Syringaresinol-di-O-β-D-glucoside (SOG; cat. no. E-0542; http://www.tautobiotech.com/BRM/ProductShow.asp?ArticleID=542) was purchased from Shanghai TAUTO BIOTECH Co., Ltd. (Shanghai, China). STZ (cat. no. S0130) and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Nitrotyrosine (NT) antibody (cat. no. DMABT-Z60522) was purchased from Creative Diagnostics, Shirley, NY, USA. Insulin (cat. no. ab100578) and interleukin-6 (IL-6; cat. no. ab100712) ELISA kits, and transforming growth factor-β1 (TGF-β1; cat. no. ab9758) and GAPDH (cat. no. ab8245) antibodies, were purchased from Abcam (Cambridge, MA, USA). Total cholesterol (TC; cat. no. CY81061), triglyceride (TG; cat. no. CY81057), high-density lipoprotein cholesterol (HDL-C; cat. no. HZL2329), total protein (cat. no. HZL2206), low-density lipoprotein cholesterol (LDL-C; cat. no. HZL2321), free fatty acid (FFA; cat. no. CY81055), malondialdehyde (MDA; cat. no. EY2), superoxide dismutase (SOD; cat. no. FY1), catalase (CAT; cat. no. HZL2444), total antioxidant capacity (T-AOC; cat. no. FG5), aspartate transaminase (AST; cat. no. HZL2382), alanine transaminase (ALT; cat. no. SEA207Ra) and alkaline phosphatase (ALP; cat. no. HZL2351) kits were produced by Shanghai Chaoyan Biological Technology Co., Ltd., (Shanghai, China; http://biosis.biogo.net/sell/typeid-5638.shtml). Bicinchoninic acid (BCA) protein assay reagent was purchased from Beyotime Institute of Biotechnology (Haimen, China). All other chemicals used in the present study were of analytical reagent grade.

A total of 50 male specific pathogen-free mice (6–8 weeks old; weight, 18–23 g) were purchased from the Laboratory Animal Center of Fujian Medical University (Fuzhou, China). The animals were maintained under controlled conditions (22±2°C, 30–70% humidity and <0.03% CO₂) with a 12-h light/dark cycle and free access to food and water. All animal treatments were strictly in accordance with international ethical guidelines and the National Institutes of Health Guide concerning the Care and Use of Laboratory Animals (16), and the experiments were performed with the approval of the Animal Experimentation Ethics Committee of the Affiliated Hospital of Nanjing University of Chinese Medicine (Nanjing, China).

A diabetic animal model was established according to the previously reported method with minor modifications (17). A total 50 male mice were divided into five groups (n=10/group): Normal group, model group, 25 mg/kg SOG-treated group, 50 mg/kg SOG-treated group and 75 mg/kg SOG-treated group. All mice excluding the normal group were injected with STZ dissolved in 0.1 mol/l citric acid buffer solution (Chengdu Chemical Co., Ltd., Chengdu, China; 100 mg/kg) intravenously. Mice in the normal group were treated with an equal volume of 0.01 mol/l citric acid buffer solution. After 3 days, the fasting blood glucose (FBG) of mice treated with STZ was measured using an Accu-Chek Performa monitor. STZ-treated mice with FBG >11.1 mmol/l were considered to be diabetic. Following successful establishment of the diabetic model, mice in the normal and the model group were treated with normal saline (containing 0.5% DMSO) and mice in SOG-treated groups were treated with SOG at 25, 50 or 75 mg/kg by means of intragastric administration daily for 2 weeks. SOG was dissolved in normal saline (containing 0.5% DMSO).

During administration of SOG for 2 weeks, the water and food intake of mice were recorded each day. On days 0 (a day prior to the administration of the first dose of SOG) and 15, the body weight of mice was measured and the weight gain was calculated. On days 0, 7 and 15, the FBG of mice was determined using glucose testing strips. Blood samples were collected from the caudal vein of mice. Following the final administration of SOG, mice were sacrificed the next day by cervical dislocation. The organs were separated and the organ index was calculated according to the following formula: Organ index (g/100 g)=W_Organ/W_Body, where W stands for weight.

