4.1 Ethical approval
All experiments were approved by the Animal Experimental Ethical Committee of the Traditional Chinese Medicine Hospital of Zhongshan (no. 2018007). All related procedures were performed in accordance with the recommendations of the National Institutes of Health Guide for the Care and Use of Laboratory Animals [National Research Council, Guide for the Care and Use of Laboratory Animals. (2011)].
4.2 Experiment of high humidity acclimation and raising ambient temperature
Male BALB/c mice (six weeks of age) were obtained from and maintained under specific pathogen-free (SPF) conditions at the Laboratory Animal Research Center of Traditional Chinese Medicine Hospital (Zhongshan City, Guangdong Province, China). All study animals were housed in sterile cages in a room maintained at 20 ± 1 ℃, with an average humidity of 60 ± 1 % and a 12/12-h light:dark cycle. The mice were given access to water and chow ad libitum. Following two weeks of acclimation, 150 SPF mice were randomly assigned to two groups (30 per group). Mice in the normal control (NTC) group were maintained as described above, whereas mice in the high temperature and humidity (HTH) groups were regularly transferred into sterilized climate chambers (model: RXZ-158A, Ningbo Jiangnan Instrument Factory, China) located in the same room. The climate chambers were set to temperatures of 32 ± 1 ℃ and humidity levels of 85-90% for 12 h per day over a period of eight consecutive weeks. Food and drinking water were sterilized and renewed daily to avoid contamination.
4.3 Blood biochemical analysis and enzyme-linked immune-sorbent assay (ELISA)
At predetermined time points, the mice were fasted for approximately 16 h and then euthanized via cervical dislocation. Next, EDTA-treated whole blood samples were collected. Plasma was separated and immediately subjected to biochemical analysis using a Hitachi 7080 biochemical analyzer (Japan). Aliquots of plasma were stored at -80 °C until use.
Mouse insulin, glucagon, GLP-1, GIP, ghrelin, and PYY ELISA kits were purchased from AndyGene Biotechnology Co. LTD (Beijing, China). These parameters were used to quantify the respective levels in the plasma in accordance with the manufacturer’s instructions. A Varioskan LUX microplate reader (Thermo Fisher, USA) was used for the assay, and a 4-parameter logistic regression was used for data analysis. Two replicates were performed for each sample. The results were then averaged.
4.4 TMT-Based Quantitative Proteomic Analysis
4.4.1 Protein extraction, digestion, and TMT labeling
Proteins were extracted from minced ileal samples with an ice-cold SDT (4% SDS, 100mM Tris/HCl pH7.6, 0.1M DTT) lysis buffer according to a published protocol [33]. Total protein concentrations were determined using a bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL, USA). An appropriate amount of protein from each sample was digested with trypsin using the filter-aided proteome preparation (FASP) method. Peptide concentrations were measured at OD280. Following trypsin digestion, the peptides were desalted using a Strata X C18 SPE column (Phenomenex), and 100 μg of each peptide was processed according to the manufacturer’s protocol of the TMT labeling kit (Thermo Fisher Scientific, Torrance, CA, USA).
4.4.2 HPLC fractionation
The pooled samples were then fractionated via high-pH reverse-phase HPLC using an Agilent 300Extend C18 column (5 μm particle size, 4.6 mm ID, 250 mm length). Briefly, peptides were first separated with a gradient of acetonitrile (8% to 32%) in 10 mM ammonium bicarbonate (pH 9.0) over 60 min into 60 fractions. The peptides were then combined into 18 fractions and dried by vacuum centrifugation.
