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.


| INTRODUC TI ON
With increasing stress factors, depression has become a commonly occurring and life-threatening neuropsychiatric disease in modern society. 1 With its high prevalence, recurrence, and enormous personal and societal costs, depression has attracted the attention of scientists all over the world. 2,3 Scholars around the globe have been devoting themselves to exploring the pathogenesis of depression in an attempt to find more effective and safer antidepressants.
A number of theories about the aetiology and course of depression have been proposed. In the 1960s, the classical monoamine hypothesis of depression was first proposed, stating that depression is due to a deficiency in one or more of three biogenic monoamines (dopamine [DA], 5-hydroxytryptamine  and norepinephrine [NE]) in the central nervous system. 4 Later, the hypotheses of neuroendocrinology 5,6 and neuroplasticity 7 and the theory of inflammation and immunity 8 on depression were put forward successively.
According to these theories, a good amount of scientific research has been performed to investigate the pathophysiological changes of depression. Thus, it can be seen that the pathogenesis of depression is complicated, involving several systems, multiple proteins and a lot of small molecules. No single theory or signal pathway can fully elucidate its pathogenesis. Therefore, we should consider all factors and pay attention to the interaction among them. Only in this way can we comprehensively and systematically elucidate the pathogenesis of depression.
In the past few years, proteomic technologies have been extensively applied to identify biomarkers, characterize complex biochemical systems and examine pathophysiological processes in various diseases. 9 However, there are still no proteomic data available on the therapy mechanism of Saikosaponin A (SA) on stress-induced depression. Therefore, in our study a quantitative proteomics technology-iTRAQ-was used to screen the differentially expressed target proteins regulated by chronic unpredictable mild stress (CUMS) and SA. The numerous differentially expressed proteins detected by iTRAQ and the bioinformatics analysis of them will help to enhance our understanding of the pathogenesis of depression and the mechanisms behind the antidepressant effects of SA.
SA, a major bioactive component isolated from Radix Bupleuri, is one kind of triterpene saponins, containing a sugar side chain of monodesmosides in an oleanane-type triterpene skeleton at C-3. 10 This saponin exhibits a wide variety of pharmacological activities, causing anti-malignant cell proliferation, anti-inflammation, anti-oxidation and liver protective effects. 11,12 Increasing studies have confirmed that SA has certain anti-tumour effects through inducing cell apoptosis. It is reported that SA triggers caspase-3 dependent and independent apoptosis mediated through the regulation of the Bcl-2 family, leading to mitochondrial dysfunction and release of apoptotic factors in hepatic stellate cells. 10 Ming Feng Chen and his team have demonstrated that Saikosaponin A and Saikosaponin D could induce cell apoptosis by inhibiting proliferation and migratory activity of rat HSC-T6 cells. 13 It is found SA induces caspase-mediated apoptosis in human colon carcinoma (HCC) cells by triggering caspase-2 and caspase-8 activation, suggesting SA may be a promising cancer therapy agent in certain types of cancer. 14 In recent years, Radix Bupleuri and saikosaponins have been demonstrated to possess antidepressant effects in in vivo and in vitro experiments. [15][16][17][18] A study published in Neuroscience Letters in 2017 demonstrates that administration of saikosaponin A for 4 weeks could produce the antidepressant-like effects in perimenopausal rats, and the potential mechanism may be the restoration of neuroendocrine, neuroinflammation and neurotrophic systems in the hippocampus during perimenopausal. 19 These results indicate that there is a strong correlation between SA and depression, but the exact therapeutic mechanisms of SA on depression need to be further explored.
A CUMS depression animal model has been widely used for investigating the pathophysiological mechanisms underlying depression and evaluating the efficacy of antidepressants. 20 This stress-induced model of depression has good validity and reliability. 21 Moreover, it can overcome the stress habituation that usually occurs with chronic restricted stress (CRS) models and induce consolidated long-lasting behavioural deficits. 22 Therefore, in the present study, we utilized the CUMS depression model to investigate whether long-term treatment with SA could prevent the CUMS-induced depressive-like behaviours and reverse monoamine neurotransmitter changes in the hippocampus. To further explore the underlying therapeutic mechanism of SA, iTRAQ was used to screen for differentially expressed proteins before and after CUMS (and SA) treatment. With reference to domestic and foreign literatures, we chose protein Proline-rich transmembrane protein 2 (PRRT2) as the key research object.
Before any experimentation, all of the rats were allowed to have identification of these 15 differentially expressed proteins, including PRRT2, provides further insight into the treatment mechanism of SA for depression.

