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Protective effect of afamin protein against oxidative stress related injury in human ovarian granulosa cells

Journal of Ovarian Research volume 17, Article number: 189 (2024) Cite this article

Abstract

Background

Ovarian granulosa cells (GCs) play crucial roles in follicular growth and development. Their normal function is influenced by various factors, including oxidative stress, which is a significant factor. Afamin protein is a vitamin E-specific binding protein that acts as a vitamin E carrier in follicular fluid. Although the mechanism of the protective effect of afamin on human ovarian GCs is still unclear, there is evidence it has an antioxidant effect in neuronal cells.

Methods

In this study, we investigated the protective effects of afamin proteins on testosterone propionate (TP)-induced ovarian GCs using a human ovarian tumor granulosa cell line (KGN).

Results

The results showed that afamin reduced TP-induced oxidative stress in KGN cells by decreasing the levels of oxidative damage markers, enhancing the activity of antioxidant enzymes, and exerting a protective effect on GCs. Supplementation with afamin repaired mitochondrial dysfunction by improving mitochondrial membrane potential damage and ATP levels. It counteracted TP-induced apoptosis by decreasing the activity of Caspase-3 and upregulating the expression of the anti-apoptotic gene (BCL-2) while downregulating the expression of the pro-apoptotic gene BCL-2-associated X protein (BAX). In addition, afamin regulated the expression of genes related to ovarian steroid hormone synthesis, ameliorating the endocrine dysfunction observed in TP-induced KGN cells.

Conclusion

Therefore, Afamin proteins may serve as important complementary factors that protect GCs from other types of damage, such as oxidative stress, and may help improve ovarian follicle quality and female reproductive function.

Introduction

Ovarian granulosa cells (GCs) regulate oocyte development and are critical for follicle growth [1]. These cells primarily secrete estrogen and progesterone, with cytochrome P450 aromatase (CYP19A1) playing a crucial role in the synthesis of estrogen. Additionally, 3β-hydroxysteroid dehydrogenase (HSD3B1) is involved not only in the production of progesterone but also in the conversion of androgens [2]. GCs and oocytes exchange substances and signals through gap junctions to provide the most nutrients required for oocyte growth and development. They also interact with each other to regulate the processes of follicular maturation, ovulation, and fertilization [3, 4]. Damage to GCs results in pathophysiological changes in ovaries. In a hyperandrogenic microenvironment, excess androgens inhibit the proliferation of GCs and promote apoptosis by acting through the androgen receptor (AR). This affects follicular development and leads to follicular atresia, resulting in polycystic changes of the ovary and causing the development of Polycystic Ovary Syndrome (PCOS) [5, 6]. Damage to ovarian GCs can negatively affect female reproductive health.

Oxidative stress (OS), which is an imbalance between the production of reactive oxygen species (ROS) and the body’s antioxidant defense system [7], can also harm ovarian function. It is important to control ROS levels to maintain normal ovarian function as GCs are sensitive to ROS. Elevated ROS levels can disrupt redox signaling and redox-regulated cellular processes, leading to an oxidative stress (OS) response in GCs. This can cause DNA damage in GCs and affect their function [8,9,10]. Antioxidants have been shown to inhibit GC progression, restore disturbances in cellular redox balance, and reduce ROS levels in GCs, improving ovarian function [11]. Thus, inhibition of OS is considered an effective method to alleviate reproductive dysfunction caused by oxidative damage to GCs.

Afamin, the fourth member of the albumin gene family, has been identified as a vitamin E-specific binding protein [12]. Under physiological conditions, afamin acts as a vitamin E carrier in body fluids such as human follicular fluid [13, 14]. Afamin proteins have multiple binding sites with specific affinities for α-tocopherol and γ-tocopherol. These tocopherols can substitute for vitamin E transport in body fluids when the lipoprotein system is inadequate [13]. Vitamin E is an essential antioxidant. Afamin protein, a specific binding protein for vitamin E, has been shown to protect cortical neurons from apoptosis and act as an antioxidant against neuronal emergence of oxidative stress in neuronal cellular assays in vitro. This effect is observed either alone or in synergy with vitamin E [15, 16]. However, the role of afamin in ovarian GCs remains unclear. Therefore, the purpose of this study is to treat KGN cells using testosterone propionate (TP) to simulate high androgen levels and induce cellular damage. This approach will help to elucidate the protective effects of afamin at the cellular level in ovarian GCs, thereby providing new insights and potential therapeutic strategies for treating ovarian diseases related to OS and other forms of cellular damage.

Materials and methods

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 sequences
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Statistical 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.

