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Avicularin Relieves Depressive-Like Behaviors Induced by Chronic Unpredictable Mild Stress in Mice

Background Avicularin (AL, quercetin-3-α-l-arabinofuranoside), a glycoside of quercetin, has been reported to display diverse pharmacological properties. The present study aimed to investigate whether AL has an anti-depressant-like effect on a mouse model of depression induced by chronic unpredictable mild stress (CUMS). Material/Methods A mouse model of depression was established and treated with different concentrations of AL (1.25, 2.5 or 5.0 mg/kg/d) and fluoxetine (20 mg/kg/d). Then, behavioral tests – sucrose preference test (SPT), forced swimming test (FST), and the tail suspension test (TST) – were performed. The levels proinflammatory cytokines – interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) – in the hippocampi of mice were detected by enzyme-linked immunosorbent assay (ELISA). Apoptosis of hippocampal neuronal cells was determined using flow cytometry. Expression levels of phosphorylated (p)-MEK1/2, p-ERK1/2, p-NF-κB (p-p65), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), Caspase3, and B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax) were measured by Western blot assay or/and quantitative real-time polymerase chain reaction (qRT-PCR), respectively. Results The results showed that AL significantly relieved CUMSinduced depressive-like behaviors. Compared with the model mice, AL treatment significantly increased the sucrose preference of the mice, and the immobility time in the FST and the TST were shortened. We also found that AL decreased CUMS-induced increases in the levels of IL-1β, IL-6, and TNF-α in the hippocampi of mice. AL significantly decreased the apoptosis rate of hippocampal neuronal cells in mice, which was increased by CUMS. Furthermore, activation of the MEK/ERK/NF-κB pathway induced by CUMS was inhibited by AL treatment. Conclusions Our results show the anti-depressant-like effects of AL on CUMS-induced depression in a mouse model.


Background
Depression is a multifactorial neurological disease and its etiology and pathogenesis are still unclear [1]. The main symptoms of depression are low mood, self-deprecation, energy and sleep deficits, psychosomatic disorders, anhedonia, and suicidal tendency [2,3]. Although depression affects more than 300 million people worldwide and will become the second leading cause of disability in the world by 2030 [4], 30-50% of depressed patients do not fully recover with currently available drug therapy [5]. Therefore, further study needs to identify new drugs with fewer adverse effects.
Dysregulation of pro-inflammatory cytokines plays a crucial role in the development of neuropsychiatric disorders, which include depression [6,7]. Antidepressant treatment can reduce these pro-inflammatory cytokines [8]. The MEK/ERK/nuclear factor-kB (NF-kB) signaling pathway is an important mechanism of inflammatory responses and has been reported to inhibit transduction of the MEK/ERK/NF-kB signaling pathway, which subsequently inhibits the inflammatory response [9]. Activation of NF-kB is mainly controlled by IkB-regulated negative feedback [10]. Once stimulated, inflammatory cytokines can be mediated by NF-kB [11].
Recently, studies have reported that drugs produced from medicinal plants show antidepressant-like effects and reduce adverse effects in refractory patients. For example, Hypericum perforatum L. (St. John's Wort) is widely used as an herbal medicine for the treatment of mild to moderate depressive episodes [12]. Lippia sidoides CHAM (Verbenaceae) essential oil and its major compound, Thymol, have an antidepressantlike effect [13]. Avicularin (the chemical structure is shown in Figure 1), which is quercetin 3-O-a-L-arabinofuranoside, is a flavonoid and quercetin derivative [14]. Flavonoids have been reported to have antidepressant activity [15]. Quercetin and its derivatives have anti-inflammatory and anti-oxidant effects [16,17]. A previous study has reported that quercetin-3-O-glucuronide can increase spontaneous activity and stimulate the nerve center to enhance excitement [18]. AL can inhibit accumulation of the intracellular lipids by decreasing C/EBPaactivated GLUT4-mediated glucose uptake in adipocytes [19]. However, the function and regulatory mechanism of AL action in depression have never been identified.
In this study, we aimed to investigate the antidepressant efficacy of AL. Furthermore, we evaluated whether the MEK/ERK/NF-kB signaling pathway is involved in its antidepressant-like effects.

