Plant material collection, samples preparation and analysis of PUFA
Walnut (Juglans regia L.) nuts were purchased from the local market in Shandong Province and harvested in October 2019 from Nanshan District, Jinan City located in Shandong Province. Walnut oil was extracted from Shandong walnuts according to the cold-press method with minor modification . The preparation process of PUFA is based on previous literature  and prepared by a professional following a standard protocol. PUFA is stored at -20℃ to prevent oxidation and contamination. And 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 192 nm 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).
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 (dissolved in 0.1 mol/L citrate buffer, pH 4.5) on GD6, GD7, and GD8, respectively . Non-diabetic control rats received an equal volume of citrate buffer by intraperitoneal injection. The rats with a fasting blood glucose value of more than 16.7 mmol/L were considered diabetic and used for further researches. The present study was performed in five groups of eight pregnant rats each: Pregnant control group (PC), rats which received the 1% carboxymethylcellulose sodium (CMC) solution by oral gavage. Gestational diabetes mellitus group (GDM), GDM rats which received the 1% CMC solution by oral gavage. LPUFA group, GDM rats which received a low dose of polyunsaturated fatty acids (225mg/kg body weight, LPUFA). MPUFA group, GDM rats which received a middle dose of polyunsaturated fatty acids (450mg/kg body weight, MPUFA). HPUFA group, GDM rats which received a high dose of polyunsaturated fatty acids (900mg/kg body weight, HPUFA). PUFA was dissolved in 1% CMC solution and administered to rats by oral gavage per day from GD 0 to GD 17. The doses of PUFA were selected according to previous study  and our pilot study. The pilot study was performed and results indicated that 900 mg/kg could be the appropriate dose to exert hypoglycemic effect in the present study.
Blood and tissue samples collection
On GD 18, rats were euthanized by light ether anesthesia after overnight fasting, the fetuses, placentas, and liver tissues were immediately weighed and stored at -80˚C until further assay. Blood samples were collected from the orbital venous plexus. The hemoglobin (Hb) and glycated hemoglobin (HbA1c) levels were measured in whole blood sample. Plasma was immediately obtained from blood after centrifugation at 10,000 rpm for 15 min at 4˚C and used for mesurment of plasma glucose, insulin, triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C). The hepatic tissues were minced and homogenized in an ice-cold saline solution. After that, the hepatic homogenates were centrifuged at 10,000 rpm for 20 min at 4˚C. The supernatant was collected for further assays.
Determination of fasting insulin concentration, fasting blood glucose levels, Hb, and HbA1c
Body weight, serum glucose, and insulin levels were assayed on GD 0, GD 9, and GD 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. Insulin concentration was measured by a rat insulin enzyme-linked immunosorbent assay (ELISA) kit (Thermo Scientific). Blood glucose, Hb, and HbA1c levels were measured using commercial kits obtained from Nanjing Jiancheng Bioengineering Institute (Jiangsu, China) according to the supplier’s instructions. Measurement of Homeostasis Model of Insulin Resistance (HOMA-IR) was assayed by the following formula: HOMA-IR = (Fasting blood glucose × fasting insulin)/22.5
Oral glucose tolerance test (OGTT) and intraperitoneal insulin tolerance test (IPTT)
OGTT and IPTT were measured on GD 17. Before the measurement, all rats were fasted overnight. For OGTT, rats were orally administered with glucose at 2 g/kg body weight. For IPTT, rats were intraperitoneally injected with insulin at 2 units/kg body weight. Blood glucose concentrations were assayed from the orbital venous plexus at baseline and after the glucose and insulin loading (30, 60, 90, and 120 min).
Measurement of lipids parameters in plasma and liver
Plasma TG, TC, LDL-C, 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. The supernatant was collected for further assays. 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.