Plant material and analysis of walnut oil
Walnut (Juglans regia L.) nuts were purchased from the local market in Shandong province. Walnut oil was extracted from Shandong walnuts according to the cold-press method with minor modification 15. The composition of PUFA in walnut oil was measured by converting them into free fatty acids by saponification 16. The free fatty acids were separated and determined by HPLC-UV equipped with Lichrosorb RP-18 column (particle size 5 µm; 150×4.6mm, Merck). The HPLC conditions were as follows: The column temperature was maintained at 30 ˚C with a flow rate of 0.9 mL/min, the UV absorption at a wavelength of 192nm and the mobile phase composition was water/acetonitrile (1:9) isocratic for 15 min. The free fatty acid peaks were identified by matching them with fatty acid standards (Sigma-Aldrich, USA). All other chemical reagents were purchased from Aladdin Reagent Co. (Shanghai, China).
Animals and treatment
Animal procedures in this experiment were approved by the Animal Care and Use Committee of the Central hospital of Linyi, by the guiding principles for the care and use of animals published by the National Institute of Health. Wistar rats (10 weeks old female rats: 180–240g; adult male rats: 300–340g) were purchased from the Experimental Animal Center of Shandong Province. All rats were housed in a controlled room with a temperature of (22–24˚C), a relative humidity of (40–60%) with a light cycle (12/12 h light/dark) and fed with a basic diet and water. After 7 days of adaptation, female rats and male rats were permitted to mate. Pregnancy was confirmed by the presence of a copulatory plug in the next morning, and the day was defined as gestational day (GD) 0.
The GDM rat model was induced by intraperitoneal injection of 40 mg/kg streptozotocin on GD6, GD7, and GD8, respectively 17. The rats with a fasting blood glucose value of more than 16.7 mmol/L were considered diabetic and used for further researches. The blood glucose levels in rats were measured at GD0, GD9, and GD18. The present study was performed in five groups of eight pregnant rats each: pregnant control group (PC), gestational diabetes mellitus group (GDM) and gestational diabetes rats fed a diet supplemented with a low dose of polyunsaturated fatty acids (225mg/kg body weight, LPUFA), a middle dose of polyunsaturated fatty acids (450mg/kg body weight, MPUFA), and a high dose of polyunsaturated fatty acids (900mg/kg body weight, HPUFA) The doses of PUFA were selected according to previous study 18.
The blood samples were obtained from the orbital venous plexus after overnight fasting. Plasma samples were obtained from blood after centrifugation at 10,000 rpm for 15 min at 4˚C. Samples were stored at -80˚C until analysis. Rats were euthanized on GD18 by light ether anesthesia after overnight fasting, fetuses, placentas, and liver tissues were immediately weighed and stored at -80˚C until further assay.
Determination of insulin concentration, blood glucose levels, and lipids parameters
Plasma insulin concentration was measured by a rat insulin enzyme-linked immunosorbent assay (ELISA) kit (Thermo Scientific). Blood glucose levels, plasma triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and hepatic levels of triglycerides (TG), cholesterol (TC), were measured, respectively, using commercial kits obtained from Nanjing Jiancheng Bioengineering Institute (Jiangsu, China) according to the supplier’s instructions.
Estimation of hepatic glycogen levels and oxidative stress
Hepatic tissues were homogenized in an ice-cold saline solution. After that, the hepatic homogenates were centrifuged at 10,000 rpm for 20 min at 4˚C. Hepatic glycogen levels were assayed with commercial kits obtained from Nanjing Jiancheng Bioengineering Institute (Jiangsu, China) according to the supplier’s protocols. The oxidative stress was also examined by the measurement of MDA level and SOD, GSH-Px and CAT activities using commercial kits obtained from Nanjing Jiancheng Bioengineering Institute (Jiangsu, China).
RNA isolation and real-time polymerase chain reaction (RT-PCR)
Total RNA was extracted from liver tissues using a commercial reagent (Invitrogen, CA, USA) according to the manufacture’s protocol. The cDNA synthesis was performed by reverse transcription of 1µg total RNA using Frist Strand cDNA Synthesis Kit (Thermo, USA). RT-PCR amplification was carried out with an SYBR Green qPCR Master Mix kit (Thermo, USA) according to the manufacturer’s protocol. The qPCR was carried out in duplicate, the condition of RT-PCR amplification reaction as follows: 45 cycles of 95°C for 10 s, 60°C for 30 s and 72°C for 30 s with the primer sequences (Table 1). The expression of target gene transcripts was related to the reference gene (GAPDH). Results were expressed as folds of control.
The data statistical analysis
All experimental results were reported as the means ± SD. The differences between groups were estimated using one-way ANOVA followed by the Tukey’s multiple comparison test for post-hoc analysis using GraphPad Prism software (GraphPad software, Inc., La Jolla, USA); P < 0.05 was usually considered as statistically significant.