Table 1.
The ingredients of the Chaihu-Shugan-San (CSS) solution.
Figure 1.
Protocol for the behavioral tests.
The interval between the blocks was 1 day.
Table 2.
Primers for quantitative real-time RT-PCR (Rat).
Figure 2.
Validation of the rat groups used in this study.
A: Changes in body weight (BW) of the rats during the entire experimental period. BW of the young normal control rats (CON) increased sharply with an increase in time, that of the normally aging rats without CSS administration (ACON without CSS) increased gradually; that of the normally aging rats with CSS administration (ACON with CSS) was the same as that of the normally aging rats without CSS administration (p>0.05), that of the UCMS rats without CSS administration (UCMS without CSS) decreased sharply, and that of the UCMS rats with CSS administration (UCMS with CSS) decreased gradually. CSS administration decreased the weight loss in the UCMS rats (*p<0.005). B: Duration of estrous cycle in the rats. Duration of estrous cycle was significantly shorter in the CON rats than in the ACON and UCMS rats, while no significant difference was found between ACON and UCMS rats and between rats who received CSS and those who did not (**p<0.001).
Figure 3.
Behavioral tests for depression-like behavior in rats.
A. Results of the sucrose consumption test. Sucrose intake was significantly decreased in the UCMS without CSS group compared with that in the ACON without CSS groups (p<0.001). On the other hand, sucrose intake in the UCMS with CSS group was significantly increased compared with that in the UCMS without CSS (p<0.001) group. B: Results of the forced swimming test (FST). The immobility time was significantly longer in the UCMS without CSS group than in the ACON without CSS group (p<0.001). However, immobility time in the UCMS with CSS group was significantly shorter than that in the UCMS without CSS group (p<0.005). The depressive state in the UCMS rats was verified (ACON without CSS vs. UCMS without CSS). CSS administration was effective for treating depressive symptoms in the UCMS rats (UCMS with CSS vs. UCMS without CSS). Values are means ± SEM. *p<0.005, **p<0.001. The p-value was established by analysis of variance (ANOVA). The post-hoc Bonferroni test was not applied when the p-value was >0.05 as per ANOVA).
Figure 4.
Open field test for anxiety-like behavior.
OFT indices, including number of squares crossed (Figure 4A), number of rearings (Figure 4B), and number of center entries (Figure 4D) were significantly lower in the UCMS without CSS group than in the ACON without CSS group (p<0.001). Moreover, the index of time spent in the center (Figure 4C) was significantly longer in the UCMS without CSS group than in the ACON without CSS (p<0.001) group, confirming the anxiety state in the UCMS rats (ACON without CSS vs. UCMS without CSS). Furthermore, the number of square crosses (Figure 4A), number of rearings (Figure 4B), and number of center entries (Figure 4D) were significantly higher in the UCMS with CSS group than in the UCMS without CSS group. Moreover, the time spent in the center (Figure 4C) was significantly shorter in the UCMS with CSS group than in the UCMS without CSS group [numbers of square crosses (Figure 4A) and center entries (Figure 4D), p<0.001; numbers of rearings (Figure 4B) and time spent in the center (Figure 4C), p<0.005], showing that CSS administration was effective for releasing the stressful state in UCMS rats (UCMS with CSS vs. UCMS without CSS). Values are means ± SEM. *p<0.005, **p<0.001. The p-value was established by analysis of variance (ANOVA). The post-hoc Bonferroni test was not applied when the p-value was >0.05 as per ANOVA).
Figure 5.
Serum and Hippocampal E2 levels.
The results for serum (Figure 5A.) and hippocampal (Figure 5B.) E2 levels were analogous. The E2 levels in the CON group were significantly higher than those in the other groups (p<0.001), but no difference was observed among the other groups. Values are means ± SEM. **p<0.001. The p-value was established by analysis of variance (ANOVA). The post-hoc Bonferroni test was not applied when the p-value was >0.05 as per ANOVA).
Figure 6.
Hippocampal mRNA expression of ERα, ERβ, and GPR30 and the ERα/ERβ mRNA ratio.
A: No significant difference was observed among all rats. B: ERβ mRNA expression showed no significant difference among all groups. C: GPR30 mRNA expression showed no significant difference among all groups. D: The ERα/ERβ ratio was significantly lower in the UCMS without CSS group than in the other groups (p<0.001). Therefore, this ratio was sensitively affected in the UCMS rats (UCMS without CSS vs. CON and ACON). The ERα/ERβ ratio was higher in the UCMS with CSS group than in the UCMS without CSS group (p<0.001). This ratio was significantly ameliorated by CSS administration. Values are means ± SEM. **p<0.001. The p-value was established by analysis of variance (ANOVA). The post-hoc Bonferroni test was not applied when the p-value was >0.05 as per ANOVA).