Materials
Non-heat edible oil (fresh canola oil, NEO) was purchased from a local supermarket. Deep-fried edible oil (canola oil following deep-frying process, DFEO) was prepared as described previously [37]. In Brief, fresh canola oil was heated at 190 ± 5 °C for 4 intermittent days (8 h each day) for a total of 32 h. Fresh canola oil (7 L) was poured into an iron saucepan with a bore of 45 cm and a depth of 20 cm, and 100 g of chicken nuggets, potato chips, bread pieces, or fish were fried for 4 or 2 min, respectively, in succession for a total of 30 min without replenish. Other chemicals were of reagent grade and used as received.
Animals and diets
Male, 6 weeks old Wistar rats of 295 ± 10 g weight were purchased from the animal house, Chinese Military Medical Science Academy. After 1 week’s adaptive feeding with the basic diet, the rats were randomly divided into three groups. Group 1: basal diet without extra oil consumption (control group); Group 2: basal diet supplemented with non-heated canola oil (NEO group); Group 3: basal diet supplemented with deep-fried canola oil (DFEO group). One point five milliliters (1.5 mL) of either non-heated or heated oil was fed by oral gavage using a feeding needle once daily for 6 consecutive weeks before animals were sacrificed for analysis. Each group had eight animals housed in plastic cages (4 rats/cage) with free access to water and food. The conditions of humidity (55 ± 5 %), light (12/12 h light/dark cycle) and temperature (at 23 °C) were controlled throughout the entire experimental period. The main ingredients of the basal diet (standard rodent chow) are shown in Additional file 1: Table S1.
Histological study
After dissection of animals, intestinal tissues were removed and fixed in 10 % neutral formalin for 48 h, washed in running tap water for 24 h. The tissues were then dehydrated using 30, 50, 70, 80, 90, 95 and 100 % ethanol, cleared in two changes of xylene, embedded in paraffin (BMJ-III embedding machine, Changzhou Electronic Instrument Factory, Jiangsu, China), and then cut into 5-μm thick sections using a microtome (Leica RM2235; Leica, Heidelberg, Germany). Slides were stained with haematoxylin and eosin (H&E) for histological examination.
Total RNA extraction and quantitative RT-PCR analysis
After the 6-week experimental trial, rats were dissected immediately with sterile scissors. The liver was removed, weighed, cut into 0.5-cm3 pieces, immediately frozen in liquid nitrogen, and then stored at−80 °C before homogenizing for total RNA extraction.
Total RNA was extracted from each liver sample using Trizol Reagent (Invitrogen, Life Technologies, Carlsbad, CA, USA) following the manufacturer’s protocol. Purified poly (A) + mRNA was extracted from the total RNA sample using Oligo (dT) magnetic beads. Total RNA and cDNA syntheses were performed as described below and the resultant cDNA was stored at−20 °C until qRT-PCR analysis, which was carried out in a 20 μL volume containing 2 μM of each primer, 40 ng of cDNA, and 10 μL of SYBR Primix ExTag. Thermal cycling conditions included an initial denaturation step at 95 °C for 5 min, and then 40 cycles of 95 °C for 30 s, 58–60 °C for 30 s and 72 °C for 30 s. Fluorescence was measured at the end of each cycle. The 18S rRNA gene was used as an internal control to normalize target gene expression. Three replicates of each reaction were carried out, and the relative transcript quantity was calculated according to the method of 2-ΔΔCT [38].
Digital gene expression tag profiling
The mRNA was sheared into short fragments by adding a fragmentation buffer. First-strand cDNA was synthesized from these short poly (A) + mRNA fragments by adding random primers and Superscript II. Buffer, dNTPs, DNA polymerase I, and RNaseH were then added to generate second-strand cDNA. The double-stranded cDNA was end-repaired by adding T4 DNA polymerase, Klenow Enzyme and T4 polynucleotide kinase. This was followed by a single ‘A’ base addition using Klenow 3–5’ exo-polymerase, and then sequencing adapters were ligated to the fragments using DNA ligase. For high-throughput sequencing, the cDNA fragments (PE200) were separated by agarose gel electrophoresis and then sequenced on the Illumina Hiseq™ 2000 platform.
