Depression and anxiety are common diseases that endanger the physical and mental health of individuals. Agarwood incense inhalation has been used as a traditional Chinese medicine for relaxation and to improve sleep for centuries. In a previous study by the authors it was demonstrated that agarwood essential oil (AEO) injection exerted anxiolytic and antidepressant effects. Therefore the present study further investigated the anxiolytic and antidepressant effects of AEO inhalation on anxiolytic mice induced by M-chlorophenylpiperazine and depressive mice induced by chronic unpredictable mild stress. The results demonstrated that AEO exerted a significant anxiolytic effect, whereby autonomous movements were inhibited during the light dark exploration test and open field test. Furthermore, the tail suspension test and the forced swimming test demonstrated that AEO also exerted an antidepressant effect, whereby the immobility times were decreased. Moreover, AEO was determined to increase the levels of 5-hydroxytryptamine, γ-aminobutyric acid (GABA) A receptor (GABAA) and glutamate (Glu) in anxiolytic mice and inhibit the levels of GABAA and Glu in depressive mice. Further investigations into how AEO affected the Glu/GABA system demonstrated that AEO markedly increased the protein expression levels of GABA transaminase (GABAT), glutamate metabotropic receptor 5 (GRM5), glutamate ionotropic receptor AMPA type subunit 1 (GluR1) and vesicular glutamate transporter 1 (VGluT1). Furthermore, AEO reduced the expression levels of GABAT, glutamate ionotropic receptor NMDA type subunit 2B and GRM5, and enhanced the expression levels of GluR1 and VGluT1. These results demonstrated that AEO potentially possesses antianxiety and antidepressant properties. The present study determined that the mechanism was related to the regulation of Glu/GABA neurotransmitter system homeostasis.
Depression and anxiety are complex neurological and psychological diseases that are rated as two of the most severe health problems by the World Health Organization (
At present, the pathological mechanisms of anxiety and depression are unclear and complex. An increasing number of studies have reported that their pathogenesis is related to the autonomic nervous system, hypothalamic-pituitary-adrenal (HPA) axis, neural circuits and the immune system (
Agarwood is an important spice that has been widely used in incense, religion and aromatherapy for centuries (
Therefore, in the present study AEO incense inhalation was used to study the antianxiety and antidepressant effects of AEO and investigate its possible underlying mechanism in regulating neurotransmitters. The effects of AEO on
A video analysis system for spontaneous activity (model: JLBehv-LAM-4) was purchased from Shanghai Jiliang Software Technology Co., Ltd. The incense box (50x50x40 cm) was formed of plexiglass with a 20x20x20 cm3 hollow cylinder inside with incense inserts in the middle. The centrifuge and microplate reader were purchased from Thermo Fisher Scientific, Inc. (model: Multiskan Go, serial no. 1510-04123). The neurotransmitter and its receptor kits glutamate (Glu; cat. no. SRA-EMS-20177), 5-hydroxytryptamine (5-HT; cat. no. SRA-ESM-00901) and γ-aminobutyric acid receptor A (GABAA; cat. no. SRA-ES-31001; all from DXY.cn; Hangzhou Lianke Meixun Biomedical Technology Co., Ltd.) were purchased from Beijing Bosheng Jingwei Biotechnology Co., Ltd. The primary antibodies GABA transaminase (GABAT; cat. no. AG1008), glutamate metabotropic receptor 5 (GRM5; cat. no. AF1744), glutamate ionotropic receptor NMDA type subunit 2B (GRIN2B) (cat. no. AF7029), and glutamate ionotropic receptor AMPA type subunit 1 (GluR1; cat. no. AF2473) were purchased from Beyotime Institute of Biotechnology. Vesicular glutamate transporter 1 (VGluT1; cat. no. bs-11167R) was purchased from BIOSS. The secondary antibodies HRP goat anti-rabbit (cat. no. A0208) and HRP goat anti-mouse (cat. no. A0216) were also obtained from Beyotime Institute of Biotechnology. Diazepam (cat. no. DZ-225336;
The agarwood raw material was artificially propagated agarwood (
A total of 96 adult male KM mice (weight, 18-20 g) were purchased from the Hainan Provincial Institute of Medicine [cat. no. SCXK (Qiong) 2020-0007]. All animals were kept in a specific pathogen-free (SPF) animal facility at a temperature of 23
The anxiolytic effects and the underlying mechanisms of AEO inhalation in an anxious mouse induced by mCPP were explored. The animals were divided into the following six groups (8 mice/group): i) Control group; ii) mCPP (8 mg/kg) model group; iii) diazepam (2.5 mg/kg) group; and iv) to vi) the AEO (2, 4 or 8 µl) inhalation groups. With the exception of the control group, the mice were administered with mCPP intraperitoneal injections for 2 days to create the anxiety animal model. The control group was given an equal volume of saline. The diazepam group was injected intraperitoneally for 7 days with 2.5 mg/kg (10 ml/kg) diazepam. The AEO groups were administered with AEO via inhalation for 7 days. After 7 days, animal behavior was assessed via the open field test (OFT) and the light-dark exploration test (LDET). Following the behavioral tests, the mice were sacrificed via cervical dislocation and blood samples and brain tissues were rapidly collected and preserved in liquid nitrogen. The blood samples were centrifugated at 314 x g for 15 min at 4˚C and stored at -20˚C.