4.4.3 LC-MS/MS analysis
Peptides from each fraction were dissolved in 0.1% formic acid, directly loaded onto a reversed-phase analytical column (Thermo Scientific EASY-Column, 10 cm, ID 75 μm, 3 μm, C18-A2) using an EASY-nLC 1000 UPLC system (Thermo Fisher Scientific, San Jose, USA). The gradient was comprised of an increase from 7% to 25% solvent B (0.1% FA in 90% acetonitrile) over 26 min, another increase 25% to 40% in 8 min, a further increase to 80% in 3 min, and then holding at 80% for the last 3 min at a constant flow rate of 300 nL/min. The peptides were subsequently analyzed via tandem mass spectrometry (MS/MS) using a Q Exactive™ Plus (Thermo Fisher Scientific). The applied electrospray voltage was 2.0 kV. Intact peptides were detected in the orbitrap at a resolution of 70,000 and ion fragments were detected in the orbitrap at a resolution of 17,500. In the MS survey scan, a data-dependent mode with automatic alteration (1 MS scan followed by 20 MS/MS scans) was used for the top 20 precursor ions above a threshold ion count of 5 × 104 with 30s exclusion. Automatic gain control (AGC) was used to prevent overfilling of the orbitrap; 5 × 104 ions were accumulated for generating MS/MS spectra. For MS scans, the m/z scan was 350–1800 and the fixed first mass was set at 100 m/z.
4.4.4. Data analysis
The resulting MS/MS data were processed using Mascot 2.2 and Proteome Discoverer 1.4. The mass tolerance for precursor ions was set to 20 ppm in the first search and 5 ppm in the main search, and that for fragment ions was set to 0.02 Da. The false discovery rate (FDR) was adjusted to < 1% at the protein, peptide, and PSM (peptide spectrum match) levels, and the minimum score for peptides was set > 40. Only unique peptides were used for the protein quantification.
4.4.5. Bioinformatics analysis
Gene Ontology (GO) annotation was derived from the UniProt-GOA database (http://www.ebi.ac.uk/GOA/). First, the identified protein ID was converted to a UniProt ID and then mapped to the GO ID. If some identified proteins were not annotated by the UniProt-GOA database, InterProScan software (a sequence analysis application) was used to annotate the protein’s GO function based on the protein sequence alignment method. The DEPs were then classified by GO annotation based on three categories (GO term level 1): biological process, cellular component, and molecular function. The Clusters of Orthologous Groups (COG) of the protein database were used for the functional classification of DEPs. The protein-protein interaction networks and pathways were annotated using the STRING (https://string-db.org/) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases (http://www.kegg.jp/), respectively. A p-value ≤0.05 was used as the threshold to determine the significant enrichments of the GO annotation and KEGG pathways.
4.5 16S rRNA gene sequence analysis
4.5.1 Fecal collection
Fecal samples were harvested from each mouse at predesigned times (weeks 1, 2, 4, 6, and 8) through abdominal pressing and collected in sterilized tubes. Samples were immediately frozen and maintained at -80℃ until processing for bacterial DNA isolation and extraction, which was performed in accordance with previously reported methods [34]. Briefly, the bead-beating method was used to collect DNA, which was dissolved in a Tris-EDTA (TE) buffer after extraction using phenol and chloroform.
4.5.2 Generation of amplicon library and 16S rRNA sequencing
A random selection of ten fecal samples per group was performed for 16S rRNA sequencing. Gene amplification, cloning, and sequencing of the bacterial 16S rRNA PCR products were conducted in a laboratory maintained by BGI-ShenZhen (Beijing Genomic Institute, Shenzhen Huada Gene Institute, China). PCR amplification of the V5-V4 regions of the bacterial 16S rRNA gene [35; 36] was conducted usin653g universal primers (515F 5′-GTGCCAGCMGCCGCGGTAA-3′ and 806R:GGACTACHVGGGTWTCTAAT; 907R 5′-CCGTCAATTCMTTTRAGT-3′), which incorporate unique sample barcode sequences. To generate an amplicon library, 20 ng of each genomic DNA sample, 1.25 U of Taq DNA polymerase, 5 μl of 10× Ex Taq buffer, 10 mM dNTPs (all reagents from TaKaRa Biotechnology Co., Ltd, Dalian, China), and 40 pmol of primer mix were added to a 50 μl reaction mixture. The PCR conditions were as follows: a 5 min initial denaturation at 95 ℃, 28 cycles of denaturation at 95 ℃ (30 s), annealing at 55 ℃ (30 s), and elongation at 72 ℃ (45 s), followed by a final extension at 72 ℃ for seven min. The PCR products were purified using magnetic beads (Axygen Biosciences, Union City, CA, USA), and the concentration of the amplicon library was estimated using a 2100 Bioanalyzer System (Agilent Technologies Inc., Waldbronn, Germany). Equal amounts of amplicons from each sample were pooled. Sequencing data were retrieved from the European Bioinformatics Institute (accession number: ERP120131).
