Free Wanderer Powder regulates AMPA receptor homeostasis in chronic restraint stress-induced rat model of depression with liver-depression and spleen-deficiency syndrome

Identification of compounds in FWP

As shown in Figure 1, the chromatographic fingerprints were constructed firstly for FWP and each herb. Then 23 peaks were identified by comparing their UV and MS spectra with published data or standard compounds (Table 1, Figure 2): D-Fructose (Po1), Lithospermic acid (M1), Rosmarinic acid (M2), Oxypaeoniflorin (P1), 3,4-Dihydroxybenzaldehyde (M3), Albiflorin (P2), Anisic acid (A1), 7-Hydroxycoumarin (AM1), Glycyrrhizic acid (G1), Angelol A (A2), Lactiflorin (P3), Ononin (G2), 4',7-Dihydroxyflavone (G3), Saikosaponin C (B1), Benzoylpaeoniflorin (P4), Saikosaponin F (B2), Formononetin (G4), Saikosaponin A (B3), 6-Gingerol (Z1), Saikosaponin E (B4), Licoflavone A (G5), Atractylenolide III (AM2) and Ligustilide (A3).

Figure 1. UHPLC-MS chemical fingerprint of FWP. D-Fructose (Po1), Lithospermic acid (M1), Rosmarinic acid (M2), Oxypaeoniflorin (P1), 3,4-Dihydroxybenzaldehyde (M3), Albiflorin (P2), Anisic acid (A1), 7-Hydroxycoumarin (AM1), Glycyrrhizic acid (G1), Angelol A (A2), Lactiflorin (P3), Ononin (G2), 4',7-Dihydroxyflavone (G3), Saikosaponin C (B1), Benzoylpaeoniflorin (P4), Saikosaponin F (B2), Formononetin (G4), Saikosaponin A (B3), 6-Gingerol (Z1), Saikosaponin E (B4), Licoflavone A (G5), Atractylenolide III (AM2) and Ligustilide (A3).

Figure 2. Chemical structure of identified components in FWP: Albiflorin, Angelol A, Licoflavone A, Atractylenolide III, D-Fructose, Glycyrrhizic acid, Formononetin, 4',7-Dihydroxyflavone, Lactiflorin, Oxypaeoniflorin, Lithospermic acid, 6-Gingerol, Anisic acid, Ononin, 7-Hydroxycoumarin, Saikosaponin A, Saikosaponin F, Ligustilide, 3,4-Dihydroxybenzaldehyde, Rosmarinic acid, Saikosaponin C, Saikosaponin E, and Benzoylpaeoniflorin.

