Kai-Xin-San series formulae alleviate depressive-like behaviors on chronic mild stressed mice via regulating neurotrophic factor system on hippocampus

Abstract

Kai-xin-san (KXS) is a famous Chinese medicinal formula applied for treating stress-related psychiatric diseases with the symptoms such as depression, forgetfulness and dizziness. In clinic, the composition ratio of KXS is always varied and KXS series formulae are created. Here, we aim to compare the anti-depressive effect of different ratios of KXS and reveal its action mechanism on regulation of neurotrophic factor system. Firstly, daily intra-gastric administration of chemically standardized extracts of KXS series formulae for seven days significantly alleviated the depressive symptoms of chronic unpredictable mild stressed mice displayed by enhanced sucrose consumptions and decreased immobile time of forced swimming coupled with increased locomotor activities. KXS might fulfill this effect by up-regulating the expressions of NGF, BDNF and Trk receptors in hippocampus, which were confirmed by the treatment of corresponding blockers tPA-stop and K252a. The ratio with higher amounts of Ginseng Radix et Rhizoma and Polygalae Radix exerted most profound effect on anti-depression and regulation enzymes in metabolic pathway of neurotrophic factors. These findings suggested that KXS was beneficial for enhancing supplies, up-regulating receptors, and restoring the dysfunction of metabolic pathway of neurotrophic factors, which might account for its anti-depression effect.

Figure 1

Effect of KXS series formulae treatment on the behaviors of CUMS-treated mice. (A) After four weeks of CUMS treatment, all groups of mice were employed for behavior evaluation tests, including the sucrose preference test, forced swimming test. Fluoxetine at daily dosage of 4 mg/kg was set as the positive control. KXS series treatment was set for three groups, K-652, K-984, D-652, all at 10 g/kg. (B) Non-stressed mice were treated with fluoxetine and KXS. The period and dosage of treatment were same to that of CUMS treated groups. Afterwards, same behavior evaluation tests were applied. Values are expressed in the percentage of non-stressed group as Mean ± SEM (n = 8). Comparisons between groups were carried out by a one-way ANOVA followed by a post-hoc Bonferroni test. ^#p < 0.05, ^##p < 0.01 (compared with non-stressed group); *p < 0.05, **p < 0.01 (compared with stressed group).

Figure 2

KXS series formulae exerted antidepressant actions in open field tests of CUMS-treated mice. After four weeks of CUMS exposure and one week of KXS series formulae treatment, all groups of mice were employed for open field tests, including time spent in central area, rearing number and total travel distance. Fluoxetine at daily dosage of 4 mg/kg was set as the positive control. KXS series formulae treatment was set for three groups, K-652, K-984, D-652, all at 10 g/kg. Values are expressed in the percentage of non-stressed group as Mean ± SEM (n = 8). Comparisons between groups were carried out by a one-way ANOVA followed by a post-hoc Bonferroni test. ^#p < 0.05, ^##p < 0.01 (compared with non-stressed group); *p < 0.05, **p < 0.01 (compared with stressed group).

Figure 3

Effect of KXS series formulae treatment on expressions of neurotrophic factors in hippocampus of CUMS-treated mice by ELISA analysis. The treatment of KXS series formulae and fluoxetine in the mice were same as that in Fig. 1. The hippocampus tissues were collected after four-week treatment and the amounts of NGF were analyzed by ELISA kits. Comparisons between groups were carried out by a one-way ANOVA followed by a post-hoc Bonferroni test. Values are expressed in the percentage of non-stressed group as Mean ± SEM (n = 8). ^#p < 0.05, ^##p < 0.01 (compared with non-stressed group); *p < 0.05, **p < 0.01 (compared with stressed group).

Figure 4

Effect of KXS series formulae treatment on the protein expressions of neurotrophic factors and its precursors in hippocampus of CUMS-treated mice by western analysis. (A) The treatment of KXS series formulae and fluoxetine in the mice were same as that in Fig. 1. The hippocampus tissues were collected after four-week treatment and the expressions of proNGF and NGF were analyzed by western blot analysis. α-tubulin was used as the loading control. Afterwards, the ratio of mNGF to proNGF was calculated. (B) Expressions of proBDNF and BDNF were determined and the ratio of BDNF to proBDNF was calculated as same as that of NGF. Comparisons between groups were carried out by a one-way ANOVA followed by a post-hoc Bonferroni test. Values are expressed in the percentage of non-stressed group as Mean ± SEM (n = 8). ^#p < 0.05, ^##p < 0.01 (compared with non-stressed group); *p < 0.05, **p < 0.01 (compared with stressed group).