Results

Afamin protein ameliorates TP-induced oxidative stress levels in KGN cells

The DCFH-DA fluorescent probe could freely pass through the cell membrane. Once inside, it is oxidized by intracellular ROS to fluorescent DCF. The average fluorescence intensity of DCF was used to analyze ROS levels. As shown in (Fig. 1A-B), the levels of ROS were significantly higher in the TP group than in the control group, suggesting that TP-induced OS in the KGN cells. Compared with the TP group, afamin protein significantly reduced the expression of ROS in KGN cells, protecting them from OS-induced damage.

DHE, one of the most commonly used fluorescent probes, was employed to detect superoxide anions. Upon dehydrogenation, the red fluorescence of the probe indicates the presence of superoxide anions. Therefore, the levels of superoxide anions in the KGN cells were examined using a DHE fluorescent probe. The data presented in (Fig. 1C-D) indicate that the level of superoxide anions was significantly increased in the TP-treated group compared to the control group, suggesting the occurrence of OS. However, the addition of afamin resulted in a significant reduction in superoxide anion levels in KGN cells compared to that in the TP group, indicating that afamin mitigated the oxidative damage caused by TP in KGN cells.

ROS induces lipid peroxidation in vivo and in vitro, leading to an increase in MDA levels. MDA is a product of lipid peroxidation and an indicator of cellular oxidative damage. MDA levels were significantly higher in the TP-treated group than in the control group (Fig. 1E). However, the addition of afamin protein to KGN cells resulted in a significant decrease in MDA levels compared to those in the TP group. It has been suggested that afamin proteins may have damaging antioxidant effects.

Fig. 1
figure 1

Afamin protein ameliorates TP-induced oxidative stress in KGN cells. (A-B) Afamin reduced TP-induced ROS levels in KGN cells. (C-D) Afamin reduced superoxide anion levels in TP-induced KGN cells. (E) Afamin reduces TP-induced MDA levels in KGN cells. The experiment was repeated for three times, results are expressed as mean ± standard deviation, *P < 0.05

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OS occurs due to the limited ability of antioxidants to scavenge excess ROS, resulting in diminished antioxidant capacity. Four antioxidant enzymes, GSH-Px, GR, SOD, and CAT, were assayed using relevant antioxidant assay kits. Our data revealed that the activities of four antioxidant enzymes (GSH-Px, GR, SOD, and CAT) decreased in the KGN cells of the TP group compared to those in the control group, indicating that TP-induced OS in KGN cells. The addition of afamin significantly elevated the activities of the four antioxidant enzymes (SOD, CAT, GSH-Px, and GR) in KGN cells compared to those in the TP group(Fig. 2A-D). These findings indicated that afamin proteins may mitigate OS damage in KGN cells by modulating the expression of antioxidant enzymes.

Fig. 2
figure 2

Afamin protects KGN cells from oxidative stress damage by increasing the activity of antioxidant enzymes. (A) GSH-Px enzymatic activity in KGN cells. (B) GR enzyme activity in KGN cells. (C) SOD activity in KGN cells. (D) CAT activity in KGN cells. The experiment was repeated for three times, results are expressed as mean ± standard deviation, *P < 0.05

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Afamin protein ameliorates TP-induced mitochondrial damage in KGN cells

MMP levels were measured using flow cytometry. The results indicate that the MMP level was significantly decreased in the TP group compared to that in the control group, suggesting that the mitochondrial function of KGN cells in the TP group may be impaired. The inclusion of afamin significantly increased MMP expression in KGN cells compared to that in the TP group (Fig. 3A-B), indicating that afamin improved mitochondrial membrane potential in KGN cells.

Mitochondria are crucial intracellular organelles involved in ATP synthesis. ATP levels in KGN cells were measured using an ATP assay kit. The results indicate that ATP levels decreased in the TP-treated group, whereas ATP levels increased significantly with the addition of afamin protein (Fig. 3C). Previous studies have suggested that TP leads to mitochondrial dysfunction in KGN cells and that afamin ameliorates this impairment.

Mitochondrial superoxide levels were measured using MitoSOX Red mitochondrial superoxide indicator and analyzed by flow cytometry. The data presented in (Fig. 3D-E) indicate that intracellular mitochondrial superoxide levels increased after TP treatment, suggesting impaired mitochondrial function. Treatment with Afamin protein reduced the elevated mitochondrial superoxide levels compared to those in the TP group, indicating the effectiveness of afamin protein in restoring mitochondrial function in KGN cells.

