Abstract
Polycystic ovarian ovary syndrome (PCOS) is the main cause of ovulatory
infertility and a common reproductive endocrine disease of women in
reproductive age. In addition, nearly half of PCOS patients are
associated with obesity, and their total free fatty acids tend to
increase. Arachidonic acid (AA) is a polyunsaturated fatty acid.
Oxidation products of AA reacting with various enzymes[cyclooxygenases
(COX), lipoxygenases (LOX), cytochrome P450s (CYP)] can change cellular
mitochondrial distribution and calcium ion concentration, and increase
reactive oxygen species (ROS) production. In this study, we analyzed
the follicular fluid fatty acids and found higher levels of C20:4n6
(AA) in PCOS patients than in normal control subjects. Also, to
determine whether AA induces oxidative stress (OS) in the human ovarian
granulosa tumor cell line (KGN) and affects its function, we treated
KGN cells with or without reduced glutathione (GSH) and then stimulated
them with AA. The results showed that AA significantly reduced the
total antioxidant capacity (TAC) and activity of antioxidant enzymes
and increased the malondialdehyde (MDA), ROS and superoxide
anion(O[2-])levels in KGN cells. In addition, AA was also found to
impair the secretory and mitochondrial functions of KGN cells and
induce their apoptosis. We further investigated the downstream genes
affected by AA in KGN cells and its mechanism of action. We found that
AA upregulated the expression of growth differentiation factor 15
(GDF15), which had a protective effect on inflammation and tissue
damage. Therefore, we investigated whether AA-induced OS in KGN cells
upregulates GDF15 expression as an OS response.Through silencing of
GDF15 and supplementation with recombinant GDF15 (rGDF15), we found
that GDF15, expressed as an OS response, protected KGN cells against
AA-induced OS effects, such as impairment of secretory and
mitochondrial functions and apoptosis. Therefore, this study suggested
that AA might induce OS in KGN cells and upregulate the expression of
GDF15 as a response to OS.
Keywords: PCOS, arachidonic acid, oxidative stress (OS), human ovarian
granulosa tumor cell line (KGN), GDF15, response
Introduction
Polycystic ovary syndrome (PCOS) is one of the most common reproductive
endocrine diseases, with a prevalence rate of 5-10% ([32]1). Its
clinical manifestations vary, and include amenorrhea, hirsutism,
obesity, hyperinsulinemia, hyperandrogenemia, presence of polycystic
ovary in ultrasound examination, etc ([33]2). Patients with PCOS
produce more oocytes after stimulation of ovulation than non-PCOS
patients. However, the growth and fertilization rate of oocytes and
embryo rate are reduced, which is consistent with other studies ([34]3,
[35]4). Follicular fluid provides a living environment for the
development and maturity of oocytes. Therefore, changes in the
composition of follicular fluid can affect the quality of oocytes,
including the maturation, fertilization, cleavage and early embryo
formation of oocytes ([36]5). In addition, the follicular fluid
contains a large amount of polyunsaturated fatty acids ([37]6). In our
study, it was found that the AA in follicular fluid of PCOS patients
after hyperovulation was higher than that of the control group. AA is
one of the most abundant, active and widely distributed polyunsaturated
essential fatty acids in the human body ([38]7, [39]8). Enzymatic
oxidation products of AA reactions, catalyzed by various enzymes (COX,
LOX, CYP, etc.) ([40]9–[41]11), are involved in almost the entire
reproductive process, including oocyte maturation, ovulation,
implantation, delivery, etc. In addition, AA plays an important role in
inflammation and is closely related to OS ([42]12). Therefore, the
study of the relationship between the two can lead to a better
understanding of the pathogenic mechanism of PCOS, which is of great
importance to the research on the etiology and treatment of the change
in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI)
assisted pregnancy of PCOS patients. Investigating the association
between AA-induced OS and the expression of GDF15 in KGN cells
established a theoretical basis for the development of a treatment
strategy to improve pregnancy outcome of PCOS patients.
