decrease in Arg1 expression was significantly increased by treatment with Kampo formulas (Bonferroni’s post-hoc test, P < 0.01). The ratio of NLRP3 to Arg1, which represents the balance between pro- and anti-inflammatory phenotypes [21], was significantly increased in the water-administered SAMP8 mice as compared with the water-administered SAMR1 mice (Figure 3D, left; Bonferroni’s post-hoc test, P < 0.05). This increase was significantly reduced in SAMP8 mice treated with Kampo formulas (Bonferroni’s post-hoc test, P < 0.01), suggesting that Kampo formulas abrogate hippocampal neuroinflammation in SAMP8 mice aged 19 weeks.

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.

Next, we examined whether Kampo formulas prevented the neuroinflammatory response to LPS injection, to assess the influence of neuroinflammatory priming on the hippocampus. NLRP3 expression was significantly increased in the water-administered SAMP8 mice as compared with the water-administered SAMR1 mice (Figure 3A and 3B, right; Bonferroni’s post-hoc test, P < 0.01), but this increase was not decreased by treatment with Kampo formulas. In contrast, Arg1 expression was significantly reduced in the water-administered SAMP8 mice as compared with the water-administered SAMR1 mice (Figure 3A and 3C, right; Bonferroni’s post-hoc test, P < 0.0001), and the Arg1 expression was significantly increased in the NKS-administered SAMP8 mice relative to the water-administered SAMP8 mice (Bonferroni’s post-hoc test, P < 0.05). The ratio of NLRP3 to Arg1 was significantly increased in the water-administered SAMP8 mice as compared with the water-administered SAMR1 mice (Figure 3D, right; Bonferroni’s post-hoc test, P < 0.0001). This increase in the NLRP3/Arg1 ratio was significantly decreased in KS- and NKS-administered SAMP8 mice (Bonferroni’s post-hoc test; KS, P < 0.05; NKS, P < 0.0001). These results suggest that hippocampal neuroinflammatory priming by LPS is buffered by treatment with KS and NKS.

The expression of Toll-like receptor 4 (TLR4), an endogenous receptor for LPS, was also unaltered in all groups, irrespective of whether LPS had been administered (Supplementary Figure 4). This suggests that the inhibitory effects of KS and NKS on hippocampal neuroinflammatory priming do not result from the downregulation of TLR4 protein.

Kampo formulas result in characteristic gene expression patterns in the hippocampus of SAMP8 mice

To investigate whether Kampo formulas have characteristic effects on hippocampal gene expression dynamics, a genome-wide RNA-seq analysis was conducted on hippocampal tissue from the treated mice. Genes that were significantly downregulated in the water-administered SAMP8 mice as compared with the water-administered SAMR1 mice and that were significantly upregulated by treatment with Kampo formulas were regarded as “V-shaped recovered genes.” Genes that were significantly upregulated in the water-administered SAMP8 mice as compared with the water- administered SAMR1 mice and that were significantly downregulated by treatment with Kampo formulas were regarded as “reverse V-shaped recovered genes.” On the basis of these criteria (Figure 4A), the V-shaped and reverse V-shaped recovered genes under physiological and LPS-stimulated conditions are summarized (Figure 4B), and a full list of significantly regulated genes is shown in Supplementary Table 1. The Venn diagrams show that there were many genes that were significantly recovered by each Kampo formula. Among the Kampo formulas, the greatest number of genes, with or without annotation, was altered by HJG treatment (Supplementary Figure 5).

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.

