Animals
We used twelve 12-week-old male Goto–Kakizaki rats (CREA Japan, Inc.). These rats were reared in a controlled environment. The procedures in this experiment were approved by the Department of Animal Research Resources, Institute of Health Biosciences, Tokushima University. All experiments were performed according to the relevant guidelines and regulations, and all authors complied with the ARRIVE guidelines.
Methods
DJB, sham operation, TU-100 treatment, and TU-100 treated DJB
The 12 twelve rats were divided into four groups: a sham group, a DJB group, a Daikenchuto (TU-100) treated group, and a TU-100 treated DJB group. Each group had three rats. These rats were fasted before the surgery and anesthetized in 2% to 3% isoflurane and air/oxygen. In the sham operated group, a midline laparotomy was performed, and this incision was simply closed. DJB was performed as reported by Rubino, et al. [9]. Briefly, DJB was performed by post-pyloric transection of the duodenum, closure of the duodenal stump, transection of the jejunum at 10 cm from the Treitz ligament, reconstruction of the alimentary passage by duodenojejunostomy, and finally reconstruction of the biliopancreatic limb to the jejunum at 15 cm distal from the duodenojejunostomy. These rats in the sham and DJB group were allowed free access to water and a basal diet, freely (MF; Oriental Yeast, Tokyo, Japan). In the TU-100 group and TU-100 treated DJB group, TU-100 mixed food by gavage for 7 days before surgery.
Blood glucose in the DJB group was measured at preoperation and 24 hours postoperation. Blood glucose in the TU-100 group was measured at pre-TU-100 treatment and post-TU-100 treatment. In the TU-100 treated DJB group, blood glucose was measured at pre-TU-100 treatment, preoperation, and 24 hours postoperation.
Body weight was measured at pre-TU-100 treatment and post-TU-100 treatment in the TU-100 group and pre-TU-100 treatment, preoperation, and 24 hours postoperation in the TU-100 treated DJB group.
Fecal pellets were harvested at pre-TU-100 treatment and post-TU-100 treatment from the TU-100 group and pre-TU-100 treatment, preoperation, and 24 hours postoperation from the TU-100 treated DJB group.
Rats in the DJB and TU-100 treated DJB groups were sacrificed 24 hours postoperation.
Figures 1 and 5 show the experimental overview of Studies 1 and 2.
Reagents
TU-100 granules were made by Tsumura & Co (Tsumura Daikenchuto Extract Granules; Tsumura & Co, Tokyo, Japan). TU-100 contains three medical herbs: processed ginger (Zingiberis Siccatum Rhizoma), ginseng (Ginseng radix), and Japanese pepper (Zanthoxylum fruit).
Ribonucleic acid (RNA) isolation and quantitative real-time reverse transcription (RT)-PCR
Total ribonucleic acid (RNA) was extracted using the RNeasy Mini kit (Qiagen, Valencia, CA, USA) and reverse transcribed with the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA). Quantitative real-time RT-PCR was performed using a 7500 Real-Time PCR system with the TaqMan Gene Expression Assay-on-Demand and TaqMan Universal Master Mix (Applied Biosystems). The levels of the liver and gut inflammatory cytokines IFNγ, IL1, IL6, and TNFα (IFNγ: Rn00594078_m1; IL1: Rn00580432_m1; IL6: Rn00561420_m1; and TNFα: Rn00562055_m1) (Applied Biosystems) were assayed, and the control gene was the TaqMan Rat GAPDH endogenous control (GAPDH; 4352338E; Applied Biosystems). The thermocycling conditions were as follows: 2 minutes at 50°C, 10 minutes at 95°C, 40 cycles of fifteen 15 seconds at 95°C, and 1 minute at 65°C. Data were analyzed using Applied Biosystems Prism 7500 Sequence Detection System Software version 1.3.1.
16S rRNA gene metagenome sequencing of stool samples
Each 10–30 mg stool sample was treated with 1 mL extraction buffer [400 μL 10% sodium dodecyl sulfate (Sigma-Aldrich Japan Inc., Tokyo, Japan) in Tris-EDTA (TE) buffer (10 mmol/L Tris (pH 7.4; Fujifilm Waco Pure Chemical Corporation, Osaka, Japan) and 1 mmol/L EDTA (pH 8.0; Fujifilm Waco Pure Chemical Corporation))], 400 μL phenol:chloroform:isoamyl alcohol (25:24:1 v/v) (Nippon Gene, Tokyo, Japan), and 200 μL 3 M sodium acetate (Fujifilm Waco Pure Chemical Corporation), added to a Lysing Matrix E tube (MP Biomedicals, Solon OH, USA), and homogenized using a FastPrep-24 automated cell disruptor (MP Biomedicals) at a speed setting of 6 meters/sec for 40 sec. The homogenization process was repeated twice. The homogenate was centrifuged at 10,000 × g for 30 min, and DNA extract was obtained as the aqueous phase, which was purified twice by adding an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1, v/v). Subsequently, an equal volume of isopropyl alcohol (Fujifilm Waco Pure Chemical Corporation) was added, and DNA was obtained as a pellet by centrifugation (10,000 × g, 5 min). After drying, the DNA was dissolved in TE. The preparation of the 16S rRNA gene metagenome library for MiSeq (Illumina, Inc., San Diego, USA) was performed according to the manufacturers’ protocol. Briefly, 10 ng DNA template were amplified using an Advantage-HF 2 PCR kit (Takara Bio Inc., Shiga, Japan) with universal primers for the 16S rRNA v3–v4 region (forward primer: 5' TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG 3'; reverse primer: 5' GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC 3'). Subsequently, index sequences for each sample were added to both ends of the purified PCR fragments. The concentrations of each amplicon were measured using a Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher Scientific, Inc., Waltham, USA) and mixed equally. The library was applied to a MiSeq Reagent Kit v3 (Illumina, Inc.), and sequences were determined in accordance with the manufacturers’ standard protocol. Sequence data were processed using the 16S rRNA sequence analysis pipeline QIIME 1.8.0 as follows [23]. Initially, both sequence reads were joined, and sequences with a Phred quality score below 20 were removed. Chimera elimination by Usearch was performed to remove contaminated sequences. Open reference operational taxonomic unit (OTU) picking was performed against Greengenes 13_8 97% OTU representative sequences. A summary of the taxonomy of each sample was obtained using the script ‘summarize_taxonomy_through_plots.py’ in QIIME 1.8.0.
Statistical analysis
Data were analyzed using an unpaired Student’s t-test or a one-way ANOVA with Bonferroni’s post hoc test. Differences were defined as significant when p < 0.05. Results were expressed as means ± standard error of the mean (SEM). All data were generated using StatView version 5.0 for Windows (SAS Institute Inc., Cary, NC, USA).