Materials and Methods
Participants
Sixteen women with PCOS were selected and enrolled in this study
according to the revised Rotterdam consensus guidelines ([43]13).
Additionally, another 16 women with infertility due to simple tubal
factors were also selected and enrolled in this study as controls.
These patients had regular menstruation, normal ovarian function, and
no androgenic clinical and biochemical characteristics. The patients in
the PCOS group were divided into PCOS normal weight group (8 cases,
18.5 kg/m^2 ≤BMI<24 kg/m^2) and PCOS overweight group (8 cases, BMI ≥
24 kg/m^2), according to the BMI of Chinese people which was developed
by the International Society for Life Sciences according to the body
type of Chinese people ([44]14). In the control group, subjects were
divided into normal weight group (8 cases, 18.5 kg/m^2 ≤ BMI<24 kg/m^2)
and overweight group (8 cases, BMI ≥ 24 kg/m^2). Ovulation induction
regimens in both groups were modified long protocols and all patients
were between 25 and 35 years old. Patients with the following diseases
were excluded from the study: (1) Patients with other conditions that
cause ovulation dysfunction, including hyperprolactinemia, early-onset
ovarian insufficiency, hypogonadotropin amenorrhea, and thyroid
dysfunction. (2) Women with other causes of hyperandrogenism, such as
congenital adrenal hyperplasia, androgen-secreting tumors, Cushing’s
syndrome, etc ([45]15). All patients signed informed consent for this
study. The study was approved by the Ethics Committee of the Second
Hospital of Jilin University (Jilin, China 2020 Review No. 123).
Controlled Ovarian Hyperstimulation and Follicular Fluid Collection
All patients were enrolled in a modified long protocol. On the second
day of menstruation, leprorelin acetate (GnRH-a, 3.75 mg/dose) was
subcutaneously injected with 0.935 mg. After administering the
injection for18-20 days, the drop regulation was adjusted according to
the level of sex hormones and the results of the ultrasound
examination. If it did not meet the criteria, another subcutaneous
injection of 1.25 mg of leprorelin acetate was given. Gn was used when
the down-regulation standard was reached ([46]16). When there were 2
dominant follicles with average diameter ≥18 mm or 3 dominant follicles
with average diameter ≥18 mm and gt, Gn was stopped at 17 mm. Then, 250
µg of recombinant human chorionic gonadotropin was injected on the same
night, and ovum pick up (OPU) was performed 34-36 h later. Follicular
fluid from 18-20 mm diameter follicles was collected during OPU(to
prevent blood contamination), centrifuged immediately (800g*10 min) and
supernatant was collected and placed in a 1.5 mL cryopreserved tube and
stored at -80°C.
Extraction, Measurement and Calculation of Fatty Acids in Follicular Fluid
Pre-Processing Method
(1) Sample Hydrolysis
Firstly, we weigh an appropriate amount of uniform sample, add about
100 mg of pyrogallic acid, add a few grains of zeolite, and then add 2
mL of 95% ethanol, and mix well. Secondly, we add 10 mL of hydrochloric
acid solution and mix. The flask was placed in a water bath at 70°C to
80°C for hydrolysis for 40 min. Then, we shake the flask every 10 min
to mix the particles adhering to the walls of the flask into the
solution. After the hydrolysis was complete, the flask was removed and
cooled to room temperature.
(2) Extraction of Fat
Firstly, we add 10 mL of 95% ethanol to the hydrolyzed sample, and mix
well. Secondly, we transfer the hydrolyzate in the flask to a
separatory funnel, rinse the flask and stopper with 50 mL of
ether-petroleum ether mixture, and incorporate the rinse into the
separatory funnel and cap it. Shake for 5min and let stand for 10min.
