Salidroside (#HY-N0109) and Compound C (#HY-13418A) were purchased from MedChemExpress (China); DHT (#S4757) was purchased from Selleck (China), and AMPK siRNA (#sc45312) and negative control siRNA (#sc-37007) were purchased from Santa Cruz Biotech. Nrf2 siRNA and RNATransMate (#E607402) was purchased from Sangon Biotech (China). The nuclear and cytoplasmic extraction reagent (#78833) was obtained from ThermoFisher (USA). The Mitochondrial Membrane Potential Assay Kit with JC-1 (#C2006), Annexin V-PE Apoptosis Detection Kit (#C1065L), and Cell Counting Kit-8 (CCK-8; #C0037) were obtained from Beyotime Biotechnology (China).
Cell culture and treatment
The KGN cells were provided by the RIKEN BioResource Research Centre (Tsukuba, #RCB1154, Japan). The cell line was cultured in DMEM/F12 medium (Gibco, #11320033, USA) in the presence of 10% FBS (Gibco, #10100, USA) and 1% PS (Servicebio, #G4003-100ML, China) at 37℃ under 5% CO2 atmosphere. The KGN cells were then treated with DHT (500 nM) as a PCOS cell model, which been used in reveral studies3, 7. To detect the effects of salidroside, the KGN cells were incubated with salidroside at different concentrations for 24 h.
Cell viability assay
Cell viability was assessed by using the Cell Counting Kit-8 (CCK-8) assay (Beyotime Biotechnology, #C0037, China). The KGN cells were seeded in 96-well flat-bottomed plates at 3 × 103 per well, treated with DHT, low or high doses of salidroside and incubated for 24 h or 48 h, followed by incubation with cck8 (10 µL/well) for 1.5 h, and the absorbance was read at 450 nm on a microplate reader (Perkin Elmer, USA).
The KGN cells were cultured at the density of 70–90%, and then AMPK siRNA, Nrf2 siRNA, or the negative control siRNA were transfected with the RNA TransMate (Sangon Biotech, #E607402, China) to silence the AMPK and Nrf2 according to the manufacturer’s protocol. Next, the cells were incubated for 48 h before testing. The siRNA sequence of Nrf2 was 5’-UUGUGUUUAGUGAAAUGCCGG-3’ (Nrf2 siRNA-1), 5’-UAUAUCCCGAAUUAAUGCAAG-3’ (Nrf2 siRNA-2), and 5’-UUGCCAUCUCUUGUUUGCUGC-3’ (Nrf2 siRNA-3).
ROS was detected with the ROS Assay Kit (Beyotime Biotechnology, #S0033S, China). The KGN cells were digested by Trypsin–EDTA (0.25%) (Gibco Life Technologies, #25200056, USA) and resuspended in a medium with 5 μM dihydroethidium (DHE). After 30 min of incubation in dark at 37℃, the KGN cells were washed with phosphate-buffered saline (PBS; Gibco Life Technologies, #10010023, USA), and the production of ROS was assessed by flow cytometry (Beckman , BC43326, USA).
Detection of cell apoptosis
Apoptosis of the KGN cells was detected with the Annexin V-PE Apoptosis Detection Kit (Beyotime Biotechnology, #C1065L). After treatment, the KGN cells were collected with 2.5% trypsin (Gibco Life Technologies, #15090046, USA) and washed twice with PBS. Next, 0.5 mL of the binding buffer was added to resuspend the KGN cells. Then, the resuspended cells were incubated with 5 μL of annexin V-FITC and 10 μL of propidium iodide (PI) in the dark at 37℃ for 15 min. The percentage of apoptotic KGN cells was then calculated by flow cytometry.
Cell immunofluorescence staining
The KGN cells were seeded into a 6-well plate and fixed with 4% paraformaldehyde for 5 min, after which 0.2% Triton X-100 in PBS was used for permeation and 10% normal goat serum was used to block the cells. Then, the cells were incubated with an anti-Nrf2 antibody (1:200; Abcam, #ab137550, UK) overnight at 4℃. The cells were washed with PBS and incubated with a secondary antibody Cy3 conjugated goat anti-rabbit IgG (H+L) for 1 h. 4',6-Diamidino-2-phenylindole (DAPI) was used to label the nuclei. Images were visualized by using a fluorescence microscope (Olympus BX6 with DP72 Camera, Japan).
Mitochondrial Membrane Potential Detection
The Mitochondrial Membrane Potential (MMP) Assay Kit with JC-1 (Beyotime Biotechnology, #C2006, China) was used to analyze the mitochondrial depolarization according to the manufacturer’s protocol. The cells were incubated with JC-1 solution for 20 min and then washed with the JC-1 staining buffer. Fluorescence microscopy (Olympus IX73P2F, Japan) was used to analyze the MMP and the corresponding images were assessed by the Image-J software. The red/green fluorescence intensity ratio was used to calculate the changes in the MMP.
The protein concentrations of the KGN cell lysates were detected by using the BCA protein assay kit (Beyotime Biotechnology, #P0010S, China). The nuclear proteins of the KGN cells were extracted by using the Nuclear and Cytoplasmic Protein Extraction Kits (Beyotime Biotechnology, #P0027, China). The proteins were separated on the SDS-PAGE gels and transferred onto PVDF- membranes. The membranes were blocked with skimmed milk for 2 h and then incubated at 4℃ overnight with primary antibodies against Nrf-2 (1:1000; Abcam, #ab62352, UK), HO-1 (1:2000; Abcam, #ab52947, UK), NQO1 (1:10000; Abcam, #ab80588, UK), AMPK (1:1000; Abcam, #ab133448, UK), phosphor-AMPK (1:1000; Abcam, #ab80039, UK), AKT (1:1000; Proteintech, #10176-2-AP, China), phosphor-AKT (1:2000; Proteintech, #66444-1-Ig, China), ERK (1:2000; Proteintech, #67170-1-Ig, China), phosphor-ERK (1:2000; Cell Signalling Technology, #4370, USA), JNK (1:1000; Proteintech, #24164-1-AP, China), phosphor-JNK (1:1000; Proteintech, #80024-1-RR, China), p38 (1:500; Proteintech, #14064-1-AP, China), phosphor-p38 (1:1000; Abcam, #ab178867, UK), cleaved caspase-3 (Abcam; 1:500, #ab32042, UK), cleaved caspase-9 (1:1000; Abcam, #ab2324, UK), Bax (1:5000; Proteintech, #50599-2-Ig, China), Bcl2 (1:1000; Proteintech, #12789-1-AP, China), Histone 3 (1:1000; Proteintech, #17168-1-AP, China), and GAPDH (1:20000; Proteintech, #60004-1-Ig, China). Then, horseradish peroxidase-conjugated secondary antibodies were used to incubate the membranes for 1 h, and the bands were visualized by enhanced chemiluminescence (ECL).
All data were analyzed with the GraphPad Prism 8 software. All quantitative data were presented as mean ± standard deviation (SD). Multiple comparisons were used by one-way ANOVA, followed by Tukey’s posthoc test. P ＜ 0.05 was considered to be statistically significant.