To explore the biological significance of these recovered DEGs, we used the DAVID functional annotation tool. GO analysis of the genes recovered by each Kampo formula was performed, and the top 25 statistically significant (likelihood ratio test, P < 0.05) terms are shown in Supplementary Figure 6. The group of genes that were recovered by KS treatment showed almost no significantly enriched GO and KEGG terms. In contrast, the group of genes that were recovered after treatment with HJG had the most enriched GO and KEGG terms. Among the V-shaped recovered genes, under physiological conditions, the most enriched GO term was chemotaxis, for both NKS and HJG treatment (Supplementary Figure 6A). In terms of the reverse V-shaped recovered genes, the most-enriched GO terms were related to the regulation of transcription (Supplementary Figure 6B). In contrast, under LPS-stimulated conditions, the V-shaped recovered genes showed that the most enriched GO term was bone mineralization in KS treatment and cell cycle in HJG treatment, while few enriched GO terms were shown in NKS treatment (Supplementary Figure 6C). In terms of the reverse V-shaped recovered genes, the most-enriched GO terms for all KS, NKS and HJG treatment were related to the immune system (Supplementary Figure 6D). Several terms related to response to virus and interferon response, as well as other immune-related terms, were significantly enriched by HJG treatment.

A KEGG pathway analysis revealed that signaling pathways related to various cellular functions (e.g., cell survival, proliferation, apoptosis, cancer) were more enriched in both V-shaped (Supplementary Figure 6E and 6G) and reverse V-shaped recovered genes (Supplementary Figure 6F and 6H) associated with HJG treatment. Additionally, in the reverse V-shaped recovered genes, virus-related terms were also observed under LPS stimulation in the groups that received KS or HJG treatment (Supplementary Figure 6H). These results indicate that LPS-induced immune, inflammatory, and viral responses in the hippocampus were downregulated by treatment with Kampo formulas, and with HJG in particular.

Next, we focused on the recovered transcripts classified into the GO category “positive and negative regulation of inflammatory response” and “positive regulation of leukocyte activation.” Under physiological conditions, significant differences in numerous genes related to the inflammatory response were found between water-administered SAMR1 and water-administered SAMP8 mice, and these were significantly regulated by treatment with Kampo formulas, particularly HJG (Figure 4C, left). Similarly, under LPS-stimulated conditions, significant differences in numerous genes related to the inflammatory response were found between water-administered SAMR1 and water-administered SAMP8 mice, but there were a number of genes that were significantly recovered by treatment with KS, NKS, and HJG (Figure 4C, right). These results imply that each Kampo formula induces a characteristic gene expression pattern in the hippocampus of SAMP8 mice.

Hippocampal tau levels are suppressed by KS and NKS treatment in SAMP8 mice

Depression with mild cognitive impairment is associated with progression to Alzheimer’s disease [56]. Consequently, we examined the influence of Kampo formulas on tau levels in the hippocampus of 19-week-old SAMP8 mice. Hippocampal tau levels were significantly higher in the water-administered SAMP8 mice than in the water-administered SAMR1 mice (Figure 5A and 5B, Bonferroni’s post-hoc test, P < 0.01). These levels were significantly reduced by treatment with KS and NKS (Bonferroni’s post-hoc test; KS, P < 0.05; NKS, P < 0.01). However, no change was found between groups in terms of tau synthesis per se, as indicated by the gene expression levels of Mapt, the gene encoding tau (Figure 5C). These results suggest that tau accumulation, but not its synthesis, is hindered by treatment with KS and NKS. This finding is consistent with observations made in coronal brain sections (Supplementary Figure 7), suggesting that KS and NKS exert a suppressive effect on tau accumulation in the brain.

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.

Next, to identify the potential sites of action of Kampo formulas for abrogating tau accumulation, we assessed the expression of genes involved in tau phosphorylation (Figure 5D). Cdkrap1, a gene encoding an inhibitor of cyclin-dependent protein kinase 5 (Cdk5) [57] that phosphorylates tau, showed V-shaped recovered expression with KS treatment. The gene encoding dual-specificity tyrosine phosphorylated and regulated kinase 2 (Dyrk2) [58] showed reverse V-shaped recovered expression with HJG treatment. NKS and HJG treatment both caused reverse V-shaped recovered of the genes encoding Krüppel-like factor-10 (Klf10) and Ngfi-A-binding protein-2 (Nab2), which mediate neurotoxicity and tau phosphorylation, respectively [59]. Furthermore, expression of the gene encoding TRA2B (Tra2b), a splicing factor that contributes to alternative splicing of tau exon 10 in the central nervous system, was also reversed with NKS and HJG treatment. The alternative usage of exon 10 results in tau isoforms containing either three or four microtubule-binding repeats (3R-tau and 4R-tau, respectively) [60]. Thus, changes in Tra2b expression in SAMP8 mice may affect the 3R-tau/4R-tau expression ratio.