Animals and reagents
Male C57BL/6 (age 8-10 weeks) mice weighting 18-22 g were purchased from the Vital River Company (Beijing, China). Animals were housed under a standard environment (temperature, 22±2°C, 55±5% humidity and 12-h light/dark cycle) and allowed food and water ad libitum. All experimental procedures were conducted in accordance with the Institutional Animal Care and Use Committee at Jiaxing University and carried out in accordance with the National Guidelines for Animal Care and Use Committees. The present study was approved by the Animal Ethics Committee of Jiaxing University.

Experimental design
Animals were adapted to the laboratory environment with a room temperature of 20±1°C, a reversed 12: 12 h light cycle and allowed food and water ad libitum for 1 week. Mice were randomly assigned into 6 groups (n=15 per group): Control group (unstressed + saline vehicle); the Model group (CUMS + saline vehicle), the CUMS +1.25 mg/kg/d AL treatment group (CUMS + AL1), the CUMS + 2.5 mg/kg/d AL treatment group (CUMS + AL2), the CUMS + 5.0 mg/kg/d AL treatment group   [20,21] and fluoxetine (20 mg/kg/d) [22] used in the present study was selected according to previous studies.

Chronic mild stress (CUMS) model
The CUMS model was performed as previously proposed by Katz [23], with slight modification. We used a series of chronic, unpredictable, mild stressors, including tail clamp (1 min), altering light/dark cycle (24 h), tilting of cage (45°, 12 h), constraint (12 h), wet bedding (12 h), food and water deprivation (12 h), swimming in cold water (4°C, 5 min), grouped housing (2 h), crowding mice into an empty bottle (6 h), and stroboscopic lighting (12 h). We randomly scheduled 2-3 types of stimuli daily, which could not be repeated for 3 consecutive days. The sucrose preference was tested once per week during the CUMS.

Sucrose preference test
After a training session, mice had 24 h of water and food for deprivation, followed by the sucrose preference test. Each mouse had free access to 2 bottles for 12 h: one bottle with 1% sucrose solution (w/v) and another bottle with tap water.
To avoid position effects, the positions of the 2 bottles were reversed. After 12 h, the volumes of tap water and sucrose solution consumed were recorded. The sucrose preference was calculated as sucrose preference (%)=(sucrose solution intake)/ (sucrose solution intake+tap water intake)×100%.

Forced swimming test
Each mouse was placed in an open cylindrical container (diameter 10 cm, height 25 cm) with 25±1°C water to a depth of 19 cm and allowed to swim for 6 min. Immobility times were measured during the last 4 min of the test. Immobility was considered as no movements other than those necessary to keep its head above water [24].

Tail suspension test
The Tail Suspension Test (TST) was conducted as previously described [25]. Each mouse was individually secured in place by adhesive tape placed 1 cm from the tip of the tail. The last 4 min was recorded for immobility time during a 6-min test.

Flow cytometric analysis
The Annexin V-FITC/PI double staining kit (Cat no. 70-AP101-100; MultiSciences, Hangzhou, China) was used to detect the hippocampal cell apoptosis. Hippocampi were regionally dissected from mice and dissociated to a single-cell suspension by enzymatic degradation using a neural tissue dissociation kit (Miltenyi Biotec, Auburn, CA) following the manufacturer's protocol. After centrifuging at 1000 rpm for 5 min, the supernatant was discarded. The cell pellet was re-suspended in Annexin V-FITC binding buffer and propidium iodide (PI) solution and incubated for 10 min in the dark at 37°C. Apoptotic cells were analyzed immediately by use of a FACScan flow cytometer (FACSCalibur, BD Biosciences, NJ).

Western blot analysis
Hippocampi were lysed on ice in buffer lysis (consisting of a cocktail of NaF and Na 3 VO 4 ), and the protein concentration was determined by Bicinchoninic acid (BCA) assay kit (Pierce Biotechnology, Rockford, IL). Protein samples (50 µg per line) were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene difluoride membrane (Millipore, Bedford, MA). The membranes were blocked with 5% skim milk at room temperature for 1.5 h. The membranes were incubated with primary antibody: anti-p-MEK1/2, p-ERK1/2, p-p65, iNOS, COX-2, Caspase-3, Bax, and b-actin overnight at 4°C, then incubated with the appropriate horseradish peroxidase-(HRP-) conjugated secondary antibody for 1 h at room temperature. Protein densities were detected by an ImageQuant LAS 4000 apparatus (GE Healthcare Life Sciences) and Image J software was used to perform quantification.

