2.1. Human Lens Extracts

Human lens samples including lens capsules (mainly composed of LECs) and the cortex were obtained from Department of Ophthalmology, Affiliated Hospital of Nantong University. The lens tissues from 390 patients with different subtypes of ARC during phacoemulsification were collected. The clinical diagnosis of ARC is based on criteria of the Lens Opacities Classification System II (LOCS II) [20]. We also included 88 age-matched individuals without cataract but had their lens extracted because of vitreoretinal diseases as control during extracapsular cataract extraction. Patients with the following conditions were excluded: other eye diseases such as high myopia, dislocated lens, glaucoma, uveitis, and diabetic retinopathy and systemic diseases such as hepatic dysfunction, diabetes, cancer, uncontrolled hypertension, hyperlipemia, obstructive sleep apnea, and nervous system diseases such as Alzheimer’s disease. All of the extracts were preserved in −80°C until further analysis. Ethical permission was obtained from the Medical Ethics Committee of Affiliated Hospital of Nantong University (No. 2012(021)), and the informed consent was received from all patients according to the tenets of the Declaration of Helsinki.

2.2. Rat Lens Extracts

96 male Sprague Dawley rats (SD rats) at approximately 4 weeks of age, weighing 150–180 g with normal lens were provided from the Laboratory Animal Center of Nantong University. Rats were housed in solid bottom cages in an environmentally controlled room (20 ± 2°C, 50 ± 15% humidity) on a 12 h light/dark cycle and freely accessed standard laboratory chow and water. Before extracting lens, the rats were anesthetized by intraperitoneal injection of 80 mg/kg pentobarbital sodium. The eye globes were collected immediately after sacrifice. All experimental procedures were performed in accordance with the requirements of Animal Care and Use Committee of Nantong University.

2.3. Western Blot

The protein of human LECs, cortex, and rat lens in each group were extracted in RIPA lysis buffer III (Sangon Biotech, China) as per the instructions. After ultrasonication, the specimens were centrifuged at 12000 rpm (15 min, 4°C), and the supernatant was collected. Equal amounts of lysates (100 μg/lane) were separated on 10% SDS-PAGE and transferred to PVDF membranes. After blocking in 5% skimmed milk (TBST as vehicle) for 2 h, the membranes were incubated with rabbit anti-LSS antibody (1 : 500, Abcam, UK) and mouse anti-GAPDH antibody (1 : 5000, ABclonal, France) overnight at 4°C. After washing, the membranes were incubated with goat HRP-conjugated secondary antibodies (Jackson ImmunoResearch, USA). Proteins were visualized using an ECL system (Cell Signaling, USA). Relative level of proteins was quantified with ImageJ software (National Institutes of Health, USA).

2.4. Enzyme-Linked Immunosorbent Assay (ELISA)

The LSS level of human LECs and the cortex of cultured lens were quantified by ELISA using human LSS ELISA kit (QIYBO, China) according to the manufacturer’s instructions. The optical density was read at 450 nm wavelength using a microplate reader (ELx800, BioTek, USA).

2.5. Lens Explant Culture and Reagent

The lenses extracted from the globes were cultured in M199 medium (Gibco, Rockville, MD) supplemented with 100 U/ml penicillin and 0.1 mg/ml streptomycin in a 75% humidified 37°C incubator with 21% O₂ and 5% CO₂ for 1 day in 24 well cell culture clusters. Lenses without opacification were then randomized into five groups with 3 lenses or more per treatment: untreated control, vehicle (DMSO) control, U18666A treatment, lanosterol treatment, and U18666A and lanosterol treatment. U18666A solution was prepared using DMSO as the solvent with the final concentration of 200 nM in culture media. Lanosterol solution was prepared using M199 medium with the final concentration of 40 μM. Final medium osmolality of each group was maintained from 295 to 310 mOsM/kg. The medium was replaced every 24 h. The opacity of lens was image analyzed at 1, 3, and 5 days. At the end of culture, the lenses were weighed and preserved in −80°C for further experiments.