Autophagy is a degradation pathway involved in the clearance of various toxic aggregated proteins associated with neurodegenerative diseases, such as tau in tauopathies [61]. Therefore, we searched for genes related to autophagy regulation among the recovered genes. As shown in Figure 5E, several autophagy-related genes were detected among both V-shaped and reverse V-shaped recovered genes associated with NKS and HJG treatment. These results suggest that reduction of tau protein caused by KS and NKS treatment is more likely due to inhibition of tau phosphorylation than to tau degradation by autophagy. However, it remains uncertain why HJG failed to reduce tau levels, regardless of marked changes in genes related to tau dynamics.

Hippocampal levels of pro- and anti-inflammatory cytokines and chemokines are modulated by NKS treatment

To verify the effects of Kampo formulas on hippocampal neuroinflammation in western blotting analysis, we measured hippocampal protein levels of pro- and anti-inflammatory cytokines and chemokines by ELISA. Under physiological conditions, IL-1β and IL-6 levels were significantly reduced in the NKS-administered SAMP8 mice as compared with the water-administered SAMP8 mice (Figure 6; Bonferroni’s post-hoc test; IL-1β, P < 0.01; IL-6, P < 0.05). The NKS-administered SAMP8 mice also showed a tendency for reduced MCP-1 levels as compared with the water-administered SAMP8 mice (Bonferroni’s post-hoc test, P < 0.1). Under LPS-stimulated conditions, IL-1β and MCP-1 levels were significantly reduced in the NKS-administered SAMP8 mice as compared with the water-administered SAMP8 mice (Bonferroni’s post-hoc test, IL-1β, P < 0.05; MCP-1, P < 0.05). The NKS-administered SAMP8 mice also showed a trend toward elevated IL-4 levels as compared with the water-administered SAMP8 mice (Bonferroni’s post-hoc test, P < 0.1). These results partially reflected data obtained from western blotting analysis, particularly in the NKS-treated group, which suggested that NKS plays a role in preventing hippocampal neuroinflammation and neuroinflammatory priming, thereby leading to antidepressant-like effects.

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.

Oral administration of HJG suppresses systemic inflammation in SAMP8 mice

To examine whether Kampo formulas affect systemic inflammation, serum levels of inflammatory factors were measured by ELISA. Under LPS-stimulated conditions, serum IL-6 levels were significantly increased in the water-administered SAMP8 mice as compared with the water-administered SAMR1 mice (Figure 7; Dunnett’s post-hoc test, P < 0.05), and this increase was significantly blocked by HJG treatment (Dunnett’s post-hoc test, P < 0.05). Likewise, the HJG-administered SAMP8 mice showed a significant decrease in serum MCP-1 levels as compared with the water-administered SAMP8 mice (Dunnett’s post-hoc test, P < 0.05). However, no significant difference in either IL-6 or MCP-1 levels was found between these groups under saline-injected conditions. These results suggest that HJG suppresses systemic inflammation stimulated by LPS.

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.

Materials and Methods

Animals

Male SAMP8 and SAMR1 mice were obtained at 5 weeks of age from Japan SLC (Hamamatsu, Japan) and were allowed to acclimate for 1 week after arrival. Mice were individually housed for avoiding fights and their concomitant injuries at a constant temperature (23 ± 2°C) and humidity (55 ± 10%) and with a 12-h light/dark cycle (lights on at 08:00) with access to food (LabDiet5058, Lab Supply, Fort Worth, TX, USA) and water ad libitum.

All animal experiments were approved by the Institutional Animal Care and Use Committee of Kitasato University and were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of Kitasato University and the National Research Council Guide for the Care and Use of Laboratory Animals in Japan. Every effort was made to minimize the number of animals used and their suffering.

