Male Sprague-Dawley rats (180–220 g) were used in the animal experiments. Animal care was in accordance with the Guidelines for Animal Experimentation of Jiangxi University of Traditional Chinese Medicine (Nanchang, China) and the protocol was approved by the Animal Ethics Committee of the institution. Rats were bred in the Laboratory Animal Science and Technology Center, Jiangxi University of Traditional Chinese Medicine. They were kept in aseptic packing box until the start of experiments. Rats were fed autoclaved chow and water ad libitum under a 12-hour light/12-hour dark cycle (lights on at 7:30 a.m.) and constant temperature (21 °C to 22 °C) and humidity (55 ± 5%) in barrier environment.
Rats were randomly assigned to either a anxiety group or a control group. The chronic unpredictable stress procedure was performed as described previously [17-19] with minor modification as indicated in Table 1. Rats in the control group were kept undisturbed in their home cages, while rats in the CUS group were exposed to various stress conditions for 30 days. Body weight was monitored every 5 days. Fresh fecal samples were collected on day 30. After the completion of the behavioral test, all samples were collected at approximately 10 h, frozen in liquid nitrogen stored at −80 °C. Then, samples were processed for 16S rRNA sequence, untargeted metabolomic, histological, PCR and western blot analyses.
Open-field test (OFT): Rats were placed individually in the corner of an open-field box (L × W × H, 100 cm by 100 cm by 40 cm) and allowed to explore freely for 6 min. Their spontaneous activities over the last 5 min were recorded. The total move distance, average moving speed, entry center frequency, spend time in the center were designated as an index of locomotor activity, while increased time , frequency or distance spent in the center indexes decreased anxiety. After testing each animal, the apparatus was cleaned with ethanol to remove olfactory cues.
Tail suspension test (TST): Rats were individually suspended by their tails using adhesive tape (distance from tip of tail was 2 cm). Test sessions lasted for 6 min with the last 5 min scored for immobility. Rats that climbed on their tails were removed from further testing. Animals were considered to be immobile when they exhibited no body movement and hung passively . Forced swimming test (FST): Individual rats were placed softly into Plexiglas cylinder (30 cm in height×18 cm in diameter) filled with 30 cm water (24 ± 1 °C). Rat’s immobility and struggling behavior during the 5min swim session were recorded and quantified by the investigators who were unaware of group assignment. Struggling was defined as vigorous movements of the forepaws breaking the water and immobility was defined when rat floated without struggling and making only those movements necessary to keep its head above the water .
Elevated plus maze test (EPMT): EPMT was a usual assessment for evaluating the anxiolytic-like effects. The apparatus was 100 cm above the ground including: two closed arms with dark walls (60 × 12 × 40 cm) and two open arms (60 × 12 cm). The arms were connected by the central platform (12 × 12 cm). Rats were placed in the center area facing one of the closed arms, and then allowed to explore the maze for 5 min and defined as entering an arm when four paws crossed the dividing line. Time and entries into the open arms were considered as the anxiolytic indices by observers who were blind to the grouping. After each test, the maze was cleaned with ethanol to remove olfactory cues .
Tube test (TT): Tube test was a very interesting experiment. Two groups of rats were randomly placed on both sides of the test tube. Push-and-shove competition occurs when rats meet in tube. The number of losers was recorded as an anxiety index by the experimenters .
Microbial DNA extraction and PCR amplification
Microbial DNA was extracted from fecal samples using the OMEGA-soil DNA Kit (Omega Bio-Tek, U.S.) according to manufacturer’s protocols. The final DNA concentration and purification were determined by NanoDrop 2000 UV-vis spectrophotometer (Thermo Scientific, Wilmington, USA), and DNA quality was checked by 1% agarose gel electrophoresis. The V3-V4 hypervariable regions of the bacteria 16S rRNA gene were amplified with primers 338F (5’- ACTCCTACGGGAGGCAGCAG-3’) and 806R (5’- GGACTACHVGGGTWTCTAAT-3’) by thermocycler PCR system (GeneAmp 9700, ABI, USA). The PCR reactions were conducted using the following program: 3 min of denaturation at 95 °C, 27 cycles of 30 s at 95 °C, 30s for annealing at 55 °C, and 45 s for elongation at 72 °C, and a final extension at 72 °C for 10 min. PCR reactions were performed in triplicate 20 μL mixture containing 4 μL of 5 × FastPfu Buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of each primer (5 μM), 0.4 μL of FastPfu Polymerase and 10 ng of template DNA. The resulted PCR products were extracted from a 2% agarose gel and further purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) and quantified using QuantiFluor™-ST (Promega, USA) according to the manufacturer’s protocol.