Fig. 3
figure 3

Afamin protein attenuates TP-induced mitochondrial damage in KGN cells. (A-B) Afamin reduced TP-induced MMP damage in KGN cells. (C) Afamin increased ATP levels in TP-treated KGN cells. (D-E) Afamin reduces TP-induced mitochondrial superoxide levels in KGN cells. The experiment was repeated for three times, results are expressed as mean ± standard deviation, *P < 0.05

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Afamin protein ameliorates TP-induced apoptosis in KGN cells

Mitochondrial dysfunction induces apoptosis. Flow cytometry was used to detect apoptosis in KGN cells. The results showed a significant increase in the apoptosis of KGN cells after exposure to TP. However, afamin protein treatment ameliorated TP-induced apoptosis(Fig. 4A).

The activity of Caspase-3, an important factor in apoptosis, was analyzed to confirm these results. The results indicate that Caspase-3 activity increased in the TP group and decreased in the afamin group(Fig. 4B), indicating that afamin protein ameliorated TP-promoted apoptosis.

To investigate the mechanisms of apoptosis in KGN cells, we analyzed the expression of the pro-apoptotic gene BAX and anti-apoptotic gene BCL-2 using fluorescence quantitative PCR. The results in (Fig. 4C-D) indicate that exposure to TP led to a significant increase in the expression of the pro-apoptotic BAX gene and a significant decrease in the expression of the anti-apoptotic BCL-2 gene in KGN cells. Conversely, pretreatment with afamin resulted in a decrease in BCL-2-associated X protein (BAX) expression and increased BCL-2 expression. These findings are objective and are based solely on the data presented. Afamin appears to ameliorate TP-induced apoptosis in KGN cells by regulating BAX/BCL-2 and acting as an inhibitor of apoptosis.

Fig. 4
figure 4

Afamin protein inhibits TP-induced apoptosis in KGN cells. (A) Flow cytometry analysis suggests that afamin can reduce TP-induced apoptosis in KGN cells. (B) Afamin decreased caspase-3 activity in TP-treated KGN cells. (C-D) Afamin protein reduced TP-induced apoptosis in KGN cells by increasing BCL-2 mRNA expression and decreasing BAX mRNA expression. The experiment was repeated for three times, results are expressed as mean ± standard deviation, *P < 0.05

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Afamin protein ameliorates TP-induced abnormal steroid hormone secretion in KGN cells

Excessive ovarian stimulation leads to impaired function of GCs, which are the main sources of steroidal estrogens (E2) and progesterone (P4). Therefore, we determined the levels of E2 and P4 secreted by KGN cells into the supernatant using ELISA. The results showed abnormal levels of E2 and P4 hormone secretion in the TP group compared to those in the control group. However, the addition of afamin restored abnormal levels of E2 and P4 secretion in KGN cells (Fig. 5A-B). To investigate the mechanism of the effect of afamin protein on steroid hormone synthesis in TP-treated KGN cells, we detected the mRNA expression of the hormone synthesis-related genes CYP19A1, CYP11A1, steroidogenic acute regulatory (STAR), and HSD3B1 using fluorescence quantitative PCR.

The results in (Fig. 5C-F) indicate a significant decrease in the mRNA expression of STAR, HSD3B1, and CYP11A1, and an increase in CYP19A1 mRNA expression in the TP group. In contrast, KGN cells treated with Afamin protein exhibited elevated STAR, HSD3B1, and CYP11A1 mRNA expression levels, along with reduced CYP19A1 mRNA expression. It has been suggested that afamin may regulate the expression of genes related to steroid hormone synthesis, ameliorating the abnormal secretory function observed in TP-treated KGN cells.

Fig. 5
figure 5

Afamin protects TP-induced secretory function of KGN cells. (A-B) Afamin ameliorates the abnormal secretion of E2 and P4 in TP-treated KGN cells. (C-F) Afamin repairs the secretory function of KGN cells by regulating the mRNA expression of CYP19A1, CYP11A1, STAR, and HSD3B1. The experiment was repeated for three times, results are expressed as mean ± standard deviation, *P < 0.05