K E Y W O R D S
depression, dopamine, proline-rich transmembrane protein 2, proteomics, Saikosaponin A 1 week to adapt to the environment of the laboratory at the Animal Center of Shandong University. Throughout the whole experiment process, the laboratory temperature was kept at 20 ± 2°C, at a humidity of 45%-65%, and on a 12 h light/12 h dark cycle. During the 1-week adaptive phase, the rats were housed in groups and had free access to water and food. All of the experimental procedures involving the animals were previously approved by the Animals Care Committee of Shandong University, in line with the US National Institute of Health Guide for the Care and Use of Laboratory Animals and conforming to the ARRIVE guidelines.

| Drugs and treatment groups
SA (Chengdu Must Bio-technology, Sichuan, China) was dissolved in normal saline. Rats were randomly divided into three groups: the control group (C, n = 15), the CUMS group (M, n = 15) and the CUMS + Saikosaponin A group (50 mg/kg) (SA, n = 15). At the end of the fourth week, we conducted behavioural tests to prove that the stressors had worked. Then, the rats were intragastrically administrated with SA or saline once a day 30 min prior to stress exposure for four weeks. That is to say, rats in the SA group were intragastrically administrated with SA at a dose of 50 mg/kg, while the C group and M group were intragastrically treated with the same volume of normal saline.

| CUMS procedure
To establish the depression animal model, we adopt the method of CUMS combined with separation as previously described with minor modifications, 23 which is generally accepted and widely used. As is mentioned above, we had divided the experimental rats into three groups: the C, M, and SA (50 mg/kg) groups. Leaving the C group aside, the other two groups were subjected to eight kinds of stressors throughout the procedure. The stressors were as follows: fasting for 48 hours, water deprivation for 24 hours, physical restraint for 6 hours (from 9:00 to 15:00), 60°C cage tilt for 24 hours, cold swimming (at 4°C) for 5 minutes, tail pinch for 1.5 min (1 cm from the end of the tail), inversion of the light/dark cycle and damp sawdust for 24 hours. One stressor was applied daily at different times, and the same stressor cannot be exerted twice in one week in case the experimental animals adapted to these stressors. The CUMS procedure lasted for 8 weeks.

| Bodyweight Measurement and Behavioural Testing
Before CUMS, we measured the experimental rat bodyweight as their initial weight (W1). Subsequently, the bodyweights were measured once a week during the whole CUMS process, recorded as W2, W3, W4, W5, W6, W7, W8 and W9. Then, we could draw the bodyweight growth curve and calculate the increase of bodyweight (W9 − W1). Behavioural tests were performed from the next day after eight weeks of CUMS exposure in sequence as follows.

| Open field test
The spontaneous exploratory behaviour was measured in the open field test (OFT), which was performed with minor modifications as described previously. 22 In accordance with the reference, we made an open box (80 × 80 × 40 cm), which was painted with black inside, and the floor was divided into 25 equal lattices by white lines. At the beginning of the test, rats were placed individually in the centre of the open box and then left to explore freely for a 5-min session.
The horizontal locomotor activity (segments crossed with four paws) and vertical activity (number of rearings) were recorded. The total number of crossing and rearing was calculated and used as a measurable indicator of experimental animals' spontaneous exploratory behaviour. The apparatus was cleaned with detergent prior to each test session to remove any olfactory cues.

