Animal study
The in vivo experiments were performed in compliance with the guidelines of the Ethics and Animal Welfare of Kyungpook National University and were approved by the Institutional Review Board (KNU-2019-0034-1). Five-week-old male C57BL/6 mice were obtained from Orient-Bio Inc. (Seongnam-si, Gyeonggi-do, Korea). The animal diets were procured from Research Diet (New Brunswick, NJ, USA). The mice were provided access to water and chow ad libitum during the 1st week of acclimatization. They were maintained under controlled conditions of 23 °C ± 1 °C and a light–dark cycle of 12 h. They were then divided into four groups of 10 animals per group and treated as follows: Low-fat diet (ND) (D12450J, 10% kcal from fat) and HFD (D12492, 60% kcal from fat) and HFD with Orlistat. Orlistat-treated mice received 200 mg of Orlistat per kg of diet for 8 weeks. The mice were then euthanized by cervical dislocation followed by excision of the whole intestine, which was immediately frozen at − 80 °C. Fecal samples were collected weekly for each group.
RNA isolation from the intestine and quantitative real-time PCR
Total RNA was isolated from the jejunum to the colon using the Takara MiniBEST universal RNA extraction kit (Takara, Kusatsu, Japan). To quantify the amount of mRNA, cDNA was synthesized from 1 μg of total RNA in a final volume of 20 μL using the Maxima H Minus First Strand cDNA Synthesis Kit (K1681; Thermo Fisher Scientific, MA). Next, quantitative real-time PCR (qRT-PCR) was done using the TOPreal™ qPCR 2× pre-mix (SYBR-Green with low ROX, Daejon, South Korea) with the Light Cycler® 96 software version 1.1 PCR Detection System (F. Hoffman-La Roche AG. Basel, Switzerland) following the manufacturer’s instructions. The results were analyzed using the 2−ΔΔCt method with GAPDH and β-actin as internal standards. All primer sequences are listed in Additional file 1: Table S1.
Extraction of aqueous secondary metabolites from fecal matter
The fecal matter from the three mouse groups was collected during the final 2 weeks of treatment and pooled. From each pooled sample, 100 mg was placed in sterile 1.5 mL conical Eppendorf tube. Then, 1 mL of phosphate buffered saline (PBS) was added to the tube and the mixture was kept on ice and vortexed every 5 min for 1 h. The final slurry was centrifuged at 10,000g for 10 min at 4 °C. The aqueous supernatants were separated and stored at − 80 °C.
Cell culture
The human gut epithelial cancer cell line, Caco-2, was used for cell mitochondrial stress analysis. Cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin (Welgene, South Korea). A stable human gut epithelial cell line, HCT-116, was transfected with the pGL4.37[luc2P/ARE/Hygro] vector (Promega, Madison, WI, USA) [15]. The resulting HCT-116-ARE-luciferase cell line was maintained in the aforementioned culture medium and included 0.4 mM hygromycin (Sigma-Aldrich, St. Louis, MO, USA). All cultures were maintained in a humidified CO2-incubator (37 °C and 5% CO2) for the designated period.
Cell mitochondrial stress analysis
Subconfluent Caco-2 cells were harvested and seeded at 1.6 × 104 cells per well in an 8-well plate for the Agilent Seahorse XFp Cell Mito-Stress assay (Agilent Technologies, DE, USA). Each well contained 100 μL of growth medium and the cells were incubated until 80% confluence was achieved. The cells were either incubated for 3 or 12 h with fecal extract from the HFDOrl and HFD group with or without a subsequent 3 h t-BHP challenge by adding 9 µL of 100 µM t-BHP. Untreated cells were used as controls. Prior to the assay, the growth medium from each well was removed, the cells were washed twice with 180 µL of pre-warmed assay medium (XF base medium supplemented with 25 mM glucose, 2 mM glutamine, and 1 mM sodium pyruvate; pH 7.4), and pre-incubated with 160 µL of the assay medium at 37 °C without CO2 for 1 h.
A pre-warmed sensor cartridge containing XF calibrant and the energy substrates, which included oligomycin, FCCP, and rotenone/antimycin A, were loaded into injector ports A, B, and C, respectively, of the Agilent Seahorse XFp analyzer (Seahorse Bioscience, Billerica, MA, USA). The assay was primed with the cartridge calibration using pre-warmed XF calibrant and was continued for the samples using the cell mito-stress test assay protocol provided by the manufacturer (Seahorse Bioscience, Billerica, MA, USA). The oxygen consumption rate was measured under basal conditions followed by the sequential addition of oligomycin, FCCP, and rotenone/antimycin A. This allowed for an estimation of the contribution of individual parameters for basal respiration, proton leak, maximal respiration, spare respiratory capacity, non-mitochondrial respiration, and ATP production using Wave software sourced from Agilent Technologies (Agilent Technologies, DE, USA). This data was then exported to Microsoft Excel for further analysis.
Luciferase activity
A luciferase reporter assay was conducted on the stable HCT-116-ARE-luciferase cells. The cells were treated with fecal extracts for 12 h, then challenged with 300 µM t-BHP for 3 h prior to harvest and lysis. Treatment with t-BHP activates apoptosis because of changes in the ion channels of the mitochondrial membrane and activation of lipid peroxidation. The luciferase activity, which corresponds to ARE activity, was measured using a luciferase assay system (Promega) according to the manufacturer’s instructions. Sulforaphane (Sigma-Aldrich, St. Louis, MO, USA), an isothiocyanate, was used as a control ARE activator. The luminescence of the assay was detected and the values were normalized based on total cell count. The values were then converted to fold-change using the blank as the baseline value.
Measurement of mitochondrial ROS generation using a superoxide indicator
Cells were seeded (5 × 103 cells) on cover slips and placed into 24-well plates (SPL Life Sciences Co., Ltd.) and treated with fecal extracts alone for 12 h or in combination with t-BHP for the final hour. Afterwards, the media was discarded. Immediately after treatment, mitochondrial ROS was measured using the MitoSOX TM Red superoxide indicator (Invitrogen, Carlsbad, CA, USA) followed by counterstaining with 4ʹ,6-diamidino-2-phenylindole (DAPI, 1 µg/mL). The cells were washed twice with PBS and then mounted with a drop of mounting medium (Vector Laboratories Inc., California, USA). Fluorescence was assessed using a fluorescence microscope (Eclipse 80i, Nikon, Tokyo, Japan) at 400× magnification and quantified densitrometrically (ImageJ Software, US National Institute of Health, Bethesda, Maryland, USA) from at least three random fields selected for each coverslip. Representative images for all conditions were selected for illustration.
Statistical analysis
In vitro and in vivo data are expressed as the mean ± standard error of the mean (SEM). A p-value < 0.05 was considered statistically significant.