2.6. Lens Transparency Assessment

For imaging, an antibody incubation box which had been high-pressure steam disinfected was placed in vertical clean bench. To observe the transparency of lens, we sprayed 75% alcohol on vertical clean bench as much as possible. 24 well cell culture clusters were taken from incubator and placed on the antibody incubation box carefully, making sure there were no air bubbles but only 75% alcohol between the clusters and antibody incubation box. The image was captured with the Nikon D90 camera under the largest magnification. In this way, the transparency of total lens area was observed, with the black image representing transparent lens, gray image representing opaque lens.

2.7. Immunofluorescence Assay

The cultured rat lens was fixed with chloroform fixative overnight and sugar dehydrated followed by frozen section. After permeabilization and blocking in 3% fetal bovine serum with 0.3% TritonX-100 for 30 min, the sections were incubated overnight at 4°C with IgG (negative control), anti-LSS antibody (1 : 500, Abcam, UK), and anti-KDEL antibody (1 : 250, Abcam, UK). After washing, tissue sections were incubated with FITC-conjugated (1 : 250) or Cy3-conjugated secondary antibody (1:1000) (Jackson ImmunoResearch) for 2 h at 37°C.

2.8. Statistical Analysis

All data were presented as means ±SD. SPSS software (SPSS 17.0; SPSS Inc., USA) was used for statistical analysis. One-way ANOVA was used for statistical comparisons of multiple groups. The difference between two groups was determined by Student’s t-test. Statistical significance was based on a P value < 0.05. Each experiment consisted of at least three replicates.

3.1. LSS and Lanosterol Level Changes in LECs and Cortex of Human ARCC

To detect LSS level in LECs and the cortex in human ARC tissues, western blot was performed, showing that LSS changed along with the cataract levels in LECs of human ARCC, increasing at C II and decreasing at C IV and C V. While in the cortex of human ARCC, LSS decreased with the aggravation of opaque degree (Figures 1(a) and 1(b)). Furthermore, ELISA showed that LSS had the similar change trends in different types of human ARC tissues (Figure 1(c)). However, there were no significant changes of LSS level in either LECs or the cortex of human ARNC and ARPSC (Figures 1(d)–1(i)).

Lanosterol, the catalyst of LSS, was down-regulated in LECs and the cortex of human ARCC, especially in C IV and C V groups, decreasing in LECs from C IV to C V and in the cortex from C III till C V (Figures 2(a) and 2(b)). However, lanosterol changed slightly in LECs and the cortex of human ARNC and ARPSC (Figures 2(c)–2(f)). The data were consistent with the results of LSS level, thus ARCC was chosen for subsequent experiments.

3.2. LSS Pathway Delays Lens Opacity

To further identify the function of LSS pathway in the formation of ARCC, LSS inhibitor U18666A was used in lens culture system in vitro. U18666A treatment adversely affected the transparency of explanted rat lens within 3 d of exposure, indicated by blurring in the cortical area, while lanosterol effectively delayed the occurrence of lens opacity, but not to prevent opacity (Figure 3). After U18666A treatment, LSS expression decreased in opaque rat lens in comparison to transparent ones (Figures 4(a) and 4(b)). LSS is a membrane-associated enzyme, which is targeted to the cytoplasmic leaflet of endoplasmic reticulum (ER) [21]. Immunostaining showed colocalization of LSS with Lys-Asp-Glu-Leu (KDEL; an ER marker, ER retention sequence) [22] and the level of LSS markedly decreased after U18666A treatment (Figure 5).

Microscopy showed that there was a breakdown of structural integrity with extensive disruption in the cortical fiber cells of the opaque lens following U18666A treatment. This disrupted cortical region was completely different from nuclear region, where the fiber cells remained in order. Lens treated with lanosterol suggested slight fracture in the cortex (Figure 6).