The systematic bioinformatics analysis for antidepressive mechanism of FWP

Totally, 17 compounds were collected, including 2 in Radix Angelicae Sinensis (a), 2 in Rhizoma Atractylodis Macrocephalae (am), 2 in Radix Bupleuri (b), 5 in Radix et Rhizoma Glycyrrhizae Praeparata cum Melle (g), 3 in Herba Menthae (m), 2 in Radix Paeoniae Alba (p), and 1 in Rhizoma Zingiberis Recens (z) (Figure 3A). Their ADME information and 283 direct targets were summarized in Supplementary Tables 1 and 2, respectively. The meta-analysis of target overlaps for each herb, as well as the functional interaction among targets, were shown in Figure 3D. The same targets shared among herbs were linked by purple lines. Different targets in the same GO/KEGG items were linked by blue lines. There were little direct overlap, whereas much functional overlap among herbs. These might be due to different parts of the same biological processes regulated by different herbs. The enriched DisGenNET items in Supplementary Table 3 were then analyzed for depression related diseases (Figure 3B). Targets of FWP and each herb were mainly enriched in mood disorders, depressive disorder, and mental depression. The enriched diseases were more significant for total targets of FWP. These might be due to the synergistic effect of herbs in FWP. The enriched GO and KEGG items of total FWP targets were summarized in Figure 3C and Supplementary Table 4. The enriched cellular components (CC) included dendrite, synapse, and synaptic membrane. Molecular functions (MF) involved activity and binding of G protein-coupled receptor (GPCR), neurotransmitter receptors, and nuclear receptor. Moreover, biological processes (BP) results indicated that FWP might regulate depression-related signaling, such as GPCR, synaptic and various neurotransmitter signaling. The top enriched KEGG items were related to several critical depression-associated pathways, such as neuroactive ligand-receptor interaction, calcium signaling and synapse. These findings demonstrated FWP regulated synapse function, and neurotransmitter receptors like glutamate receptor to exhibit anti-depressive effects. The complex relationships between compounds in FWP and glutamate-related signaling were analyzed using a Compound-Target-Pathway Network (Figure 3E). There were 51 nodes (9 compounds, 38 targets, and 4 pathways) in this network connected by 103 interaction. Compounds in FWP regulated glutamate receptor signaling, glutamatergic synaptic transmission, glutamatergic synapse, and glutamate secretion. Saikosaponin A (B3) regulated glutamate receptor signaling. Hydroxycoumarin (AM1) regulated glutamatergic synaptic transmission and glutamate receptor signaling. 6-Gingerol (Z1) regulated glutamatergic synaptic transmission and glutamate secretion. Compounds from herb g and m could regulate all 4 related signaling.

Figure 3. The systematic bioinformatics analysis for antidepressive mechanism of FWP. (A) compounds of FWP included (green) or unreported (red) in NPASS and PubChem database. (B) Enrichment analysis for herb-disease correlation (total: FWP). (C) Functional enrichment analysis on GO and KEGG for targets of FWP. (D) Target overlaps (purple line) and functional interaction (blue line) among targets for each herb in FWP. (E) Compound-Target-Pathway Network for compounds of FWP.

Evaluation of rat model of depression with LDSDS

Normal rats kept a stable mental and physical state on day 1, 7, 14 and 21. During 21-day procedure, model rats showed sleep latency, reduced sexual/aggressive behaviours and self-care, impairments in place preference conditioning and brain stimulation reward, and frequent loose stool. The above changes were reversed on FWP-treated rats. Model rats exhibited significantly lower body weight increase compared with normal controls. FWP treatment could significantly reversed this lower pattern on day 21 (P<0.01, Figure 4A). When exposed to the OFT, model rats showed significant decreases in grid crossing counts, standing times and grooming times on day 21, which were alleviated after FWP treatment (P<0.01, Figures 4B–D). On day 21, compared with normal group, urinary excretion rate of xylose in model group decreased significantly, which were inhibited in FWP group (P<0.01, Figure 4E). Moreover, decreased sucrose preference is a anhedonia symptom representing clinical depression feature. Compared with normal rats, there was a significant decreased sucrose consumption in model rats, which was inhibited by FWP treatment (P<0.05, Figure 4F). Taken together, changes in model rats paralleled characteristic depression with LDSDS (i.e., depressed mood, psychomotor activity changes, anorexia, uncomfortable loose bowels).

Figure 4. Evaluation of chronic restraint stress induced rat model representing depression with liver-depression and spleen-deficiency syndrome, as well as effects of FWP on behavior changes. For model evaluation experiment: (A) Body weight increase (g). (B–D) Number of grid crossing, standing times, and grooming times in open field test. (E) Urinary excretion rate of xylose test (%). (F) Sucrose consumption (mL). For FWP treatment experiments: (G) Body weight increase (g). (H–K) Number of grid crossing, median residence time (s), grooming times, and standing times in open field test. (L) CNQX injection sites in amygdaloid nucleus (marked by methylene blue). n=19 or 20 for (A–F), and n=13-15 for G-K. *P<0.05, **P<0.01 compared with the normal group; ^#P<0.05, ^##P<0.01compared with the model group; ^♦P<0.05, ^♦♦P<0.01 compared with the sham-operated group.