Following treatment with SOG for 2 weeks, the mice were fasted for 12 h with free access to water. A total of 50 µl serum was obtained from whole blood by centrifugation for 5 min at 10,000 × g at 4°C and serum fasting insulin of mice was measured using a commercial insulin ELISA kit (Abcam; cat. no. ab100578), according to manufacturer's protocol. In addition, the pancreas were treated with lysis buffer (Abcam; cat. no. ab156035) for 10 min and centrifuged at 4°C for 5 min (10,000 × g) and then the pancreatic levels of insulin and IL-6 in were also detected using insulin and IL-6 ELISA kits (Abcam; cat. no. ab100712), respectively.

The levels of TC, TG, HDL-C, LDL-C and FFA in the serum of STZ-induced diabetic mice after 12 h of fasting were measured using TC, TG, HDL-C, LDL-C and FFA kits, respectively. The value of VLDL-C in the serum was calculated according to the Friedewald formula reported by previous study (17): VLDL-C=TG/5.

Kidneys were treated with lysis buffer (Abcam; cat. no. ab156035) for 10 min and centrifuged at 4°C for 5 min (10,000 × g) and then the levels of kidney TC and TG in STZ-induced diabetic mice were determined using TC and TG kits, according to the manufacturer's protocols. The content of kidney protein in STZ-induced diabetic mice was measured using a BCA protein assay reagent.

Following the final administration of SOG, mice were fasted for 12 h and the blood samples were collected from the eyeballs and centrifuged for 10 min (3,500 × g) at 4°C to obtain the serum. The levels of MDA, SOD, CAT, T-AOC, AST, ALT and ALP in the serum of STZ-induced diabetic mice were measured using commercial ELISA kits for MDA (cat. no. EY2), SOD (cat. no. FY1), CAT (cat. no. HZL2444), T-AOC (cat. no. FG5), AST (cat. no. HZL2382), ALT (cat. no. SEA207Ra) and ALP (cat. no. HZL2351) kits, respectively following the manufacturers' protocol.

The kidney tissues of mice were cut into small pieces and treated with RIPA lysis buffer (Abcam; cat. no. ab156035). The supernatant was obtained by centrifugation at 12,000 × g for 15 min at 4°C. Protein concentration was determined using BCA protein assay reagent (Beyotime Institute of Biotechnology; cat. no. P0010). Subsequently, 35 µg proteins were separated by 12% SDS-PAGE and transferred to polyvinylidene difluoride membranes. Membranes were blocked with 5% fat-free dry milk in 1X TBS-Tween-20 (containing 0.1% Tween-20) for 2 h at room temperature. Membranes were subsequently incubated with primary antibodies specific to NT (1:1,000), TGF-β1 (1:1,000) and GAPDH (1:2,000) at 4°C overnight. Membranes were further incubated with horseradish peroxidase-conjugated secondary antibodies (1:2,000; Beyotime Institute of Biotechnology; cat. no. A0286) at room temperature for 1 h. The proteins were detected using the Beyo ECL Star reagents (Beyotime Institute of Biotechnology; cat. no. P0018A). Signals were quantified using ImageQuant LAS 4000 Imaging system (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA). To normalize for protein loading, an antibody against GAPDH was used and protein expression levels were expressed relative to GAPDH.

All tests were conducted in triplicate and all data are presented as the mean ± standard deviation. The significance of differences between groups was analyzed by one-way analysis of variance followed by a Dunnett's multiple comparisons post-hoc test using SPSS software (SPSS for Windows version 19.0; IBM Corp., Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference.

