Cell culture
Human retinal microvascular endothelial cells (HRMECs)) were cultured in M199 medium supplemented with 20% fetal bovine serum in a humidified incubator containing 5% CO2 and maintained at 37°C. 30mmol/L D-glucose was added to the medium to form a HG environment to construct a DR cell model. 5mmol/L NAC and/or 0.5mmol/L TAU were added to treat DR.
Mtt Assay
Cells were collected and resuspended, and seeded at a density of 5×104/ml in 96-well plates treated with HG, NAC and/or TAU. After incubation in the incubator for 24, 48 or 72h, 10ul of MTT solution was added to each well and incubated for 4h. The culture supernatant was discarded, Formazan lysate was added and shaken for 10min, and the absorbance at 570 nm was measured with a microplate reader.
Tube Formation Assay
Melt Matrigel at 4°C, spread 10ul Matrigel in the lower well of the angiogenic slide, put the wet box with slides into the incubator for 30min and wait for the gel to solidify. Prepare a cell suspension with a density of 2×105 cells/ml, add 50ul the suspension to the upper well of the slide, incubate at 37°C, collect images regularly and analyze the tube formation ability of the cells.
Wound-healing Assay
Cells were seeded in 6-well plates at a density of 5×105 cells per well, and when cells reached 90% fusion, monolayers were disrupted along a straight line with a 20ul pipette tip, washed with PBS, and replaced with fresh medium. After 24h incubation, cells were observed under a microscope and photographed.
Flow Cytometry Analysis For Apoptosis
Cells were treated with appropriate amount of trypsin, washed with pre-cooled PBS and collected. 195µl binding buffer was added to gently resuspend the cells, then 5µl Annexin V-FITC and 10ul PE were added and gently mixed. The cells were incubated for 20min at room temperature and protected from light, and then placed in an ice bath for timely detection on a flow cytometer.
Western Blot
Cells were lysed using RIPA lysate and total protein was retained. Proteins were separated by SDS-PAGE electrophoresis and transferred to PVDF membranes. The membrane was subsequently closed with 5% skim milk for 2h and incubated with primary antibody at 4°C overnight. The bands were washed three times with TBST for 15 min each time and incubated with secondary antibody at room temperature for 2 h. The bands were analyzed using Image Lab software. GAPDH was used as an internal reference.
Establishment Of Dr Rat Models
50 healthy adult rats were randomly divided into 5 groups: control (Con), untreated (UD), NAC-treated (NAC), TAU-treated (TAU) and combined-treated (N + T) groups. DR model rats were injected intraperitoneally at a dose of 60mg/kg of streptozotocin (STZ) (dissolved in 0.1mol/L sodium citrate buffer). Control rats were given the same dose of sodium citrate buffer. 7 days after STZ injection, rats with blood glucose concentration ≥ 16.7 mmol/L were used as DR rats.
He Staining
The rat retinas were prepared into paraffin sections. The sections were first dewaxed using xylene and then rehydrated with different concentrations of alcohol. Hematoxylin staining was done for 5 min followed by eosin staining for 1min. Then the sections were dehydrated with alcohol, treated with xylene for 1min, dried naturally and sealed with neutral resin.
Tunel Assay For Apoptosis
Fix rat retinal cryosections with 4% paraformaldehyde for 1h. Wash 2 times with PBS for 10min each. PBS containing 0.5% Triton X-100 was added and incubated for 5min at room temperature. Prepare TUNEL solution according to the instructions, cover the tissue with the solution, and incubate at 37°C for 1h. After sealing the slice with anti-fluorescence quenching sealing solution, observe the slice under a fluorescence microscope.
Statistical analysis
The data are presented as the mean ± SD. SPSS 20.0 was used for statistical analysis. Comparisons between groups were analyzed by one-way ANOVA and between two groups by t-test. P < 0.05 was considered to be statistically significant.