Effects of FWP on depressive behaviors in rats

The general state, body weight increase and behaviour in OFT were evaluated on day 21. Compared with model rats, there was not difference in sham-operated and CNQX groups, but significant alleviation in FWP group (P<0.01). On day 25, compared with normal rats, model rats exhibited depression-like behaviours with body weight increasing slowly (P<0.01, Figure 4G–4K). Compared with model, sham-operated rats suffered acute stress of operation and were irritable and hyperactive, with increased grid crossing counts, standing times and grooming times, as well as decreased residence times (P<0.01). These behavior changes were reversed significantly after FWP and CNQX treatment (P<0.05). In detail, FWP showed mild callback effect, whereas CNQX had an excessive effect. Moreover, compared with model, operation or CNQX treatment showed similar slow weight gain, while FWP treatment showed rapid weight gain on day 25 (Figure 4G). Therefore, FWP seemed to have a better regulatory effect.

Effects of FWP on distribution of c-Fos, GluR1 and GluR2/3 immunoreactivity

The immediate early gene c-Fos is critical for neuronal plasticity. Neurons with c-Fos immunoreactivity were seen in hippocampus, bed nucleus of stria terminalis, amygdala, cingulate gyrus, marginal area and cortex. The stained neurons were round or oval-shaped, with the reaction product concentrated in nuclei. The membrane and cytoplasm were never stained (Figure 5A). Compared with control, there were significantly more neurons with c-Fos immunoreactivity in hippocampus (CA1, CA3 and DG) and amygdala (BLA) in model and sham-operated groups (P<0.05 or P<0.01), and slightly increased neurons with c-Fos immunoreactivity in FWP group (P>0.05). Compared with model and sham-operated groups, there were significantly less neurons with c-Fos immunoreactivity in CNQX and FWP group (P<0.05 or P<0.01, Figure 5B).

Figure 5. Effects of FWP on c-Fos, GluR1 and GluR2/3 expression in hippocampus CA1, CA3 and DG, and amygdala BLA, in depressive rats with liver-depression and spleen-deficiency syndrome (×400, scale bar = 200 μm). (A, B) The positive expression and positive cell of c-Fos; (C, D) The positive expression and positive cell of GluR1; (E, F) The positive expression and positive cell of GluR2/3. Results are presented as mean ± SD from 25 sections. *P<0.05, **P<0.01 compared with the normal group; ^#P<0.05, ^##P<0.01compared with the model group; ^♦P<0.05, ^♦♦P<0.01 compared with the sham-operated group.

Specific GluR1 staining (brownish yellow) was concentrated in hippocampus and amygdala, while almost colourless in other parts (Figure 5C). The stained neurons were round, with orderly and closely arrangement. The membrane and fiber were darkly stained, and the cytoplasm and nuclei were not stained. Within the hippocampus, staining was especially prominent in the first and radiation layers of CA1, slight in CA3, and almost colourless in the transparent layer. The amygdala was more staining than cortex. Compared with control, there were significantly more neurons with GluR1 immunoreactivity in CA1 and DG, while significantly less in CA3 and BLA in model and sham-operated groups (P<0.05 or P<0.01). Compared with model and sham-operated groups, there were significantly less neurons with GluR1 immunoreactivity in CA1 and DG, while significantly more in CA3 and BLA in CNQX and FWP group (P<0.05 or P<0.01, Figure 5D). The results were similar in positive area change and integrated optical density (IOD) (Figure 6A, C), while a slightly different in mean optical density (MOD) (Figure 6B).

Figure 6. Effects of FWP on AMPA receptor homeostasis in hippocampus and amygdala, in chronic restraint stress induced rat model representing depression with liver-depression and spleen-deficiency syndrome (×400). (A–C) Change of GluR1 positive area (μm²), MOD, and IOD (×10³) values. (D–F) Change of GluR2/3 positive area (μm²), MOD, and IOD (×10³) values. Results are presented as mean ± SD from 25 sections. *P<0.05, **P<0.01 compared with the normal group; ^#P<0.05, ^##P<0.01compared with the model group; ^♦P<0.05, ^♦♦P<0.01 compared with the sham-operated group.