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Discussion

GCs play crucial roles in follicle growth and development. They provide nutrients and maturation factors to maintain oocyte maturation and protect oocytes from oxidative damage [18, 19]. In recent years, damage caused by OS, which is characterized by the excessive production of ROS, has garnered significant attention from researchers. OS is considered a potential inducing factor in the pathogenesis of PCOS. For patients with PCOS, elevated androgen levels and insulin resistance could exacerbate oxidative stress, adversely affecting the function of granulosa cells (GCs) and the normal growth and development of follicles [20, 21]. ROS, an important marker of OS, can lead to DNA damage in GCs, affecting female oocyte maturation and follicle growth and development [22, 23]. Excessive ROS production can induce lipid peroxidation both in vivo and in vitro. This process commonly results in the formation of malondialdehyde (MDA) [24, 25], which is a harmful stimulus that can cause protein misfolding and exacerbate the OS response [26]. Studies have shown that patients with PCOS typically exhibit elevated levels of MDA, indicating significant lipid peroxidation and oxidative damage [27]. Furthermore, the body relies on four antioxidant enzymes (SOD, CAT, GSH-Px, and GR) to defend against OS by neutralizing excess ROS and preventing damage to cellular structures [28, 29]. SOD serves as the first line of defense in the antioxidant defense system by converting superoxide anions into hydrogen peroxide, thereby reducing levels of ROS. In PCOS, SOD activity is commonly reduced, indicating a weakened antioxidant defense capability. CAT further decomposes hydrogen peroxide into water and oxygen, thus reducing its toxicity to cells. Reduced CAT activity leads to the accumulation of hydrogen peroxide, resulting in cellular damage and dysfunction. GSH-Px reduces lipid peroxides and hydrogen peroxide, protecting cells from oxidative damage. In PCOS, decreased GSH-Px levels indicate compromised antioxidant capacity, exacerbating cellular oxidative damage. Additionally, GR maintains intracellular glutathione levels by reducing oxidized glutathione to its reduced form, thereby enhancing cellular antioxidant capacity. Reduced GR activity weakens cellular antioxidant defenses, increasing the risk of cellular damage [8, 30, 31]. However, it is well known that vitamin E is an important antioxidant. Afamin, as a specific binding protein for vitamin E, can take over the role of transporting vitamin E in body fluids when the lipoprotein system is inadequate [13]. In in vitro experiments, researchers have discovered that afamin protects cortical neurons from apoptosis and acts as an antioxidant against neuronal OS [16]. In this study, exposure to TP led to elevated levels of the OS biomarkers ROS, superoxide anions, and MDA in KGN cells. Furthermore, the levels of four antioxidant enzymes (SOD, CAT, GSH-Px, and GR) decreased. However, afamin protein could downregulate the levels of ROS, superoxide anions, and MDA while increasing the activities of the antioxidant enzymes GSH-Px, GR, SOD, and CAT. Therefore, afamin protects KGN cells from oxidative damage by reducing OS and increasing their antioxidant capacity.

Mitochondria are highly susceptible to oxidative damage due to their proximity to ROS-generating sources, lack of histone and DNA repair mechanisms, and presence of a circular genome known as mitochondrial DNA (mtDNA) between the inner and outer mitochondrial membranes [32]. When ROS are produced in large quantities, they can damage mitochondria through free radical reactions, resulting in impaired mitochondrial function. This impairment is mainly characterized by alterations in MMP. MMP deficiency can cause defects in the mitochondrial electron transport chain, leading to an increase in ATP consumption and a decrease in energy metabolism [33, 34]. This study demonstrates that mitochondrial function in KGN cells may be impaired after TP treatment. Afamin supplementation resulted in a significant increase in MMP and ATP levels as well as a significant reduction in mitochondrial superoxide levels, indicating that afamin may improve mitochondrial dysfunction in KGN cells. Furthermore, oxidative damage to mitochondria can induce mitochondrial outer membrane permeabilization (MOMP), resulting in a reduction in the mitochondrial transmembrane potential. This opens the mitochondrial membrane permeability transition pore (mPTP), leading to the release of cytochrome c from the mitochondria into the cytoplasm. Cytochrome c binds to apoptotic protease activating factor 1 (APAf-1) to form the “apoptosome” complex, which sequentially activates a series of cysteine caspases, leading to apoptosis [34, 35]. BAX is a key regulator of mitochondrial apoptosis. Although it physiologically maintains tissue homeostasis, dysregulation of BAX can cause aberrant cell death [36]. However, BCL-2 prevents MOMP by maintaining mitochondrial integrity, which prevents cytochrome c release. BCL-2 overexpression inhibits cell apoptosis [37]. The study results indicated that exposure to TP significantly increased apoptosis of KGN cells, elevated BAX expression, decreased BCL-2 expression, and significantly increased caspase-3 activity. The addition of afamin reversed these effects, suggesting that afamin can ameliorate apoptosis in KGN cells.

