A total of 90 Male Wistar rats (6–8 weeks, 180–220 g) were purchased from The Shanghai Animal Center, and kept under specific pathogen-free conditions. The rats were housed at 22±2°C, under a 12-h light/dark cycle, at 50±10% relative humidity, and with free access to water and food. Experiments were performed according to the guidelines for the care and use of experimental animals established by the Ministry of Science and Technology of the People's Republic of China (26), and the study was approved by the Laboratory Animal Management Committee of Linyi Central Hospital (approval no. 2006-398). After 2 weeks of acclimatization, animals were administered 65 mg/kg STZ (Sigma-Aldrich; Merck KGaA) in 0.1 M citrate buffer (pH 4.5), as previously described (27–29). Rats in the control group were administered 0.1 M citrate buffer (vehicle control). Blood glucose was measured every week using a GlucoLeader automatic analyzer. In the present study, animals exhibiting blood glucose levels ≥16 mmol/l were considered diabetic (30).

Subsequently, animals treated with STZ were randomly divided into four experimental groups. Diabetic rats (18 rats in each group) were administered with 0 (DM group; untreated diabetic rats), 25, 50 or 100 mg/kg puerarin (Aladdin Reagent) by intraperitoneal injection, as previously described (31,32). Animals were treated with puerarin for 12 weeks daily, and they were provided a standard rodent diet with free access to water. Untreated non-diabetic rats were also used as the control group with an equal volume of 0.1 M citrate buffer. Blood was collected from the tail vein, and blood glucose levels were monitored in all rats.

After 12 weeks, the development of cataract was assessed using an ophthalmoscope. Eye inspection was preceded by topical administration of 1% tropicamide drops. Cataract formation was graded based on the classification described by Varma (33): Grade 0, clear normal lens; grade I, peripheral vesicles; grade II, peripheral vesicles and cortical opacities; grade III, diffuse central opacities; and grade IV, mature cataract. Cataract formation was considered complete (grade IV) when the red fundus reflex was not visible through any part of the lens and the lens appeared completely opaque to the naked eye.

After 12 weeks, the rats were sacrificed and the eye lenses were collected. Each pair of lenses was homogenized in prechilled 0.2 M potassium phosphate buffer (pH 7.0). This homogenate was used to assess the activity of glutathione peroxidase (GPx), the concentrations of GSH and malondialdehyde (MDA), and the protein expression levels of various factors. Samples were stored at −80°C prior to use in biochemical assays.

Antioxidant capacity (AOC) was measured using total Antioxidant capacity assay kit (A015-1-2, Nanjing Jiancheng Bioengineering Institute) according to the manufacturer's protocol. The assay is based on the ability of the sample to inhibit oxidation of 2,2′-azino-di(3-ethylbenzthiazoline-6-sulphonate) (ABTS) by metmyoglobin. The antioxidants in the sample cause a decrease in absorbance at 750 nm, corresponding to the concentration of ABTS (34). Each sample was measured in duplicate.

MDA levels were measured using a thiobarbituric acid (TBA) assay kit (A003-1-2, Nanjing Jiancheng Bioengineering Institute) according to the manufacturer's protocol. Briefly, 0.1 ml sample was mixed with 1,1,3,3-tetramethoxypropane, 0.75 ml TBA working solution (0.37%) and perchloric acid. The resulting solution was incubated at 95°C for 45 min. After cooling (10 min in ice water bath), the flocculent precipitate was removed by centrifugation (4,000 × g 10 min at room temperature). The supernatant was analyzed at 532 nm by multi-scan spectrum microplate spectrophotometer at room temperature.

GPx activity was assayed using an hydrogen peroxide assay (A005-1-2, Nanjing Jiancheng Bioengineering Institute), as previously described (35). The decrease in NADPH was assessed spectrophotometrically at 340 nm. The reaction mixture consisted of 240 mU/ml glutathione disulfide reductase, 1 mM GSH, 0.15 mM NADPH in 0.1 M potassium phosphate buffer (pH 7.0) containing 1 mM EDTA. In total, 50 µl sample was added to this mixture and incubated at 37°C for 3 min. Subsequently, 1.5 mM hydrogen peroxide was added to adjust the final volume of the assay mixture to 1 ml.

GSH content of lens homogenate was assayed by spectrophotometric method, using the reduced GSH assay kit (A006-1-1, Nanjing Jiancheng Bioengineering Institute) according to the manufacturer's protocol. The detection was performed at 420 nm by multi-scan spectrum microplate spectrophotometer at room temperature.

Interleukin (IL)-1β levels were quantified in the retina using a rat IL-1β ELISA kit (H002, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's protocol. The concentration of IL-1β was calculated as pg IL-1β/mg total retina protein in each sample according to the manufacturer's protocol. VEGF levels were quantified in the retina using a rat VEGF ELISA kit (H044, Nanjing Jiancheng Bioengineering Institute) according to the manufacturer's protocol. The standard solution or the samples were added in a 96-well plate that was coated with a monoclonal antibody from the kit. The samples were incubated for 2 h 37°C. After washing, the samples were detected at the wavelength of 450 nm using a VERSA max tunable microplate reader (Molecular Devices, LLC). The ELISA assay could detect concentrations of VEGF ≥15 ng/ml.