The ether layer extract was collected into a 250 mL flask. Then, we
repeat the extraction of the hydrolyzate 3 times according to the above
steps, and finally rinse the separatory funnel with a mixture of ether
and petroleum ether, and collect it into a flask with constant weight,
put the flask on a water bath and evaporate to dryness and dry it in an
oven at 100°C ± 5°C for 2 hours.
(3) Saponification of Fat and Fatty Acid Methylation
Firstly, in the fat extraction, we continue to add 2 mL of 2% sodium
hydroxide methanol solution, water bath in a water bath at 85°C for 30
min and add 3 mL of 14% boron trifluoride methanol solution. Water bath
at 85°C for 30 min. Secondly, after the water bath is completed, we
wait for the temperature to drop to room temperature, add 1 mL of
n-hexane to the centrifuge tube, shake and extract for 2 min, and let
it stand for one hour to wait for stratification. Then, we take 100 μL
of the supernatant, dilute to 1 mL with n-hexane and use 0.45 μm filter
membrane to test on the machine. Finally, after hydrolyzing the sample,
the fat was extracted and saponified, and the fatty acids were methyl
esterified.
Then, the fatty acids were detected by gas chromatography-mass
spectrometry (GC-MS). The contents of each fatty acid in the sample
were calculated using the corresponding formula.
Origin and Culture of the Human Ovarian Granulosa Tumor Cell Line (KGN)
The immortal human ovarian granulosa tumor cell line cell line (KGN)
was used in this study. This cell line was derived from granulosa cells
of a patient with ovarian cancer and immortalized. It has the ability
to synthesize steroid hormones and the growth characteristics of
granulosa cells, so it is often used to study the function,
proliferation and hormone regulation of granulosa cells. The frozen KGN
cells were quickly thawed in a 37°C water bath and quickly transferred
to a centrifuge tube containing 10 mL of serum-free medium. After
centrifugation at 1,200 rpm for 7 min, the precipitated cells were
transferred to Dulbecco’s modified Eagle’s medium (DMEM) containing 10%
fetal bovine serum and well mixed. The cells were cultured in a cell
incubator at 37°C with 5% CO[2] and the cell morphology and density
were observed. When the cell confluence reached 80-90%, cells were
detached by digestion with 0.05% trypsin containing EDTA and passaged.
Treatment of KGN Cells
KGN cells were stimulated with different concentrations (0, 25, 50, 100
and 200 μM) of AA and the optimum concentration was selected for
subsequent experiments. After treating KGN cells with different
concentrations of AA, we examined whether these concentrations have
different effects on mitochondrial function and cell viability. In
addition, KGN cells were exposed to 50 μM AA for 12 h with or without
GSH (5 mmol/L). KGN cells pretreated with or without buthionine
sulfoximine (BSO) (10 μM)for 1h, and added with or without recombinant
GDF15 (50ng/ml)(rGDF15; Abnova, Taiwan, China) for 4 h in advance, were
also exposed to 50 μM AA for 12 h. KGN cells transfected with GDF15
small-interfering RNA (siRNA) for 6 h and then culture was continued by
replacing the medium. Finally, KGN cells were also exposed to 50 μM AA
for 12 h in the presence or absence of 20 μM Mito-TEMPO.
Cell Viability
Briefly, the KGN cells were seeded into a 96-well plate and treated
with AA at different concentration for 12 h. Then, 10 μL of Cell
Counting Kit-8 (CCK-8; Beyotime Biotechnology, Shanghai, China)
solution was added into each well and the plate was incubated at 37°C
for 1 h. The optical density (OD) of each well was measured at 450 nm
on a microplate reader(BioTek Instruments, Winooski, VT, USA).
Measurement of ROS, O[2-] and mitoSOX Levels
KGN cells were treated as described above, and the cells were incubated
with 20 μM dichlorodihydrofluorescein diacetate (DCFH-DA; Beyotime
Biotechnology) for 20 min, followed by 10 μM dihydroethidium (Beyotime
Biotechnology) for 30 min or 5 μM MitoSOX Red mitochondrial superoxide
indicator (Invitrogen, Carlsbad, CA, USA) for 10 min. Intracellular
ROS, O[2-] and mitoSOX levels were measured by flow cytometry.