Specific GluR2/3 staining (brownish yellow) was concentrated in cerebral cortex, hippocampus, amygdala and hypothalamus, while almost colourless in other parts (Figure 5E). The stained neurons were circular, with darkly stained surface of nerve fiber and cell body. The cytoplasm and nuclei were not stained. Within the hippocampus, staining was especially prominent in soma layer, concentrated in the granular layer of DG, obvious in the initial/radiation/molecular layers, and almost colourless in the transparent layer of CA3. The amygdala was slightly more staining than cortex. Compared with control, there were significantly less neurons with GluR2/3 immunoreactivity in CA1, CA3 and DG, and significantly more in BLA in model and sham-operated groups (P<0.05 or P<0.01). There were slightly decreased neurons with GluR2/3 immunoreactivity in FWP group (P>0.05). Compared with model and sham-operated groups, there were significantly more neurons with GluR2/3 immunoreactivity in CA1, CA3 and DG, while significantly less in BLA in CNQX and FWP group (P<0.05 or P<0.01, except for result in DG of CNQX group: P>0.05, Figure 5F). For positive area change, IOD, and MOD, there were similar results in CA1, CA3 and BLA region, while no expression difference in DG region (Figure 6).

The similar immunohistochemical changes between FWP and CNQX groups suggested that FWP had a positive and balanced effect via antagonizing the AMPA receptor in amygdala. FWP inhibited AMPA receptor excitability, reduced the intensity of hippocampal stimulation and related damage.

Effects of FWP on protein expression and phosphorylation of GluR1, GluR2 and GluR3

Compared with normal group, CRS in model group decreased GluR1 and p-GluR1 protein expression in CA3, GluR2 in CA3 and DG, p-GluR2 in CA1, CA3, and DG, and GluR3 in CA1 (P<0.05 or P<0.01), which were reversed by FWP treatment (P<0.05 or P<0.01, except for GluR2 in DG). Meanwhile, CRS increased p-GluR1 in CA1, p-GluR2 in BLA, which were also reversed by FWP treatment (P<0.05 or P<0.01). Compared with normal rats, rats in sham-operated group showed decreased expression of GluR1 and p-GluR1 in CA3, p-GluR2 in CA1 and CA3, and GluR3 in CA1 (P<0.05 or P<0.01), which were reversed by FWP treatment (P<0.05 or P<0.01, except for GluR2 in DG). Meanwhile, rats in sham-operated group showed increased expression p-GluR1 in CA1, p-GluR2 in BLA, which were also reversed by FWP treatment (P<0.05 or P<0.01, Figure 7). Results showed that FWP mainly regulated the phosphorylated form of protein GluR1 and GluR2.

Figure 7. Effects of FWP on protein expression and phosphorylation of GluR1, GluR2 and GluR3 in hippocampus and amygdala, in chronic restraint stress induced rat model representing depression with liver-depression and spleen-deficiency syndrome. (A, D–G) protein expression and phosphorylation of GluR1. (B, H–K) protein expression and phosphorylation of GluR2 expression. (C, L) GluR3 expression (n=8). *P<0.05, **P<0.01 compared with the normal group; ^#P<0.05, ^##P<0.01compared with the model group; ^♦P<0.05, ^♦♦P<0.01 compared with the sham-operated group.

Effects of FWP on gene expression of GluR1, GluR2 and GluR3

Compared with control, gene expression of the AMPA receptor subunits GluR1 were increased in CA1 and DG, and decreased in CA3 and BLA in model and sham-operated groups (P<0.01 in CA1 and CA3). The gene expressions of GluR2 and GluR3 were decreased in CA1, CA2, and CA3, and increased in BLA (P<0.01 in CA1 and CA3). The dysregulated expressions of GluR1, GluR2 and GluR3 were alleviated after FWP and CNQX treatment (P<0.05 or 0.01 in CA1 and CA3, Figure 8). Patterns of gene expression were consistent with protein expression and distribution for different AMPA receptor subunits.

