Materials

The KGN human ovarian granulosa cell line used in this study was purchased from Wuhan Punosai Life Science and Technology Co., Ltd. The afamin protein was purchased from BIO-techne (USA), and fetal bovine serum was purchased from Clark (USA). Dulbecco’s Modified Eagle’s Medium/Nutritional Mixture F-12Ham, trypsin, dimethyl sulfoxide, and TriPure Isolation Reagent were purchased from Sigma (USA). Trypsin, dimethyl sulfoxide (DMSO), and TriPure Isolation Reagent were purchased from Sigma. Takara (Japan) supplied the RNA reverse transcription kit.

Cell culture and processing

The experiment used cells derived from GCs of patients with invasive ovarian cancer, specifically the human ovarian granulosa tumor cell line (KGN). KGN cells maintain the physiological properties of normal ovarian GCs and are widely used in various functional GC studies. KGN cells that had been preserved in liquid nitrogen were thawed by rapid shaking in warm water, transferred to sterile centrifuge tubes containing a serum-free medium, and then centrifuged at 1000 rpm for 7 min at room temperature. The culture flask was pre-loaded with DMEM containing 10% fetal bovine serum. After centrifugation, the supernatant was discarded, and the cells were resuspended by pipetting. Transfer 2 × 106 cells into the pre-loaded T75 culture flask. The flask was gently shaken horizontally and vertically to ensure proper mixing and then placed in a cell culture incubator at 37 °C with 5% CO2 for growth. The morphology and density of the cells were observed under a microscope. Once the cell density reached approximately 80% and the morphology was satisfactory, trypsin containing 0.05% EDTA was used to digest the cells, which were passaged. Then, seed the KGN cells into six-well plates at a density of 2.5 × 105 cells per well for subsequent experiments.

The experiments were randomly divided into three groups: control, TP, and Afamin + TP. Based on our previous experimental study and relevant literature [17], we selected 50 μmol/L TP and 50 ng/mL afamin protein for the experiment. Once the KGN cell density reached 80% or higher, the medium was replaced with DMEM containing 2% fetal bovine serum. Afamin protein at a concentration of 50 ng/mL was added to the Afamin + TP group, whereas the remaining two groups were incubated with DMEM containing 2% fetal bovine serum. After 4 h, KGN cells in the TP and Afamin + TP groups were treated separately with TP at a concentration of 50 μmol/L for 24 h. Subsequent experiments were then conducted.

ROS level detection

KGN cells were treated as previously described. Intracellular ROS levels were detected using a dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescent probe from Beyotime Biotechnology (Shanghai, China). DCFH-DA was diluted with a serum-free medium to a final concentration of 10 μmol/L. The old medium in the six-well plate was discarded, and the cells were washed with PBS three times before aspirating the liquid. DCFH-DA (1 mL) was added to each well. After incubation at 37 °C in the dark, the working solution was discarded. The cells were washed three times with PBS and collected by trypsin digestion. After centrifugation, the supernatant was discarded, and the fluorescence intensity of the cells was measured by flow cytometry.

Superoxide anion level detection

Superoxide anion levels were measured using flow cytometry and dihydroethidium (DHE) fluorescent probe (Beyotime Biotechnology). The superoxide assay working solution was prepared, and the DHE probe was diluted to a final concentration of 10 μmol/L using serum-free medium at a ratio of 1:1000. The cells were washed three times with PBS, and the old medium was aspirated. Add 1 mL of DHE working solution to each well. The working solution was discarded at the end of the incubation period at 37 °C and was protected from light. The cells were washed with PBS buffer three times. The cells were collected by trypsin digestion and centrifuged, and the supernatant was discarded. The fluorescence intensity of each group of cells was determined by flow cytometry.

Malondialdehyde (MDA) level detection

MDA levels were determined using an MDA Assay Kit (Beyotime Biotechnology) to evaluate the presence of oxidative damage in the cells. The TBA storage solution and MDA assay working solution were prepared, the standards were diluted, and the reaction system and conditions for each step of the assay were established. The samples in each group were reacted, and the absorbance at 532 nm was measured using an enzyme marker. The MDA content of the samples was calculated based on protein concentrations.

Antioxidant system detection

This study measured the activities of four antioxidant enzymes: glutathione peroxidase (GSH-Px), glutathione reductase (GR), superoxide dismutase (SOD), and catalase (CAT). The corresponding kits from Beyotime Biotechnology were used to assay enzymes. After the appropriate treatment of the cells, the proteins in the samples were extracted. The protein concentration of each sample was determined using a BCA Protein Concentration Assay Kit (Beyotime Biotechnology). After treating the cells appropriately, the proteins were extracted, and their concentrations were determined using a BCA Protein Concentration Assay Kit (Beyotime Biotechnology). The instructions for each antioxidant enzyme kit were followed to measure the amount of each enzyme using an enzyme labeler.