Measurement of Parameters Related to Pro-Oxidation and Anti-Oxidation
After subjecting the KGN cells to the appropriate treatment, their
proteins were extracted. Then, the contents of malondialdehyde (MDA)
and reduced glutathione (GSH) were measured using to the corresponding
assay kit (Beyotime Biotechnology). In addition, the activities of
superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase
(GSH-Px) and glutathione reductase (GR) were determined using to the
corresponding assay kit (Beyotime Biotechnology).
Measurement of Mitochondrial Membrane Potential and ATP Content
After the appropriate treatment, KGN cells were respectively incubated
with JC-1 and tetramethylrhodamine ethyl ester (TMRE) fluorescent
probes and hoechst staining using the corresponding mitochondrial
membrane potential detection kit (Beyotime Biotechnology). Then, probes
and dye were detected by flow cytometry and immunofluorescence
microscopy. The ATP content was measured according to the instructions
and the corresponding kit (Beyotime Biotechnology)
Measurement of Levels of Hormones
After treatment, culture supernatants were collected. The estradiol
(E[2]) and progesterone (P) levels were measured using the
corresponding enzyme-linked immunosorbent assay (ELISA) kit (Cusabio
Technology LLC, Houston, TX, USA).
Cell Apoptosis Analysis
After the appropriate treatment, KGN cells were incubated with annexin
V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) according
to the manufacturer’s instructions (Beyotime Biotechnology). Then, cell
apoptosis rate was determined by flow cytometry. In addition, Caspase3
activity was determined using the corresponding assay kit (Beyotime
Biotechnology).
RNA Sequencing and Bioinformatics Analysis
Each group of 3 KGN cells RNA samples with RNA Integrity Number (RIN) ≥
7 were sequenced by Sangon Biotech Co, Ltd. (Shanghai, China). We used
the Gene Ontology (GO) database to functionally categorize the
differentially expressed genes. The possible signaling pathways
associated with these differentially expressed genes were identified
using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database.
RNA Interference
After introducing the GDF15 siRNA into KGN cells using the
Lipofectamine 3000 protocol cells were collected at 48 h with or
without Mito-TEMPO.Primer sequences are listed in [47]Table 1 .
Table 1.
The primer sequences for small-interfering RNAs (siRNAs) targeting
GDF15.
siRNAs Primer
siRNA-1(sense) 5’-CUCAGAGUUGCACUCCGAATT-3’
siRNA-1(antisense) 5’-UUCGGAGUGCAACUCUGAGTT-3’
siRNA-2(sense) 5’-CCGGAUACUCACGCCAGAATT-3’
siRNA-2(antisense) 5’-UUCUGGCGUGAGUAUCCGGTT-3’
siRNA-3(sense) 5’-GCUCCAGACCUAUGAUGACTT-3’
siRNA-3(antisense) 5’-GUCAUCAUAGGUCUGGAGCTT-3’
negative control(sense) 5’-UUCUCCGAACGUGUCACGUTT-3’
negative control(antisense) 5’-ACGUGACACGUUCGGAGAATT-3’
[48]Open in a new tab
Quantitative Real-Time Quantitative Polymerase Chain Reaction (qRT-PCR)
Analysis
Total RNA was extracted from KGN cells using the appropriate RNA
extraction kit (Beyotime Biotechnology) according to the manufacturer’s
instructions. The extracted RNA was reverse transcribed using a reverse
transcription kit. Polymerase chain reaction (PCR) was used to measure
the expression of target genes, and quantitative real-time PCR
(qRT-PCR) was used to determine the dynamic abundance of target genes.
Relative mRNA expression of the following genes was analyzed by 2^−ΔCT
method. Primer sequences are listed in [49]Table 2 .