Figure 8. Effects of FWP on gene expression of GluR1, GluR2 and GluR3 in hippocampus and amygdala, in chronic restraint stress induced rat model representing depression with liver-depression and spleen-deficiency syndrome. (A) Electrophoresis of GluR1, GluR2 and GluR3. (B–D) relative quantitation of GluR1, GluR2, and GluR3 (n=5). *P<0.05, **P<0.01 compared with the normal group; ^#P<0.05, ^##P<0.01 compared with the model group; ^♦P<0.05, ^♦♦P<0.01 compared with the sham-operated group.

Animals and reagents

Male SD rats (230±10 g, clean grade) were purchased from Vital River Laboratory Animal Technology Co. Ltd (SCXK (Beijing) 2002-0003, Beijing, China). Animals were housed in a quiet room on a 12-h light/dark cycle, maintaining the temperature (20–24°C) and relative humidity (30–40%). Standard laboratory chow and water were provided ad libitum. All animal experiments were approved by the Laboratory Animal Management and Ethics Committee of the Dongzhimen Hospital, and Beijing University of Chinese Medicine.

FWP consisted of eight herbs (Table 2), which were purchased from the Beijing Tongrentang Yinpian Co., Ltd. (Bozhou, China). The voucher specimens were identified by experts, and deposited at Beijing University of Chinese Medicine. The preparation method for FWP dry extract was shown in Supplementary Figure 1 with an extraction yield of 21.74%. FWP extract was dissolved in distilled water at a concentration of 0.3854 g/mL for intragastric administration. CNQX (Sigma, Milwaukee, USA) was dissolved in 20% DMSO/saline to a concentration of 1 mg/mL.

UHPLC-MS

Five microliter of FWP solution, filtered through 0.22 μm membrane, was injected into an UltiMate 3000 LC system (Thermo Fisher Scientific, Bremen, Germany) on a C18 column (100 × 2.1 mm, 2.6 μm, Phenomenex Inc., Torrance, USA) at 35 °C. The mobile phase consisted of 0.1% formic acid (v/v) (A) and acetonitrile (B), and flow rate was 1 mL/min. The gradient used was: 0-30min 5%- 70%B, 30-35 min 70%-100% B, 35-39 min 100% B, 39-45 min 100%-5% B. The wavelength for detection was 254 nm. The mass spectrometer measurements were performed on a HR-ESI-MS detector with Thermo Q-Exactive system. The positive and negative electrospray voltages were 3.5 kV and 2.5 kV, and the capillary temperature was set at 300 °C.

Systematic bioinformatics analysis for FWP

Construction of FWP constituent and target database

Compounds identified by UHPLC-MS, were searched in NPASS [43] and PubChem Databases (https://pubchem.ncbi.nlm.nih.gov/). ADME properties were collected, comprising molecular weight (MW), lipophilicity descriptors (ALogP, MLogP, XLogP), hydrogen bond donor, hydrogen bond acceptor, polar surface area, rotatable bond, aromatic rings, heavy atoms and LipinskiFailure. Corresponding protein targets were retrieved, and then classified into each herb in FWP. Target source was mapped into Homo sapiens for further analysis.

Interactions between targets

Protein–protein interaction data analysis was useful to characterize the molecular basis of disease. Interactions between targets were searched using Kyoto Encyclopedic of Genes and Genomes (KEGGs) [44], and Gene Ontology (GO) [45]. The correlation among targets of each herb was demonstrated by Circos diagrams using Metascape.

Enrichment analysis for herb- disease correlation

Targets of FWP and each herb were mapped into DisGenNET database [46] through Metascape [47]. The accumulative hypergeometric p-values were calculated to filter the statistically enriched terms. Then, the enriched diseases related to depression were analyzed for each herb in FWP and visualized using heatmap of -Log P value.

