All patients were treated at the Second Affiliated Hospital of Nanchang University (Nanchang, China) between January 2014 and December 2015 and provided written informed consent. Patients were divided into the POAG group (patients with POAG) and the control group (patients with senile cataracts). Patients with comorbidities, including hypertension, diabetes mellitus, liver and kidney dysfunction, cerebral infarction or thyroid dysfunction, were excluded from the present study. In the control group, patients with a history of cataracts, eye trauma or eye diseases were also excluded. The POAG group comprised 41 patients (22 men, 19 women; mean age 59.20±12.8 years). The control group comprised 53 patients (30 men, 23 women; mean age 62.51±8.49 years). The present study was performed in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of the Second Affiliated Hospital of Nanchang University.

The diagnostic criteria for POAG were as follows: i) IOP >21 mmHg at any time; ii) fundus examination or optical coherence tomography (OCT) examination revealing glaucomatous optic nerve damage; iii) visual field examination revealing characteristic visual field loss; iv) gonioscope and ultrasound biomicroscope (UBM) examinations revealing an open anterior chamber angle; and v) no signs of secondary glaucoma or a non-glaucomatous cause for the optic neuropathy.

A total of 3 ml of fasting blood was collected from study participants and centrifuged (1,000 × g) for 15 min at room temperature to separate the serum. Serum Hcy levels were measured using an AU-500 automatic biochemical analyzer (Beckman Coulter, Inc., Brea, CA, USA) according to the manufacturer's protocol. The reference range of normal Hcy values was ≤10 mmol/l. During trabeculectomy (POAG group) or phacoemulsification cataract surgery (control group), the anterior chamber was punctured to aspirate 0.2–0.3 ml of the aqueous humor and Hcy levels were detected using the AU-500 automatic biochemical analyzer.

Primary HTMCs (cat. no. HUM-iCell-n012) were purchased from iCell Bioscience, Inc. (Shanghai, China). Primary HTMCs were cultured at 37°C in Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) containing 15% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) in an incubator containing 5% CO₂ with 100% humidity. The culture medium was replaced every 3–4 days. HTMCs were monitored under optical microscope at a magnification of ×100 and ×400. When cell growth reached ~80% confluence at the bottom of the culture flasks, cells were dissociated using 1–2 ml 0.25% EDTA-trypsin, passaged and continuously cultured. Cells at the third and fourth generations were used in the experiments.

HTMCs at the logarithmic growth phase were dissociated using 0.25% EDTA-trypsin to prepare the cell suspension. Cells were diluted using DMEM containing 15% FBS to a density of 1×10⁴ cell/ml, inoculated into 96-well plates and cultured in an incubator containing 5% CO₂ with 100% humidity at 37°C. When the cell monolayer at the bottom of the wells reached confluence, the culture medium was aspirated and cells were washed 3 times with PBS. The culture medium was replaced and Hcy was added at a concentration of 30, 100, 300 or 1,000 µmol/l. Vehicle wells contained cells in DMEM supplemented with 15% FBS, while mock wells only contained DMEM supplemented with 15% FBS. Each treatment was performed in triplicate. Cells were continuously cultured for 48 h and washed with PBS, following which 100 µl DMEM containing 15% FBS was added to each well. A total of 10 µl CCK-8 solution (Sigma-Aldrich; Merck KGaA) was added to each well, samples were thoroughly mixed and the cells were incubated for 4 h at 37°C. The absorbance of each well at 450 nm was measured using a microplate reader.

Following treatment, MMP detection was performed using the JC-1 method (12). Cells were treated with Hcy for 48 h at 37°C, washed with PBS, dissociated with trypsin, centrifuged (1,000 × g) for 15 min at room temperature and collected. Cells were resuspended in DMEM supplemented with 15% FBS, mixed thoroughly with 0.5 ml JC-1 staining working solution (Abcam, Cambridge, UK) and incubated at 37°C for 20 min. Cells were then centrifuged (600 × g) at 4°C for 4 min, washed twice with ice cold JC-1 staining buffer (1X) and resuspended in 1 ml JC-1 staining buffer (1X). The cells were centrifuged (600 × g) at 4°C again for 4 min, resuspended in JC-1 staining buffer (1X) and loaded onto a flow cytometer for qualitative analysis. More than 10,000 cells were observed.

Cells were treated with Hcy for 48 h, dissociated and centrifuged as described above. Cells were mixed thoroughly with 1 ml DMEM and 1 µl DCFH-DA (Abcam) and incubated in a 37°C incubator for 20 min. The cells were washed 3 times with serum-free DMEM culture medium and loaded onto a flow cytometer for analysis. More than 10,000 cells were observed.

