Human HLE-B3 lens epithelial cell line was obtained from the American Type Culture Collection (Manassas, VA, USA), and chlorogenic acid (purity, 98.7%) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products of China (Beijing, China). MTT and H₂O₂ (30%) were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). A 500 µM H₂O₂ solution was prepared in phosphate-buffered saline (PBS) immediately prior to application. The Annexin V/propidium iodide (PI) apoptotic detection kit was purchased from Nanjing KeyGen Biotech Co., Ltd. (Nanjing, China). All other chemicals used were purchased from Sigma-Aldrich (Merck KGaA) unless otherwise stated.

HLE-B3 cells were cultured in RPMI-1640 medium (HyClone; GE Healthcare Life Sciences, Logan, UT, USA) containing 1 g/l glucose, 10% fetal bovine serum (HyClone; GE Healthcare Life Sciences), 100 U/ml penicillin and 100 µg/ml streptomycin (HyClone; GE Healthcare Life Sciences) under a humidified atmosphere with 5% CO₂ at 37°C. The cells were seeded into a 60 mm culture dish (Corning Incorporated, Corning, NY, USA). When at 75–80% confluence, the cells were treated with H₂O₂ (10–500 µM) for 24 h or pretreated with CGA for 2 h prior to H₂O₂-treatment. At the indicated time-points, the cells were collected for different assays.

The concentrations of CGA and H₂O₂ were optimized using MTT assay. Briefly, hLECs were cultured in 96-well plates and treated with a broad range of concentrations for each reagent (H₂O₂ or CGA) for 24 h. To examine the effect of CGA, hLECs were incubated with 100 µM H₂O₂ in the absence or presence of different concentrations of CGA for 24 h. Subsequently, 20 µl MTT solution (5 mg/ml) was added into each well. After another 4 h incubation at 37°C, the medium was removed and the formazan crystals formed by oxidation of the MTT dye were dissolved with 150 µl DMSO. The absorbance was measured at 490 nm using the spectrophotometer [Unico (shanghai) Science Instruments Co., Ltd., Shanghai, China]and the cell survival ratio was expressed as a percentage of the blank.

To obtain further evidence for the protective effect of CGA against H₂O₂ induced oxidative stress, alterations of intracellular ROS levels were determined. The production of intracellular ROS was measured using 2′,7′-dichlorofluorescin diacetate (DCFH-DA; Invitrogen, Carlsbad, CA, USA) by flow cytometry. Briefly, hLECs were incubated either with 100 µM H₂O₂ alone or treated with different concentrations (10, 30 and 50 µM) of CGA for 2 h prior to treatment with 100 µM H₂O₂ for 24 h. After harvest of hLECs, the cells were incubated with DCFH-DA solution (10 µM) in the dark at 37°C for 30 min, washed with PBS (pH 7.4), and analyzed within 30 min using a flow cytometer (Accuri C6; Accuri Cytometers Inc., Ann Arbor, MI, USA). The specific fluorescence signals that correspond to DCFH-DA were collected with a 525 nm band pass filter. For each determination, 2.0×10⁴ cells were counted.

For quantification of the apoptosis rate in hLECs, cells were cultured on a 6-well plate at 5.0×10⁵ cells/well and treated with 100 µM H₂O₂ with or without CGA (0, 10, 30 and 50 µM) for 24 h. Subsequently, hLECs were collected and stained using Annexin V/PI kit and assessed by a flow cytometer (Accuri C6; Accuri Cytometers), following the instructions of the manufacturer.

The effect of CGA on the gene expression of BCL2 associated X, apoptosis regulator (Bax) and BCL2, apoptosis regulator (Bcl-2) mRNA using RT-qPCR in the presence and absence of H₂O₂. The cells (6×10⁵) were incubated either with 100 µM H₂O₂ for 24 h or with CGA for 2 h prior to treatment with 100 µM H₂O₂. Total RNA was extracted with TRIzol reagent (Life Technologies; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and cDNA was generated using a Superscript cDNA kit (Thermo Fisher Scientific, Inc.) based on the manufacturer's protocol. Following quantification with a microspectrophotometer (Beijing Kaiao Technology Development Co., Ltd., Beijing, China), cDNA was synthesized using total RNA. qPCR was performed with SYBR-Green Master Mix (Takara Biotechnology Co., Ltd., Dalian, China) in a Stratagene Mx3000P sequence detection system (Agilent Technologies, Inc., Santa Clara, CA, USA). GAPDH was used as a positive control, and a negative control without template RNA was also included. The primer sequences are presented in Table I. The reactions were performed in a total volume of 20 µl using the SensiMix One-Step kit (http://www.quantace.com; Quantace, Finchley, UK). The conditions of PCR amplification for Bcl-2 and Bax were as follows: 95°C for 10 min, followed by 40 cycles of a 95°C denaturation for 15 sec, annealing at 55°C for 30 sec, and 72°C extension for 50 sec. Each experiment was carried out four times and the ΔΔCq values were calculated by normalizing the gene expression levels to the expression of GAPDH (25). The relative expression level of each gene was expressed as a fold change.

