1.1.1 Experimental animals
Specific pathogen free (SPF), 6-week old female C57BL/6J mice were purchased from Beijing Huafukang Biotechnology co., LTD. (Beijing, China) and the license batch number of the animals was SCXK (Beijing) 2016-0002. The animal experiments were conducted in strict accordance with the regulations on animal experimentation of Guangdong Ocean University and the experimental procedures were approved by the University’s Ethics Committee on Experimentation with Animals (GDOU-20190724). The experimental animals were raised in Guangdong Ocean University’s Laboratory Animal Center (License No. SYXK 2014-0053). The mice were fed with adequate supply of Co60 irradiation-sterilized feed with the nutrient composition according to the Chinese standard GB 14924.3-2001 and sterile water under controlled environment: temperature 20-25℃, relative humidity 40-70%, pressure gradient 20-50 Pa, one-way flow of fresh air, and 12 h light-dark cycle. Water bottle, cage and pad materials used were sterilised by high-pressure steam. Water bottles and pad materials were changed three times a week.
1.1.2 Materials, chemicals and reagents
Food grade litchi extract powder was purchased from Shenzhen Antai Biotechnology Co., Ltd (Shenzhen, China). Metronidazole (purity ≥ 99%) was from Dalian Meilun Biotechnology co., LTD (Dalian, Liaoning Province, China); neomycin sulfate from Beijing Jintai Hongda Biotechnology co., LTD (Beijing, China); vancomycin (purity ≥ 98%) from Hefei Bomei Biotechnology co., LTD (Hefei, Anhui Province, China); microplate quantitative chromogenic matrix limulus kit from Xiamen Limulus Reagent Biotechnology co., LTD (Xiamen, Fujian Province, China); mouse TNF-α, IFN-γ, IL-1β and IL-6 ELISA kits from Neobioscience Technology co., LTD (Shenzhen, China).
1.2 Treatment of experimental animals
1.2.1 Raising and grouping of human fecal associated mice
The raising of human fecal associated (HFA) mice followed the procedure of Hirayama K, et al (1999) with minor modifications . Briefly, the experimental mice were firstly acclimated for one week under the feeding conditions described above to ensure that they adapted to the feeding environment and reached the same base line of gut microbiota, After acclimation, the mice were gavaged a mixture of antibiotics (vancomycin 400 mg/kg·d-1, neomycin 400mg/kg·d-1, and metronidazole 400 mg/kg·d-1) for 3 days to obtain germ free mice.
Fresh feces were collected from a healthy volunteer (male, 19 years old, without digestive tract or metabolic diseases and had not taken antibiotics in the previous 3 months) for the first bowel movement in the morning. Under the condition of anaerobic asepsis, the mass was measured, and 0.1m PBS buffer was added to dilute the content in a mass/water ratio of 1:9. The mixture was stirred to break up the fecal mass and then votexed for 2 min to obtain a homogenous suspension, which was stood for 10 min, and the supernatant was collected as the human fecal microbial suspension. Twelve of the germ free mice, obtained as described above, were gavaged with 0.3 ml of the fecal suspension once every other day for three weeks to allow the microbiota to colonize the intestinal tract of the mice. The mice obtained were regarded as human fecal associated mice (HFA) .
The 12 HFA mice were randomly divided into four groups with three mice in each group. The first three groups were gavaged with litchi powder solutions at the concentration of 400 mg/kg·d-1, 800 mg/kg·d-1 or 1600 mg/kg·d-1 daily alongside normal feeding for 7 days, and were regarded as low, medium and high dose groups, respectively. The fourth group was gavaged with sterile water instead of litchi solution as control.
1.2.2 Collection of mice blood and fecal samples
Mice blood (about 1 ml) collected by eyeball extirpating was centrifuged at 1200×g and 4℃ for 5 min, the supernatant taken, and the serum samples were stored at -80℃ until use. For collection of fecal sample, mice were massaged in the abdomen, and fresh fecal particles were collected into sterile centrifuge tubes which were immediately covered with ice bath before being stored at -80℃ until use.
1.3 Measurement of inflammatory markers
Mice TNF-α, IFN-γ, IL-1β and IL-6 in serum samples were used as inflammatory markers and were measured by respective ELISA kits according to the manufacturer’s instructions. Concentration of lipopolysaccharides (LPS) was determined by the microplate quantitative chromogenic matrix limulus kit according to manufacturer’s instructions.
1.4 Gut microbiota DNA extraction
Total gut bacterial genomic DNAs were extracted from fecal samples using the PowerMax (stool/soil) DNA isolation kit (MoBio Laboratories, Carlsbad, CA, USA), following the manufacturer’s instructions, and stored at -20°C prior to further analysis. The quantity and quality of extracted DNAs were measured using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and agarose gel electrophoresis, respectively.