| Sucrose preference test
All of the rats included in the blank control group were placed individually in the cages during the sucrose preference test (SPT). The entire SPT lasted for 4 days. The first two days were used to train experimental rats to drink sucrose water, that is, every rat was offered two bottles of 1% sucrose water for 24 hours. Then, one bottle of sucrose water was replaced by tap water. All of the rats were deprived of food and water on the third day. At 20:00 of the fourth day, each rat was provided with two bottles of water, one contained 400 mL sucrose water and the other 400 mL tap water. After 12 hours, the consumed volumes of sucrose solution and tap water were recorded.
The sucrose preference, which is used as an index of anhedonia, was calculated using the following formula:

| Measurement of 5-HT, NE and DA levels in the hippocampus
All of the animals were deeply anesthetized with chloral hydrate and decapitated after the last behavioural test. Their brains were rapidly removed and put on ice. Adhered blood was rinsed by ice-cold normal saline, and then, the hippocampi were dissected, frozen in liquid nitrogen and stored at −80°C. 5-HT, NE and DA content in the hippocampus were determined by high-performance liquid chromatography-mass spectrometry (HPLC-MS), using an Agilent 1200 series HPLC system (Agilent, USA). The chromatographic separation was carried out on an Agilent XDB C18 column (50 × 4.6 mm, 5 m; Waters) at 30°C. The samples were separated using a gradient mobile phase consisting of 5% Sp = sucrose consumption ÷ (water consumption + sucrose consumption) × 100% methanol and 95% water at a flow rate of 0.2 mL/min. The injection volume was 10 μL. The mass spectrometer (Agilent, 6410B, USA) was operated in the positive ion electrospray mode with MRM. Data were acquired and processed using Agilent Mass Hunter software.

| Sample preparation
In the study, we prepared eight sample pools: C1, C2, M1, M2, M3, SA1, SA2 and SA3. For the C group, the hippocampus of every four rats was mixed together as a sample pool; for the M group and the SA group, the hippocampus of every three rats was mixed together as a sample pool. Then, we add the right amount of SDT buffer, quartz

| Preliminary experiment
Twenty micrograms of proteins for each sample were taken out and mixed with 5× loading buffer, followed by boiling for 10 minutes.
Then, the proteins were separated on 12.5% SDS-PAGE gel. Protein bands were visualized by Coomassie Blue R-250 staining.

| FASP digestion and 8-plex iTRAQ labelling
Thirty microlitres of protein solution from each sample was sequentially lysed, washed, blocked and digested, and finally, the peptides of each sample were desalted on C18 Cartridges (Empore ™ SPE Cartridges C18 (standard density), bed ID 7 mm, volume 3 mL, Sigma),  Figure 3A. Subsequently, the eight labelled samples were pooled, centrifuged and dried.

| HPLC and LC-MS/MS analysis
The iTRAQ-labelled peptide mixtures were separated using an Easy nLC Liquid Chromatograph (Thermo Scientific). The peptide mixture was loaded onto a reverse-phase trap column (Thermo Scientific Acclaim PepMap100, 100 μm × 2 cm, nanoViper C18) connected to the C18 reversed-phase analytical column (Thermo Scientific Easy Column, 10 cm long, 75 μm inner diameter, 3 μm resin), followed by a mobile phase elution with buffer A (0.1% formic acid) and buffer B (84% acetonitrile and 0.1% formic acid). Peptides were then eluted in a linear gradient with buffer B from 0% to 100% over 60 min at a flow rate of 300 nL/min. LC-MS/MS analysis was performed on a Q Exactive mass spectrometer (Thermo Scientific) that was coupled to an Easy nLC (Proxeon Biosystems, now Thermo Fisher Scientific) for 60 min. The mass spectrometer was operated in positive ion mode.
MS data were acquired using a data-dependent top 10 method dynamically choosing the most abundant precursor ions from the survey scan (300-1800 m/z) for HCD fragmentation. The instrument was run with peptide recognition mode enabled.

