Asiaticoside (purity >98%) was purchased from Nanjing Zelang Medical Technology Co., Ltd. Fluoxetine (FLX) hydrochloride was purchased from Changzhou Siyao Pharmaceuticals Co., Ltd. All other chemicals and reagents used were of analytical grade.

In total, 60 male ICR mice (age, 2 months old; weight, 18–22 g) were purchased from The Experimental Animal Center in Jiangsu Province. Animals were randomly divided into five treatment groups (n=12 mice/group) and housed in cages at room temperature (22±2°C) under a 12-h light/dark cycle (lights on at 8:00 a.m.), with ad libitum access to food and water. The mice were allowed to acclimate for 1 week before the experiment commenced. The groups were as follows: i) Normal control group; ii) CMS model group; iii) CMS + FLX (20 mg/kg, i.g) group; iv) CMS + asiaticoside (20 mg/kg, i.g) group; and v) CMS + asiaticoside (40 mg/kg, i.g) group. Asiaticoside and FLX were prepared in double-distilled water. Mice were exposed to the CMS stimuli for 4 weeks (weeks 1–4), followed by 4 weeks (weeks 5–8) of asiaticoside or FLX treatment during which CMS stimulation continued. For the control and CMS model groups, mice were given an equal volume of double-distilled water. Asiaticoside doses were selected based on a previous study (28). All experiments were conducted as per the Guidelines of the Institutional Animal Care and Use Committee of China and the present study was approved by Experimental Animal Ethics Committee of Xuzhou Medical University (Lianyungang, China).

Behavioral tests were performed after the last administration of asiaticoside or FLX (Fig. 1). CMS was introduced as previously described (17,30) with minor modifications (the order of stressors was random). The normal control group animals were left undisturbed in their cages in a separate room throughout the 8 weeks of treatment. The other four groups were individually housed and subjected to a variety of stresses for 8 weeks: i) Soiled cage (200 ml water in 100 g sawdust bedding; ii) foreign object exposure; iii) light/dark perversion; iv) physical restraint for 2 h; v) food deprivation for 24 h; vi) water deprivation for 24 h; vii) overnight illumination; viii) cage tilt (45°) for 10 h; ix) white noise; x) 3-min oscillation; and xi) 1-min tail pinch (1 cm from the root of the tail). All the stressors were applied randomly to ensure the unpredictability of the experiment.

Sucrose preference test was conducted every week during the experimental period (17). Mice were first deprived of water and food for 10 h before the test, and were then allowed to freely choose between two bottles (one with tap water, and another with 1% sucrose solution) for 10 h. To prevent possible effects of side preference on drinking behavior, the positions of the bottles were switched after 5 h. The consumption of the sucrose solution and tap water was estimated by weighing the bottles. Sucrose preference was calculated as sucrose preference (%) = sucrose intake (g)/[sucrose intake (g) + water intake (g)] ×100.

TST was performed as described previously (17). Individual mice were acoustically and visually isolated, and suspended ~50 cm above the floor by placing adhesive tape ~1 cm from the tail tip. Mice were considered immobile when they were passively suspended and remained completely motionless. Each animal was suspended for 6 min, and total immobility was recorded during the last 4 min of the test.

FST was carried out as described previously on the day after TST (17). Mice were forced to swim in a cylinder (20 cm height × 14 cm diameter) containing fresh water (25±1°C) to a height of 10 cm. The immobility time was recorded as the time the mice spent floating in the water without struggling or only making movements necessary to keep their heads above the water. Each animal was forced to swim for 6 min, and total immobility time was recorded during the last 4 min of the test.

Mice were sacrificed after the final behavioral tests were concluded. Ketamine and xylazine (100 and 10 mg/kg, respectively) were intraperitoneally injected to anesthetize the mice and the mice were then sacrificed by decapitation. The whole hippocampus was rapidly dissected on an iced-plate and weighed. The hippocampus was identified as described in Paxinos and Watsons Atlas (30).