1.5 16S rDNA amplicon pyrosequencing
PCR amplification of the bacterial 16S rRNA genes V4 region was performed using the forward primer 515F (5’- GTGCCAGCMGCCGCGGTAA -3’) and the reverse primer 806R (5’- GGACTACHVGGGTWTCTAAT -3’). Sample-specific paired-end 6-bp barcodes were incorporated into the TrueSeq adaptors for multiplex sequencing. The PCR components contained 25 μl of Phusion High-Fidelity PCR Master Mix, 3 μl (10 uM) of each forward and reverse primer, 10 μl of DNA template, 3μl of DMSO, and 6 μl of ddH2O. Thermal cycling consisted of initial denaturation at 98 °C for 30 s, followed by 25 cycles consisting of denaturation at 98 °C for 15 s, annealing at 58 °C for 15 s, and extension at 72 °C for 15 s, with a final extension of 1 min at 72 °C. PCR amplicons were purified with Agencourt AMPure XP Beads (Beckman Coulter, Indianapolis, IN) and quantified using the PicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA). After the individual quantification step, amplicons were pooled in equal amounts, and pair-end 2×150 bp sequencing was performed using the Illlumina NovoSeq6000 platform at GUHE Info technology Co., Ltd (Hangzhou, China).
1.6 Sequence analysis
The Quantitative Insights Into Microbial Ecology (QIIME, v1.9.0) pipeline was employed to process the sequencing data, as previously described . Briefly, raw sequencing reads with exact matches to the barcodes were assigned to respective samples and identified as valid sequences. The low-quality sequences were filtered through the following criteria : sequences that had a length of <150 bp, sequences that had average Phred scores of <20, sequences that contained ambiguous bases, and sequences that contained mononucleotide repeats of >8 bp. Paired-end reads were assembled using Vsearch V2.4.4 (--fastq_mergepairs--fastq_minovlen 5). Operational taxonomic unit (OTU) picking using Vsearch V2.4.4 included Dereplication (--derep_full length), cluster (--cluster_fast,--id 0.97), detection of chimeras (--uchime_ref) . A representative sequence was selected from each OTU using default parameters. OTU taxonomic classification was conducted by VSEARCH searching the representative sequences set against the greengen database.
An OTU table was further generated to record the abundance of each OTU in each sample and the taxonomy of the OTUs. OTUs containing less than 0.001% of total sequences across all samples were discarded. To minimize the difference of sequencing depth across samples, an averaged, rounded rarefied OTU table was generated by averaging 100 evenly resampled OTU subsets under the 90% of the minimum sequencing depth for further analysis.
1.7 Bioinformatics analysis
Sequence data analyses were mainly performed using QIIME and R packages (v3.2.0). OTU-level alpha diversity indices, ACE metric (abundance-based coverage estimator), PD whole_tree, Shannon diversity index, and Simpson index, were calculated using the OTU table in QIIME.
OTU-level ranked abundance curves were generated to compare the richness and evenness of OTUs among samples. Beta diversity analysis was performed to investigate the structural variation of microbial communities across samples using UniFrac distance metrics [17, 18] and visualized via principal coordinate analysis (PCoA), nonmetric multidimensional scaling (NMDS) .
Principal co-ordinates analysis (PCoA) was conducted based on the genus-level compositional profiles . Venn diagram was generated to visualize the shared and unique OTUs among samples or groups using R package “VennDiagram”, based on the occurrence of OTUs across samples/groups regardless of their relative abundance . Taxa abundances at the phylum, class, order, family, genus and species levels were statistically compared among samples or groups by Kruskal test from R stats package. Microbial functions were predicted by PICRUSt (Phylogenetic investigation of communities by reconstruction of unobserved states), based on high-quality sequences . The output file was further analyzed using Statistical Analysis of Metagenomic Profiles (STAMP) software package v2.1.3 . FAPROTAX is a database that maps prokaryotic clades (e.g. genera or species) to established metabolic or other ecologically relevant functions .
1.8 Hematoxylin and eosin (H&E) staining of intestine tissues
At the end of the experimental period, the mice were sacrificed and dissected. The last 5 cm of the intestine was cut off and then sliced longitudely. The specimen was immediately immerged in 10% formalin, and then stained with H&E. Sections of the specimen were examined under a light microscope and assessed for histological damage. At least 3 sections from each animal were examined.
1.9 Statistical analysis
Results were presented as means with standard error of the mean (SEM), analyzed by SPSS 21.0 (SPSS, Chicago, IL, USA). Comparisons between groups were made by one-way analysis of variance (ANOVA) followed by LSD test, with p < 0.05 considered significant.