Enzyme linked immunosorbent assay (ELISA)
After drug treatment, mice were sacrificed. Hippocampi were dissected quickly and then stored at -80°C. After being homogenized and centrifuged at 5000×g for 15 min, the supernatant was collected. The levels of tumor necrosis factor-a (TNF-a), Interleukin-6 (IL-6), and Interleukin-1b (IL-1b) were detected using Quantikine ELISA Kits (R&D Systems; Minneapolis, MN) in accordance with the manufacturer's instructions.

Statistical analysis
All data were analyzed using SPSS version 20.0 (SPSS Inc., Chicago, IL). The statistical significance of differences between groups was evaluated using one-way analyses of variance (ANOVA) with post hoc least square difference (LSD) test. Data are expressed as mean ±SD and p<0.05 was considered a significant difference.

AL reduced body weight of CUMS mice
As shown in Figure 2, before treatment, there was no significant difference in body weight of mice between the CUMS group and the control group. After 2 weeks of CUMS modeling, the body weight of the mice was significantly lower than in the control group. AL treatment for 3 weeks significantly increased the body weight of CUMS-induced mice in a dose-dependent manner and similar effects were obtained after fluoxetine treatment.

AL ameliorated depressive-like behaviors of CUMS mice
Three weeks after AL treatment, sucrose consumption test, forced swimming test (FST), and tail suspension test (TST) were performed to evaluate the antidepressant activity of AL. Compared with the control group, sucrose consumption was significantly decreased in the CUMS group. Three weeks of AL treatment significantly increased sucrose consumption compared with the CUMS model group ( Figure 3A). CUMS significantly increased the immobility time in both FST ( Figure 3B) and TST ( Figure 3C) compared with the control group. AL or fluoxetine treatment significantly decreased immobility time during the FST ( Figure 3B) and TST ( Figure 3C) in a dose-dependent manner compared to untreated mice. These results indicate that AL treatment can reverse CUMS-induced depressive-like behavior.

AL attenuated inflammation in the hippocampi of CUMS mice
Next, we examined the effect of AL treatment on neuroinflammation. ELISA analysis of cytokines expression showed that the levels of the pro-inflammatory cytokines TNF-a, IL-6, and IL-1b were significantly higher in the hippocampi of CUMS mice than that in the unstressed mice ( Figure 4A-4C). AL treatment significantly decreased the expression of these pro-inflammatory cytokines in a dose-dependent manner ( Figure 4A-4C). Likewise, qRT-PCR results showed that the mRNA levels of TNF-a, IL-6, and IL-1b were also significantly higher in the hippocampi of CUMS mice than in unstressed mice ( Figure 4D-4F). AL treatment also downregulated the mRNA levels of these pro-inflammatory cytokines in a dose-dependent manner ( Figure 4D-4F). Furthermore, the expression of iNOS and COX-2 was detected by qRT-PCR and Western blot analysis. As shown in Figure 4G-4I, the protein and mRNA levels of iNOS and COX-2 were higher in the hippocampi of CUMS mice than in the unstressed mice. AL or fluoxetine treatment markedly decreased the expression of iNOS and COX-2 in a dose-dependent manner compared with the untreated group.

AL suppressed cell apoptosis in the hippocampi of CUMS mice
As shown in Figure 5A and 5B, CUMS significantly induced cell apoptosis in the hippocampus, while AL and fluoxetine treatment significantly attenuated hippocampal cell apoptosis. In addition, Western blot assay and qRT-PCR analysis were performed to assess the protein expression of apoptosis markers Bax and Caspase-3. As shown in Figure 5C, the protein levels of pro-apoptotic proteins Bax and Caspase-3 in the CUMS group were higher than in the control group. Compared with the CUMS group, AL treatment significantly decreased the protein levels of Bax and Caspase-3 in a dose-dependent manner and similar effects were obtained after fluoxetine (20 mg/kg) treatment.

