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other·endocrinology, reproductive endocrinology, gynecology, nutrition·PMC10145554

n-3 PUFA Promotes Ferroptosis in PCOS GCs by Inhibiting YAP1 through Activation of the Hippo Pathway

Nutrients · 14 authors, 4 centres

AI SUMMARY

FIDELITY 92%

POPULATIONDHEA-induced PCOS mouse model (ICR mice) and KGN ovarian granulosa cell line

INTERVENTIONn-3 PUFA supplementation (DHA 180 mg/mL + EPA 120 mg/mL, 0.2 mL daily by gavage for 60 days in vivo; DHA 1.8 ng/mL + EPA 1.2 ng/mL for 48 h in vitro)

COMPARISONPCOS model group without n-3 PUFA treatment; n-3 PUFA + Ferrostatin-1 (ferroptosis inhibitor) group

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TL;DR

This study demonstrates that n-3 polyunsaturated fatty acids (PUFA) inhibit the over-proliferation of ovarian granulosa cells in polycystic ovary syndrome (PCOS) by activating the Hippo signaling pathway, which promotes YAP1 nuclear exclusion and disrupts YAP1-Nrf2 crosstalk, ultimately triggering ferroptosis. In a DHEA-induced PCOS mouse model, n-3 PUFA supplementation reduced body weight, restored estrous cycles, and improved serum hormone profiles including testosterone, estradiol, and AMH. These findings identify the Hippo-YAP1-Nrf2-ferroptosis axis as a potential therapeutic target for PCOS and support n-3 PUFA as a dietary intervention for clinical symptom relief.

Full summary

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**Background:** Polycystic ovary syndrome (PCOS) is an endocrine disorder affecting 6–10% of women of reproductive age, characterized by hyperandrogenemia, insulin resistance, anovulation, and polycystic ovarian morphology. Current treatments target clinical symptoms but yield unsatisfactory results. n-3 polyunsaturated fatty acids (PUFA) have demonstrated anti-inflammatory, anti-hyperlipidemic, and insulin-sensitizing properties, but the molecular mechanisms underlying their therapeutic effects in PCOS remain poorly understood. Ferroptosis, a non-apoptotic form of cell death driven by lipid peroxidation and iron-dependent oxidative stress, has been implicated in various diseases. The Hippo signaling pathway and its downstream effector YAP1 regulate cell proliferation and differentiation, and YAP1 crosstalk with Nrf2 has been shown to modulate ferroptosis susceptibility. This study investigates whether n-3 PUFA alleviates PCOS by promoting ferroptosis in ovarian granulosa cells via the Hippo-YAP1-Nrf2 axis.

**Methods:** Three-week-old ICR mice were injected with DHEA (6 mg/100 g body weight) for 21 consecutive days to induce PCOS. Dietary intervention with n-3 PUFA (DHA 180 mg/mL + EPA 120 mg/mL, 0.2 mL daily by gavage) began 21 days after DHEA treatment and continued for 60 days. Vaginal epithelial cells were stained with toluidine blue to assess estrous cycles. Serum hormone levels (testosterone, estradiol, LH, progesterone, FSH, AMH, prolactin, SHBG) were measured by ELISA. KGN ovarian granulosa cells were treated with n-3 PUFA (DHA 1.8 ng/mL + EPA 1.2 ng/mL) for 48 h, with or without Ferrostatin-1 (20 μM), a ferroptosis inhibitor. Cell proliferation was assessed by CCK-8 and EDU assays. Ferroptosis was evaluated by measuring MDA, GSH, Fe²⁺ levels, transmission electron microscopy for mitochondrial morphology, and expression of ferroptosis marker genes (TFRC, ACSL4, SLC7A11, FTH, FTL, GPX4) by qPCR and western blot. Hippo pathway activation and YAP1 subcellular localization were assessed by immunohistochemistry, immunofluorescence, and western blot for MST1/2, LAST1/2, and YAP1. Nrf2 expression was also measured.

**Key Results:** n-3 PUFA treatment significantly reduced body weight in PCOS mice and restored serum hormone levels: testosterone was suppressed, estradiol and progesterone were increased, and AMH was significantly reduced compared to the PCOS group. n-3 PUFA restored the disrupted estrous cycle, increased the ovarian index, and significantly elevated SHBG levels. Ovarian histomorphology showed increased corpora lutea and reduced cystic follicles. In KGN cells, n-3 PUFA significantly inhibited proliferation as measured by CCK-8 and EDU assays, and flow cytometry confirmed cell cycle arrest. n-3 PUFA promoted ferroptosis, evidenced by increased MDA and Fe²⁺, decreased GSH, upregulated TFRC and ACSL4 expression, and downregulated SLC7A11, FTH, FTL, and GPX4 expression. Transmission electron microscopy revealed mitochondrial shrinkage and increased bilayer density characteristic of ferroptosis. JC-1 staining showed a shift from high to low mitochondrial membrane potential, and BODIPY 581/591 staining confirmed increased lipid peroxidation. Ferrostatin-1 rescued these effects. n-3 PUFA activated the Hippo pathway, as demonstrated by increased expression of MST1/2 and LAST1/2 kinases, and promoted YAP1 nuclear exclusion, with significantly reduced nuclear YAP1 in treated cells. Nrf2 expression was downregulated following n-3 PUFA treatment and upregulated with ferroptosis inhibitor addition, consistent with disrupted YAP1-Nrf2 crosstalk.