Table 2.
The primer sequences for CYP11A1, CYP19A1, STAR, HSD3B1, BAX, BCL2,
Caspase3 and GAPDH.
Gene Primer
CYP11A1(sense) 5’-CCCTGTTGGATGCAGTGTCT-3’
CYP11A1(antisense) 5’-TTGAGCACAGGGTACTTTA-3’
CYP19A1(sense) 5’-GGACCCCTCATCTCCCACG-3’
CYP19A1(antisense) 5’-CCCAAGTTTGCTGCCGAAT-3’
STAR(sense) 5’-CAGACTTCGGGAACATGCCT-3’
STAR(antisense) 5’-GGGACAGGACCTGGTTGATG-3’
HSD3B1(sense) 5’-AGCATCCGAGGACAGTTCTAC-3’
HSD3B1(antisense) 5’-AGGGCGGTCGATAGGTGTAA-3’
BAX(sense) 5’-ACGGCCTCCTCTCCTACTTT-3’
BAX(antisense) 5’-GCCTCAGCCCATCTTCTTC-3’
BCL-2(sense) 5’-GCCGGTTCAGGTACTCAGTC-3’
BCL-2(antisense) 5’-GCCGGTTCAGGTACTCAGTC-3’
Caspase3(sense) 5’-CTGGACTGTGGCATTGAGAC-3’
Caspase3(antisense) 5’-GCAAAGGGACTGGATGAACC-3’
GAPDH(sense) 5’-ATTTGGCTACAGCAACAGG-3’
GAPDH(antisense) 5’-TTGAGCACAGGGTACTTTATT-3’
[50]Open in a new tab
Steroidogenic acute regulatory protein (STAR), cytochrome P450 family
11 subfamily a member 1 (CYP11A1), hydroxy-delta-5-steroid
dehydrogenase, 3β-hydroxysteroid dehydrogenase(HSD3B1), cytochrome P450
family 17 subfamily a polypeptide 1 (CYP19A1), cytochrome P450 family
19 subfamily a member 1 (CYP19A1).
Western Blot Analysis
After the appropriate treatment, the proteins from KGN cells were
extracted and the protein concentration was determined using the
bicinchoninic acid (BCA) Protein Assay Kit (Beyotime Biotechnology)
according to the manufacturer’s instructions. Then, the samples were
subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) and then transferred to polyvinylidene fluoride (PVDF
membranes. Antibodies against GDF15 (1:1,000; Cell Signaling
Technology, Danvers, MA, USA), or β-actin (1:2,000; Proteintech Group,
Rosemont, IL, USA) were incubated overnight with the membranes followed
by incubation with the appropriate horseradish peroxidase
(HRP)-conjugated secondary antibody. The signal was visualized using an
enhanced chemiluminescence substrate kit.
Statistical Analysis
All the results are presented as the means ± standard error of mean
(SEM) and each experiment was performed in triplicate. Statistical
analyses were performed with the GraphPad Prism8 software (GraphPad
Software Inc., San Diego, CA, USA) and the SPSS v.17.0 software (IBM
Corporation, Armonk, NY, USA). When a one-way analysis of variance
(ANOVA) indicated significant differences among groups, a value of P <
0.05 was considered statistically significant.
Results
Analysis of IVF Cycle Parameters of the Study Population
The general characteristics of the patients are shown in [51]Table 3 .
There were no significant differences between the groups in terms of
individual characteristics and basic clinical data, including age, BMI,
or serum FSH, E[2], P on days 3–5 of the menstrual cycle. However, the
serum testosterone level on days 3–5 of the menstrual cycle and
duration of infertility was higher level in the PCOS patients than in
the control subjects (P<0.01)([52] Table 3 ). Furthermore, the E[2] and
testosterone levels on the day of administration were significantly
higher in PCOS patients than those in control subjects (P<0.05) ([53]
Table 4 ). In addition, embryo implantation rate was considerably lower
in the PCOS patients than in the control subjects (P < 0.05) ([54]
Table 4 ).