Functional enrichment analysis on GO and KEGG

Enrichment analysis was an effective method to increase the reliability of the identification of biological phenomena, resulting in meaningful annotation information. The “clusterProfiler” package in R was used to analyze enriched targets groups on KEGGs and GO items. P < 0.05 were considered statistically significant.

Construction of compound-target-pathway network

To clarify the relationship between compounds in FWP and glutamate signaling, glutamate signaling-related genes were retrieved from the KEGG database. Intersections among compound-related genes and genes-related pathways were analyzed to create a Compound-Target-Pathway Network in CytoScape. The node correlation degree in the network was calculated using the STRING database [48]. The number of connections in network were calculated using Network Analyzer in Cytoscape and expressed as node size.

Chronic restraint stress-induced rat model and treatment

Model evaluation experiment

After 3 days of adaptive feeding, overactive or quiet rats were excluded. Rats were randomly divided into three groups (n=20): normal, model and FWP groups. All groups except normal received the chronic restraint stress procedure (8-11 a.m., 3h/day, 21 days, no access to food or water). The chest/ abdomen and four limbs were fixed by a soft bonds on a T-shaped bilayer restraint platform. Thirty minutes before restraint, rats received 3.854 g/kg•d FWP solution through gastric gavage in FWP group, or equal volume of saline in normal and model groups.

FWP treatment experiment

Rats were randomly divided into 5 groups (n=15): normal, model, sham-operated, CNQX, and FWP groups. Rats received an above-mentioned restraint and treatment for 25 days. Rats in sham-operated and CNQX group received restraint and saline. On day 22, rats in CNQX group were injected with CNQX (0.5 μg in 0.5 μL) into bilateral amygdala, rats in sham-operated and FWP group were injected with 0.5 μL saline, while rats in normal and model group received no injection. During operation, 1 rat in sham-operated, 2 in CNQX, and 1 in FWP group died due to deep anaesthesia. For 300-350g SD male rats, the injection sites of amygdaloid nucleus (Figure 3L) were AP=-2.5, L=±4.4, DV =-7.9 [49]. Penicillin (160,000 units) were intra-peritoneally injected to prevent infection. On day 25, behaviour observation was performed. On day 26, rats were anaesthetized and transcardially perfused with heparinized saline (Supplementary Figure 2).

Behavioral tests

General observation

Before daily administration, rats’ mental state, posture, skin color, mobility, reactivity to restraint, eye fissure mucosa color, auricle color and feces were carefully observed.

Weight gain

Rats was weighed on day 1, 7, 14, and 21. Weight gain was calculated with weight on day 1 as baseline.

Open field test

On day 1, 7, 14 and 21, the open-field test was performed in a four-sided wooden enclosure, which was divided into 25 equal squares by black lines with grey floor and side walls (100cm×100cm×40 cm). Rat was gently placed in the central square. The number of grid crossing/median residence time, standing times, and grooming times were recorded by video tracking system for 5 min. The Observer 5.0 software (Noldus Information Technology B.V., Wageningen, Netherlands) was used to evaluate the autonomous activities, learning and memory, anxiety and other behaviour of rats.

Sucrose consumption test

After 24h of water deprivation on day 1, 7, 14 and 21, rat was subjected to an individual metabolic cage in which 100 mL of 1% sucrose solution were placed. The consumption of 1% sucrose solution was calculated within 1 h.

Urinary excretion rate of xylose test

On day 1, 7, 14 and 21, D-xylose-free urine was collected after 11h of food deprivation for each rat in an individual metabolic cage. Then rat was given 10% D-xylose solution (0.15g/100g). The urine was collected for the following 5h, and analysed using phloroglucinol method with absorbance measured at 554 nm [50]. Urinary D-xylose metabolic rate = D-xylose excreted from urine for 5 h (g)/amount of D-xylose administered (g) × 100%.