The levels of 5-HT and NE in the mouse hippocampus were determined by high-performance liquid chromatography (HPLC). The hippocampus was homogenized in extract solution, which consisted of 0.1 mM EDTA and 0.1 M HClO₄ buffer, and the mixture was centrifuged at a speed of 20,000 × g for 30 min at 4°C. Then, 50 µl of the resulting supernatant was injected into the liquid chromatography system equipped with a reversed phase C18 column (2.2 µm, 120 Å, 2.1×100 mm; Dionex; Thermo Fisher Scientific, Inc.). The mobile phase was composed of solutions A (0.1% formic acid, v/v) and B (acetonitrile), with a gradient elution as follows: 0–6 min, 90–65% A; 6–8 min, 65–0% A; 8–10 min, 0% A. The flow rate was maintained at 0.4 ml/min, and the column temperatures were maintained at 30°C, and was detected by ESA Coulochem^® III Electrochemical Detector (Dionex; Thermo Fisher Scientific, Inc.). The detector was set at 350 mV. The identification and purity were evaluated by the chromatographic peaks as well as their quantitative evaluation, which was measured by comparing their retention times and peak areas with those of standard solutions. 5-HT (cat. no. H9523) and NE (cat. no. N-069-1ML) standards were purchased from Sigma-Aldrich (Merck KGaA).

Cytokine and cAMP concentrations in the mouse hippocampus were measured by ELISA, according to the manufacturers instructions. The following kits were used: Mouse IL-1β ELISA kit (cat. no. 1210122); mouse IL-6 ELISA kit (cat. no. 1210602); mouse TNF-α ELISA kit (cat. no. 1217202; all purchased from Dakewei Biotechnology Co., Ltd.); and mouse cyclic adenosine monophosphate, cAMP GENLISA™ ELISA kit (cat. no. KLM0071; Krishgen BioSystems). The concentrations were measured using a microplate reader (450 nm absorbance). The results were reported in picograms per milliliter (pg/ml).

Protein extracts were obtained by homogenizing hippocampal tissue in lysis buffer [50 mM Tris HCl (pH 7.2) containing 1% sodium deoxycholate, 1% NP-40, 0.15 mM NaCl and 0.1% SDS; Roche Applied Science). A bicinchoninic acid assay was used to determine protein concentrations (Sigma-Aldrich; Merck KGaA). Equal amounts of protein (30 µg/µl) were separated by SDS-PAGE on 10% gels and were then transferred to PVDF membranes (0.2 µm; EMD Millipore). The membranes were blocked for 2 h with 5% non-fat dry milk at room temperature, then incubated at 4°C overnight with the following antibodies: Rabbit anti-phosphorylated (p)NF-κBp65^Ser536 (1:1,000; cat. no. orb501609; Biorbyt Ltd.), rabbit anti-NF-κBp65 (1:1,000; cat. no. orb453023; Biorbyt Ltd.), rabbit anti-NLRP3 (1:1,000; cat. no. orb101128; Biorbyt Ltd.), rabbit anti-caspase-1 (1:1,000; cat. no. ab179515; Abcam), rabbit anti-PKA (1:1,000; cat. no. ab75991; Abcam), rabbit anti-p-VASP^ser157 (1:1,000; cat. no. ab47268; Abcam) and rabbit anti-VASP (1:1,000; cat. no. ab205952; Abcam), rabbit anti-pCREB^Ser133 (1:1,000; cat. no. ab32096; Abcam), rabbit anti-CREB (1:1,000; cat. no. ab32515; Abcam), rabbit anti-BDNF (1:1,000; cat. no. ab108319; Abcam) and rabbit anti-β-actin (1:1,000; cat. no. ab8227; Abcam). After three washes in TBS-Triton X-100 (1%) buffer, the membranes were incubated for 1 h at 24°C, with horseradish peroxidase-labeled anti-rabbit IgG (1:5,000; cat. no. BS13271; Bioworld Technology, Inc.). Densitometric measurements were performed using the ECL Western detection system (EMD Millipore) and the Quantity One imaging program (v7.1; Bio-Rad Laboratories, Inc.).

Experiments were repeated three times. Data are presented as the mean ± SEM. Statistical significance was assessed by one-way ANOVA followed by Tukeys test. All statistical analyses were performed using GraphPad Prism 8 software (GraphPad Software, Inc.). P<0.05 was considered to indicate a statistically significant difference.