GCs play a crucial role in steroid hormone synthesis. Oxidative damage to GCs can lead to disorders of steroid hormone secretion in vivo [38, 39]. Steroid hormone synthesis involves several enzymes and proteins. Changes in these key enzymes can affect the production of steroid hormones [40]. The STAR facilitates cholesterol transfer from the outer mitochondrial membrane to the inner mitochondrial membrane. This process is the rate-limiting step in steroid hormone formation. The P450 cholesterol side-chain cleavage enzyme (CYP11A1) is situated on the stromal side of the inner mitochondrial membrane. It can convert cholesterol to pregnenolone. After leaving the mitochondrion, pregnenolone is converted to progesterone by the 3β-hydroxysteroid dehydrogenase (HSD3B1) in the mitochondrial compartment [41, 42]. Furthermore, the conversion of androgens to estrogens is primarily conducted by cytochrome P450 aromatase (CYP19A1), which is widely expressed in the ovarian GCs. This enzyme is a crucial component of estrogen synthesis [43]. In this study, KGN cells exposed to TP and damaged by OS exhibited abnormal secretion of E2 and P4, and abnormal expression of genes related to steroid hormone synthesis. Oxidative damage to KGN cells may affect the expression of genes involved in steroid hormone synthesis, leading to abnormal synthesis. However, Afamin protein reduced TP-induced OS damage in KGN cells, reversed the expression levels of steroid hormone synthase genes, and improved the secretory function of KGN cells. These results indicate that afamin protein inhibits intracellular ROS levels, increases antioxidant enzyme activities in KGN cells, and protects against OS and related injuries in human ovarian GCs.

Conclusions

In summary, the findings of this study demonstrate that the protective effect of Afamin protein against TP-induced damage in KGN cells is achieved by enhancing the antioxidant capacity of these cells. This enhancement helps to prevent oxidative stress (OS), reduce mitochondrial damage and apoptosis, and improve hormone secretion functions in KGN cells(Fig. 6). This study presents a theoretical foundation for further investigation into the function and regulatory mechanism of Afamin protein, as well as a potential molecular basis for the early treatment of ovarian reproductive endocrine disorders, such as PCOS, in women.

Fig. 6
figure 6

Afamin protects against TP-induced oxidative stress-related damage in KGN cells

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Data availability

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Abbreviations

ATP:

Adenosine triphosphate

BAX:

BCL2-Associated X

BCL2:

B-cell lymphoma-2

CAT:

Catalase

CYP11A1:

Cytochrome P450 Family 11 Subfamily A Member 1

CYP19A1:

Cytochrome P450 Family 19 Subfamily A Member 1

GR:

Glutathione reductase

GSH-Px:

Glutathione peroxidase

HSD3B1:

Hydroxy-delta-5-steroid dehydrogenase, 3beta- and steroid delta-isomerase 1

MDA:

Malondialdehyde

ROS:

Reactive oxygen species

SOD:

Superoxide dismutase

STAR:

Steroidogenic acute regulatory protein

References

  1. Jozkowiak M, Piotrowska-Kempisty H, Kobylarek D, Gorska N, Mozdziak P, Kempisty B, Rachon D, Spaczynski RZ. Endocrine disrupting chemicals in polycystic ovary syndrome: the relevant role of the theca and granulosa cells in the pathogenesis of the ovarian dysfunction. Cells. 2022;12(1). https://doiorg.publicaciones.saludcastillayleon.es/10.3390/cells12010174

  2. Havelock JC, Rainey WE, Carr BR. Ovarian granulosa cell lines. Mol Cell Endocrinol. 2004;228(1–2):67–78. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.mce.2004.04.018

    Article  CAS  PubMed  Google Scholar 

  3. Herlands RL, Schultz RM. Regulation of mouse oocyte growth: probable nutritional role for intercellular communication between follicle cells and oocytes in oocyte growth. J Exp Zool. 1984;229(2):317–25. https://doiorg.publicaciones.saludcastillayleon.es/10.1002/jez.1402290217

    Article  CAS  PubMed  Google Scholar 

  4. Acuña-Hernández DG, Arreola-Mendoza L, Santacruz-Márquez R, García-Zepeda SP, Parra-Forero LY, Olivares-Reyes JA, Hernández-Ochoa I. Bisphenol A alters oocyte maturation by prematurely closing gap junctions in the cumulus cell-oocyte complex. Toxicol Appl Pharmacol. 2018;344:13–22. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.taap.2018.02.011

    Article  CAS  PubMed  Google Scholar 

  5. Rodriguez Paris V, Bertoldo MJ. The mechanism of androgen actions in PCOS etiology. Med Sci (Basel). 2019;7(9). https://doiorg.publicaciones.saludcastillayleon.es/10.3390/medsci7090089