Table 3.
Clinical characteristics of the study participants between control and
PCOS group
[MATH: X¯±S :MATH]
.
Variables Control (n=16) PCOS (n=16) P-value
Age(y) 31.81 ± 3.94 31.88 ± 4.10 0.965
Duration of infertility(y) 3.06 ± 1.34 6.00 ± 3.95 <0.01*
BMI^a (Kg/m^2) 23.04 ± 1.87 23.69 ± 1.75 0.313
Basal FSH^b(U/L) 4.35 ± 2.30 5.16 ± 2.42 0.341
Basal LH^c (U/L) 4.21 ± 1.22 3.79 ± 2.10 0.502
Basal E2(pg/ml) 53.39 ± 19.12 48.88 ± 18.73 0.505
Basal Testosterone(ng/mL) 0.38 ± 0.15 0.65 ± 0.09 <0.01^*
Total gonadotropin dose (IU) 2812.5 ± 978.26 2343.75 ± 812.64 0.151
LH level on the day of hCG administration(mIU/m L) 1.65 ± 0.30 1.73 ±
0.74 0.627
E2 level on the day of administration(pg/ml) 2394.28 ± 597.18 3909.52 ±
984.34 <0.01^*
P level on the day of administration (ng/mL) 1.06 ± 0.56 1.03 ± 0.40
0.857
Testosterone level on the day of administration(ng/mL) 0.34 ± 0.12 0.82
± 0.42 0.025^*
[55]Open in a new tab
^aBMI, body mass index; ^bFSH, Follicle- stimulating hormone; ^cLH,
Luteinizing Hormone; ^dE2, estradiol.
Table 4.
Laboratory and pregnancy outcomes of the study participants between
control and PCOS group
[MATH: X¯±S :MATH]
.
Variables Control (n=16) PCOS (n=16) P-value
Cycles(n) 16 16 —
Number of oocytes retrieved (n) 7.50 ± 1.32 8.00 ± 3.38 0.586
Number of mature oocytes (n) 6.75 ± 1.29 7.75 ± 3.26 0.262
Number of normal fertilized oocytes (n) 6.13 ± 1.01 6.50 ± 2.76 0.616
Fertilization rated^d (%) 81.67 (98/120) 81.25 (104/128) 0.933
Number of cleaved embryos Cleavage rate^e (%) 72.45 (71/98) 64.42
(67/104) 0.159
Number of available embryos 4.44 ± 2.42 4.19 ± 1.17 0.712
Number of good quality embryos 3.06 ± 1.69 3.13 ± 1.09 0.902
Good quality embryo rate^f (grade I and II) (%) 69.01 (49/71) 74.63
(50/67) 0.587
Embryo implantation rate^g (%) 50.00 (26/48) 28.57 (18/56) 0.039*
Cumulative clinical pregnancy rate^h (%) 58.30 (14/24) 39.29 (11/28)
0.275
[56]Open in a new tab
Data are reported as mean ± standard deviation.
^dFertilization rate, number of fertilized oocytes/number of oocytes
retrieved; ^eCleavage rate, number of cleaved embryo/number of
fertilized oocytes; ^fGood quality embryo rate, grade I and II/number
of cleaved embryo ^gEmbryo implantation rate, Number of gestational
sac/number of embryos transferred. ^hCumulative clinical pregnancy
rate, presence of embryonic heartbeat visualized by ultrasound at 6
weeks after embryo transfer/number of transplantation cycles.