Preparation of Kampo extract

The component herbs of KS, NKS, and HJG are shown in Table 1. Each Kampo formula was decocted with 600 ml of distilled water until the volume was reduced by half. The water extract was immediately filtered with a commercial tea strainer and centrifuged at 1000 × g for 10 min at 4°C, and the supernatant was lyophilized. The total yield of KS, NKS, and HJG extracts was approximately 28%, 25%, and 19.6%, respectively, relative to the dry weight of the herbal mixture. The preparation of KS and NKS extracts and their component analysis were performed by Kotaro Pharmaceutical Co., Ltd (Osaka, Japan).

Table 1. Component herbs and daily quantities for kososan (KS), nobiletin-rich kososan (NKS), and hachimijiogan (HJG).

Herb	KS	NKS	HJG
a. Cyperus rhizome	4 g	4 g
b. Perilla herb	2 g	2 g
c. Citrus unshiu peel	3 g
d. Citrus reticulata peel		3 g
e. Glycyrrhiza	2 g	2 g
f. Ginger	0.5 g	0.5 g
g. Rehmannia root			6 g
h. Alisma rhizome			3 g
i. Poria sclerotium			3 g
j. Dioscorea rhizome			3 g
k. Cornus fruit			3 g
l. Moutan bark			3 g
m. Cinnamon bark			1 g
n. Aconite root			1 g
Total herbal weight per day	11.5 g	11.5 g	23 g
Herbs (a–c, f) were purchased from Tsumura and Co. (Tokyo, Japan). Herb (d) was purchased from Kotaro Pharmaceutical Co., Ltd. (Osaka, Japan). Herbs (e, g, h, n) were purchased from Uchida Wakan-yaku Co., Ltd. (Tokyo, Japan). Herbs (i, j, k, l, m) were purchased from Tochimoto Tenkaido Co., Ltd. (Tokyo, Japan).

Drug treatment and measurement of body weight

Each extract of the Kampo formula was dissolved in distilled water. The extract (1.0 g/kg body weight) or distilled water was administered once daily by oral gavage to mice from 7 weeks of age for 13 weeks (Figure 8). The dose of KS extract in this study was selected based on our previous studies, which showed that KS extract has an antidepressant-like effect in mouse models of stress-induced depression [35–37, 39, 102]. Other Kampo extracts (NKS and HJG) were set to the same dose as KS for comparison. The dose of each Kampo formula used in mice was equivalent to approximately 15-fold of that in humans. Body weight was measured in mice once a week before the drug treatment.

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.

Monitoring of locomotor activity

Locomotor activity of mice in their home cages was simultaneously recorded for 24 h using a Nanotag device (Kissei Comtec Co., Ltd., Nagano, Japan), an implantable three-axis accelerometer (weight, 2.6 g). The nanotag was intraperitoneally implanted in half of mice per group aged 6 weeks under inhalation anesthesia with isoflurane (Figure 8). After recovery for 1 week, the locomotor activity of mice at 7, 9, 11, 13, 15, and 19 weeks of age was recorded in 4-min bins over 24 h, and the data were analyzed using the Nanotag viewer program (Kissei Comtec Co., Ltd.).

Open field test

For the open field test (OFT), individual mice were introduced into an opaque gray open field box (40 × 40 × 40 cm) and were allowed to explore freely for 5 min under high light conditions (120–130 lux). Time spent in three zones (center, intermediate, and peripheral zones) and the total distance traveled were recorded using a video tracking system (EthoVision 3.0; Noldus, Wageningen, Netherlands). Anxiety-like behaviors were evaluated based on the time spent in the three zones. The OFT was performed between 12:00 and 17:00 after acclimation to a habituation room for 1 h.

Sucrose preference test

The sucrose preference test (SPT) was performed to evaluate anhedonia, a depression-like behavior, as described [21, 103] with some modifications. Briefly, mice were trained to adapt to the presentation of two bottles of water in their home cages for 2 days. After adaptation, each mouse had free access to two bottles, one containing a 2% sucrose solution and the other containing water, with a counterbalance in its home cage from 17:00 to 9:00 over 2 days. The sucrose preference ratio was calculated as follows: (2% sucrose solution intake [ml]/total fluid intake [ml]) × 100, and the average of the ratio per mouse over the 2 days of the experiment was used for analysis.