  6. McFee RM, Romereim SM, Snider AP, Summers AF, Pohlmeier WE, Kurz SG, Cushman RA, Davis JS, Wood JR, Cupp AS. A high-androgen microenvironment inhibits granulosa cell proliferation and alters cell identity. Mol Cell Endocrinol. 2021;531:111288. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.mce.2021.111288

    Article  CAS  PubMed  Google Scholar 

  7. Pizzino G, Irrera N, Cucinotta M, Pallio G, Mannino F, Arcoraci V, Squadrito F, Altavilla D, Bitto A. Oxidative stress: harms and benefits for human health. Oxid Med Cell Longev. 2017;2017:8416763https://doiorg.publicaciones.saludcastillayleon.es/10.1155/2017/8416763

    Article  CAS  Google Scholar 

  8. Zeber-Lubecka N, Ciebiera M, Hennig EE. Polycystic ovary syndrome and oxidative stress-from bench to bedside. Int J Mol Sci. 2023;24(18). https://doiorg.publicaciones.saludcastillayleon.es/10.3390/ijms241814126

  9. Jones DP. Redefining oxidative stress. Antioxid Redox Signal. 2006;8(9–10):1865–79. https://doiorg.publicaciones.saludcastillayleon.es/10.1089/ars.2006.8.1865

    Article  CAS  PubMed  Google Scholar 

  10. Luderer U. Ovarian toxicity from reactive oxygen species. Vitam Horm. 2014;94:99–127. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/B978-0-12-800095-3.00004-3

    Article  CAS  PubMed  Google Scholar 

  11. Liu S, Jia Y, Meng S, Luo Y, Yang Q, Pan Z. Mechanisms of and potential medications for oxidative stress in ovarian granulosa cells: a review. Int J Mol Sci. 2023;24(11). https://doiorg.publicaciones.saludcastillayleon.es/10.3390/ijms24119205

  12. Lichenstein HS, Lyons DE, Wurfel MM, Johnson DA, McGinley MD, Leidli JC, Trollinger DB, Mayer JP, Wright SD, Zukowski MM. Afamin is a new member of the albumin, alpha-fetoprotein, and vitamin D-binding protein gene family. J Biol Chem. 1994;269(27):18149–54.

    Article  CAS  PubMed  Google Scholar 

  13. Voegele AF, Jerković L, Wellenzohn B, Eller P, Kronenberg F, Liedl KR, Dieplinger H. Characterization of the vitamin E-binding properties of human plasma afamin. Biochemistry. 2002;41(49):14532–8. https://doiorg.publicaciones.saludcastillayleon.es/10.1021/bi026513v

    Article  CAS  PubMed  Google Scholar 

  14. Dieplinger H, Dieplinger B. Afamin–A pleiotropic glycoprotein involved in various disease states. Clin Chim Acta. 2015;446:105–10. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.cca.2015.04.010

    Article  CAS  PubMed  Google Scholar 

  15. Azzi A. Oxidative stress: what is it? Can it be measured? Where is it located? Can it be good or bad? Can it be prevented? Can it be cured? Antioxid (Basel). 2022;11(8). https://doiorg.publicaciones.saludcastillayleon.es/10.3390/antiox11081431

  16. Heiser M, Hutter-Paier B, Jerkovic L, Pfragner R, Windisch M, Becker-André M, Dieplinger H. Vitamin E binding protein afamin protects neuronal cells in vitro. J Neural Transm Suppl. 2002(62):337–45. https://doiorg.publicaciones.saludcastillayleon.es/10.1007/978-3-7091-6139-5_32

  17. Jin J, Ma Y, Tong X, Yang W, Dai Y, Pan Y, Ren P, Liu L, Fan HY, Zhang Y, et al. Metformin inhibits testosterone-induced endoplasmic reticulum stress in ovarian granulosa cells via inactivation of p38 MAPK. Hum Reprod. 2020;35(5):1145–58. https://doiorg.publicaciones.saludcastillayleon.es/10.1093/humrep/deaa077

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Kranc W, Budna J, Kahan R, Chachuła A, Bryja A, Ciesiółka S, Borys S, Antosik MP, Bukowska D, Brussow KP, et al. Molecular basis of growth, proliferation, and differentiation of mammalian follicular granulosa cells. J Biol Regul Homeost Agents. 2017;31(1):1–8.