The antidepressant effects of AEO were assessed in depressed mice induced via CUMS
The LDET was used to observe the exploratory behavior of mice when they were added to a new environment. As the rodents were innately averse to brightly lit areas, they developed anxiety and the animal activity was disordered. The apparatus contained two parts, a light box and a dark box, which were used to detect the movement of the animals. The apparatus was connected to a computer to record the time spent, distance moved and number of transitions in each part during a 6-min session. For this experiment, the mice were administered diazepam as aforementioned, to detect the anxiolytic effects of AEO. In the present study, the data from the last 4 min were selected for analysis.
The OFT evaluated the general exploratory behaviors of mice. For this experiment, the mice were administered diazepam as aforementioned, to detect the anxiolytic effects of AEO. Briefly, 1 h after the last treatment was administered, OFT real-time detection analysis was performed. The mice were placed into the open field along the barrel wall for 10 min of observation and the system automatically recorded the spontaneous activities of the animals, such as movement distance and movement time. The time spent and distance moved in the central area were collected to reveal the anxiolytic effect of AEO.
The FST was performed according the Porsolt swim test method. In brief, 1 h following the last treatment administration, forced swimming real-time detection and analysis were performed. After setting the experimental parameters, geometry and illumination calibration, the mice were placed in a thermostatic swimming apparatus (height, 20 cm; diameter, 18 cm; water depth, 12 cm; water temperature, 23-25˚C). The system automatically recorded the activity status of the mice over 6 min and assessed the accumulated immobility time of the mice within the last 4 min.
In this experiment, the mice were administered with diazepam and paroxetine as aforementioned, to detect the anxiolytic and antidepressant effects of AEO, respectively. Next
The precipitate of hippocampal tissues was added to RIPA (cat. no. BL504A; Biosharp; Beijing Lianshi Yunshang Network Technology Co., Ltd.) buffer supplemented with protease and phosphatase inhibitors. The homogenate was centrifuged at 1,364 x g, for 10 min at 4˚C. The protein concentration was determined using a BCA Protein Assay Kit. According to the molecular weight of the target protein, the protein samples (10 µg) were separated using 10% gel electrophoresis. Subsequently, the separated proteins were transferred to a PVDF membrane and blocked with 5% skimmed milk powder for 1 h at room temperature. The membranes were then incubated with the following primary antibodies; GABAT, GRIN2B, GRM5, GluR, VGluT1 (all at 1:1,000) and β-actin (1:2,000; cat. no. AF0003; Beyotime Institute of Biotechnology) at 4˚C overnight. Following the primary incubation, the membranes were incubated with secondary antibodies (1:2,000) for 2 h at room temperature. The membrane was treated with an ECL kit (cat. no. BL520A; Biosharp; Beijing Lianshi Yunshang Network Technology Co., Ltd.) and was observed using a gel imager. The grayscale of the protein bands were scanned and quantitative analysis was performed using the Gel-Pro Analyzer 4.0 (Media Cybernetics, Inc.). Subsequently, histograms were produced.