| Data analysis
A MASCOT engine (Matrix Science, London, UK; version 2.2) embedded into Proteome Discoverer was used to search for MS/MS spectra.

| Bioinformatics analysis
Gene Ontology (GO) annotation was used to annotate the target proteins, and the progress was roughly divided into four steps: blast, mapping, annotation and annotation augmentation. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation of target proteins was performed by using KEGG Annotation Automatic Server (KAAS) software. The FASTA protein sequences of differentially measured proteins were blasted against the online KEGG database to retrieve their KOs and were subsequently mapped to pathways in the KEGG. Then, the corresponding KEGG pathways were extracted.

| Measurement Of PRRT2 Expression At tissue level
Western blot was performed to verify the protein expression level of PRRT2 in hippocampus. The Western blotting analysis was carried out as previously described 24 with minor modifications. Briefly, the rat hippocampus tissues were homogenized in 500 μL ice-cold RIPA lysis buffer (Beyotime, Shenzhen, Guangdong, China) using a tissue ho-

| Measurement of PRRT2 expression at cell level
To further explore the relationship between SA and PRRT2 at cellular level, we used the corticosterone solution to stimulate PC12 cell, Thirdly, we use the optimal concentration SA to pretreat PC12 cell in appropriate time, after that the optimal concentration of corticosterone was added to PC12 cell. After 24 hours, we extracted cell proteins and measured the protein expression level of PRRT2 using western blot.

| Data analysis
Statistical analysis was carried out using SPSS version 22.0. All of the data are expressed as mean ± SEM. The data were analysed in a one-way ANOVA followed by the LSD test. The level of significance was set at P < .05.

| CUMS depression model was successfully established and SA could reverse depressive behaviours
The method of 8-week CUMS exposure combined with separation successfully established the rat depression model, resulting in significant behavioural changes. The experimental animals' grouping and administration details are presented in Figure 1A. The whole process of the CUMS procedure is schematically presented in Figure 1B.

| SA reverted the diminished bodyweight gain induced by CUMS
Rat bodyweights were measured to assess the efficacy of CUMS and the antidepressant efficacy of SA. Figure 2A shows the weight growth F I G U R E 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 curves of the three groups (C, M and SA) during the CUMS procedure.
The initial bodyweight (W1), the last measured weight (W9) and the weight gain (W9 − W1) of the three groups are shown in Figure 2B-D.
These data indicate that rats suffering CUMS reveal a decreased bodyweight gain relative to the control group of animals (P < .01), while chronic administration of SA (50 mg/kg daily) significantly increased the bodyweight gain compared with the M group (P < .01).

| SA improved the animal performance in OFT
To explore the experimental animals' spontaneous exploratory behaviour and anti-anxiety effects, horizontal (number of crossings) and vertical (number of rearings) exploratory activity was measured in the OFT.
Test results revealed that there was no significant difference among three groups in the total number of crossings and rearing before CUMS ( Figure 3A). Figure 3B shows that CUMS exposure significantly reduced the total number of crossing and rearing (P < .01) in rats as compared to the C group. Chronic treatment with SA (50 mg/kg daily) significantly reversed the reduction as compared to the M group (P < .05).

| SA reverted the CUMS-induced decrease in SP
The reduced consumption of sucrose solution is an indicator of anhedonia-like behaviour. Figure 3C presented the effects of SA treatment on SPT performance in CUMS-exposed rats. Eight-week CUMS exposure significantly reduced the percentage of sucrose consumption as compared to the C group (P < .01), while chronic treatment with SA (50 mg/kg daily) significantly increased the percentage of sucrose consumption as compared to the M group (P < .01).

| SA Increases the content of dopamine in hippocampus of rats exposed to CUMS
The effect of SA on 5-HT, NE and DA levels in the hippocampus of CUMS rats is shown in Figure 4, Tables 1 and 2. The data indicate that CUMS significantly reduced DA concentrations in the hippocampus of the M group compared with that in the C group (P < .01), while it had no significant influence on the 5-HT and NE concentrations (P > .05). Administration of SA (50 mg/kg daily) was able to reverse the effects of CUMS on DA (P < .05).

