Materials
Curcumin, Gastrodin, Propylgallate, Adenosine and H2O2 were purchased from Sigma (C1386, SBM00313, 48710, A9521, 323381, St. Louis, MO, USA).Dulbecco’s modified Eagle’s medium (DMEM, Gibco™,11965092,USA),Fetal bovine serum (FBS, Gibco™,10099141,USA),penicillin/streptomycin(Gibco™,15140122,USA) were purchased from Thermo Fisher Scientific.Trypsin was ordered from Invitrogen (Grand Island, 15050065,NY, USA). Cytotoxicity LDH Assay Kit(abam,ab65393,UK) and MTT t(abam,ab211091,UK) were purchased from abcam and Apoptosis Detection kit was purchased from Beyotime Institute of Biotechnology (Beyotime ,C1062,Nantong, China).
Culture of RGC-5 Cells
RGC-5 cells(ATCC,2CM3085) were grown in DMEM medium, supplemented with 10% FBS, 1% penicillin/streptomycin. RGC-5 cells were cultured in growth medium and incubated at 37°C in 5% CO2.
Cellular oxidative stress model
The oxidative stress model of RGC-5 cells was established by treating RGC-5 cells with 20µM, 25µM, 100µM, 500µM H2O2 treated for 24 hours.
Proliferation Assay
The effects of curcumin, gastrodin, propylgallate, adenosine on the proliferation of RGC-5 cells under oxidative stress condition were detected by MTT tests. The RGC-5 cells were seeded in 96‑well plates at concentration of 2000 cells/ml. The cells were pretreated with different concentration of Curcumin (10, 20, 40µM), gastrodin (20, 40, 60µM), propylgallate (5, 10, 20 µM), adenosine (10, 20, 40 µM) for 2 hours, then treated with 500µM H2O2 for 24 hours. Cells without any treatment and cells with the treatment of 500µM H2O2 for 24 hours as control. Following, 10µm MTT was added to each well and cultured for 3 hours. Then the medium was removed and 100 µm DMSO was added to each well. Finally, the plates were read immediately on a plate reader at a test wavelength of 490 nm.
Cytotoxicity Assay
The cytotoxicity of curcumin, gastrodin, propylgallate, adenosine were detected by Cytotoxicity LDH Assay Kit following the manufacturer’s instructions. The RGC-5 cells were seeded in 96‑well plates at concentration of 2000 cells/ml. The cells were pretreated with different concentration of Curcumin (10, 20, 40µM),Gastrodin(20, 40, 60µM ),Propylgallate(5, 10, 20 µM), Adenosine(10, 20, 40 µM) for 2 hours, then treated with 500µM H2O2 for 24 hours.Following, LDH release agent was added, and the cells were incubated for another 30 minutes, and then 50µL of stop solution to each sample well and mix by gentle tapping. Finally, the plates were read immediately on a plate reader at a test wavelength of 490 nm.
Apoptosis assay
The apoptosis levels of the RGC-5 cells treated with different concentration drugs were subsequently analyzed using an Annexin V‑fluorescein isothiocyanate (FITC) Apoptosis Detection kit according to the manufacturer's instructions. The cells were seeded in 6‑well plates at a density of 1x105 cells/well and pretreated with different concentration of Curcumin (10, 20, 40µM),Gastrodin (20, 40, 60µM),Propylgallate (5, 10, 20 µM),Adenosine (10, 20, 40 µM) for 2 hours, then treated with 500µM H2O2 for 24 hours.Then cells were then digested with 0.25% trypsin and resuspended in 300µl binding buffer containing 5µl Annexin V‑FITC and 5µl propidium iodide solution, and incubated at room temperature in the dark for 20 min. The stained cells were analyzed by flow cytometry (FACScan; BD Biosciences, Franklin Lakes, NJ, USA).
Apoptosis PCR array
An mouse Apoptosis PCR array (PAMM-012A,Qiagen,Frederick, MD., USA) was used to screen a panel of 84 genes representative of apoptosis in RGC-5 cells cell under Propylgallate treated. Total RNA was isolated from RGC-5 cells,RGC-5 cells treated with H2O2, RGC-5 cells treated with H2O2 + Propylgallate groups using Qiagen RNeasy Mini Kit by following manufacturer’s protocol.The first-strand cDNA was mixed with 2 × RT² SYBR Green qPCR Master Mix and ddH2O.The qPCR was performed on an Applied Biosystems (ABI) 7500 according to the RT 2 Profiler PCR Array instructions under the following conditions: 95°C for 10 min, then 40 cycles at 95°C for 15 sec and 60°C for 1 min.Microarray data was normalized against the house keeping genes by calculating the ΔCt for each gene of interestin the plate.Fold changes of gene expression, scatterplot and heatmap were analyzed and generated by using RT2PCR array data analysis web portal version 3.5(http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php).
Western Blot
Whole protein was isolated from RGC-5 cells,RGC-5 cells treated with H2O2, RGC-5 cells treated with H2O2 + Propylgallate groups using Protein extraction kit(Beyotime,P0028,China) by following manufacturer’s protocol.The expression of Caspase-3, Caspase-8(19677-1-AP,66093-1-Ig,proteintech) and Caspase-9(CST,#9508) in RGC-5 cells,RGC-5 cells treated with H2O2, RGC-5 cells treated with H2O2 + Propylgallate groups were detected by SDS-PAGE.
Statistical analyses
Data are presented as mean ± standard deviation. SPSS software (v. 20.0, SPSS, Chicago, IL) was used for analysis, and differences among the groups were assessed using one-way ANOVA. p values < 0.05 indicated statistical significance. p values < 0.01 indicated a significant difference.