The following high-grade chemicals and reagents were purchased: Urea from GE Healthcare (Chicago, IL, USA) and thiourea from Nacalai Tesque, Inc. (Kyoto, Japan). All other chemicals and reagents were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).

Healthy Male Wistar rats (mean weight, 220 g; n=10) were provided from the Kiwa Laboratory Animals Co., Ltd. (Wakayama, Japan) and the 6-week old rats were injected with STZ for 2 days (100 mg/kg/day via i.p. injection). As lens opacification was initially observed 2–3 weeks after STZ treatment, the rats were evaluated 3 weeks after the injection of STZ to elucidate the early mechanism of diabetic cataracts. All of the experiments were performed in compliance with the regulations approved by the Ethics Committee of the Kindai University Faculty of Pharmacy (Osaka, Japan). The rats were housed in a room at 25°C under a 12-h light/dark cycle (2–3 rats/cage). All rats had access to food and water ad libitum.

Blood (50 ml) was sampled without anesthesia from the tail vein of each rat after fasting for 12 h (10:00 a.m.). The plasma glucose level was measured using an Accutrend GCT (Roche Diagnostics GmbH, Mannheim, Germany), and plasma insulin levels were assayed using an ELISA Insulin kit (cat. no. M1103; Morinaga Institute of Biological Science, Inc., Kanagawa, Japan) according to the manufacturer's protocol (24). The dynamic range of the ELISA Insulin kit is 0.1–6.4 ng/ml.

The rats were administered with 0.1% pivalephrine (Santen Pharmaceutical Co., Osaka, Japan) without anesthesia, and monitored using an EAS-1000 (Nidek Co., Ltd., Gamagori, Japan). The EAS-1000 conditions were as follows: Flash power index, 2,300±73; flash level, 100 W/sec; slit length and width, 5.0 mm.

Three weeks after injection, the STZ rats were euthanized, and the lenses of the STZ rats were removed and homogenized in urea lysis buffer (7 M urea, 2 M thiourea, 5% CHAPS and 1% Triton X-100). The protein concentration was measured using a Bio-Rad Protein Assay kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

A gel-free digestion approach was performed in accordance with the protocol described by Bluemlein and Ralser (25). In brief, 10 µg protein extract from each sample was reduced by the addition of 45 mM dithiothreitol and 20 mM Tris(2-carboxyethyl)phosphine and alkylated using 100 mM iodoacetic acid. Following alkylation, samples were digested with trypsin gold, MS grade (Promega Corporation, Madison, WI, USA) at 37°C for 24 h. Subsequently, the digests were purified using PepClean C-18 Spin Columns (Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's protocol.

Approximately 2 µg peptide samples were injected onto a peptide L-trap column (Chemicals Evaluation and Research Institute, Tokyo, Japan) using an HTC PAL Autosampler (CTC Analytics AG, Zwingen, Switzerland) and further separated through a Paradigm MS4 (AMR, Inc., Tokyo, Japan) using a reverse-phase C18-column (L-column, gel particle diameter, 3 µm; 120 Å; pore size, 0.2×150 mm; Chemicals Evaluation and Research Institute). The mobile phase consisted of solution A (0.1% formic acid in water) and solution B (acetonitrile). The column flow rate was 1 µl/min with a concentration gradient of acetonitrile, from 5% B to 40% B, for 120 min. Gradient-eluted peptides were analyzed using an LTQ ion-trap mass spectrometer (Thermo Fisher Scientific, Inc.). The results were acquired in a data-dependent manner in which MS/MS fragmentation was performed on the two most intense peaks of every full MS scan.

All MS/MS spectral data were searched against the SwissProt Rattus Norvegicus database using Mascot (version_2.4.01; Matrix Science, London, UK). The search criteria were set as follows: Enzyme, trypsin; allowance of up to two missed cleavage peptides; mass tolerance ± 2.0 Da and MS/MS tolerance ± 0.8 Da; and modifications of cysteine carbamidomethylation and methionine oxidation.

The fold changes in expressed proteins, on a base-2 logarithmic scale, were calculated using the RSC, based on spectral counting (26). Relative quantities of identified proteins were also calculated using the normalized spectral abundance factor (NSAF) (27). Differentially expressed proteins were selected when their RSC was >1 or <-1, which corresponded to fold changes of >2 or <0.5.