Transcript abundance and differential gene expression were calculated with the program Cufflinks [39]. The P value threshold was determined by the false discovery rate (FDR) to account for multiple tests of significance. In this study, a FDR threshold ≤ 0.01 and a Fold change ≥ 2 were considered as significant differences in gene expression.
Gut microbiota analysis
Fecal sample collection, DNA extraction and purification
After 6 weeks feeding, all rats were transferred to fresh sterilized cages and the fresh feces were collected, immediately frozen in liquid nitrogen, and then stored at−80 °C until DNA extraction. Six fecal samples of each group were taken.
Microbial DNA was extracted from 200 mg samples using the E.Z.N.A. DNA Stool Mini Kit (Omega Biotek, Germany) according to the manufacturer’s protocols. For each sample, DNA was extracted in duplicate to avoid bias, and the extracts from the same sample were pooled. DNA purity and concentration were analyzed spectrophotometrically using the e-Spect ES-2 (Malcom, Japan). The extracted DNA was stored at−20 °C until use.
PCR amplification of 16S rDNA V4 hypervariable regions
Sequences encompassing V4 16S rDNA hypervariable regions were PCR amplified from DNA samples using fusion primers (515 F and 806R). All PCR reactions were carried out with Phusion® High-Fidelity PCR Master Mix (New England Biolabs). PCR products were analyzed by mixing the same volume of 1X loading buffer (contained SYB green) with PCR product and separated by electrophoresis on 2 % agarose gel. Samples with a bright main band between 400 and 450 bp were chosen for further experiments. PCR products were then purified with a Qiagen Gel Extraction Kit (Qiagen, Germany). Sequencing libraries were generated using TruSeq® DNA PCR-Free Sample Preparation Kit (Illumina, USA) following manufacturer's recommendations and index codes were added. The library quality was assessed on a Qubit@ 2.0 Fluorometer (Thermo Scientific) and Agilent Bioanalyzer 2100 system. Finally, the library was sequenced using an Illumina HiSeq2500 platform and 250 bp paired-end reads were generated.
Sequence analysis
All of the raw reads were treated according to the standard protocols and effective Tags were acquired [40–42]. Based on the HiSeq Illumina sequencing platform, the double end sequencing (Paired-End) method was used. Terminal sequencing was constructed by a small fragment library. Sequences analysis was performed by Uparse software (Uparse v7.0.1001). Sequence data were processed by read trimming and identification of V4 sequences, followed by filtering and assigning the operational taxonomic units (OTUs). Sequences with ≥97 % similarity were assigned to the same OTUs. Sample species composition was revealed by OTUs cluster, species annotation and abundance analysis.
The species richness and diversity of microbial communities in different samples were analyzed by Chao and Shannon indices. Sample index was calculated with QIIME (Version 1.7.0) and displayed with R software (Version 2.15.3). Beta diversity analysis was used to comparatively analyze the microbial community diversity in different samples. Beta diversity on both weighted and unweighted unifrac was calculated by QIIME software (Version 1.7.0). The difference between samples in terms of species was shown by PCoA (Principal Co-ordinates Analysis, PCoA) and NMDS (Non-Metric Multi-Dimensional Scaling, NMDS). MRPP analysis was used to compare the differences in community structure within and between the groups.
Statistical analysis
Body mass was expressed as mean ± SD and analyzed using the Fisher test. T-test and LDA Effect and Size analysis were used to determine statistically significant differences in biomarkers and in gut microbiota among the different groups. P < 0.05 was considered as significant difference.
Ethics statement
All animal trial procedures were reported and approved by the Ethical Committee for the Experimental Use of Animals in Center for Drug Safety Evaluation, Tianjin University of Science & Technology (approval No:17/055/MIS).