SPSS 17.0 software (SPSS, Inc.) was used for data analysis. Experimental data are presented as the mean ± SD and experiments were perfromed in triplicate. All the data were first analyzed for normal distribution and variance homogeneity. The statistical comparisons between more than three groups were performed using one-way ANOVA followed by Tukey's post hoc tests. P<0.05 was considered to indicate a statistically significant difference.
The distance and velocity of the animals were markedly enhanced in the model group compared with the control group in the dark box (P<0.01), which suggested that the anxiety model was successful. However, AEO significantly shortened the distance travelled in a dose-dependent manner (P<0.05 in the 8-µl group) (
The distance and velocity were significantly enhanced in the model mice (P<0.01) compared with the control mice, which demonstrated that the mice exhibited symptoms of anxiety. The results further demonstrated that AEO markedly shortened these distance increases in a dose-dependent manner (
The immobility time of TST was prolonged in the model mice, which indicated that the mice exhibited depression-like symptoms. AEO treatment markedly shortened the immobility time (
The immobility time in the FST was significantly prolonged in the model mice (P<0.05), which indicated that the animals were exhibiting depression-like symptoms. AEO significantly reduced the immobility time (
The levels of 5-HT, GABAA and Glu were assessed in mCPP-induced anxious mice and the results revealed that the level of 5-HT was significantly reduced (P<0.001), the level of GABAA was also reduced (P<0.01) and the level of Glu was enhanced (P<0.05) in the model mice. AEO treatment significantly increased the levels of 5-HT and GABAA and decreased the levels of Glu in a dose-dependent manner (
The levels of 5-HT, GABAA and Glu were assessed in CUMS-induced depressant mice and the results revealed that the levels of 5-HT were significantly reduced (P<0.001) and the levels of GABAA and Glu were enhanced (P<0.05) in model mice. AEO significantly increased the levels of 5-HT and decreased the levels of GABAA and Glu (
The protein expression levels of GABAT, GRM5, GluR1 and VGluT1 were assessed in mCPP-induced anxious mice and the results revealed that AEO inhalation significantly upregulated the protein expression levels of GABAT, GRM5, GluR1 and VGluT1 (P<0.05, P<0.01 and P<0.001). Furthermore, the results demonstrated that AEO treatment potentially served a role in relieving anxiety via regulating the protein expression levels and transport of GABA and Glu, and therefore the balance of the Glu/GABA system (
The protein expression levels of GABAT, GRIN2B, GRM5, GluR1 and VGluT1 were assessed in CUMS-induced depressant mice and the results revealed that AEO inhalation markedly decreased the protein expression levels of GABAT, GRIN2B and GRM5, and increased the protein expression levels of GluR1 and VGluT1. The results demonstrated that AEO treatment potentially served an antidepressant role via the regulation of the protein expression levels of GABA and Glu and the balance of the Glu/GABA system (
The present study suggested that AEO displays anxiolytic and antidepressant effects via balancing the E/I Glu and GABA neurotransmissions in anxious and depressant mice. Behavioral evaluation further confirmed that AEO not only significantly inhibited the anxiety of mice as assessed by LDET and OFT, but also exerted a significant antidepressant effect by decreasing the immobility time of mice as determined using TST and FST. In summary, the results indicated that AEO may represent a potential treatment of anxiety and depression by regulating the homeostasis of the Glu-GABA system.
Spontaneous locomotor activity tests, including the LDET and OFT, are used to generate general parameters to study the effects of a drug (
The abnormal secretion of neurotransmitters is important in the pathogenesis of anxiety, depression and comorbid disorders (
The hippocampus may play a critical role in the pathophysiology and treatment of anxiety and depression. Its functions correspond to those altered in anxiety and depression (
In conclusion, the present study demonstrated that AEO incense exerted potential antianxiety and antidepressant effects via the regulation of their receptors and via the GABA/Glu system. These effects were similar to those of treatment with diazepam and paroxetine. The inhalation of AEO resulted in two-way effects in regulating the balance of the GABA/Glu system, which suggested that AEO could serve as a potential therapeutic candidate aiding in the treatment of anxiety, depression and CNS diseases. Furthermore, the present study may also provide a theoretical basis for the development and utilization of agarwood. However, the specific underlying molecular mechanism of AEO needs to be further explored.