    CAS  PubMed  Google Scholar 

  19. Rimon-Dahari N, Yerushalmi-Heinemann L, Alyagor L, Dekel N. Ovarian folliculogenesis. Results Probl Cell Differ. 2016;58:167–90. https://doiorg.publicaciones.saludcastillayleon.es/10.1007/978-3-319-31973-5_7

    Article  CAS  PubMed  Google Scholar 

  20. Zeng X, Xie YJ, Liu YT, Long SL, Mo ZC. Polycystic ovarian syndrome: correlation between hyperandrogenism, insulin resistance and obesity. Clin Chim Acta. 2020;502:214–21. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.cca.2019.11.003

    Article  CAS  PubMed  Google Scholar 

  21. Kaufman MJ, Kanayama G, Hudson JI, Pope HG Jr. Supraphysiologic-dose anabolic-androgenic steroid use: a risk factor for dementia? Neurosci Biobehav Rev. 2019;100:180–207. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.neubiorev

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Mello Filho AC, Hoffmann ME, Meneghini R. Cell killing and DNA damage by hydrogen peroxide are mediated by intracellular iron. Biochem J. 1984;218(1):273–5. https://doiorg.publicaciones.saludcastillayleon.es/10.1042/bj2180273

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Agarwal A, Gupta S, Sharma RK. Role of oxidative stress in female reproduction. Reprod Biol Endocrinol. 2005;3:28. https://doiorg.publicaciones.saludcastillayleon.es/10.1186/1477-7827-3-28

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Liu S, Navarro G, Mauvais-Jarvis F. Androgen excess produces systemic oxidative stress and predisposes to beta-cell failure in female mice. PLoS ONE. 2010;5(6):e11302. https://doiorg.publicaciones.saludcastillayleon.es/10.1371/journal

    Article  PubMed  PubMed Central  Google Scholar 

  25. Sousa BC, Pitt AR, Spickett CM. Chemistry and analysis of HNE and other prominent carbonyl-containing lipid oxidation compounds. Free Radic Biol Med. 2017;111:294–308. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.freeradbiomed.2017.02.003

    Article  CAS  PubMed  Google Scholar 

  26. Wang L, Zhang M, Fang Z, Bhandari B. Gelation properties of myofibrillar protein under malondialdehyde-induced oxidative stress. J Sci Food Agric. 2017;97(1):50–7. https://doiorg.publicaciones.saludcastillayleon.es/10.1002/jsfa

    Article  CAS  PubMed  Google Scholar 

  27. Tan W, Dai F, Yang D, Deng Z, Gu R, Zhao X, Cheng Y. MiR-93-5p promotes granulosa cell apoptosis and ferroptosis by the NF-kB signaling pathway in polycystic ovary syndrome. Front Immunol. 2022;13:967151. https://doiorg.publicaciones.saludcastillayleon.es/10.3389/fimmu

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Lv H, Liu Q, Wen Z, Feng H, Deng X, Ci X. Xanthohumol ameliorates lipopolysaccharide (LPS)-induced acute lung injury via induction of AMPK/GSK3β-Nrf2 signal axis. Redox Biol. 2017;12:311–24. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.redox.2017.03.001

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Valko M, Rhodes CJ, Moncol J, Izakovic M, Mazur M. Free radicals, metals and antioxidants in oxidative stress-induced cancer. Chem Biol Interact. 2006;160(1):1–40. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.cbi.2005.12.009

    Article  CAS  PubMed  Google Scholar 

  30. Ma Y, Zheng L, Wang Y, Gao Y, Xu Y. Arachidonic acid in follicular fluid of PCOS induces oxidative stress in a human ovarian granulosa tumor cell line (KGN) and upregulates GDF15 expression as a response. Front Endocrinol (Lausanne). 2022;13:865748. https://doiorg.publicaciones.saludcastillayleon.es/10.3389/fendo

    Article  PubMed  Google Scholar 

  31. Abudawood M, Tabassum H, Alanazi AH, Almusallam F, Aljaser F, Ali MN, Alenzi ND, Alanazi ST, Alghamdi MA, Altoum GH, et al. Antioxidant status in relation to heavy metals induced oxidative stress in patients with polycystic ovarian syndrome (PCOS). Sci Rep. 2021;11(1):22935https://doiorg.publicaciones.saludcastillayleon.es/10.1038/s41598-021-02120-6

    Article  CAS  Google Scholar 

  32. Mustafa MF, Fakurazi S, Abdullah MA, Maniam S. Pathogenic mitochondria DNA mutations: current detection tools and interventions. Genes (Basel). 2020;11(2). https://doiorg.publicaciones.saludcastillayleon.es/10.3390/genes11020192