The mRNA expression levels of Nrf2 and HO-1 were determined by RT-qPCR. Total RNA was extracted from rat retinas using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). RT was performed using 1 µg RNA. Moloney Murine Leukemia Virus reverse transcriptase kit (cat. no. 28025013, Thermo Fisher Scientific, Inc.) was used to synthesize the cDNA. The synthesized cDNA was diluted by adding 75 µl DNase-free water and stored at −20°C. qPCR was performed using the SYBR-Green PCR kit (Takara Biotechnology Co., Ltd.) and gene-specific primers were synthesized by Nanjing Sunshine Biotechnology Co., Ltd. Primer sequences are listed in Table I. The thermocycling conditions were: 95°C for 10 min, 95°C for 10 sec and 55°C for 10 sec, with a final step of 72°C for 30 sec. Then steps 2–4 were repeated for 40 cycles followed by a melt curve program for 60 min. GAPDH served as an internal control. Relative gene expression levels of Nrf2 and HO-1 were measured using the 2^−ΔΔCq method (36). The expression levels of Nrf2 and HO-1 were normalized to the control group.

For immunoblot analysis, total protein was extracted using radioimmunoprecipitation buffer (P0013D, Beyotime Institute of Biotechnology) and nuclear extracts were isolated with a Nuclear and Cytoplasmic Protein Extraction kit (BioTeke Corporation), according to the manufacturer's instructions. Equal amounts of protein samples (~30 µg) were separated by 10% SDS-PAGE. The protein concentration was determined using a bicinchoninic protein assay according to the manufacturer's protocol (Thermo Fisher Scientific, Inc.). The proteins were electro-blotted onto a PVDF membrane (thickness, 0.45 µm; EMD Millipore). The membranes were incubated with blocking solution (5% dried milk) for 1 h at room temperature and incubated overnight at 4°C with specific primary antibodies anti-Nrf2 (1:1,000; cat. no. ab137550; Abcam), HO-1 (1:1,000; cat. no. ab13248; Abcam), GAPDH (1:5,000, cat. no. A0208; Beyotime Institute of Biotechnology) and LaminB (1:5,000, cat. no. AF1408; Beyotime Institute of Biotechnology). After washing with TBS/0.05% Tween 20 three times, the blots were incubated with horseradish peroxidase-conjugated immunoglobulin G, horseradish peroxidase-labeled mouse anti-rabbit IgG (1:5,000; cat. no. 5127, Cell Signaling Technology, Inc.) and horseradish peroxidase-labeled rabbit anti-mouse IgG (1:5,000; cat. no. 58802, Cell Signaling Technology, Inc.), for 1 h at room temperature. The signals were developed using the Pierce ECL western blotting substrate (Thermo Fisher Scientific, Inc.). Immunoreactive protein bands were visualized using the ChemiDoc MP Imaging system and quantified using Image Lab 3.0 software (Bio-Rad Laboratories, Inc.).

Data are presented as the mean ± standard error of the mean. Comparisons were performed using one-way ANOVA followed by Tukey's post hoc test using GraphPad Prism software 6.0 (GraphPad Software, Inc.). P<0.05 was considered to indicate a statistically significant difference.

Blood glucose levels in all experimental animal groups were measured every week. Puerarin administration significantly decreased blood glucose levels in diabetic rats, between 6 and 12 weeks of STZ administration (Fig. 1).

The effects of puerarin on cataract development in hyperglycemic rats were examined (Fig. 2). First, the incidence of cataract in diabetic rats following treatment with puerarin was investigated (Table II). Each group consisted of 18 rats. A total of 15 rats developed cataract in the diabetic group (rats induced withSTZ and not treated with puerarin). Following treatment with puerarin at 25, 50 and 100 mg/kg, the number of rats with cataracts was 13, 11 and 7, respectively (Table II). All animals presented clear lenses (baseline cataract score, 0) at week 0. However, after 12 weeks, the cataract scores varied among groups (Table III). The average cataract scores at the end of the experiment were decreased in the diabetic rats treated with puerarin compared with the untreated group, particularly in the 100 mg/kg group (P<0.01).

The untreated diabetic group exhibited significantly different levels of AOC, GSH, GPx and MDA compared with the control group (Fig. 3). Moreover, in the groups treated with 50 and 100 mg/kg puerarin, AOC and GSH levels were significantly higher compared with the untreated diabetic group (Fig. 3A and B). The levels of GPx in groups treated with 50 and 100 mg/kg puerarin were significantly higher compared with the untreated diabetic group. (Fig. 3C). Finally, MDA levels were significantly decreased by treatment with 50 and 100 mg/kg puerarin, compared with the untreated diabetic group (Fig. 3D).

To assess the protein levels of IL-1β and VEGF in the retina, retinas were collected from the different experimental groups at the end of the experiment and ELISA was performed. The untreated diabetic group exhibited significantly higher levels of VEGF and IL-1β levels compared with the control group (Fig. 4). Notably, the protein levels of IL-1β were significantly decreased in the 50 or 100 mg/kg puerarin group compared with the untreated diabetic group (Fig. 4B). Similarly, treatment with puerarin at 50 and 100 mg/kg decreased the VEGF levels compared with the untreated diabetic group (Fig. 4A).

The effects of puerarin on the mRNA expression levels of Nrf2 and HO-1 were determined in the retinas in the different groups (Fig. 5). The mRNA expression levels of Nrf2 and HO-1 were significantly increased in the diabetic rats compared with the control group. In addition, puerarin treatment upregulated the mRNA expression levels of Nrf2 and HO-1; however, the higher doses of puerarin (50 or 100 mg/kg) significantly increased the mRNA expression levels of Nrf2. Treatment with puerarin at 50 and 100 mg/kg upregulated the mRNA expression levels of HO-1 in diabetic rats.

Additionally, the protein expression levels of Nrf2 in the nuclei and the protein expression levels of HO-1 were examined by western blotting. The protein expression levels of Nrf2 and HO-1 were higher in the untreated diabetic group compared with the control group, but not statistically significant (Fig. 6). Treatment with puerarin significantly increased the protein expression levels of these factors. In conclusion, treatment with puerarin could activate the Nrf2/HO-1 signaling pathway in retinal tissues from STZ-induced diabetic rats.