The Mechanism of Ferroptosis Regulating Granulosa Cell Apoptosis and Oxidative Stress Through the NF-κB/PTGS2 Axis in Porcine Atretic Follicles
Abstract
1. Introduction
2. Materials and Methods
2.1. Follicle Collection and Initial Processing
2.1.1. Sample Acquisition and Handling
2.1.2. Preparation of Transcriptome Sequencing Samples
2.2. Reagent Sources and Ingredients and Preparation of Commonly Used Reagents
2.2.1. Reagent Sources and Ingredients
2.2.2. Preparation of Commonly Used Reagents
2.3. Isolation and Culture of Primary Ovarian Granule Cells
2.4. Dissection, Culture, and Treatment of Porcine Follicles
2.5. Immunofluorescence Staining
2.6. Cell Cycle Detection
2.7. Criteria for Determining the Degree of Follicular Atresia
2.8. Real-Time PCR
2.9. Detection of Indicators
2.9.1. ROS Detection Assay
2.9.2. Mitochondrial Membrane Potential (ΔΨm) Assay
2.9.3. MitoTracker Staining
2.9.4. CCK-8
2.9.5. EDU
2.9.6. Reduced Glutathione (GSH) Assay
2.9.7. MDA Detection Method
2.9.8. Ferrous Ion Detection
2.9.9. BODIPT581/591 Labeling Experiment
2.10. Data Statistics and Analysis
3. Results
3.1. Different Levels of Ferroptosis in Healthy and Atretic Follicles of Pigs
3.2. Ferroptosis Inhibits Granulosa Cell Proliferation and Induces Oxidative Stress
3.3. The NF-κB in the Nuclear Inhibitors Inhibits Granular Cell Death and Oxidative Stress and Promotes Proliferation
3.4. Inhibition of PTGS2 Promotes the Proliferation of Granulosa Cells and Alleviates Ferroptosis
3.5. Melatonin Rescued Erastin-Induced Ferroptosis
3.6. Melatonin Receptor Inhibitors Weaken the Ferroptosis-Inhibiting Effect of Melatonin in GCs
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Abbreviations
LH | luteinizing hormone |
FSH | follicle-stimulating estrogen |
PCD | programmed cell death |
EA | primary atretic follicles |
PA | atretic follicles |
H | healthy follicles |
GC | granular cells |
ROS | reactive oxygen species |
GSH | glutathione |
ALOX | arachidonate lipoxygenase |
MUFA | monounsaturated Fatty Acids |
PUfas | polyunsaturated fatty acids |
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The Interfering Chain of PTGS2 | Sequence |
---|---|
si-ssc-PTGS2_001 | CCACGAGTACAACTATCAA |
si-ssc-PTGS2_002 | GCACTTCACCCATCAGTTT |
si-ssc-PTGS2_003 | CACCAACTTACAATATGCA |
Follicular State | Diameter | P4/E2 | Color | Vascular Distribution | Transparency |
---|---|---|---|---|---|
Healthy follicle | 4–5 mm | ≤5 | pink | more | transparent |
Primary atretic follicle | 4–5 mm | 5–10 | light pink | less | translucency |
Atretic follicles | 4–5 mm | ≥10 | milky white and yellow | none | turbid |
Gene Accession Number | Primer | Sequence (5′→3′) |
---|---|---|
NM_214407 | GPX4-F | GAGGCAAGACCGAAGTAAACTAC |
GPX4-R | CCGAACTGGTTACACGGGAA | |
NM_214001 | TFRC-F | ACCATTGTCATATACCCGGTTCA |
TFRC-R | CAATAGCCCAAGTAGCCAATCAT | |
XM_005660803 | FTH1-F | ACTTTGACCGCGATGATGTG |
FTH1-R | GCTCTCCCAGTCATCACAGT | |
NM_001244131 | FTL-F | ATGGGGTGCGGACTTAGAAAG |
FTL-R | CTTGCGGTCTCTTCAGGGTAG | |
XM_005673820 | ACSL4-F | CATCCCTGGAGCAGATACTCT |
ACSL4-R | TCACTTAGGATTTCCCTGGTCC | |
Z35483 | IκBα-F | TGACCTTGTTCACGGGTCTG |
IκBα-R | TCTTGGACTCCGTTCCCTCT | |
NM_001114281 | p65-F | GGCACCGGATTGAGGAGAAA |
p65-R | GCCTCTGTCAGTGTCCCTTC | |
GQ339058 | TNFα-F | CAAGCCACTCCAGGACCC |
TNFα-R | GAAAACGTTGGTGGAAGGGC | |
NM_213821 | SLC7A11-F | GACATCCGAGAAGCCAACAT |
SLC7A11-R | AGGTTGTTCCTGGGGAAGAT | |
NM_214131 | caspase 3-F | GCCATGGTGAAGAAGGAAAA |
caspase 3-R | CACGCCATGTCATCTTCAGT | |
AM233489 | BAX-F | AAGCGCATTGGAGATGAACT |
BAX-R | AAAGTAGAAAAGCGCGACCA | |
XM_021099602 | Bcl2-F | GACTTTGCCGAGATGTCCAG |
Bcl2-R | ACGCTCTCCACACACATGAC | |
NM_213824 | p53-F | CCTCACCATCATCACACTGG |
p53-R | GGCTTCTTCTTTTGCACTGG | |
XM_013977858 | p21-F | GAGAGCGATGGAACTTCGAC |
p21-R | GGACAGCAACAGGTCCACAT | |
XM_021086047 | ACTB-F | GGCTGTATTCCCCTCCATCG |
ACTB-R | CCAGTTGGTAACAATGCCATGT |
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Yang, Y.; He, Y.; Gan, M.; Zhao, X.; Liao, T.; Lei, Y.; Chen, L.; Niu, L.; Zhao, Y.; Wang, Y.; et al. The Mechanism of Ferroptosis Regulating Granulosa Cell Apoptosis and Oxidative Stress Through the NF-κB/PTGS2 Axis in Porcine Atretic Follicles. Antioxidants 2025, 14, 1071. https://doi.org/10.3390/antiox14091071
Yang Y, He Y, Gan M, Zhao X, Liao T, Lei Y, Chen L, Niu L, Zhao Y, Wang Y, et al. The Mechanism of Ferroptosis Regulating Granulosa Cell Apoptosis and Oxidative Stress Through the NF-κB/PTGS2 Axis in Porcine Atretic Follicles. Antioxidants. 2025; 14(9):1071. https://doi.org/10.3390/antiox14091071
Chicago/Turabian StyleYang, Yiting, Yuxu He, Mailin Gan, Xue Zhao, Tianci Liao, Yuhang Lei, Lei Chen, Lili Niu, Ye Zhao, Yan Wang, and et al. 2025. "The Mechanism of Ferroptosis Regulating Granulosa Cell Apoptosis and Oxidative Stress Through the NF-κB/PTGS2 Axis in Porcine Atretic Follicles" Antioxidants 14, no. 9: 1071. https://doi.org/10.3390/antiox14091071
APA StyleYang, Y., He, Y., Gan, M., Zhao, X., Liao, T., Lei, Y., Chen, L., Niu, L., Zhao, Y., Wang, Y., Shen, L., Liu, Y., & Zhu, L. (2025). The Mechanism of Ferroptosis Regulating Granulosa Cell Apoptosis and Oxidative Stress Through the NF-κB/PTGS2 Axis in Porcine Atretic Follicles. Antioxidants, 14(9), 1071. https://doi.org/10.3390/antiox14091071