**Clinical Implications:** This study provides mechanistic evidence that n-3 PUFA supplementation may serve as a dietary intervention for PCOS by targeting the Hippo-YAP1-Nrf2-ferroptosis axis to inhibit abnormal granulosa cell proliferation and restore ovarian function. The findings identify YAP1-Nrf2 crosstalk as a potential therapeutic target for regulating granulosa cells in PCOS. However, these results are derived from a DHEA-induced mouse model and KGN cell line; clinical translation requires validation in human studies. The study does not report specific p-values or confidence intervals for key comparisons, limiting quantitative assessment of effect sizes. Further research is needed to determine optimal dosing, long-term safety, and efficacy in PCOS patients.

PICO

PPOPULATION

DHEA-induced PCOS mouse model (ICR mice) and KGN ovarian granulosa cell line

IINTERVENTION

n-3 PUFA supplementation (DHA 180 mg/mL + EPA 120 mg/mL, 0.2 mL daily by gavage for 60 days in vivo; DHA 1.8 ng/mL + EPA 1.2 ng/mL for 48 h in vitro)

OOUTCOME

Body weight, serum hormone levels (testosterone, estradiol, LH, FSH, progesterone, AMH, prolactin), estrous cycle regularity, ovarian index, SHBG levels, granulosa cell proliferation (CCK-8, EDU), ferroptosis markers (MDA, GSH, Fe²⁺, TFRC, ACSL4, SLC7A11, GPX4), mitochondrial membrane potential, YAP1 nuclear localization, Hippo pathway kinase expression (MST1/2, LAST1/2), Nrf2 expression

STUDY TYPE

other

SPECIALTY

endocrinology

SUMMARISED BY

AI pipeline

FIDELITY CHECK

92% · A

ORIGINAL PAPER

PMC10145554

n-3 PUFA Promotes Ferroptosis in PCOS GCs by Inhibiting YAP1 through Activation of the Hippo Pathway

Nutrients

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ABSTRACT

Polycystic ovary syndrome (PCOS) is an endocrine disorder characterized by hyperandrogenemia with multiple suspended sinus follicles, thickened cortical tissue, and excessive proliferation of ovarian granulosa cells that severely affects the fertility and quality of life of women. The addition of n -3 PUFA to the diet may slightly reduce body weight and greatly alleviate disturbed blood hormone levels in PCOS mice. We treated KGN as a cell model for n -3 PUFA addition and showed that n -3 PUFA inhibited the proliferation of GCs and promoted ferroptosis in ovarian granulosa cells. We used CCK-8, fluorescence quantitative transmission electron microscopy experiments and ferroptosis marker gene detection and other methods. Furthermore, n -3 PUFA was found to promote YAP1 exocytosis by activating Hippo and weakening the cross-talk between YAP1 and Nrf2 by activating the Hippo signaling pathway. In this study, we found that n -3 PUFA inhibited the over proliferation of granulosa cells in ovarian follicles by activating Hippo, promoting YAP1 exocytosis, weakening the cross-talk between YAP1 and Nrf2, and ultimately activating the ferroptosis sensitivity of ovarian granulosa cells. We demonstrate that n -3 PUFA can alleviate the hormonal and estrous cycle disorder with PCOS by inhibiting the YAP1-Nrf2 crosstalk that suppresses over proliferating ovarian granulosa cells and promotes iron death in GCs. These findings reveal the molecular mechanisms by which n -3 PUFA attenuates PCOS and identify YAP1-Nrf2 as a potential therapeutic target for regulation granulosa cells in PCOS.

CITATION EXPORT

Peiwen Zhang, Yuheng Pan, Shuang Wu et al.. n-3 PUFA Promotes Ferroptosis in PCOS GCs by Inhibiting YAP1 through Activation of the Hippo Pathway. Nutrients. (2023). https://doi.org/10.3390/nu15081927

AUTHORS

14 · 4 CENTRES

Peiwen Zhang, Yuheng Pan, Shuang Wu, Yuxu He, Jinyong Wang, Lei Chen, Shunhua Zhang, Hui Zhang, Ye Zhao, Lili Niu, Mailin Gan, Yan Wang, Linyuan Shen, Li Zhu

1Key Laboratory of Livestock and Poultry Multi-Omics, Ministry of Agriculture and Rural Affairs, College of Animal and Technology, Sichuan Agricultural University, Chengdu 611130, China; [email protected] (P.Z.); [email protected] (Y.P.); [email protected] (S.W.); [email protected] (Y.H.); [email protected] (L.C.); [email protected] (S.Z.); [email protected] (Y.Z.); [email protected] (L.N.); [email protected] (M.G.); [email protected] (L.Z.)
2Farm Animal Genetic Resource Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu 611130, China
3Chongqing Academy of Animal Science, Chongqing 402460, China; [email protected]
4Sichaun Center for Animal Disease Control, Chengdu 610041, China; [email protected]

EDITOR / REVIEWER

James H. Swain, Carlo Agostoni