Tail suspension test

The tail suspension test (TST) was performed to evaluate apathy, which is a depression-like behavior, as described [21, 104] with some modifications. Briefly, for 6 min the mice were suspended by their tails 50 cm above the floor using experimental clips (Yamashitagiken, Tokushima, Japan) connected to a hook that was connected to a steel bar. A mouse was considered immobile only when it ceased struggling and hung motionless. All behaviors were videotaped, and the duration of immobility during the last 4 min of the TST was determined. The TST was conducted between 12:00 and 14:00 after acclimation to a habituation room for 1 h.

Lipopolysaccharide injection

After the TST, mice were injected i.p. with lipopolysaccharide (LPS, serotype O55:B5, 0.33 mg/kg body weight; Sigma, St. Louis, MO, USA) or saline. Intraperitoneal LPS injection was performed at a volume of 10 ml/kg body weight.

Brain and serum collection

Twenty-four hours after LPS or saline injection, the mice were decapitated, and their brains and trunk blood samples were collected. The brains were frozen in liquid nitrogen. Two hours after blood sampling, trunk blood was centrifuged twice at 3,000 × g for 1 min at 4°C to collect serum. Brain and serum samples were stored at −80°C until further processing.

Western blotting

Frozen brains were sliced into ~1-mm-thick coronal sections (bregma −1.5 to −2.5 mm), and hippocampi were dissected on dry ice. Proteins were extracted from the hippocampi by sonication of the tissue samples in lysis buffer (CelLytic MT, Sigma) containing 1% (v/v) protease inhibitor cocktail (Sigma) on ice, followed by centrifugation at 18,000 × g for 10 min at 4°C and collection of the resulting supernatants. Total protein content was determined using a BCA protein assay kit (Pierce, Rockford, IL, USA). Proteins (10 μg) were separated on 4–15% SDS-PAGE gels (Bio-Rad, Tokyo, Japan) and were transferred to polyvinylidene difluoride membranes (Bio-Rad) using a Trans-Blot Turbo™ Transfer System (Bio-Rad). After being blocked with Blocking One solution (Nacalai Tesque, Kyoto, Japan) for 30 min at room temperature (RT), the membranes were incubated with the following primary antibodies at RT for 1 h or 4°C overnight, as indicated: rabbit anti-nod-like receptor family, pyrin domain-containing (NLRP)3 (1:1000, overnight; Cell Signaling Technology, Tokyo, Japan), rabbit anti-arginase (Arg)1 (1:3000, overnight; GeneTex, Irvine, CA, USA), rat anti-tau (1:2000, overnight; Fujifilm Wako Pure Chemical Corporation, Osaka, Japan), or horseradish peroxidase (HRP)-conjugated mouse anti-GAPDH (1:10000, 1 h; Proteintech, Rosemont, IL, USA). The membranes were then rinsed in Tris-buffered saline containing 0.05% (v/v) Tween 20 and were incubated with appropriate HRP-linked secondary antibodies (except in the case of anti-GAPDH) for 1 h at RT. The blots were visualized with Clarity Western ECL Substrate or Clarity Max Western ECL Substrate (Bio-Rad) using a ChemiDoc™ Imaging System (ChemiDoc Touch, Bio-Rad). Band intensities were analyzed using ImageLab software (Ver. 6.0, Bio-Rad).