Mitochondrial membrane potential (MMP) level detection

The cells were stained with JC-1 using a Mitochondrial Membrane Potential Assay Kit (Beyotime Biotechnology). JC-1 staining the working solution was prepared according to the manufacturer’s instructions. Then, the prepared staining working solution was added. The old cell culture solution in the six-well plate was discarded, and the cells were washed with PBS. Finally, the working solution was discarded at the end of the incubation at 37 °C and protected from light. The cells were washed with a pre-prepared JC-1 staining buffer, collected via trypsin digestion, and centrifuged. The supernatant was discarded, and the MMP levels of each group of cell samples were measured using a flow cytometer.

ATP level detection

ATP levels were measured using an ATP Assay Kit (Beyotime Biotechnology). The cell culture solution in the six-well plate was discarded, and 200 μL of the cell lysate was added to each well. After sufficient lysis, supernatants were collected for subsequent assays. The standard was diluted and an ATP assay working solution was prepared. To each well of a 96-well plate, 100 μL of ATP Assay Work Solution was added and allowed to sit at room temperature for 3 to 5 min to consume any background ATP and reduce background noise. Next, the standard and sample were added to each well, mixed quickly and thoroughly, and RLU values were determined using a chemiluminescence meter. Finally, ATP concentrations of the samples were calculated based on their respective protein concentrations.

Mitochondrial superoxide level detection

Mitochondrial superoxide levels were measured using MitoSOX Red mitochondrial superoxide indicator (Thermo Fisher, USA). To prepare a 5 mM MitoSOX storage solution, DMSO was added, and MitoSOX was diluted with serum-free culture solution at a ratio of 1:1000 to obtain a final concentration of 5 μM. The old culture was aspirated, and the cells were washed with PBS before adding 1 ml of diluted MitoSOX working solution to each well. After incubation at 37 °C, the working solution was discarded, and the cells were washed with PBS. Next, the cells were collected by trypsin digestion and the supernatant was discarded after centrifugation. Finally, the fluorescence intensity of each group of cells was determined by flow cytometry.

Apoptosis flow cytometry detection

Apoptosis was detected using the Annexin V-FITC Apoptosis Detection Kit (Beyotime Biotechnology). The previous cell culture was collected in a 15 ml centrifuge tube, washed with PBS buffer, trypsin-digested, and the cell suspension was mixed with the previous culture in the centrifuge tube. The cells from the old culture were collected in a 15 mL centrifuge tube and washed with PBS buffer. The collected cells were trypsin-digested and the resulting cell suspension was mixed with the old culture in a centrifuge tube. After centrifugation, the supernatant was discarded, and the cells were washed again with PBS. Add 195 μL of Annexin V-FITC conjugate and 5 μL of Annexin V-FITC dye. Mix well and add 10 μL of propidium iodide staining solution. The assay was completed within 1 h after incubation, protected from light.

Caspase-3 activity detection

Caspase-3 activity was measured using the Caspase-3 Activity Assay Kit (Beyotime Biotechnology). Standard dilutions were prepared, the pNA standards were diluted, and A405 was measured using an enzyme labeler. The results are plotted on a standard curve. The cell culture was collected, the adherent cells were digested with trypsin, transferred to the same centrifuge tube as the collected culture, and the supernatant was discarded after centrifugation. After washing, lysis solution was added, and the cells were lysed in an ice bath for 15 min. The resulting mixture was centrifuged to collect the supernatant in a pre-cooled EP tube. A portion of the supernatant was collected to determine protein concentration. Next, each well of a 96-well plate was sequentially added to the assay buffer, the samples to be tested, and Ac-DEVD-pNA (2 mM). The mixture was thoroughly mixed and incubated at 37 °C. The absorbance at 405 nm was measured using an enzyme labeler after a significant change in color was observed. Caspase-3 activity was calculated based on the protein concentration in each sample.

Steroids hormone secretion level detection

The levels of E2 and P4 secretion in the cells were determined using human estrogen and progesterone ELISA kits (Cusabio Technology LLC, Houston, TX, USA). Once the cell density reached 70–80%, the medium was changed to serum-free medium, and the cells were treated according to the above grouping. The culture supernatant of each group of cells was collected, centrifuged, and stored at -80 °C. The levels of E2 and P4 secreted in the supernatant were measured using ELISA, following the manufacturer’s instructions.

Real-time polymerase chain reaction (PCR)

RNA was extracted from KNG cells using an RNA extraction kit and reverse-transcribed to cDNA using an RNA reverse transcription kit. Gene expression levels were analyzed by RT-qPCR using FS Universal SYBR Green Real Master (Roche) and the 2−ΔΔCt method, based on the sample cycling reaction threshold (Ct). The primer sequences used are listed in Table 1.

Table 1 Gene primer sequencesStatistical analysis

Data were analyzed and plotted using SPSS software (version 25.0) and GraphPad Prism 9.4.0 software. Significant differences were analyzed using one-way analysis of variance (ANOVA), and the data were tested for normality using chi-square tests. The mean ± standard deviation (Mean ± SD) was used to express the data, and differences were considered statistically significant when P < 0.05.