Proteomics-based screening of the target proteins associated with antidepressant-like effect and mechanism of Saikosaponin A

Abstract

Depression is a commonly occurring neuropsychiatric disease with an increasing incidence rate. Saikosaponin A (SA), a major bioactive component extracted from Radix Bupleuri, possesses anti-malignant cell proliferation, anti-inflammation, anti-oxidation and liver protective effects. However, few studies have investigated SA's antidepressant effects and pharmacological mechanisms of action. Our study aimed to explore the anti-depression effect of SA and screen the target proteins regulated by SA in a rat model of chronic unpredictable mild stress (CUMS)-induced depression. Results showed that 8-week CUMS combined with separation could successfully produce depressive-like behaviours and cause a decrease of dopamine (DA) in rat hippocampus, and 4-week administration of SA could relieve CUMS rats' depressive symptoms and up-regulated DA content. There were 15 kinds of significant differentially expressed proteins that were detected not only between the control and CUMS groups, but also between the CUMS and SA treatment groups. Proline-rich transmembrane protein 2 (PRRT2) was down-regulated by CUMS while up-regulated by SA. These findings reveal that SA may exert antidepressant effects by up-regulating the expression level of PRRT2 and increasing DA content in hippocampus. The identification of these 15 differentially expressed proteins, including PRRT2, provides further insight into the treatment mechanism of SA for depression.

Keywords: Saikosaponin A; depression; dopamine; proline-rich transmembrane protein 2; proteomics.

Figure 1

A, The details of experimental animals’ grouping and drug treatment. Forty‐five rats were divided into three groups: the C group, the M group and the SA group. The SA group was treated with SA at a dose of 50 mg/kg for 4 weeks, while the C and M groups were given the same volume (2 mL) of saline. B, Scheme of the CUMS protocol. The whole process lasted for 10 weeks: one week for adaptation, eight weeks for the CUMS procedure and one week for behaviour tests. After the last behaviour test, all of the rats were decollated and the tissue of the hippocampus was obtained

Figure 2

Effects of SA on experimental animal bodyweight. A, Weight growth curve during the whole process of building the depression model. With time, the bodyweight of each group gradually increased. The weight growth rate of rats in the M group was significantly lower than that of the C group, and the rate of the SA group was between those of the M group and the C group. B, Bodyweight of each group before CUMS procedure (W1). There was no significant difference between the groups (P > .05). C, The final bodyweight (W9) of each group. D, The increase of bodyweight relative to the initial weight (W9 − W1). SA (50 mg/kg) reversed the weight changes induced by CUMS exposure. All of the values are presented as means ± SEM. **P < .01 as compared with the C group. ##P < .01 as compared to the M group. Data was analysed in a one‐way ANOVA followed by the LSD test

Figure 3

Effects of SA on behavioural tests. A and B, Effects of SA on the amount of locomotor activity (horizontal and vertical activity) in the OFT before and after the CUMS procedure. C, Effects of SA on the percentage of sucrose consumption. All of the values are presented as means ± SEM. **P < .01 as compared with the C group. #P < .05 as compared with the M group. ##P < .01 as compared with the M group in C. Data were analysed in a one‐way ANOVA followed by the LSD test

Figure 4

Effect of SA on 5‐HT, NE and DA levels in hippocampus. A, Chromatogram of monoamine neurotransmitter standard substance. B, Representative chromatogram of monoamine neurotransmitters from hippocampus of a control rat. C‐E, Standard curves of DA standard, NE standard and 5‐HT standard. F‐H, The concentration changes of monoamine neurotransmitters from the hippocampus of three rat groups. All of the values are presented as means ± SEM. **P < .01 as compared with C group. #P < .05 as compared with M group. Data were analysed in a one‐way ANOVA followed by the LSD test

Figure 5

A, The analysis result of the GO annotation of differentially expressed proteins between the C and M groups. Biological process, molecular function and cellular component are marked, respectively, with the colours red, blue and orange. B, The statistical results of the top 20 KEGG pathways associated with the differentially expressed proteins between the C and M groups. C, GO annotation of differentially expressed proteins between the M and SA groups. D, The top 20 KEGG pathways associated with the differentially expressed proteins between the M and SA groups

Figure 6

The expression level of PRRT2 in rat hippocampus. Normalized intensity bands of PRRT2 expression levels were presented as the means ± SEM

Figure 7

SA could reduce the neurotoxic effect of corticosterone on PC12 cells. A, With the increase of corticosterone concentration, the survival rate of PC12 cells decreased gradually. B, The survival rate of PC12 cells was 50% of the control group when the corticosterone concentration was 400 μmol/L. C, The effect of different concentrations of SA on the survival rate of PC12 cells when the pre‐treatment time of SA was 0 h, ##P < .01 compared with C group. D, The effect of different concentrations of SA on the survival rate of PC12 cells when the pre‐treatment time of SA was 1 h, #P < .05 compared with C group, **P < .01 compared with Cort group, *P < .05 compared with Cort group. E, The effect of different concentrations of SA on the survival rate of PC12 cells when the pre‐treatment time of SA was 2 h, #P < .05 compared with C group, **P < .01 compared with Cort group, *P < .05 compared with Cort group. F, The effect of different concentrations of SA on the survival rate of PC12 cells when the pre‐treatment time of SA was 4 h, ##P < .01 compared with C group, **P < .01 compared with Cort group, *P < .05 compared with Cort group. G, The effect of different concentrations of SA on the survival rate of PC12 cells when the pre‐treatment time of SA was 8 h, ##P < .01 compared with C group. Data were analysed in a one‐way ANOVA followed by the LSD test

Figure 8

The expression levels of PRRT2 in PC12 cell were measured by Western blotting. 400 μmol/L corticosterone was able to significantly down‐regulated the expression of PRRT2 in PC12 cell, and 5 μmol/L SA had the trend of up‐regulating the expression of PRRT2, #P < .05 compared with C group. Data were analysed in a one‐way ANOVA followed by the LSD test

Figure 9

A, The mechanism diagram of the present study. Our study indicated that SA could significantly relieve depressive symptoms of CUMS rats. On one hand, SA could up‐regulate the expression of protein PRRT2 and increase the content of DA in hippocampus. On the other hand, SA can reduce the toxic effects of excessive corticosterone on nerve cells. Therefore, PRRT2 is expected to be a potential target protein for SA to exert anti‐depressive effects. B, PRRT2 played a key role in DA release process. PRRT2 is mainly located in presynaptic terminals of neurons. It acts as a catalyst and a regulator in the fusion and Ca2⁺‐sensing apparatus for fast synchronous release processes by interacting with SNARE proteins, including VAMP2, SNAP‐25 and the Ca2⁺ sensor Syt1. PRRT2 of rats subjected to the CUMS procedure was down‐regulated and the proteins Syt1, VAMP2 and SNAP‐25 could not interact well with each other. As a result, the release probability and amount of released DA were reduced in the hippocampus. Chronic administration of SA (50 mg/kg) could up‐regulate the PRRT2 expression level and then increase the release probability and the amount of released DA