Animals and experimental design
Fifty-five male Wistar rats were purchased from the Pasteur Institute (Tehran, Iran) at 4 weeks of age and weight of 55 ± 4 g then animals were individually kept in cages then acclimatized under controlled conditions (rooms with 21-24 °C, 50-60% relative humidity and12/12 h reverse light-dark cycle) for 1 week. All procedures handling and using animals have been approved by the Ethics Committee of the Iran University of medical sciences and were based on National Institutes of Health guide for the care and use of laboratory animals (23).
First, rats were randomly placed on the ad libitum HFD group (n=50) comprising 60% kcal from fat and normal diet (ND) group which fed standard laboratory chow diet (n=5) for 17 weeks. The animals were weighed every week. When the mean weight of HFD-fed rats became significantly more than rats in the ND group and obesity model induced, rats were randomly divided into one of the following five groups for an additional 8 weeks (n= 10/group): I. RJ fed during CRD II. TRF fed during CRD III. RJ+TRF fed during CRD IV. CRD with no supplementation and V. HFD with no supplementation (remain on HFD). The sample size determined according to previous similar studies (24).
The composition of CRD was similar to HFD with 30% calorie restriction (Appendix1). RJ group was treated with 100 mg/kg lyophilized RJ powder containing 6% of trans-10-hydroxy-2-decenoic acid and TRF group was treated with 85 mg/kg TRF. The TRF is composed of 11.9% (w/w) α-tocopherol, 12% α-tocotrienol, 2% β-tocotrienol, 19.3% γ-tocotrienol, 5.5% δ-tocotrienol, and 23.5% α-tocopherol. Administered doses of RJ and TRF were chosen considering previous studies based on no observed adverse effects (25, 26)
Finally, animals were subjected to 12 h fasting and anesthetized with an injection of xylazine (xylazine 2%, 20 mg ml−1, Alfasan, Netherlands), and ketamine (ketamine 10%, 100 mg ml−1, Alfasan, Netherlands) intraperitoneally, then blood was collected via cardiac puncture. Blood samples were centrifuged at 2500 × g for 15 min and serum samples were frozen at −80 °C. The inguinal WAT, interscapular BAT, and hypothalamus were removed, washed with phosphate-buffered saline (PBS), and were frozen at −80 °C in RNAlater stabilization solution until gene expression analyses.
Biochemical assay
The concentrations of TNF-α and MCP-1 in serum were measured using Rat TNF-α and MCP-1 Immunoassay kit (MyBioSource, Inc., San Diego, USA) according to the manufacturer's protocols
RNA isolation and quantitative real-time PCR
Total RNA of the WAT, BAT, and hypothalamus was extracted with Trizol Reagent. Synthesized complementary DNA (cDNA) via reverse transcription was amplified by real-time PCR on a quantitative PCR System. PCR amplification was conducted with a fluorescence thermal cycler system using SYBR green kit and rat specific primer sequences for GRP78, TNF-α, and MCP-1 as target genes and β-actin as the housekeeping gene. All of the mentioned experiments were performed with experts who were blinded to treatment groups
Statistical Analysis
All data were expressed as the mean ± SEM. t-test and one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test performed to compare the difference between two and more than two groups respectively. P-value <0.05 was considered statistically significant. The Prism software, version 6·0 (GraphPad, CA, USA) was used for drawing figures.