Fungal identification by PCR assay
The Reishi mushroom strain 022 (Fig. 1A) used in this study was maintained at the Taiwan Agricultural Research Institute (TARI), Taichung, Taiwan. The mycelia were subcultured on potato dextrose agar (PDA, Difco, Maryland USA) at 24° C without light. To identify the Reishi species, 100 mg of 2-week-old dried mycelial mass of the fungus was homogenized in liquid nitrogen. The total of genomic DNA of the fungus was extracted by using plant gDNA kit (GPG1002, Viogene), and stored in 50 µl elution solution until being used. The universal primer pairs ITS-A (5’-GGA AGG AGA AGT CGT AAC AAG G-3’) and ITS-B (5’- CTT TTC CTC CGC TTA TTG ATA TG-3’) were used to amplify fungal gDNA for the region containing ITS1, 5.8S, and ITS2 rDNA [12]. The PCR assay was performed with 1 ul of DNA template in a total reaction volume of 50 µl. The PCR reaction was performed in an ABI 2720 Thermal Cycler (Applied Biosystems) by Faith BioTechnology Co., Ltd. Taiwan, with the following program: hot start at 95° C for 5 min, followed by 40 cycles at 95° C for 1 min, 55° C for 1 min and 72° C for 1 min, and incubated at 72° C for 7 min for final extension. The PCR-amplified products were then sequenced by Genomics BioTechnology Co., Ltd. Taiwan with the ABI 3730 XL DNA Analyzer (Applied Biosystems). Phylogenetic analysis of the aligned sequences was then performed by Neighbor-Joining phylogenetic tree method with MEGA software (2.0).
Preparation Of Extract Of The G. Lucidum
The fruiting bodies of G. lucidum cultivated on sterilized sawdust were harvested by the Mushroom Laboratory of TARI (Fig. 1B). After oven-drying, 2 kg of the dried mushroom samples were milled and submitted to extracting equipment (Chin Ying Fa Mechanical Ind. Co., Ltd. Taiwan) at 90∘C for 60min (40x 50% ethanol). The supernatants were filtered through Whatman No. 1 filter paper, and the residue was then continually extracted with 50% ethanol as described above. The filtered extracts were combined, mixed well and evaporated to a small volume, then lyophilized. The final yield ratio of Gl-E was 10 percent. The dry extract (Gl-E) was stored frozen at − 20° C until being used.
Animals And Experimental Conditions
A total of 98 male Sprague-Dawley rats (6 ~ 8 weeks) obtained from the BioLASCO Taiwan Co., Ltd. were kept in the animal center of China Medical University (Taichung, Taiwan). All rats were housed in groups (four rats per cage) on a 12/12-hr light/dark cycle with ad libitum access to food and water except during behavioral tests. The project was approved by the Institutional Animal Care and Use Committee of China Medical University (IACUC 2016 − 273). All animal experiments were performed according to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85 − 23, revised 1996).
Unpredictable Chronic Mild Stress (Ucms) Procedure
The UCMS-induced depressive animal model was used to screen new antidepressants through a series of behavioral tests. Weight 160 ~ 180 g male Sprague-Dawley (SD) rats were used in this experiment. The trial began at the age of 4 weeks and went through a duration of 6 to 8 chronic stress recommendations per week. Experimental rats were randomly exposed to unpredictable mild stressors every day in per week under one of the following stressors: physical restraint (1h), 1 min tail pinch (2.5 cm from the end of the tail), reversed light/dark cycle (24 h), overnight illumination (12 h), soiled cage (12h), and cage tilt (18 h, 45°). Each stressor was randomly assigned two or three times over the experimental period. The non-stressed control rats were normally housed in groups (four rats per cage) in the other room, and the stressed rats were singly housed. At least 12 h of rest was provided between a stressor and a test to avoid effects of acute stress [13].
Experimental Design
To investigate whether the depressive-like effect of Gl-E, rats were randomly divided into six groups as follows: control group, UCMS-vehicle group, UCMS + fluoxetine (0.02 g/kg) group, and UCMS + three doses of Gl-E groups (0.02 g/kg, 0.1 g/kg, and 0.5 g/kg).
Sucrose Preference Test (Spt)
The procedure was composed of training and testing parts. In the training course, the rats were exposed to two bottles filling with 1% (w/v) sucrose; after 24 h, the rats were exposed to two bottles filling with 1% sucrose and tap water, respectively, for another 24 h. After the rats were deprived of food and water for 24 h, a 3-h SPT was performed, in which the rats could freely choose between a bottle with 1% (w/v) sucrose and a bottle with tap water. Then the sucrose preference was evaluated according to the following formula: sucrose preference (%)=sucrose intake (g) / [sucrose intake (g)་water (g)] × 100% [14, 15].
Open Field Test (Oft)
The rat was moved to the animal room at least 1 hour before starting behavioral experiments. During the experiment, the rats were placed in the middle of a black acrylic empty box (90 x 90 x 40 cm, L x W x H). A video camera was set up above the box, video recording for 5 minutes. After the end of each rat behavioral experiment, 75% alcohol solution was sprayed and the observation box was wiped for conducting the next set of animal behavior experiments [16, 17]. All assays were conducted in eight replicates for each experiment.
Measurement Of Monoamines And Their Metabolites
After oral administration (treatment) of Gl-E for 28 days, rats were conducted the UCMS test. At the end of the experimental period, all rats were killed immediately by decapitation. Rat’s brains, divided into cortex, hippocampus, striatum and amygdala area, respectively, were added ice-cold homogenizing solution (8.8 mg of ascorbic acid and 122 mg of EDTA in 1000 ml of per chloric acid 0.1 M), and homogenized with ultrasound at 12,000 rpm. After centrifugation at 4 ° C for 30 minutes, the supernatant was taken and stored in a freezer at -80°C.
Preparation of standard stock solutions: 5 mg of NE, MHPG, 5-HT, 5-HIAA, DA and DOPAC was put in a brown bottle respectively, added by 12.5 mL LC grade pure water, and mixed well; the 25 µL of the above solution with 1.25 mL LC grade pure water was used as the standard solution.
Mobile phase preparation: Weighed 20 g of NaH2PO4 H2O, 185 mg of EDTA and 130 mg of Sodium S-1-octane sulfonate (SOS) into 1 L of LC grade pure water and calibrated to pH = 3 with H3PO4. Added 8 mL of the solution to 140 mL of methanol (14%). Nylon membrane filter with 0.45µm was used for suction filtration.
Determination of monoamine concentration: The monoamines levels (NE, DA, 5-HT) and their metabolites (MHPG, DOPAC, 5-HIAA) of samples (brain cortex, hippocampus, striatum and hypothalamus) or standard solution were detected by using LiChrospher® 100 RP-18 Endcapped HPLC Columns (4 × 125 mm; Merck Millipore, Burlington, MA, USA) and electrochemical detector (electrochemical detector (oxidation potential of + 0.75 V). The concentrations of monoamines were determined by using Shimadzu CLASS-VPTM Version 6.1 record analysis [18].
Statistical analysis
All data are expressed as mean ± SEM. Data were analyzed by one-way ANOVA followed by Scheffe’s multiple range test. The criterion for statistical significance was p < 0.05. All statistical analyses were carried out by using SPSS for Windows (SPSS Inc.).