RNA extraction and RNA-sequencing

For RNA extraction, frozen brains were sliced into ~1-mm-thick coronal sections (bregma −2.5 to −3.5 mm), and the hippocampi were dissected on dry ice. Samples were immersed in RNAlater solution (Thermo Fisher Scientific, Carlsbad, CA, USA) and cut into small pieces (~30 mg each) using scissors. RNA extraction was performed using a Pure Link RNA Mini kit (Invitrogen, Carlsbad, CA, USA). To this end, 300 μL of lysis buffer and 450 μL of TRIzol-LS (Thermo Fisher Scientific) were added to the tissue. The tissue was then homogenized on ice using a BioMasher disposable homogenizer (Nippi, Tokyo, Japan). After the sample was incubated for 10 min at RT, it was centrifuged at 12,000 × g for 15 min. The supernatant was treated with DNase and the resultant RNA was purified using spin columns. The quality of the total RNA was assessed using a Qubit 4.0 (Thermo Fisher Scientific) and a 4200TapeStation system (Agilent Technologies, Santa Clara, CA, USA). Each fresh-frozen total RNA sample had a RNA Integrity Number (RIN) > 7, indicating that they were of sufficient quality to prepare sequencing libraries.

Total RNA was used for RNA-sequencing (RNA-seq) analysis. Library preparation and Illumina HiSeq sequencing were conducted by GENEWIZ (South Plainfield, NJ, USA), which generated ~20 million (150-basepair, paired-end) reads per library.

Quality check and filtering of RNA-seq data and mapping analysis

For quality filtering of the RNA-seq data, cutadapt v.1.16 [105] was used to remove Illumina adapter sequences, followed by removal of the poly-A sequence using fastx_clipper software in the fastx toolkit software package v.0.0.14 (FASTX-Toolkit, http://hannonlab.cshl.edu/fastx_toolkit/index.html). To remove low-quality bases or sequences, we trimmed the sequences using fastq_quality_trimmer software (parameters: -t 20 -l 30 -Q 33) and fastq_quality_filter software (parameters: -q 20 -p 80 -Q 33), both of which were included in the fastx toolkit. During the above processing, any read in which one of the pairs was missing was removed using Trimmomatic v.0.38 [106]. Then, reads containing mouse rRNA, tRNA, or phiX sequences, which are the control sequences from Illumina, were removed using Bowtie 2 v. 2.3.4.1 [107]. We then performed a second round of removal of any unpaired reads using bam2fastq (https://gslweb.discoveryls.com/information/software/bam2fastq).

After completion of these filtering steps, 20 million reads of each of the forward and reverse sequences per sample were mapped to the mouse genome sequence to build GRCm38, using HISAT2 v2.1.0 [108]. The mouse genome sequence was downloaded from Illumina iGenomes (https://support.illumina.com/sequencing/sequencing_software/igenome.html). Multiple-mapped reads were removed using samtools (parameters: samtools view -q 4) [109]. Uniquely mapped reads were counted by gene annotation (Ensembl release 81) using featureCounts v.1.6.2 [110]. The counted values were normalized using the Trimmed mean of M values (TMM) method [111] using the edgeR library [112] in R v.3.5.0 (https://cran.r-project.org/bin/windows/base/old/3.5.0/) and were used for expression analysis. The resultant RNA-seq data were subjected to multidimensional scaling analysis. The original RNA-seq datasets were deposited in the DDBJ Sequence Read Archive under accession numbers DRR310804–DRR310853 which are linked to the BioProject accession number PRJDB015423.

Analysis of differentially expressed genes

P-values for the differentially expressed genes (DEGs) were obtained using the likelihood ratio test in the edgeR (https://cran.r-project.org/bin/windows/base/old/3.5.0/) package. First, we extracted genes whose expression levels were significantly down- or up-regulated (P < 0.05) in the water-administered SAMP8 mice (SAMP8/water) as compared with the water-administered SAMR1mice (SAMR1/water). Among the genes whose expression levels were significantly down-regulated (SAMR1/water > SAMP8/water), those showing a pattern of significant up-regulation in Kampo- administered SAMP8 mice (SAMP8/Kampo; KS, NKS, or HJG) as compared with SAMP8/water were referred to as "V-shaped recovered" genes (SAMR1/water > SAMP8/water, and SAMP8/water < SAMP8/Kampo) (Figure 4A, green line). Conversely, among the genes whose expression levels were significantly up-regulated (SAMR1/water < SAMP8/water), those that showed a pattern of significant down-regulation in SAMP8/Kampo as compared with SAMP8/water were referred to as "reverse V-shaped recovered" genes (SAMR1/water < SAMP8/water, and SAMP8/water > SAMP8/Kampo) (Figure 4A, orange line).