The FBG levels in model mice were higher compared with mice in other groups, indicating that the diabetic animal model was established successfully (Fig. 2A). However, treatment with SOG at 25, 50 and 75 mg/kg significantly decreased the levels of FBG in STZ-induced diabetic mice in a dose-dependent and time-dependent manner compared with the model group (P<0.01; Fig. 2A). In addition, the body weight of the model significantly decreased compared with the normal mice (P<0.01; Fig. 2B). The body weight of mice in SOG-treated groups (25, 50 and 75 mg/kg) was significantly increased compared with model group mice (P<0.05; Fig. 2B). Overeating is a primary characteristic of mice with diabetes (16). Water and food intake of model mice with diabetes was increased compared with normal mice (Fig. 2C and D). Following treatment with SOG (25, 50 and 75 mg/kg), the water and food intake of mice with diabetes was significantly decreased compared with the model group (P<0.05; Fig. 2C and D). The effect of treatment with SOG on organ indexes (kidney, pancreas, spleen and liver) of STZ-induced diabetic mice are presented in Fig. 3. Kidney, pancreas and liver indexes of diabetic mice induced by STZ were increased compared with mice in the normal group. SOG treatment (25, 50 and 75 mg/kg) significantly reduced the kidney, pancreas and liver indexes, compared with the model group, to levels similar to that in the normal group. However, no significant effects of diabetes induction or SOG treatment were observed on the spleen index.

Serum fasting insulin and pancreatic insulin levels of STZ-induced diabetic mice in the model group were significantly decreased compared with normal mice (P<0.01; Fig. 4). However, treatment with SOG (25, 50 and 75 mg/kg) significantly increased serum fasting insulin and pancreatic insulin levels in STZ-induced diabetic model mice dose-dependently (P<0.01; Fig. 4). The pancreatic levels of IL-6 in diabetic mice in the model group were significantly increased compared with the normal group (P<0.01); however, following treatment with SOG at doses of 25, 50 and 75 mg/kg, the level of pancreatic IL-6 in diabetic mice was significantly reduced compared with mice in the model group (P<0.01; Fig. 4).

TC, TG, LDL-C, VLDL-C and FFA levels in the serum of mice in the model group were significantly increased compared with the normal mice (P<0.01; Fig. 5). However, HDL-C levels in the serum of model mice were decreased compared with normal mice (P<0.01; Fig. 5). In all SOG-treated groups (25, 50 and 75 mg/kg), TC, TG, LDL-C and FFA levels in the serum of mice were significantly decreased compared with model group mice (P<0.05; Fig. 5). Similar effects were observed for VLDL-C levels at 50 and 75 mg/kg SOG; however, 25 mg/kg SOG did not induce a significant reduction in VLDL-C levels in diabetic model mice (Fig. 5). By contrast, all three concentrations of SOG led to significant increases in the HDL-C levels in the serum of diabetic model mice (P<0.01; Fig. 5).

TC and TG levels in the kidneys of model group mice were markedly increased compared with mice in the normal group (P<0.01), while total kidney protein levels were significantly decreased (P<0.01; Fig. 6). SOG (25, 50 and 75 mg/kg) significantly decreased kidney TC and TG levels, and increased levels of total kidney protein, in diabetic mice induced by STZ in a dose-dependent manner (P<0.01; Fig. 6).

Levels of MDA, SOD, CAT, AST, ALT and ALP in the kidneys of model group mice were increased significantly, and T-AOC was decreased significantly, compared with normal mice (P<0.01; Fig. 7). Following treatment with SOG (25, 50 and 75 mg/kg), the levels of MDA, SOD, CAT, AST, ALT and ALP in the kidneys of diabetic mice were significantly decreased, and T-AOC was significantly increased, compared with model group mice (P<0.05; Fig. 7).

The results of western blot analysis demonstrated that the protein expression of NT and TGF-β1 were significantly increased in diabetic model mice compared with normal mice (P<0.01; Fig. 8). Treatment with SOG at 25, 50 and 75 mg/kg in diabetic model mice resulted in a significant decrease in the protein expression of NT, compared with the model group (P<0.01; Fig. 8). SOG treatment at 50 and 75 mg/kg also significantly decreased the expression levels of TGF-β1 compared with mice in the model group (P<0.05), but no significant reduction in TGF-β1 levels was observed at 25 mg/kg SOG in diabetic model mice (Fig. 8).