Immunohistochemistry

The hippocampal CA1, CA3, DG and amygdala BLA regions were fixed in ice-cold 4% paraformaldehyde, cryoprotected with 30% sucrose solution, and sectioned into 30 μm slices. Sections were exposed to 3% H₂O₂, blocked with 10% normal goat serum, and incubated with c-Fos, GluR1, and GluR2/3 antibodies diluted in blocking sera (1:5000, 1:1000, and 1:1000). Then sections were incubated with biotinylated secondary antibodies (1:300, 1:200 and 1:200), stained using avidin-biotin-peroxidase complex (ABC, Sigma) and DAB solution, and dehydrated. Five coronal sections for each group were randomly chosen from equal levels of consecutive sections and viewed at 400× magnification (Nikon 4500 digital microscope). Image was processed as described in recent publications [51]. Positive cells were counted in a 200×100 μm² region in CA1, and a 200×200 μm² region in CA3, DG and BLA. Mean optical density (MOD), integral optical density (IOD) and area of positive substance (μm²) were quantified by the Image-Pro Plus 6.0 software.

Western blot

The hippocampus and amygdala were dissected and CA1, CA3 and DG regions were separated, lysed by RIPA containing PMSF (Beyotime, Nanjing, China), homogenized and centrifuged. A total of 50 μg protein were resolved by 8% SDS-PAGE (Nanjing Jiancheng, Nanjing, China), transferred to a PVDF membrane, blocked with 5% milk, incubated with antibodies against pGluR1 (Ser845, Invitrogen, Carlsbad, USA), GluR1 (1:400, Invitrogen), pGluR2 (Ser880, Invitrogen), GluR2 (1:400, Invitrogen), GluR3 (1:400, Invitrogen), GluR2/GluR3 (Invitrogen), β-actin (1:5000, Cell Signaling Technology, Danvers, USA) at 4°C overnight, and hybridized with biotinylated-conjugated secondary antibody (1:5000, Vector Laboratories, Burlingame, USA) for 1 hours. The immunoreactive bands were visualized using an ECL system. The relative intensities of bands were quantified using Image J.

RT-PCR

The hippocampal CA1, CA3, DG and amygdala BLA regions were collected, and RNA was extracted using TRIzol extraction Kit according to the instructions (Invitrogen). The cDNA was synthesized using cDNA reverse transcription kit (Fermentas, Burlington, Canada). Primer sequences were synthesized by SBS Genetech Co., Ltd. (Beijing, China) as follows: GluR1, Forward: 5'- GTCG TCCT CTTC CTGG TCAG CC -3', Reverse: 5'-GTGT CACA GGCT TTCG TTGC T-3', 541bp; GluR2, Forward: 5'-TTCC GTAA CCTT CGGA AGCA-3', Reverse: 5'-CAAG CCCA GACG TGTC ATTT C-3', 304bp; GluR3, Forward: 5'-TAGT CAGC AGAT TTAG CCCT TA-3', Reverse: 5'-TTTC CACC AACT TTCA TCGT AT-3', 558bp; β-actin, Forward: 5'-ATCA TGTT TGAG ACCT TCAA CA-3', Reverse: 5'-CATC TCTT GCTC GAAG TCCA-3', 500bp. PCR were performed following the instructions of AccessQuick RT-PCR system onestep kit (Promega, Madison, USA). Expression of target genes was corrected by the expression of β-actin and calculated using 2^{-(Δ ΔCt)} method. Finally, PCR product was electrophorized using 2.5% agarose gel and scanned by ImageMaster VDS system (Pharmacia Biotech, Inc., Uppsala, Sweden). The experiment was repeated three times.

Statistical analysis

Data were expressed as mean ±SD, and analyzed using GraphPad Prism 7.0 (GraphPad Software Inc., La Jolla, United States). Normal distribution and homogeneity test between groups were performed. One-way analyses of variance (One-Way ANOVA) was used for comparison of multiple groups, followed by the least significant difference (LSD) post hoc test. The Kruskal-Wallis non-parametric H test was used when there was one inconsistency. P<0.05 was considered as statistically significant.