A CO₂ incubator was purchased from Shanghai Aoxin Technology Instrument Co., Ltd. (Shanghai, China); the chemical luminous gel-imaging system was purchased from Hach Company (Loveland, CO, USA); and the cryogenic desk centrifuge was purchased from Beijing Jingli Centrifuge Co., Ltd. (Beijing, China).

Laboratory Bcl-2 knockout (K.O) mice and normal control mice were provided by Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China). The present study was approved by the Ethics Committee of The Second Hospital of Jilin University (Changchun, China) and was performed according to the Care and Use of Laboratory Animals Guidelines of China.

In total, 12 normal control mice served as the control group and 12 Bcl-2 K.O mice served as the experimental group. The mice were aged ~12 weeks in the two groups. One eye of each mouse was selected randomly for UV irradiation, while the other eye was covered and used as the control. Thus, the mice were further divided into four groups, which included the normal-nonUV, normal-UV, Bcl-2 K.O-nonUV and Bcl-2 K.O-UV groups.

In the mice, one eye was exposed to the UV radiator (SP3-250D; Ushio Electric Co., Tokyo, Japan), while the other eye was covered with a lead sheet patch. The total UV radiation dosage was 8 kJ/m² and the radiation time was 15 min. UV radiation was produced by the UV radiator. The radiation level was determined using an energy meter (GTFP200A7J; Meba Electric Co., Ltd., CA, USA) at the cornea plane, and the UV spectrum was recorded using a spectrometer (F4600; Hitachi, Ltd., Tokyo, Japan). The UV spectra ranged between 280 and 320 nm.

Mice were anesthetized with diethyl ether and sacrificed by cervical fracture following irradiation with UV for 24 h. The two eyeballs were enucleated (optic nerve with an approximate length of 2 mm was retained). Samples of the mouse crystalline lens were microscopically obtained for reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and caspase-3 activity determination. The samples were also weighed. The crystalline lens was washed three times with diethylpyrocarbonate-phosphate-buffered saline (PBS) for use in subsequent experiments.

Glacial acetic acid solution, formaldehyde solution, normal saline solution and 75% ethanol solution were mixed in the ratio 1:2:7:10 to form a fixation solution. The entire eyeball was immersed in the fixation mixture and fixed for 0.5 h at room temperature. Once the eyeball surface had become hard, the eyeballs were injected with the fixation solution from the posterior chamber and fixed for 2 h. Samples were incised from the upper and lower sections using a sharp blade or ophthalmological scissors, and subsequently fixed for 2 h at room temperature. The samples were then immersed in 10% neutral formaldehyde and fixed for a further 20 h. Following a series of dehydrations using gradient concentrations of ethanol, the samples were blocked with dimethylbenzene solution, embedded in paraffin at 60°C and cut into 4 µm sections.

Following drop-wise addition of 50 µ1 alkali Triton X-100 (0.1%; prepared by 0.1% sodium citrate), the samples were incubated for 8 min at room temperature, and washed twice with PBS for 5 min. Next, 50 µl H₂O₂ (3%) was added in a drop-wise manner and the samples were left for 10 min at room temperature, followed by two washes with PBS for 5 min. Following drop-wise addition of 50 µl TUNEL reaction solution, the samples were incubated for 2 h at 37°C in the dark, and washed twice with PBS for 5 min. The staining results were observed microscopically following subsequent procedures, as described in the relevant literature (11).

An appropriate volume of radioimmunoprecipitation assay lysis buffer was added according to the mass and volume of each specimen. The samples were placed on ice, pipetted into a single-cell suspension, and subsequently placed on ice for a further 5 min. The samples were centrifuged for 10 min at 10,000 × g at 4°C. Protein extract was obtained by separating the supernatant and a caspase-3 detection kit was used to determine the activity of caspase-3, as described in a previous study (12).

SPSS l2.0 statistical analysis software (SPSS, Inc., Chicago, IL, USA) was used for the statistical analyses, and the data are expressed as the mean ± standard deviation. An independent t-test was conducted for the comparison of mean values between samples, where P<0.05 was considered to indicate a statistically significant difference.

Total RNA was extracted from the crystalline lens of mice by using the Total RNA Extraction kit (Tiangen Biotech Co., Ltd., Beijing, China). Total RNA (0.2 µg) was subjected to RT-qPCR and the amplification products were subjected to agarose gel electrophoresis. The results demonstrated that the mRNA expression level of Bcl-2 was similar to that of the control gene, β-actin, in the normal-UV and normal-nonUV groups. Thus, a considerable level of Bcl-2 transcription was observed in the crystalline lens of the adult mice (Fig. 1).

A statistically significant difference was not observed in the expression levels of Bcl-2 between the normal-UV and normal-nonUV groups (Fig. 1), indicating that the UV irradiation did not trigger Bcl-2 mRNA transcription. In addition, UV radiation did not induce any changes in the mRNA expression of Bcl-2 in the Bcl-2 K.O groups (Fig. 1).

UV radiation did not cause lens opacities in the various specimens of the normal and Bcl-2 K.O mice. Furthermore, no characteristic changes of cell apoptosis, including hydrofracturing and vacuoles, were detected. Thus, the results indicated that the UV irradiation dose used in the experiment was insufficient to cause lens opacities prior to the sacrifice of the mice.

A TUNEL assay kit was used to investigate the rate of apoptosis in the tissue samples of the mouse crystalline lens of the various groups. The results of the present study indicated that large doses of UV radiation were able to induce a significant level of apoptosis in the epithelial cells of the crystalline lens (Fig. 2). The apoptotic rate was very low in the normal-nonUV group; however, a significant level of apoptosis was observed in the normal-UV and Bcl-2 K.O-UV groups (Fig. 2A; P<0.05). Furthermore, the apoptosis rate in the Bcl-2 K.O-nonUV group was not significant (Fig. 2B). Notably, the apoptotic rate in the normal-UV group was significantly higher, as compared with that of the Bcl-2 K.O-UV group (Fig. 2B; P<0.05).

Caspase-3 is an important member in the family of cysteine-aspartic acid proteolytic enzymes. The enzyme is able to catalyze reactions of itself and other substrates, including caspase-7. The mRNA expression of caspase-3 was quantitatively determined using RT-qPCR analysis. The results indicated that the mRNA expression of caspase-3 in the normal-UV group was significantly higher compared with the normal-nonUV group (P<0.05), while the mRNA expression in the Bcl-2 K.O-UV group was also significantly higher compared with the Bcl-2 K.O-nonUV group (P<0.05). In addition, the mRNA expression of caspase-3 in the normal-UV group was significantly higher compared with that in the Bcl-2 K.O-UV group (P<0.05), as demonstrated in Fig. 3.

Further analysis of caspase-3 activity indicated that UV radiation significantly increased the activity of the proteolytic enzyme. In addition, caspase-3 activity levels were significantly higher in the normal-UV group when compared with the Bcl-2 K.O-UV group (P<0.05), under the same dose of UV radiation (Fig. 4). Therefore, following the determination of mRNA expression and the proteinase activity of caspase-3, the results indicated that the expression of the Bcl-2 protein aggravates UV-induced apoptosis in the crystalline lens.