Sucrose preference in mice is considered a measure of anhedonia (17). It was revealed that the CMS model mice exhibited significantly reduced sucrose consumption compared with the control group (P<0.01; Fig. 2A). Moreover, asiaticoside (20 and 40 mg/kg) or FLX (10 mg/kg) administration significantly reversed the decreased sucrose consumption in the CMS model group (P<0.05, P<0.01, respectively). It was also demonstrated that CMS significantly increased the immobility time during TST (P<0.001; Fig. 2B) and FST (P<0.01; Fig. 2C). Asiaticoside (20 and 40 mg/kg) or FLX (10 mg/kg) treatment significantly reversed the increase in the immobility time in both TST (P<0.01, P<0.001, respectively; Fig. 2B) and FST (P<0.01; Fig. 2C).

The present study also examined the levels of neurotransmitters in the hippocampus. Fig. 3 shows the effect of asiaticoside on the hippocampal levels of 5-HT and NE. The present results suggested that CMS significantly decreased the levels of 5-HT and NE in the hippocampus (P<0.01), whereas asiaticoside or FLX administration significantly alleviated the levels of both 5-HT (P<0.01; Fig. 3A) and NE (P<0.01; Fig. 3B) in the hippocampus of CMS model mice.

The anti-inflammatory effect of asiaticoside in CMS mice was identified in the present study (Fig. 4). It was found that treatment with asiaticoside (20 and 40 mg/kg) or FLX (10 mg/kg) significantly decreased the levels of IL-1β in CMS mice (P<0.01, P<0.001, respectively; Fig. 4A). In the hippocampus of the CMS mice, IL-6 and TNF-α levels were significantly higher compared with the control group mice (P<0.01, P<0.001, respectively). Asiaticoside (20 and 40 mg/kg) or FLX (10 mg/kg) administration significantly attenuated the increase in IL-6 (P<0.05, P<0.01, respectively; Fig. 4B) and TNF-α (P<0.01, P<0.001; respectively; Fig. 4C) levels in these mice.

The present study measured the protein expression levels of pNF-κBp65 and NLRP3 inflammasome complex in the mice hippocampus. It was identified that the protein expression of pNF-κBp65 was significantly elevated (P<0.01) in the CMS mice (Fig. 5A). Furthermore, asiaticoside (20 and 40 mg/kg) or FLX administration significantly reduced the expression of pNF-κBp65 (P<0.05, P<0.01, respectively) in these mice. In addition, the protein expression levels of NLRP3 and mature caspase-1 (Fig. 5B) were significantly increased in the hippocampus of CMS mice (P<0.01). The present results also indicated that asiaticoside (20 and 40 mg/kg) or FLX (10 mg/kg) treatment significantly reduced the expression levels of NLRP3 and mature caspase-1 in mice (P<0.05, P<0.01, respectively).

The protein expression levels of cAMP, PKA and pVASP^ser157 were significantly decreased in the CMS mice (Fig. 6) compared with the control group (P<0.01). Moreover, treatment with asiaticoside (20 and 40 mg/kg) or FLX (10 mg/kg) reversed the CMS-induced decrease in the expression levels of cAMP (P<0.01; Fig. 6A), PKA (P<0.05, P<0.01, respectively; Fig. 6B) and pVASP^ser157 (P<0.05, P<0.01, respectively; Fig. 6C). Therefore, the present results suggested that cAMP/PKA signaling may be involved in asiaticoside-mediated inhibition of neuroinflammation.

It has been reported that the antidepressant-like effect of asiaticoside is mediated via the activation of BDNF signaling (29). However, the mechanism underlying asiaticoside-mediated alterations in BDNF levels is yet to be elucidated. cAMP/PKA activation has been reported to increase CREB phosphorylation and induce the expression of CRE-regulated genes, such as BDNF (24). Therefore, the present study examined the protein expression levels of pCREB^Ser133 and BDNF in CMS mice (Fig. 7). It was found that the ratio of pCREB^Ser133 to CREB was significantly decreased in the hippocampus of CMS mice (P<0.01), which was reversed by asiaticoside or FLX treatments (P<0.05, P<0.01, respectively; Fig. 7A). Furthermore, BDNF expression was significantly lower in the hippocampus of the CMS mice compared with the control mice (P<0.01). However, this effect was completely reversed upon asiaticoside or FLX treatment (P<0.01, P<0.05, respectively; Fig. 7B), thus supporting the role of asiaticoside in activating the cAMP/PKA signaling pathway.