Gene ontology analysis and pathway analysis

The DEGs were mapped to the relevant biological annotation from Gene Ontology (GO; http://www.geneontology.org/) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (https://www.genome.jp/kegg/) using the DAVID functional annotation online database v.6.8 (https://david.ncifcrf.gov/). Fisher’s exact test was applied to identify significant GO terms and pathways, and the threshold of significance was defined by the P-value and false discovery rate.

Enzyme-linked immunosorbent assay

Cytokine and chemokine levels in the samples were detected using a commercially available enzyme-linked immunosorbent assay (ELISA) kit for interleukin (IL)-1β and IL-6 (BD OptEIA™ ELISA set, BD Biosciences, San Diego, CA, USA) and for tumor necrosis factor (TNF)-α, IL-4, IL-10, and monocyte chemoattractant protein (MCP)-1 (Mouse DuoSet ELISA, R&D Systems, Minneapolis, MN, USA).

Statistical analysis

All data are presented as the mean ± SEM and were analyzed using Prism 7 (GraphPad Software, San Diego, CA, USA). We used the paired t-test for comparisons between two groups. Data were also analyzed by one-way ANOVA, followed by Bonferroni’s or Dunnett’s post-hoc test for comparisons among multiple groups. Correlation analysis was also performed using Pearson’s correlation coefficient. For gene expression analysis based on RNA-seq, P-values were obtained using the likelihood ratio test. In all cases, P < 0.05 was considered statistically significant.

Abbreviations

Arg1: Arginase1; Cdk5: Cyclin-dependent protein kinase 5; DEGs: differentially expressed genes; Dyrk2: Dual-specificity tyrosine phosphorylated and regulated kinase 2; GO: Gene Ontology; HJG: Hachimijiogan; HRP: Horseradish peroxidase; IL: Interleukin; KEGG: Kyoto Encyclopedia of Genes and Genomes; Klf10: Krüppel-like factor-10; KS: Kososan; LPS: Lipopolysaccharide; MCP-1: Monocyte chemoattractant protein-1; MDD: Major depressive disorder; Nab2: Ngfi-A-binding protein-2; NKS: Nobiletin-rich kososan; NLRP3: Nod-like receptor family, pyrin domain-containing 3; OFT: Open field test; RIN: RNA integrity number; RNA-seq: RNA-sequencing; RT: Room temperature; SAMP8: Senescence-accelerated mouse prone 8; SAMR1: Senescence-accelerated mouse resistant 1; SPT: Sucrose preference test; TLR4: Toll-like receptor 4; TNF-α: Tumor necrosis factor-α; TST: Tail suspension test.

Author Contributions

NI and AM designed and performed the experiments, analyzed the data, and drafted the manuscript. KO helped with RNA-seq experiments. MY prepared KS and NKS extracts and performed their component analysis. KH and CS assisted with behavioral experiments. TN, YK, HO, and NO discussed the results and edited the manuscript. All authors read and approved the final manuscript.

Acknowledgments

We thank Dr. Haruki Yamada for his advice on this research. We would like to thank Ms. Harumi Koguchi for her technical assistance. We are grateful to Ms. Kano Koike, Mr. Tomonori Tsuda, Mr. Takahiko Sakagami, and Mr. Hiroyoshi Kikuta for their financial support through crowdfunding.

Conflicts of Interest

NI, TN, YK, and HO declare financial conflicts of interest from Tsumura & Co. Norihiro Okada’s research group (AM, KO, and NO) has an ongoing collaboration with Tsumura & Co., and this collaboration has included financial support. Tsumura & Co. had no role in the design of the research, analysis or interpretation of the results, or in writing the manuscript.

Funding

This work was supported by JSPS KAKENHI (grant number 17K09320 and 21K07376 to NI), JST COI (Grant number JPMJCE1301 to NI and HO), and crowdfunding (NI).

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