Study participants and preparation of the central area of the anterior lens capsule
We selected cortical cataract (the most common clinical phenotype of ARC) patients as the study subjects. A total of 30 eyes of patients (male: 13 cases of 13 eyes, female: 17 cases of 17 eyes) with age-related cortical cataract treated by operation in our hospital from January 2016 to December 2017 were collected under the following standards: (1) the age ranged from 55 to 65; (2) non-congenital cataract, non-metabolic cataract, and non-secondary cataract; (3) without hypertension, diabetes mellitus, fundus lesions, uveitis, and glaucoma; (4) no eye trauma and the history of intraocular surgery. In the study, the normal subjects (the total of 30 eyes, male: 18 cases of 18 eyes, female: 12 cases of 12 eyes) with transparent lens were enrolled from corneal transplant donors and ocular trauma patients with lens detachment, which the age range was from 46 to 55. We declared that the study followed the tenets of the Declaration of Helsinki, and was approved by the Ethics Committee of Mudanjiang Medical University. All subjects in this study knew and understood the content and risk of the research and signed the informed consent.
The central area of the anterior lens capsule (including normal people and ARC patients) were obtained by a single ophthalmologist during surgery using the intact continuous curvilinear capsulorhexis method. After the tissue samples were separated, it was immediately placed in 4% paraformaldehyde or liquid nitrogen. Tissue samples placed 4% paraformaldehyde at room temperature were used for HE analysis and immunohistochemical staining, while those stored in liquid nitrogen were used for protein and RNA extraction.
Establishment Of Animal Model And Isolation Of Lenses
The animal model is based on our previous research (Su et al. 2017a) and has been modified. 30 Young (3 months) and 30 old (> 24 months) male BALB/c mice were housed with free access to food and water at a mean ± SD constant temperature of 22 ± 2℃, the humidity of 55 ± 5%, and a 12 h light/12 h dark cycle, and their eyes were observed daily. Partial opacity of lens was observed over 24 months, and the eyes photos were taken. This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The experimental protocol was approved by the National Institutes of Health Guide for Care and Use of Laboratory Animals.
The mice were euthanized by cervical dislocation. The entire eye of each mouse was removed, its cuticles were placed on sterile gauze face down, and fixed with tweezers. An incision is then made on the of the optic nerve that enters the eye, and the sclera is pulled back to expose the lens. Before staining, use a pair of tweezers to gently lift the ciliary body fragments attached to the equator plane of the lens.
Hematoxylin and eosin (H&E) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay
Samples were fixed with 4% paraformaldehyde and embedded in paraffin, and the thickness of the sections was 4 µm. The prepared paraffin sections were dewaxed and hydrated with xylene and graded alcohols. The sections were washed with phosphate-buffered saline (PBS, South Logan, UT, USA), and then stained with hematoxylin and eosin, and images were captured using Nikon Eclipse microscope.
In the TUNEL assay, a kit (Roche, Basel, Switzerland) was used for in situ cell death detection according to the manufacturer's instructions. Briefly, sections were incubated with TUNEL labeling solution at 37℃ for 1 hour and stained with Hoechst 33342 (Sigma-Aldrich, St. Louis, MO, USA). Sections were analyzed with a Nikon Eclipse fluorescence microscope.
Immunohistochemical (IHC) staining
The specific method of Immunohistochemical (IHC) staining has been described in our previous article (Su et al. 2017b). After paraffin sections were incubated at 60 degrees for 2 hours, they were dewaxed and hydrated with xylene and graded alcohol. After washing three times with PBS, it was boiled in 0.1 M citric acid (pH 6.1) for 30 minutes and cooled to room temperature. PBS has washed again and soaked with 0.3% H2O2 to inhibit endogenous peroxidase activity. Sections were incubated with normal fetal bovine serum (FBS, GIBCO, Grand Island, NY, USA) for 30 min and then treated with anti-ROCK1 (1:1000, Abcam, ab45171), anti-p53 (1:500, Cell Signaling Technology, 2524), and anti-phospho-p53 (1:20, Abcam, ab1431) antibodies overnight. The next day, the corresponding secondary antibody conjugated to horseradish peroxidase were incubated for 1 hour. Sections were incubated in a peroxidase substrate solution (diaminobenzidine hydrochloride, DAB, Cell Signaling Technology, Danvers, MA, USA). The control group and the experimental group were simultaneously dripped with an equal amount of DAB solution. After coloring, the two were placed in PBS at the same time to stop the color development, to ensure the same staining time, and avoid the differential expression of results due to the time interval. After that, xylene and fractionated alcohol were dehydrated again, and then the sample was covered with paramount. Immunostaining images were captured using a Nikon Eclipse microscope.