Figure 5

KXS series formulae regulate mRNA expressions of proteins relating to metabolic pathway of neurotrophic factors in hippocampus of CUMS-treated mice. (A) The treatment of KXS series formulae and fluoxetine in the mice were same as that in Fig. 1. The hippocampus tissues were collected after one-week treatment and mRNA expression levels of proteins relating to synthesis of neurotrophic factors were analyzed. (B) Drug treatment and sample preparations were same as (A) and mRNA expression levels of proteins relating to degradation of neurotrophic factors were analyzed. Comparisons between groups were carried out by a one-way ANOVA followed by a post-hoc Bonferroni test. Values are expressed in the percentage of non-stressed group as Mean ± SEM (n = 8). ^#p < 0.05, ^##p < 0.01 (compared with non-stressed group); *p < 0.05, **p < 0.01 (compared with stressed group).

Figure 6

The blocker of tPA attenuates anti-depressive actions of KXS in the behavioral tests and expressions of neurotrophic factors of hippocampus of CUMS-treated mice. (A) At the end of CUMS exposures, one group of the mice were treated with D-652 and tPA-stop simultaneously. D-652 was orally treated for 7 days and tPA-stop (dissolved in artificial CSF) was transcranial injected into hippocampus for 3 days. Then, sucrose preference test and forced swimming test were carried out. The CUMS mice only injected with artificial CSF and treated with combination of CSF and D-652 were applied for contrast. (B) Amounts of NGF and BDNF were determined in hippocampus of mice as treated in (A). Comparisons between groups were carried out by a two-way ANOVA followed by a post-hoc Bonferroni test. Values are expressed in the percentage of non-stressed group as Mean ± SEM (n = 8). ^#p < 0.05, ^##p < 0.01 (comparison between group treated with CUMS/CSF plus D-652 and group treated with CUMS/CSF plus D-652 and tPA-stop); *p < 0.05, **p < 0.01 (comparison between group treated with CUMS/CSF and group treated with CUMS/CSF plus D-652; comparison between group treated with CUMS/CSF plus tPA-stop and group treated with CUMS/CSF plus D-652 and tPA-stop).

Figure 7

Effect of KXS series formulae treatment on the protein expressions of Trk receptors in hippocampus of CUMS-treated mice. The treatment of KXS series formulae and fluoxetine in the mice was same as that in Fig. 1. The hippocampus tissues were collected after one-week treatment and protein levels of TrkA and TrkB were analyzed. Comparisons between groups were carried out by a one-way ANOVA followed by a post-hoc Bonferroni test. Values are expressed in the percentage of non-stressed control group as Mean ± SEM (n = 8). ^#p < 0.05, ^##p < 0.01 (compared with non-stressed group), *p < 0.05, **p < 0.01 (compared with stressed group).

Figure 8

The blocker of Trk attenuates antidepressant actions of KXS in the behavioral tests of CUMS-treated mice. At the end of CUMS exposures, one group of the mice were treated simultaneously with D-652 and K252a for 7 days. D-652 was orally treated and K252a (dissolved in DMSO) was administered intraperitoneally. Then, sucrose preference test and forced swimming test were carried out. The CUMS mice only injected with DMSO and treated with combination of DMSO and D-652 were applied for contrast. Comparisons between groups were carried out by a two-way ANOVA followed by a post-hoc Bonferroni test. Values are expressed in the percentage of non-stressed control group as Mean ± SEM (n = 8). ^#p < 0.05, ^##p < 0.01 (comparison between group treated with CUMS/DMSO plus D-652 and group treated with CUMS/DMSO plus D-652 and K252a). *p < 0.05, **p < 0.01 (comparison between group treated with CUMS/DMSO and group treated with CUMS/DMSO plus D-652).