The levels of Bcl-2/Bax (anti-/pro-apoptotic) proteins were determined using commercially available ELISA kits [BCL-2 kit (JYM0302Hu), BAX kit (JYM0265Hu); Colorfulgene Biological Technology, Co., Ltd., Wuhan, China]. The hLECs (6×10⁵) were incubated with 100 µM H₂O₂ for 24 h alone or treated with CGA for 2 h prior to treatment with 100 µM H₂O₂ for 24 h. The cells were then collected and cell concentration was diluted to 10⁶/ml with PBS (pH 7.2–7.4). Following three freeze-thaw cycles, damaged cells were centrifuged at 5,000 × g at 4°C for 20 min. The supernatant was carefully collected and stored at −80°C prior to use. The protein in the cell lysate was determined using the ELISA kit according to the manufacturer's instructions. Subsequently, the plates were read at 450 nm using a microplate reader (BioTek ELX800; BioTek Instruments, Inc., Winooski, VT, USA). The protein level of each sample was determined by comparison to a standard curve.

All animal procedures were in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research (26), and the animal experiments were approved by Shandong University of Traditional Chinese Medicine Animal Care and Ethics Committee (Jinan, China). In the present study, New Zealand White rabbits (n=30; weighing 1.8–2.0 kg) aged 10–12 weeks were euthanized with an overdose of sodium pentobarbital injection through the marginal ear vein. The eyes were removed and the lenses were carefully dissected by a posterior approach. Lenses were immediately transferred into a 24-well culture plate containing 2 ml Dulbecco's modified Eagle's medium (HyClone, Beijing, China), 1 g/l glucose, 100 U/ml penicillin and 100 µg/ml streptomycin (HyClone; GE Healthcare Life Sciences). Approximately, 24 h after the preparation of organ cultures, transparent lenses were selected for further experimentation. During the experiment, H₂O₂ and CGA were maintained at indicated concentrations for a period of 12 h, and the medium was changed. Conditioned medium was stored for further culture. Lenses were cultured in a 5% CO₂ incubator at 37°C for 48 h, and were images using a stereomicroscope under a cross background (1.0×1.0 cm). Each sample contained three lenses and lens opacity was analyzed using ImageJ-1.46 software (National Institutes of Health, Bethesda, MD, USA). This experiment was repeated three times independently.

The apoptosis rate of lens epithelial cells was assessed by flow cytometry with Annexin V/PI staining. The lenses exposed to 500 µM H₂O₂ were cultured with various concentrations (i.e., 0, 10, 30 and 50 µM) of CGA for 48 h. At the indicated time-points (0, 12, 24 and 48 h, respectively), lens epithelial explants were carefully detached from rabbit lens under an operation microscope (YZ20P5; 66 Vision Tech Co., Ltd., Suzhou, China). Lens epithelial explants were then sheared, digested with trypsin, and RPMI-1640 medium was added to terminate the digestion (27). The cell suspension was then passed through a cell strainer and centrifuged (300 × g) at 4°C for 5 min. The cells were resuspended in RPMI-1640 medium and collected by centrifugation at 300 × g at 4°C for 10 min. The collected cells were washed with cold PBS and stained with Annexin V/PI apoptotic detection kit. Finally, the cells were resuspended in 500 µl PBS for further analysis using a flow cytometer (Accuri C6; Accuri Cytometers, Inc.).

All experiments were repeated three times and data are expressed as the mean ± standard deviation, and were analyzed by one-way analysis of variance, followed by Tukey's honest significant difference post hoc test, using SPSS software (version 17.0; SPSS Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

To determine the effect of CGA on hLECs, cells were treated with a broad range of CGA concentrations for 24 h. Cell viability was presented as a percentage of the blank value. The results indicated that CGA had no cytotoxicity in hLEC cells when the CGA concentration was <100 µM (Fig. 2A). H2O2-treated cells exhibited lower cell viability at H2O2 concentrations ≥50 µM (Fig. 2B). Co-treatment with CGA resulted in a dose-dependent reduction in cytotoxicity induced by H2O2 (100 µM; Fig. 2C).