Processing of 16S rRNA gene sequences
Purified amplicons were pooled in equimolar and paired-end sequenced (2 × 300) on an Illumina MiSeq platform. Raw fastq files were demultiplexed, quality-filtered by Trimmomatic and merged by FLASH with the following criteria: (i) The reads were truncated at any site receiving an average quality score < 20 over a 50 bp sliding window. (ii) Primers were exactly matched allowing 2 nucleotide mismatching, and reads containing ambiguous bases were removed. (iii) Sequences whose overlap longer than 10 bp were merged according to their overlap sequence. Operational taxonomic units (OTUs) were clustered with 97% similarity cutoff using UPARSE (version 7.1 http://drive5.com/uparse/) and chimeric sequences were identified and removed using UCHIME. The taxonomy of each 16S rRNA gene sequence was analyzed by RDP Classifier algorithm (http://rdp.cme.msu.edu/) against the Silva (128) 16S rRNA database using confidence threshold of 70%. These taxonomies were used to construct summaries of the taxonomic distributions of OTUs, which can then be applied to calculate the relative abundances of microbiota at different levels. Distance matrices (Beta diversity) between samples were generated on the basis of weighted (Bray-Curtis similarity) reported according to principal coordinate analysis (PCoA).
Untargeted metabolomics and measurement of metabolites
The plasma samples and brain tissue samples were analyzed respectively by a 2.1 ×100 mm ACQUITYTM 1.7 µm C18 and 2.1 ×100 mm ACQUITYTM 1.8 µm HSS T3 using an orbitrap mass spectrometer (LTQ ORBITRAP VELOS PRO, Thermo Fisher Scientific, San Jose, CA, USA) equipped with a heated electrospray ionization (HESI) probe was coupled to the UHPLC system. The metabolomic procedure including sample preparation, metabolite separation and detection, data preprocessing and statistical analysis for metabolite identification was performed following previous protocols with minor modifications [24-26]. The methods of chromatographic separation and mass spectrometry were described in detail as follow: The UPLC analysis was performed with an UltiMate 3000 UHPLC system (Thermo Scientific, San Jose, CA, USA) equipped with a LTQ ORBITRAP VELOS PRO (Thermo Fisher Scientific, San Jose, CA, USA). Chromatographic separation was carried out at 40 °C on an ACQUITYTM UPLC C18 column (2.1×100 mm, 1.7 µm, UK) and an ACQUITYTM UPLC HSS T3 column (2.1×100 mm, 1.8 µm, UK). For plasma sample: the mobile phase consisted of water (A) and methanol (B), each containing 0.1% formic acid. The optimized UPLC elution conditions were: 0~3.0 min, 5.0%~5.0% B; 3.0~7.0 min, 5.0~54.0% B; 7.0~11.0 min, 54.0~77.0% B; 11.0~33.0 min, 77.0~100.0% B; 33.0~38.0 min, 100.0% B; 38.0~39.0 min, 100.0~5.0% B; 39.0~44.0 min, 5.0% B. For brain tissue sample: the mobile phase consisted of water (A) and acetonitrile (B), each containing 0.1% formic acid. The optimized UPLC elution conditions were: 0~3.0 min, 10.0%~60.0% B; 3.0~10.0 min, 60.0~80.0% B; 10.0~16.0 min, 80.0~90.0% B; 16.0~18.0 min, 90.0~100.0% B; 18.0~20.0 min, 100.0% B; 20.0~20.1 min, 100.0~10.0% B; 20.1~23.0 min, 10.0% B, The flow rate was 0.3 ml/min. The autosampler was maintained at 4 °C. Every 2 µl sample solution was injected for each run. Mass spectrometry was performed on a LTQ ORBITRAP VELOS PRO (Thermo Fisher Scientific, San Jose, CA, USA). The scan range was from 100 to 1200 m/z. For HESI source, positive ion mode, Heater Temp, 350 °C; Capillary Temp, 320 °C, Sheath Gas Flow Rate, 35 arb; Aux Gas Flow Rate, 10 arb; I Spray Voltage, 4 kV; Capillary Voltage, 35 V; Tube Lens Voltage, 110 V; sample first full scan, resolution of 30000. Data dependent scan (DDS) was used to select the top three peaks of the first abundance for CID fragment scanning and dynode detection. For HESI source, negative ion mode, Heater Temp, 300 °C; Capillary Temp, 320 °C, Sheath Gas Flow Rate, 35 arb; Aux Gas Flow Rate, 10 arb; I Spray Voltage, 3.6 kV; Capillary Voltage, 35 V; Tube Lens Voltage, 110 V; sample first full scan, resolution of 30000. Data dependent scan (DDS) was used to select the top six peaks of the first abundance for CID fragment scanning and dynode detection. Dynamic exclusion was set to exclude a precursor ion for repeated MS/MS analysis within 15 s. The activation type was collision induced dissociation (CID) and the intensity threshold was set at 1000. All the acquisition and extraction of data were controlled by Thermo Xcalibur Roadmap software and SIEVE × 64 2.1.377 RELEASE. Then, the peak list was imported into SIMCA-P 14.0 software (Umetrics AB, Umea, Sweden) to acquire clustering information and important variables between the CUSP groups and the control group. Metabolites selected as biomarker candidates for further statistical analysis were identified on the basis of variable importance in the projection (VIP) threshold of 3 from the tenfold cross-validated PLS-DA model. The online HMDB database (http://www.hmdb.ca) (version: 4.0), KEGG database (http://www.genome.jp/kegg/) and Lipid maps Structure Database (LMSD) were used to align the molecular mass data (m/z) to identify metabolites. MetaboAnalyst (https://www.metaboanalyst.ca) (version 4.0) was used for the identification of metabolic pathways.