Frozen lens tissues from humans and mice were extracted in RIPA lysed buffer with protease inhibitor cocktail (Pierce, USA). Total protein (40 µg) was applied to a 10% SDS-polyacrylamide electrophoresis gel. We use molecular mass marker protein standard (Biotides, China) as a guide, and cut the gel horizontally to approximate the size of the protein to contain the protein of interest. To save protein, we cut different blots on the same group of protein gels to detect proteins of different molecular weight. The polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA) was incubated with the primary antibody. Although Western blotting uses the same antibodies as IHC, the use ratio has changed, as follows: anti-ROCK1 (1:2000), anti-p53 (1:500), anti-phospho-p53 (1:100) and anti-β-actin (1:1500, Sigma-Aldrich, St. Louis, MO, USA). Observation of immune response bands by chemiluminescence substrate method with a super signal western pico kit (Pierce Co, USA). Three independent experiments were performed. Protein quantitative was analyzed using Image J software.
Cell culture and plasmids
Human lens epithelial B3 (HLE-B3) cells were donated by Professor Qi (Harbin Medical University) and cultured in Dulbecco's modified Eagle's medium (DMEM, GIBCO, Grand Island, NY, USA) with 20% FBS, and penicillin-streptomycin (1:100, Sigma-Aldrich, St. Louis, MO, USA) in a humidified atmosphere containing with 5% CO2 at 37℃. ROCK1 over-expression plasmids were purchased from the Polepolar Research Company (China). The small interfering RNA (siRNA) targeting sequences of ROCK1 was 5’-GAAGAAACATTCCCTATTC-3’ (Liebig et al. 2009), and the sequences of p53siRNA was 5’-GACTCCAGTGGTAATCTAC-3’ (Ding et al. 2007). The control siRNA sequence was 5’-CGTCAACATGGCTTTCACC-3’. The ROCK siRNA and p53 siRNA plasmids were constructed as we did in the previous articles (Su et al. 2017b).
H2O2 treatment and transfection
Human lens epithelial cells B3 cells at 80% confluency were treated with H2O2 at the indicated concentrations (200 µM). Transient transfection B3 cells were performed using the Lipofectamine 3000 (Invitrogen). DNA plasmids were transfected into B3 cells by using lipofectamine 3000 under manufacture procedures. Cells were starved when transfected with a plasmid (1.25 µg/ml), H2O2 (200 µM) was added after 1 hour and 20% FBS was added 4 hours later.
As our previous study (4), the 3- (4,5-dimethylthiazole-2-yl) -2,5-diphenyltetrazole ammonium bromide (MTT) assay was used to determine the cell survival rate. After treatments or transfection, cells were incubated with 20 µl MTT (5 mg/mL) solution for 4 h. Then, the medium was aspirated and cells were dissolved with 150 µl of dimethyl sulfoxide. The absorbance was examed with a microplate reader at 490 nm.
Hoechst 33342 (Sigma-Aldrich, St. Louis, MO, USA) was used to observe the morphological changes of the nucleus and evaluate the apoptosis. Briefly, cells were cultured in a 6-well plate for a certain period time and then stained with Hoechst 33342. Images were selected using a fluorescence microscope, 5 fields were randomly selected to count the number of apoptotic nuclei.
Rna Extraction, Reverse Transcription, And Quantitative Rt-pcr
Total cellular RNA was extracted from B3 cells using the Promega Total RNA Isolation System according to the manufacturer’s instructions. Reverse transcription-polymerase chain reaction (RT-PCR) was performed using the Access RT-PCR System (Promega). The ROCK1 gene primer pairs for quantitative PCR assay were as follows: sense, 5′- TGTGACTGGTGGTCGGTT-3′ and antisense, 5-′GGTTTTTTGCTTCTTTT′G-3. Primer pairs for β-actin were as follows: sense, 5′- TCGTGCGTGACATTAAGGAG-3′, and antisense, 5′-ATGCCAGGGTACATGGTGGT′-3.
Student t-test and ANOVA analyses were used to calculate the statistical significance of the experimental data. Significance level were set as *p < 0.05; #p < 0.05; **p < 0.01; ##p < 0.01. Error bars denote SD.