hLECs treatment with 100 µM H2O2 alone for 24 h resulted in the production of ROS with a ~3-fold increase compared with 50 µM CGA-treated cells (Fig. 3). Pretreatment with CGA prior to H2O2 exposure markedly reduced the ROS levels. When hLECs were treated with CGA (0, 10, 30 and 50 µM) for 2 h prior to treatment with 100 µM H₂O₂ for 24 h, the ROS generation reduced from 90.7±7.75 to 73.5±5.98%, 51.9±4.74 and 35.8±3.53% (Fig. 3B–E), respectively. These findings demonstrated that with the increase of concentrations of CGA, the intracellular ROS level induced by H₂O₂ was reduced and the ROS reduction was in a concentration-dependent manner. In addition, there was a significant difference in the level of H₂O₂-induced ROS compared with that of untreated cells (Fig. 3).

The use of Annexin V/PI double staining allows distinction between live cell populations, cells entering early apoptosis and those in late-stage apoptosis/necrosis. Q2-LL quadrant indicates healthy cells, Q2-UR quadrant indicates necrosis cells (Fig. 4). Any late-stage apoptosis cells were considered as necrosis as this technique is not sensitive enough to differentiate between the two. The data in Fig. 4 demonstrated that following treatment with 100 µM H₂O₂ and co-treatment with CGA (10, 30 and 50 µM) for 24 h, the late apoptotic rate of HLE-B3 cells decreased from 44.5 to 35.2, 32.8 and 16.6%, respectively (Fig. 4).

The members of the Bcl-2 protein family are pivotal role in the regulation of the mitochondrial apoptotic pathway (28). Both pro-apoptotic and anti-apoptotic Bcl-2 family members can affect the execution of apoptosis. As presented in Fig. 5, treatment with 100 µM H₂O₂ for 24 h decreased the expression of Bcl-2 (0.47-fold higher compared with blank) and increased the expression of Bax (2.09-fold higher compared with blank), whereas pretreatment with CGA inhibited the downregulation of Bcl-2 and upregulation of Bax. The ratio of Bax/Bcl-2 in the 100 µM H₂O₂-treated group was significantly higher than that in the blank group (P<0.01). With elevated CGA concentrations, the ratio of Bax/Bcl-2 in the CGA-treated (i.e., 10, 30 and 50 µM) groups decreased significantly compared with the H₂O₂-treated group (P<0.01). Following treatment with 100 µM H₂O₂ and co-treatment with different concentrations of CGA (10, 30 and 50 µM) for 24 h the ratios of Bax/Bcl-2 were 4.50±0.61, 3.73±0.46, 2.22±0.47 and 1.73±0.33, respectively.

Bcl-2 and Bax levels were assessed using commercially available ELISA kits to determine whether these key regulators of apoptosis were involved in the H₂O₂-induced apoptosis mechanism. As presented in Fig. 5, Bax protein levels were 110.91±3.97, 155.49±4.30, 145.59±4.17, 136.78±3.07 and 124.19±3.11 ng/l (Fig. 6A) and Bcl-2 levels were 3.24±0.10, 1.87±0.07, 2.15±0.06, 2.64±0.09 and 2.77±0.10 ng/l (Fig. 6B) in the blank, 100 µM H₂O₂ and H₂O₂ + CGA 10, 30 and 50 µM groups, respectively. These results demonstrated that CGA increased Bcl-2 protein expression and decreased Bax protein expression in hLECs following incubation with 100 µM H₂O₂ for 24 h.

The protective effects of CGA against cataractogenesis of rabbit lenses induced with H₂O₂ (500 µM) were investigated further. As presented in Fig. 7, loss of transparency in lenses exposed to 500 µM H₂O₂ was first noted in the equatorial region, spreading throughout the superficial cortex by 24 h and into deeper regions by 48 h. By contrast, there was little change in transparency of untreated lenses during the entire exposure period. The opacities of rabbit lenses incubated in H₂O₂ (500 µM) media containing various concentrations of CGA were measured every 12 h and compared with the control and blank samples. The opacities of the lenses began to increase after 12 h treatment with H₂O₂, and were gradually improved by CGA treatment in a dose-dependent manner.

At the indicated time-point, lens epithelial explants were detached. LECs were harvested and stained with Annexin V/PI for further analysis using a flow cytometer. Lenses treated with 500 µM H₂O₂ for 12, 24 and 48 h, caused a time-dependent increase of apoptosis rates (51.8±3.81, 77.2±4.12 and 94.57±4.77% of control value, respectively) whereas co-treatment with different concentrations of CGA [10 µM (III), 30 µM (IV), 50 µM (V)] the decreased the apoptosis rates (Fig. 8).