Fecal microbiota transplantation experiments (FMT)
To obtain samples for microbiota transplantation, fecal samples were collected 1 h after 30 days of chronic unpredictable stress. Then on days 1–14, rats were fed freely with water containing antibiotics as Germ-free (GF) and record the weight. Another group of rats was administered normal water daily. Fecal samples from randomly chosen anxiety group and control group were used to colonize the guts of GF rat. The procedures of preparing the fecal samples for microbiota transplantation were as described in a previous study . Fecal samples were freshly collected from adult SPF rats and homogenized in pre-reduced PBS at 2 mL per pellet. 1 mL of the settled suspension was administered by oral gavage to recipient GF rats. For mock treatment, rats were gavaged with pre-reduced PBS. The anxiety and control microflora receptor rats were cultured in a single cage in a sterile environment to prevent the normalization of intestinal flora. The weights of the rats were measured on day 1 and day 15 of FMT experiment. The behavioral tests (including FST and TST) were performed on 15 day after microbiota transplantation. Tissue samples were collected at the time the rats were killed and immediately snap-frozen in liquid nitrogen and stored at −80 °C.
Antibiotic cocktails in drinking water containing, 50 mg/ml streptomycin, 100 mg/ml ampicillin, 50 mg/ml vancomycin, 100 mg/ml metronidazole, 125 mg/ml ciprofloxacin, and 100 mg/ml ceftazidime were freshly prepared every two days and were given ad libitum for 2 week to rats. This protocol was chosen since it was reported not to promote loss of body weight [27, 28].
Protein expression was analyzed by western blot analysis. Protein concentration was measured by PierceTM BCA Protein Assay Kit (23227, Thermo Scientific, USA). Liver tissues were homogenized in RIPA buffer with protease and phosphatase inhibitors. The protein extracts were separated by SDS-PAGE electrophoresis and transferred to a PVDF membrane. The membrane was incubated with antibodies against ADCY5/6 (Affinity, Cat# DF3508), PKA (Affinity, Cat# AF7746), the secondary antibodies of goat anti-rabbit (ZSGB-BIO, Cat# ZB-2301), The membrane was then visualized by enhanced chemiluminescence western blotting detection reagent and protein concentrations were normalized by GAPDH expression. Shooting and analysis of signal intensities from immunoblots using AnalytikJena VisionWorks systems.
Quantitative real-time PCR analysis
Rat liver tissue was frozen in liquid nitrogen and stored at -80 °C. Extraction of total RNA from frozen tissue with Trizol reagent. cDNA was synthesized from 1 μg of total RNA with a Reverse Transcription Kit (TRANS, Cat# AE311). The real-time PCR primer sequences are summarized in Supplementary Table 2.
The following primary antibodies were used: rabbit anti-S1PR2 (Proteintech, Cat# 21180-1-AP, 1:100), rabbit anti-TNF-a (Proteintech, Cat# 17590-1-AP, 1:500). Paraffin-embedded rat brain sections (5-μm thickness) were prepared as a routine procedure. The sections were deparaffinized by three xylene washes, hydrated by alcohol and washed with distilled water. Antigen retrieval was performed by a microwave oven for 15 min in the citrate buffer (10 mM, pH 6.0). The sections were added with 3% hydrogen peroxide to blockade endogenous peroxidase activity. 5% BSA were incubated for 1 h at room temperature. Next, the sections were incubated with the primary antibody at 4 °C overnight. After being washed, the sections were incubated with the secondary antibody (FITC) at 37 °C for 45 min. DAPI were added to visualization of nuclei, then the sections were mounted by antifluorescence quencher. Shooting and analysis of slices using Leica TCS STED CW systems.
Laser Doppler imaging of brain
Laser Doppler images were acquired after CUSP using a laser Doppler imaging system (moorLDI2-HIR, Moor Instruments, DE, UK). During the procedure, the core temperature of the animals was monitored to ensure euthermia.
Culture of fecal samples
Fecal samples of antibiotic and vehicle group were collected on the 14th day. Similarly, FMT group fecal samples were collected on the 30th day. Each group fecal samples (100 mg) were inoculated in 5 ml broth medium and incubated 24 h under strict anaerobic conditions in constant temperature incubator at 37 °C respectively.
The number of repeats per group was provided in the legend and results were presented as mean ± SEM. GraphPad Prism 7 (GraphPad software, San Diego, CA, USA) and R were used for statistical analysis of the data. The significance of the differences between two groups was analysed using unpaired Student’s t test, and multiple comparisons was performed by one-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test. All tests were two-tailed. Significant differences emerging from the above tests are indicated in the figures by *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Notable non-significant (and non-near significant) differences are indicated in the figures by “n.s”. For all behavioral analyses, experimenters were blind to group assignment.