4.5.3 Analysis of sequencing data
Raw data were treated using an in-house pipeline developed based on Mothur v.1.31.2 [37]. The primers were removed, low-quality sequences (average quality scores of the 30 bp window < 20) were truncated, and all high-quality reads from individual samples (lengths > 250 bp) were pooled. Thereafter, operational taxonomic units (OTUs) were clustered with a 97% identity cutoff using USEARCH (v7.0.1090) [38]. The relative abundances of the OTUs were then calculated and those lower than 0.001% were removed. This was followed by an analysis of the OTU distribution in each sample. Both weighted and unweighted UniFrac distance analyses were performed based on OTU abundances and the phylogenetic tree, and a principal component analysis (PCA) was conducted based on the OTU abundance profiles obtained using customized R (3.0.2) scripts. Based on the OTU abundances and taxonomic annotations, we obtained relative abundance profiles at the phylum, class, order, family, genus, and species levels [39]. Alpha diversity, beta diversity, and rarefaction curve analyses were conducted based on the relative OTU abundance table. The Kruskal-Wallis test was conducted to explore the enrichment of bacterial species and functions in different subgroups. The FDR was used to control type I errors and enable the identification of the most reliable candidates for multiple group comparisons. We also used PICRUSt to perform functional classification of the Kyoto Encyclopedia of Genes and Genomes (KEGG) Orthology (KOs) and Clusters of Orthologous Groups (COGs) [40].
4.6 Causal Mediation analysis
Mediation analysis was conducted to identify gut bacteria through which environmental HTH might affect bio-clinical parameters using the “mediation” package v4.5.0. The abundances of the gut microbiota at the genus level were applied separately as “mediators”. To obtain confidence intervals and statistical significance of the mediated and direct effects, 1000 Monte Carlo simulations were employed for a quasi-Bayesian approximation.
4.7 Bile acid detection.
Liver samples (30 mg) were homogenized with 200 μl pre-cooled ultrapure water, 800 μL pre-cooled methanol, and 10 μL internal standard, then mixed thoroughly by vortex, followed by incubation at -20 °C for 20 min to allow the protein precipitation. Then, the samples were centrifuged at 14,000 rcf for 15 min at 4 °C, and the supernatant was collected and dried under vacuum. Methanol–water (100 μL, 1:1, v/v) was added for reconstitution.
The standard was diluted to a series of gradient concentration standard working solutions with methanol aqueous solution, the standard curve solution was prepared according to the above method, and a standard curve was established using the isotope internal standard method.
The samples were analyzed using an Acquity UPLC system (Waters Ltd.) coupled online to a 5500 QTRAP mass spectrometer (AB SCIEX, USA). The samples (2 μl) were injected onto an ACQUITY UPLC BEH C18 1.7 μm, 2.1 mm × 100 mm) column (Waters Ltd.). The samples were eluted at a flow rate of 250 μL/min with phases A (0.1% formic acid in water) and B (methanol). The separation was performed as follows: linear gradient from 60 to 85% B (0–15 min), isocratic at 85% B (15–17 min), linear gradient from 85 to 60% B (17–17.1 min), and isocratic at 60% B (17.1–20 min). The column temperature was 45 °C.