Cells were treated with Hcy for 48 h, dissociated and centrifuged as described above. The cell pellet containing 5×10⁶ cells was mixed with 0.5 ml lysis buffer (Bio-Rad Laboratories, Inc., Hercules, CA, USA), vortexed and suspended. Each 0.5 ml lysis buffer was mixed with 1 ml protein extraction reagent (Bio-Rad Laboratories, Inc.) and centrifuged (10,000 × g) at 4°C for 10 min. The protein layer was extracted, mixed with anhydrous alcohol and centrifuged (10,000 × g) at 4°C for 3 min. The protein pellet and protein concentration was determined using the BCA method. Equal amounts (40 µg/lane) of protein were separated by SDS-PAGE (10% stacking gel and 5% separating gel). Proteins were transferred onto a polyvinylidene difluoride membrane and incubated with the anti-SOD1 primary antibodies (cat. no. sc-271014; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and anti-β-actin antibodies (cat. no. ab8226; Abcam) at a dilution of 1:2,000 overnight at 4°C. β-actin expression was used to normalize SOD1 expression. The membrane was subsequently washed and incubated with a horseradish peroxidase-conjugated rabbit anti-mouse Immunoglobulin G secondary antibody (cat. no. ab97046; Abcam) with dilution of 1:2,000 for 1 h. Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, Inc.) was then added in a dark room and subsequent steps performed in accordance with manufacturer's protocol. Gel image analysis was performed using UVP GelDoc-It Imager (UVP LLC, Upland, CA, USA) and the relative densities of the blots were measured by VisionWorks^® Image Acquisition and Analysis Software (version 7.0; UVP LLC).

Data are presented as the mean ± standard deviation and were analyzed using SPSS 19.0 (IBM Corp., Armonk, NY, USA). Comparisons between groups were performed using the Mann-Whitney U test. The results of in vitro experiments were compared using the Kruskal-Wallis test followed by a Mann-Whitney post hoc test with Bonferroni correction. P<0.05 was considered to indicate a statistically significant difference.

The results demonstrated that plasma Hcy concentrations in the POAG and control groups were 14.44±0.86 and 10.82±0.29 µmol/l, respectively. Plasma Hcy was significantly increased in the POAG group compared with patients with senile cataracts (P<0.01; Fig. 1A). In the aqueous humor, plasma concentrations were 1.60±0.27 and 0.69±0.39 µmol/l in the POAG and control groups, respectively. Compared with the control group, Hcy was significantly upregulated in the aqueous humor of patients with POAG (P<0.05; Fig. 1B).

Normal HTMCs were generally wide, large and flat, with many processes. A number of morphologies, including star-shaped, spindle-shaped and polygonal-shaped, were observed. Cells overlapped, the cytoplasm was clear and bright and nuclei were large and round or oval-shaped (Fig. 2A and B). During passaging, the cells grew faster during the third and fourth generations and began to become adherent at 10–12 h. Cells were completely attached within 24 h. Generally, cells completely fused to form a closely connected cell monolayer within 1 week. After the 6th generation, the cell growth rate decreased and the cells were spindle-shaped. Furthermore, the cytoplasm-to-nucleus ratio decreased, pigment granule deposition increased, growth speed decreased and the cells were not readily adherent. Therefore, the third to fifth generation of HTMCs were used in the present study.

The cell viability in control wells was considered to be 100% and cell inhibition rates in the other groups were calculated using the following formula: [optical density (OD) of vehicle wells-OD of experimental wells)/(OD of vehicle wells-OD of mock wells) × 100. The results revealed that cell viability was significantly decreased in all wells treated with Hcy compared with the vehicle (P<0.01; Table I and Fig. 2C). Hcy appeared to inhibit cell proliferation, with higher Hcy concentrations inducing higher inhibition rates.

The JC-1 fluorescent probe, which has an excitation wavelength of 488 nm and a monomer emission wavelength of 525 nm, is able to enter cells and localize to the mitochondrial membrane (13). Depending on changes in the MMP, the probe emits different colors of fluorescence. In cells with higher MMPs, JC-1 forms an aggregate in the mitochondrial matrix and produces red fluorescence. At lower MMPs, the probe produces green fluorescence. The results of flow cytometry revealed good HTMC activity and normal MMPs in vehicle wells (Fig. 3); in these cells, JC-1 accumulated in the AY2 region and exhibited strong red fluorescence. Compared with the vehicle group, JC-1 accumulation in the AY2 area decreased in HTMCs following treatment with Hcy for 48 h; these changes occurred in 71.2, 55.6, 44.8 and 23.9% of total cells in the 30, 100, 300 and 1,000 µmol/l Hcy group, respectively. These results indicated that the concentration of JC-1 aggregates decreased and the MMPs decreased.

ROS were detected using flow cytometry and the results demonstrated that the ROS levels in groups treated with 30–1,000 µmol/l Hcy significantly increased compared with the vehicle group (P<0.05; Fig. 4A).

Furthermore, the expression of the oxidative stress marker SOD1 in HTMCs was detected following treatment with different concentrations of Hcy. The results revealed that SOD1 expression in HTMCS decreased following Hcy treatment in a dose-dependent manner (Fig. 4B; P<0.05).