Fatty Acid Composition in Follicular Fluid
A total of 35 fatty acids in follicular fluid were found in different
amounts in normal weight control, normal weigh PCOS, overweight control
and overweight PCOS. However, some fatty acids were not detected at low
levels, so a total of 18 fatty acids were detected. The content of
Palmitic acid (C16:0),Palmitoleic acid (C16:1),stearic acid (C18:0),
coleic acid (C18:1n9),linoleic acid(C18:2n6c) were significantly higher
in the overweight PCOS women than in the overweight control women
(P<0.01)([57] Figures 1A–C and [58]Supplementary Table 1 ). In
addition, the normal weight PCOS patients had a significantly higher
content of arachidonic acid(C20:4n6) than the normal weight control
subjects (P<0.01) ([59] Figure 1C and [60]Supplementary Table 1 ). At
the same time, saturated, monounsaturated, and polyunsaturated fatty
acids in follicular fluid differed among the four groups ([61]
Supplementary Table 1 ).
Figure 1.
[62]Figure 1
[63]Open in a new tab
Fatty acid composition in follicular fluid. (A–C)
C16:0、C16:1、C18:0、C18:1n9c、C18:2n6c、C20:4n6 between the overweight PCOS
patients 、the overweight control subjects、the normal PCOS patients and
the normal control subjects. (D–I) Representative GC-MS chromatograms
of follicular fluid (FF) speciens from patients that underwent IVF. (D)
FF from a normal control (B) FF from a normal PCOS (E, H) Chromatograms
of 35 standard fatty acid in a standard mixture (G). FF from a
overweight control (I). FF from an overweight PCOS. Different lowercase
letters at the top of each bar denote significant differences among
groups. Data represent mean ± standard error, **P < 0.01.
Complete GC-MS lipidomic datasets were obtained for the follicular
fluid of normal weight control subjects, normal weight PCOS patients,
overweight control subjects and overweight PCOS patients ([64]
Figures 1D–I ).
AA Exhibited Dose-Dependent Cytotoxicity in KGN Cells
As shown in [65]Figures 2A, B , the mitochondrial membrane potential,
as determined by measurement of the relative red and green fluorescence
intensity at all time points, was decreased with increasing AA
concentration after 12 h treatment. In particular, these results reveal
that AA, between 0 and 50 μM, only slightly affected mitochondrial
membrane potential, but between 100 and 200 μM it caused obvious
impairment of mitochondrial membrane potential in KGN cells. The
purpose of using the CCK-8 cell viability assay was to screen the
optimal concentration of AA for the stimulation of KGN cells. AA from 0
to 25 μM did not affect the viability of KGN cells, and 50 μM only
slightly affected their viability. However, AA between 100 and 200 μM
showed cytotoxic effects on KGN cells. Thus, based on these two
experiments, the 50 μM concentration of AA was chosen as the optimal
does for subsequent experiments ([66] Figure 2C ).
Figure 2.
[67]Figure 2
[68]Open in a new tab
(A–C) AA caused KGN cells toxicity in a dose-dependent manner. (A, B)
Mitochondrial membrane potential was decreased with increasing AA
concentration. (C) The viability of KGN cells was decreased with
increasing AA concentration. (D–N) AA caused damage to antioxidant
capability of KGN cells. (D, E) AA enhanced the accumulation of the
mitoSOX level. (F, G) AA enhanced the accumulation of the intracellular
O[2-] level. (H–M) AA weakened the activities of antioxidantenzymes
SOD, CAT, GSH-Px and GR and the GSH content. (N) AA promoted the
expression of GDF15 protein. Data represent mean ± standard error, ^*P
< 0.05, ^* #P < 0.05.
AA Impaired the Antioxidant Capacity of KGNs
When exposed to AA, the levels of the OS biomarkers MDA, ROS and O[2-]
levels were higher than those in the control KGN cells. After exposure
to GSH, the levels of MDA, ROS and O[2-] were restored to normal in KGN
cells ([69] Figures 2D–H ). It is well known that OS occurs due to the
limited ability of antioxidants to remove excess ROS and restore the
balance between the antioxidant system and the pro-oxidant system in
cells. After exposure to AA, the activities of antioxidant enzyme,
including SOD, CAT,GSH-Px and GR, in KGN cells decreased, while the
content of GSH also decreased([70] Figures 2I–M ). Also, GSH can
attenuate the oxidative damage of AA to KGN cells, due to its ability
to enhance the antioxidant capacity of KGN cells and reduce the
accumulation of intracellular ROS. In addition, after exposure to AA,
the level of GDF15 protein increased while the level of GDF15 protein
decreased after GSH supplementation ([71] Figure 2N ).