Not applicable.
The datasets used during the present study are available from the first author or corresponding author upon reasonable request.
CW and JW designed the study. CW, BG and YW performed the experiments. YL and DC extracted the AEO, and assisted with the experiments. CW contributed to the preparation of the manuscript. CW and JW revised the manuscript. CW and BG analyzed the data and confirm the authenticity of all the raw data. All authors read and approved the final manuscript and agree to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the work are appropriately investigated and resolved.
The animal care and experimental protocol was approved by the Institutional Animal Care and Use Ethics Committee of Hainan Institute of Materia Medica Co., Ltd., Haikou, China (approval no. 2021HL014).
Not applicable.
The authors declare that they have no competing interests.
Effects of agarwood essential oil inhalation determined using a light-dark exploration test. (A) Black box distance and (B) black box velocity. All values are expressed as the means ± SD (n=8). **P<0.01 vs. the normal group; #P<0.05 vs. the model group.
Effects of agarwood essential oil inhalation determined using an open field test. (A) Total distance, (B) average velocity, (C) central area route, (D) central area velocity, (E) stick-a-wall distance and (F) rest time were assessed. All values are expressed as the means ± SD (n=8). *P<0.05 and **P<0.01 vs. the normal group; #P<0.05 vs. the model group.
Effects of agarwood essential oil inhalation determined using a tail suspension test. All values are expressed as the means ± SD (n=8). P>0.05 vs. the normal group; #P<0.01 vs. the model group. TST, tail suspension test.
Effects of agarwood essential oil inhalation determined using a forced swimming test. All values are expressed as the means ± SD (n=8). *P<0.05 vs. the normal group; #P<0.05 vs. the model group. FST, forced swimming test.
Effects of agarwood essential oil inhalation on the levels of 5-HT, GABAA and Glu in M-chlorophenylpiperazine-induced anxious mice. The levels of (A) 5-HT, (B) GABAA and (C) Glu. All values are expressed as the means ± SD (n=8). *P<0.05, **P<0.01 and ***P<0.001 vs. the normal group; #P<0.05 and ##P<0.01 vs. the model group. 5-HT, 5-hydroxytryptamine; GABAA, γ-aminobutyric acid A receptor; Glu, glutamate.
Effects of agarwood essential oil inhalation on the levels of 5-HT, GABAA and Glu in chronic unpredictable mild stress-induced depressant mice. The levels of (A) 5-HT, (B) GABAA and (C) Glu. All values are expressed as the means ± SD (n=8). *P<0.05 and ***P<0.001 vs. the normal group; #P<0.05 and ##P<0.01 vs. the model group. 5-HT, 5-hydroxytryptamine; GABAA, γ-aminobutyric acid A receptor; Glu, glutamate.
Effects of inhalation of agarwood essential oil on the protein levels of GABAT, GRM5, GluR1 and VGluT1 in M-chlorophenylpiperazine-induced anxious mice. The protein levels of GABAT, GRM5, GluR1 and VGluT1 were assessed using western blotting. All values are expressed as the means ± SD (n=3). **P<0.01 and ***P<0.001 vs. the normal group; #P<0.05, ##P<0.01 and ###P<0.001 vs. the model group. GABAT, GABA transaminase; GRM5, Glu metabotropic receptor 5; GluR1, Glu ionotropic receptor AMPA type subunit 1; VGluT1, vesicular Glu transporter 1; AEOI, agarwood essential oil inhalation.
Effects of inhalation of agarwood essential oil on the protein levels of GABAT, GRIN2B, GRM5, GluR1 and VGluT1 in chronic unpredictable mild stress-induced depressant mice. The protein levels of GABAT, GRM5, GluR1 and VGluT1 were assessed using western blotting. All values are expressed as the means ± SD (n=3). *P<0.05 and ***P<0.001 vs. the normal group; #P<0.05, ##P<0.01 and ###P<0.001 vs. the model group. GABAT, GABA transaminase; GRIN2B, glutamate receptor N2B subunit; GRM5, Glu metabotropic receptor 5; GluR1, Glu ionotropic receptor AMPA type subunit 1; VGluT1, vesicular Glu transporter 1; AEOI, agarwood essential oil inhalation.