A total of 10 µg lens extract was added to each well and subjected to 10% SDS-PAGE under reducing conditions, and the separated proteins were transferred to polyvinylidene fluoride membranes for 30 min at 15 V. Following blocking in Tris-buffered saline Tween-20 (TBST) buffer (0.1%) with 5% skimmed milk for 2 h at room temperature, the membranes were incubated at 4°C overnight with anti-phosphorylated (p)-p38 mitogen-activated protein kinase (MAPK; 1;1,000; cat. no. 4511) and anti-p38 MAPK (cat. no. 8690) antibodies (both from Cell Signaling Technology, Inc., Danvers, MA, USA), along with anti-β-actin antibody (cat. no. sc-47778; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) to confirm equal loading of the proteins. The membranes were washed three times with TBST and incubated with horseradish peroxidase-conjugated anti-rabbit immunoglobulin G antibody (cat. no. A106PU; American Qualex, San Clemente, CA, USA) for 1 h at room temperature. Following washing, the blots were visualized using SuperSignal West Dura Extended Duration substrate (Thermo Fisher Scientific, Inc.) and bands were detected using a myECL Imager system (version 2.0; Thermo Fisher Scientific, Inc.). Subsequently, the intensity of p-p38 and p38 were quantified using myImage Analysis software (version 2.0; Thermo Fisher Scientific, Inc.) and the relative luminescence level of p-p38 over p38 was used to represent the signal strength of p-p38. All western blot analyses were performed in triplicate.

The unpaired Student's t-test was used and P<0.05 was considered to indicate a statistically significant difference. All data are expressed as the standard error of the mean. The analyses were performed using GraphPad Prism software (version 5; GraphPad Software, Inc., La Jolla, CA, USA).

Initially, the changes in plasma glucose and insulin were investigated in the rats following injection of STZ and whether the hyperglycemia caused lens opacification in the STZ rats was demonstrated. The plasma glucose levels in STZ rats (247.1±10.6 mg/dl; n=5) were significantly higher than in the normal rats (81.3±4.1 mg/dl; n=5). Furthermore, insulin was not detected in the STZ rats and the body weight in the STZ rats (217.3±11.9 g; n=5) was significantly decreased when compared with that of the normal rats (319.5±8.8 g; n=5). These results demonstrate that the STZ rats developed diabetes mellitus with hyperglycemia and hypoinsulinemia. In addition, opacification in the cortical epithelium was observed in the lenses of the STZ rats (Fig. 1).

To examine the effect of hyperglycemia on lens damage, the molecular profile of proteins whose expression level was changed in the diabetic rat model was investigated using shotgun proteomics. In the lenses of the STZ rats, 235 proteins were identified and 229 were identified in that of the normal rat (Normal) using the search parameters (Fig. 2). Among the 348 proteins identified in the rat lenses, 116 (33.3%) were identified in the two groups; while, 119 (34.2%) and 113 (32.5%) proteins were unique to STZ and normal rats, respectively (Fig. 2).

Subsequently, a label-free semi-quantitative method based on spectral counting was used to identify proteins with expression levels regulated by hyperglycemia. The R_SC value was plotted against the corresponding protein (x-axis) from left to right for proteins identified in the STZ and normal rats (Fig. 3). The positive and negative R_SC values indicate increased and decreased expression levels, respectively in the STZ rats. The NSAF value was plotted against the corresponding protein. The NSAF of proteins in the STZ and normal rats are indicated above and below the x-axis, respectively (Fig. 3). Proteins with either a high positive or negative R_SC value were considered to be candidate proteins, whose expression was likely regulated by hyperglycemia. A total of 52 differentially expressed proteins were identified in the lenses of the STZ rats (Table I). The expression levels of housekeeping proteins, such as β-actin and glyceraldehyde-3-phosphate dehydrogenase, did not change in the lenses of the normal and STZ rats (Fig. 3).

To determine whether the oxidative stress response by active oxygen is affected by downregulation of superoxide dismutase (SOD) expression, the phosphorylation of p38, which is important in the signaling pathways involved in oxidative stress response, were examined. The phosphorylation of p38 in the lenses of STZ rats was significantly increased when compared with the lenses of normal rats (P<0.05; Fig. 4).