AA Impaired the Secretory and Mitochondrial Functions in KGN Cells
Mitochondria have previously been reported to be important organelles
that control the production of energy necessary for cell survival. When
exposed to AA, the ATP level and mitochondrial membrane potential
decreased, while intracellular mitoSOX level increased, suggesting that
mitochondrial function was impaired ([72] Figures 3A–F ). GSH
supplementation ameliorated the AA-induced mitochondrial dysfunction.
The contents of E[2] and P in the supernatant, secreted by KGN cells in
vitro, were determined to evaluate the effect of AA on the function of
ovarian granulosa cells. KGN cells exposed to AA exhibited an impaired
secretion function, including increased estrogen secretion and
decreased P secretion concomitant with the upregulation of CYP19A1 and
downregulation of STAR, CYP11A1,HSD3B1. Supplementation of GSH restored
the secretion function of KGN cells impaired by AA ([73] Figures 3G–J
). TMRE is an orange-red cationic fluorescent probe that permeates cell
membranes and can aggregate in intact mitochondria, but decreased
depolarization or inactive mitochondrial membrane potential leads to
decreased accumulation of TMRE. Staining of living cells with TMRM
revealed that AA-induced mitochondrial dysfunction
reduced intracellular accumulation of TMRM. Also, GSH supplementation
can protect the mitochondrial function in AA-treated KGN cells ([74]
Figure 3E ).
Figure 3.
[75]Figure 3
[76]Open in a new tab
AA caused damage to secretory and mitochondrial functions of KGN cells.
(A, B) After AA treatment, mitochondrial membrane potential was
distinctly decreased. (C) The mitochondrial function indicators ATP
level was decreased. (D, F) the level of intracellular mitoSOX was
increased. (E) The TMRE level in KGN cells was measured after AA
inhibition and addition of exogenous GSH. (G–J) KGN cells exposing to
AA exhibited an aberrant secretion function including promoting
estrogen secretion and inhibiting progesterone secretion concomitant
with the upregulation of CYP19A1 and the downregulation of STAR,
CYP11A1,HSD3B1. (K, L) Exposing to AA promoted KGN cells apoptosis rate
together with abnormal expression of Caspase3、BAX and BCL2. (M)
Exposing to AA promoted Caspase3 activity. Data represent mean ±
standard error, ^*P < 0.05, ^* #P < 0.05.Bar = 100μm.
AA Promotes Apoptosis in KGN Cells
It is well established that mitochondrial dysfunction is closely
related to cell apoptosis. Exposure to AA enhanced the apoptosis rate
of KGN cells and led to abnormal expression of Caspase3, BAX and BCL2.
In contrast, supplementation of GSH decreased cell apoptosis rate and
restored the normal expression levels of Caspase3, BAX and BCL2.
Furthermore, exposure to AA increased Caspase3 activity, while
supplementation of GSH had the opposite effect ([77] Figures 3K–M ).
RNA Sequencing and Bioinformatics Analysis
Analysis of gene expression profile after AA treatment was performed. A
total of 267 genes were differentially expressed between the AA-treated
group and normal control (NC) group (absolute log[2] (fold change) ≥1,
P<0.05). As shown in the stratified cluster heat map and volcano map in
[78]Figures 4A, B , among the 267 differentially expressed genes, 178
were upregulated and 89 were downregulated in the AA-treated group
([79] Figures 4A, B ). According to the references and our interest, 7