Cell Culture and Treatment
Ovarian GCs were obtained from patients undergoing IVF-ET due to fallopian tube factors in the Reproductive Department of the Second Hospital of Hebei Medical University, and were approved by the ethics committee (Shijiazhuang, China) and informed consent of the patients. The follicular fluid containing GCs were centrifuged at 4˚C (2000rpm, 10min). Remove the upper follicular fluid, and 5ml PBS was added into the below sediment and mixed them. 5ml human lymphocyte separation fluid (Lympholyte-H, Cedarlane Laboratories, Canada) was added to another 10ml centrifuge tube and inclined angle of 45°. PBS suspension was slowly added to the surface of the human lymphocyte separation solution, and then centrifuged at 4˚C (2500rmp, 10min). The white floc in the middle layer is GCs.
The GCs were cultured in DMEM/F12 medium (Gibco, Thermo, USA) at 37˚C in a 5% CO2 incubator. After the cells adhered to the wall, the cells were incubated with H2O2 for 24 h after the different concentrations morroniside ranging from 5 to 20μM pretreated for 24 h.
GCs were pretreated with different concentrations of morroniside and H2O2 which determined by CCK-8 assay kits (MCE, China). 96-well plate cells were added with 10μl CCK-8 reagent per well and incubated at 37℃ for 2 h. The OD value of each hole was read at 450nm with a microplate reader (Vesar Max, Santak, USA).
Intracellular ROS Detection
Intracellular ROS levels in each group were detected with ROS assay kit (Beyotime, China). The culture medium containing serum was removed, the cells were incubated with diluted DCFH-DA (1:1000) for 20 min at 37℃ in 5% CO2 incubator and then washing three times with serum-free medium. ROS content was detected by fluorescence microscope (EVOS® FL, Thermo, USA) and fluorescence intensity was analyzed by Image J software.
The ovarian GCs sample lysis fluid was diluted to the optimal concentration. The biomarkers related to oxidative stress and apoptosis, including ROS, malondialdehyde (MDA), 8-OHdG, total antioxidant capacity (T-AOC), SOD, NAD(P)H quinone oxidoreductase 1 (NQO1), and caspase-3, were detected by ELISA assay kit (Jianglai Biological Co., Ltd, Shanghai, China; Jiancheng Bioengineering Institute, Nanjing, China; Abcam, USA; Tongwei Industrial Co., Ltd, Shanghai, China) following the manufacturer’s instructions. The absorbance values were measured by the microplate reader (VersaMax, Molecular Devices, USA).
Western blot analysis
The collected GCs were lysed to extract total protein and the protein concentration was determined using BCA protein assay kit (SolarBio, Beijing, China). The same amount of total protein in each well (10-15 μg) was separated by 10% SDS-PAGE and transferred to PVDF membrane (Millipore, Merck KGAA, Darmstadt, Germany) at the end of electrophoresis. The PVDF membrane was sealed in 5% skimmed milk powder for 2 h and incubated with different primary antibodies overnight at 4℃. The primary antibodies used in this study mainly included 1:1000 SOD (ab68155, Abcam, USA), 1:1000 NQO1(ab80588, Abcam, USA), 1:1000 Bax (ab32503, Abcam, USA), 1:1000 Bcl-2 (26593-1-AP, Proteintech, USA), 1:1000 caspase-3 (YT6113, Immunoway, USA), 1:1000 caspase-9 (ab202068, Abcam, USA), 1:500 p-Nrf2 (ab76026, Abcam, USA), 1:500 Nrf2 (ab62352, Abcam, USA), 1:500 p-ERK (YP0101, Immunoway, USA), 1:500 ERK (16443-1-AP, Proteintech, USA), 1:500 JNK (66210-1-lg, Proteintech, USA), 1:500 p-JNK (YP0156, Immunoway, USA), 1:500 p38 (ab181602, Abcam, USA) and 1:500 p-p38 (ab4822, Abcam, USA). The PVDF membrane was washed with TBST for 3 times and incubated with the secondary antibody (SA00001-2, Proteintech, USA) and incubated for room temperature for 1 h. After the PVDF membrane was washed three times with TBST, antibody-antigen complexes were visualized using a Chemiluminescence Plus Western immunoblot analysis kit (Millipore, USA).
The treated GCs were fixed with 4% paraformaldehyde for 20min, drilled with 1% Triton and sealed with 10% goat serum for 30min, and then incubated overnight with Nrf2 antibody (1:200) at 4℃. After the cells were incubated with fluorescence-labeled secondary antibodies at room temperature for 2 h, the cells were stained with DAPI for 10min. Finally, it was observed under a laser scanning confocal microscope (Leica, Germany).
All data were expressed as mean ± standard deviation and statistical analysis was performed using SPSS 21.0 software (SPSS Inc., Chicago, IL, USA). Multiple groups comparison was performed by ANOVA and post-hot analysis. Values of P < 0.05 were considered statistically significant.