| Differential expression of proteins in hippocampus screened by iTRAQ before and after the CUMS
To identify the proteins related to the CUMS stimulus, which may play a critical role in the pathogenetic process of depression, the quantitative F I G U R E 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 proteomics technique iTRAQ was adopted in this study. Our results show that 4779 differentially expressed proteins were screened between the C group and the M group. According to the standard of ratio (M/C or SA/M) ≥ 1.2 and P-value ≤ .05, 391 significantly differentially expressed proteins were selected (shown in Table S1). Among

| Differentially Expressed Proteins in Hippocampus Regulated by SA
The same method was adopted to analyse the hippocampus proteome of rats exposed to CUMS and treated with SA or not. There

| Significantly differentially expressed proteins regulated by both CUMS and SA
By comparative analysis, 15 proteins were identified that not only were influenced by CUMS but were also influenced by SA.
The specific information about these 15 proteins is presented in Table 3. Among these proteins, PNMA2 and CPNE7 were up-regulated when rats were exposed to CUMS and chronic administration with SA (50 mg/kg daily) markedly decreased their expression lev- were not only down-regulated during the CUMS procedure, but they were also down-regulated by SA (50 mg/kg). These proteins, located F I G U R E 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 in different parts of the cell, have different physiological functions and are involved in various signalling pathways.

| PRRT2 expression level changes in hippocampus
Western blot was performed to confirm the results of iTRAQ. As shown in Figure 6, the expression level of PRRT2 was indeed significantly down-regulated by CUMS (P < .05), and 4-week administration with SA (50 mg/kg daily) dramatically reversed the change (P < .05).

| D ISCUSS I ON
In the present study, the method of 8-week CUMS combined with separation induced depressive-like behaviours and caused a decrease of DA content in the hippocampus, as expected. Importantly, chronic administration of SA at a dose of 50 mg/kg could significantly    TA B L E 3 These 15 kinds of proteins had different expression levels both between groups M and C and between groups M and SA. Two proteins marked with red were up-regulated during the CUMS procedure but down-regulated by SA. Ten proteins, shown in blue, were down-regulated during the CUMS procedure and were up-regulated after the chronic administration of SA. The other three proteins were down-regulated during the CUMS procedure and also after SA treatment; they are presented in green.

F I G U R E 6
The expression level of PRRT2 in rat hippocampus. Normalized intensity bands of PRRT2 expression levels were presented as the means ± SEM pathway enrichment analysis results suggest that these differentially  30 The fusion and release processes of DA are regulated by many factors, such as DA synthesis, uptake and vesicular transport, as well as by Ca 2+ homeostasis and regulatory exocytotic proteins. 29 The main neurotransmitter release-associated proteins include soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins 31,32 and synaptotagmin 1 (Syt1). 33,34 The SNARE proteins are divided into t-SNAREs and v-SNAREs, according to their location. The protein VAMP2 is just one kind of v-SNARE, which is located on the synaptic vesicle membrane.
Synaptosomal-associated protein 25 kD (SNAP-25) and syntaxin 1 are members of t-SNAREs, which are located on the presynaptic membrane. 35 Of course, the concrete interaction mechanism between SA and PRRT2 needs to be further explored.
In conclusion, the results of the present study suggest that the 4-week administration of SA significantly increases the hippocampal content of DA. Moreover, we confirmed that the protein PRRT2, one of 15 differential expressed proteins, plays a crucial role in CUMS-induced stress. The possible interaction mechanism between CUMS, SA, DA and PRRT2 was shown in Figure 9. The identification of PRRT2 and other differentially expressed proteins will provide us with novel candidate targets in the study of the anti-depression mechanism of SA.

F I G U R E 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

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

O RCI D
Shilian Liu https://orcid.org/0000-0002-2881-8918 F I G U R E 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