AL suppressed MEK/ERK/NF-kB pathway activation in the hippocampi of CUMS mice
To understand the potential anti-inflammatory mechanisms of AL, we assessed activation of the MEK/ERK/NF-kB pathway   Figure 6A-6C, the protein levels of p-MEK1/2, p-ERK1/2, and p-p65 were significantly increased in the hippocampi of CUMS mice compared with the normal mice, suggesting that the MEK/ERK/NF-kB signaling pathway was activated. AL or fluoxetine treatment significantly decreased the protein levels of p-MEK1/2, p-ERK1/2, and p-p65 in a dose-dependent manner. These results indicate that AL exerts an anti-inflammatory effect via inhibition of the MEK/ERK/NF-kB signaling pathway.

Discussion
AL is a plant flavonoid and quercetin derivative. Previous studies have reported that flavonoids have multiple biological effects, such as antidepressant, anticancer, and anti-inflammatory effects [26][27][28]. Quercetin has a variety of beneficial effects, such as suppression of neuronal apoptosis [29]. A recent report showed that Polygonum aviculare L. extract (PAE) reduced fatigue by suppressing neuroinflammation and the expression of fatigue-related hormones [30]. Avicularin has been reported to reverse multidrug resistance in human gastric cancer through increasing the expression levels of Bax and BOK [20]. Vo et al. indicated that avicularin plays an antiinflammatory role in lipopolysaccharide-stimulated RAW 264.7 macrophages [31]. These results indicate that avicularin has a specific regulatory effect in the inflammatory response and cell growth, suggesting that it affects the development of depression. However, the antidepressant effect of AL is still unclear. The present study demonstrates that AL treatment can ameliorate depressive-like behavior. We also found that AL inhibited inflammation and apoptosis in the hippocampi of CUMS mice. Moreover, AL inhibited activation of the MEK/ ERK/NF-kB signaling pathway in mice with depression induced by CUMS. CUMS-induced animal models are generally considered to be one of the best animal models of depression [32]. CUMS exposure can induce depression-like behavior in animals, such as behavioral despair, anhedonia, and less locomotion [32].
The present study showed that CUMS induced depressivelike behavior, manifested by a decrease of sucrose consumption and increase of immobility time in both FST and TST. AL significantly alleviated the depressive-like behaviors induced by CUMS. These results indicate that AL exerts anti-depressant-like effects.
Increasing evidence has shown that inflammation is the main pathological mechanism of depression [6], and psychological and physical stressors can activate immune and inflammation processes [33]. Numerous reports have shown that the rise of pro-inflammatory cytokines IL-b, IL-6, and TNF-a in some brain areas, such as the hippocampus of depressive mice, is involved in the pathophysiology of depression [7]. In this study, our results showed that CUMS significantly increased the expression of pro-inflammatory cytokines IL-1b, IL-6, and TNF-a in the hippocampi of mice, which is consistent with a previous study [7]. AL treatment significantly decreased expression of CUMS-induced pro-inflammatory cytokines. NF-kB, composed of the p50-p65 heterodimer, regulates the inflammatory response [9]. Under normal conditions, NF-kB presents in the cellular cytoplasm in an inactive state. Once activated by an activator, p65 is phosphorylated to p-p65 and translocates into the nucleus, promoting the expression of pro-inflammatory cytokines [34]. A previous study reported that the Raf-1/MEK/ERK/NF-kB signaling pathway is activated after cerebral ischemia-reperfusion injury [9]. AL can attenuate the TNF-a-induced formation of inflammatory mediators and inhibit activation of the ERK and NF-kB pathways [35]. In the present study, we showed that CUMS induced dramatic activation of the MEK/ERK/NF-kB signaling pathway. AL significantly inhibited activation of the MEK/ERK/NF-kB signaling pathway induced by CUMS.
In summary, our findings show that AL treatment effectively reduced CUMS-induced depressive-like behaviors in mice. Our data also suggest that AL can alleviate the CUMS-induced neuroinflammation and hippocampal cell apoptosis. Furthermore, AL significantly inhibited activation of the MEK/ERK/NF-kB signaling pathway induced by CUMS. The present results provided insight into the antidepressant effect of AL.

Conclusions
Our data show the anti-depressant-like effects of AL on CUMSinduced depression in a mouse model. AL may be a promising agent for the treatment of depression.