  33. Zeng X, Huang Q, Long SL, Zhong Q, Mo Z. Mitochondrial dysfunction in polycystic ovary syndrome. DNA Cell Biol. 2020;39(8):1401–9. https://doiorg.publicaciones.saludcastillayleon.es/10.1089/dna.2019.5172

    Article  CAS  PubMed  Google Scholar 

  34. Kim SH, Kim H. Inhibitory effect of astaxanthin on oxidative stress-induced mitochondrial dysfunction-a mini-review. Nutrients. 2018;10(9). https://doiorg.publicaciones.saludcastillayleon.es/10.3390/nu10091137

  35. Caroppi P, Sinibaldi F, Fiorucci L, Santucci R. Apoptosis and human diseases: mitochondrion damage and lethal role of released cytochrome C as proapoptotic protein. Curr Med Chem. 2009;16(31):4058–65. https://doiorg.publicaciones.saludcastillayleon.es/10.2174/092986709789378206

    Article  CAS  PubMed  Google Scholar 

  36. Spitz AZ, Gavathiotis E. Physiological and pharmacological modulation of BAX. Trends Pharmacol Sci. 2022;43(3):206–20. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.tips.2021.11.001

    Article  CAS  PubMed  Google Scholar 

  37. Green DR. At the gates of death. Cancer Cell. 2006;9(5):328–30. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.ccr.2006.05.004

    Article  CAS  PubMed  Google Scholar 

  38. Jozkowiak M, Hutchings G, Jankowski M, Kulcenty K, Mozdziak P, Kempisty B, Spaczynski RZ, Piotrowska-Kempisty H. The stemness of human ovarian granulosa cells and the role of resveratrol in the differentiation of MSCs-a review based on cellular and molecular knowledge. Cells. 2020;9(6). https://doiorg.publicaciones.saludcastillayleon.es/10.3390/cells9061418

  39. Karabulut S, Korkmaz O, Kutlu P, Gozel HE, Keskin I. Effects o follicular fluid oxidative status on human mural granulosa cells, oocyte competency and ICSI parameters. Eur J Obstet Gynecol Reprod Biol. 2020;252:127–36. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.ejogrb.2020.06.021

    Article  CAS  PubMed  Google Scholar 

  40. Payne AH, Hales DB. Overview of steroidogenic enzymes in the pathway from cholesterol to active steroid hormones. Endocr Rev. 2004;25(6):947–70. https://doiorg.publicaciones.saludcastillayleon.es/10.1210/er.2003-0030

    Article  CAS  PubMed  Google Scholar 

  41. Stocco DM. StAR protein and the regulation of steroid hormone biosynthesis. Annu Rev Physiol. 2001;63:193–213. https://doiorg.publicaciones.saludcastillayleon.es/10.1146/annurev.physiol.63.1.193

    Article  CAS  PubMed  Google Scholar 

  42. Miller WL, Auchus RJ. The molecular biology, biochemistry, and physiology of human steroidogenesis and its disorders. Endocr Rev. 2011;32(1):81–151. https://doiorg.publicaciones.saludcastillayleon.es/10.1210/er.2010-0013

    Article  PubMed  Google Scholar 

  43. Zhang Y, Chen X, Zhou Z, Tian X, Yang P, Fu K. CYP19A1 may influence lambing traits in goats by regulating the biological function of granulosa cells. Anim (Basel). 2022;12(15). https://doiorg.publicaciones.saludcastillayleon.es/10.3390/ani12151911

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Funding

This study was supported by the Natural Science Foundation of Jilin Province (No. YDZJ202301ZYTS434).

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Authors and Affiliations

  1. Reproductive Medical Center, The Second Hospital of Jilin University, 4026 Yatai Street, Changchun, 130022, China

    Qiang Zhang, Xueying Zhang & Lianwen Zheng

  2. Department of Gynecology, Dongguan Songshan Lake Tungwah Hospital, NO.1 Songshan Lake Science Development Seven Road, DongGuan, 523822, China

    Xiaoyu Zheng

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Qiang Zhang reviewed the literature and statistical analyses of the data and wrote the manuscript. Xiaoyu Zheng and Xueying Zhang contributed to the conception and design of the study, and Professor Lianwen Zheng supervised and revised the manuscript. All authors read and approved the final manuscript.

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Zhang, Q., Zheng, X., Zhang, X. et al. Protective effect of afamin protein against oxidative stress related injury in human ovarian granulosa cells. J Ovarian Res 17, 189 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13048-024-01511-3

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Journal of Ovarian Research

ISSN: 1757-2215

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