A QC sample was used for each set of experimental samples in the sample queue to test and evaluate the stability and repeatability of the system. The samples were separated using a Waters UPLC system. Mobile phase: 0.1% FA aqueous solution was used in phase A, with methanol in phase B.
The sample was placed in an 8 °C autosampler with a column temperature of 45 °C, a flow rate of 250 μL/min, and an injection volume of 2 μL. The relevant liquid phase gradient is as follows: 0–7 min, phase B linearly changed from 60 to 70%; 7–15 min, and phase B linearly changed from 70 to 85%. At 15–17 min, phase B was maintained at 85%, then from 17–17.1 min, the phase changed linearly from 85 to 60%. From 17.1–20 min, phase B was maintained at 60%. A QC sample was used for each set of experimental samples in the sample queue to test and evaluate the stability and repeatability of the system. Mass spectrometry was performed as follows: source temperature: 550 °C, ion source Gas1 (Gas1): 55, Ion Source Gas2 (Gas2): 55, curtain gas (CUR): 40, and ionSapary Voltage Floating (ISVF): − 4500 V. The MRM mode was used to detect the transitions to be measured.
The raw LC–MS/MS data were analyzed using Multiquant software to obtain the calibration equations and quantitative concentrations of each BA in the samples. Differences in BA measurements between the groups were analyzed using Student’s t-test, with p < 0.05 considered significant. The regression equation is as follows:
derived from the standard curve, the relative deviation of the sample’s lower limit of quantification (LLOQ), was ≤ 20%, the relative deviation of the other concentrations and quality control relative standard deviation (QC) RSD) was ≤ 30%, and the square of the correlation coefficient R was > 0.99. The contents of each test sample are as follows:
calculated from the standard curve.
Spearman’s correlation analysis was used to analyze the correlation between fecal microflora and metabolomics. Using R software (version 3.3.1) to generate graphics, we obtained results with p < 0.05, representing statistical significance.
4.8 Experiment involving fecal microbiota transplantation (FMT)
At week 4, fecal samples were collected from the mice in each group for FMT preparation. The stool samples of all animals from the same group were mixed in equal proportions. Mixed fecal samples (1 g) were suspended in 10 mL sterile PBS via vortexing for 5 min and then settled via gravity for 5 min. The clarified supernatant was transferred to a clean tube and an equal volume of 20% (w/v) skim milk (LP0031, Oxoid, UK) was added. The inoculum was freshly prepared on the day of the experiment, and the remainder was stored at -80 °C until the second inoculation. All experimental animal procedures were approved by the Institute of Zoology Institutional Animal Care and Use Committee of the Chinese Academy of Sciences. All related procedures were conducted according to the committee guidelines. Weaned and germ-free male Balb/c mice (n = 20) were maintained in flexible-film plastic isolators under a regular 12 h light cycle (lights on at 06:00). The mice were fed a sterilized normal chow diet (10% energy from fat, 3.25 kcal/g, SLAC). Surveillance for bacterial contamination was carried out through periodic bacteriological examinations of the feces, food, and padding. Samples of feces, food, water, and padding were also collected before transplantation. At six weeks of age, germ-free mice were housed in individual cages and randomly divided into four groups (each group was kept in an individual isolator). After two weeks of acclimatization, mice in all three groups (NTC-FMT, HH-FMT, and HTH-FMT) received oral gavages of 200 µL fecal suspension inoculum, whereas the four mice in the blank group received a vehicle. Inoculations were repeated three times every alternate day to reinforce microbiota transplantation. All the mice were euthanized on day 14 after 16 h of fasting. Fecal and blood samples were collected and processed as described above.
4.9 Statistical analysis
The results are presented as mean ± SD values. Multiple group comparisons were assessed through one-way analysis of variance (ANOVA) with the least significant difference (LSD) or Kruskal-Wallis multi-comparisons test using R software (version 3.4.3). Statistical significance was set at P ≥ 0.05.