Nuciferine protects hyperandrogen-injured ovarian granulosa cells by inhibiting ferroptosis via SOX2-mediated activation of the SLC7A11/GPX4 axis.

Polycystic ovary syndrome (PCOS) is a common endocrine disorder that can cause menstrual irregularities, infertility, polycystic ovaries, and metabolic abnormalities. Female reproductive health and quality of life are significantly affected by PCOS, which has recently been associated with ferroptosis in granulosa cells (GCs). Nuciferine (NF) is a naturally extracted substance with multiple pharmacological activities, which is reported with anti-ferroptosis function. Herein, the influence of NF for androgen-induced ferroptosis in GCs was investigated to explore the potential value of NF on treating PCOS. 10 μM NF and 20 μM NF were employed for treating KGN cells according to cell viability results. KGN cells were treated with 10 μM dehydroepiandrosterone (DHEA) for 1 day, followed by introducing 10 μM NF and 20 μM NF for 24 h. Strikingly reduced cell viability, increased lactate dehydrogenase release and reactive oxygen species (ROS) production, enhanced apoptosis, upregulated Bax, downregulated Bcl-2, restrained malondialdehyde contents, and declined superoxide dismutase activity were observed in DHEA-treated KGN cells, which were significantly reversed by NF. Significantly repressed GPX4, SLC7A11, and SOX2 levels, as well as increased ACSL4 levels and Fe2+ levels in DHEA-treated KGN cells, were notably rescued by NF. Furthermore, the inhibitory effect of NF on ROS production and ferroptosis in DHEA-treated KGN cells was partially abrogated by silencing SOX2. Collectively, NF protected DHEA-injured ovarian GCs by inhibiting ferroptosis via upregulating SOX2.

Yang H ，Chen S ，Yin S ，Ding Q ... -  《-》

Atractylodin alleviates polycystic ovary syndrome by inhibiting granule cells ferroptosis through pyruvate dehydrogenase kinase 4-mediated JAK-STAT3 pathway.

Polycystic ovary syndrome (PCOS) is a common endocrine disorder, and its close relationship with oxidative stress has been well-documented. Atractylodin (ATR) plays a role in the treatment of many diseases through its antioxidant function. However, its function in PCOS remains unexplored. In this study, the function and underlying mechanisms of ATR in mitigating PCOS symptoms were investigated. A mouse model of PCOS induced using DHEA and a high-fat diet was established, and many factors such as hormone levels (FSH, LH, testosterone, and progesterone), the estrous cycle, and ovarian shape were evaluated. In vitro, PCOS model was established by DHEA-induced KGN cell, and the effects of ATR on ferroptosis and oxidative stress markers were explored. Specifically, the viability of KGN cells treated with ATR was assessed using the CCK-8 assay, and the levels of malondialdehyde (MDA), glutathione (GSH), and reactive oxygen species (ROS) were measured to evaluate oxidative stress. Expression of ferroptosis-related genes (NRF2, GPX4, SLC7A11) and PDK4 was analyzed by qRT-PCR and Western blotting. PDK4's interaction with ATR was examined through molecular docking and confirmed by surface plasmon resonance (SPR) analysis. Our data show that the treatment of ATR markedly increased hormone levels and improved normal estrous cycles. Moreover, ATR was found to improve ovarian morphology by decreasing cystic dilatation and increasing the number of corpora lutea. Mechanistically, our research found that ATR regulates the expression of PDK4 by binding to its GLY331 and inhibits granulosa cell ferroptosis by regulating the JAK-STAT3 pathway mediated by PDK4. In conclusion, our study suggest that ATR may be a therapeutic option for managing PCOS and PDK4 could be a target for the development of new drugs for PCOS.

Zhou Q ，Ouyang X ，Tang H ，Wang Y ，Hua Y ，Li L ... -  《-》

Erratum: Eyestalk Ablation to Increase Ovarian Maturation in Mud Crabs.

《Jove-Journal of Visualized Experiments》

Icariin promoted ferroptosis by activating mitochondrial dysfunction to inhibit colorectal cancer and synergistically enhanced the efficacy of PD-1 inhibitors.

A controlled type of cell death called ferroptosis is linked to increased reactive oxygen species (ROS), lipid peroxidation, and iron buildup. Furthermore, evidence indicates that ferroptosis may act as an immunogenic form of cell death with potential physiological functions in tumors and immunosuppression. Inducing ferroptosis in tumor cells may have the potential to complement cancer immunotherapy strategies. The development of colorectal cancer (CRC) and the poor efficacy of immunotherapy are associated with the crosstalk of cellular ferroptosis. Currently, Icariin (ICA), the main bioactive component extracted from Epimedium, has been shown to inhibit a variety of cancers. However, the specific role and potential mechanism of ICA in regulating ferroptosis in CRC remains unclear. The aim of this investigation was to clarify the mechanism underlying the anti-CRC cancer properties of ICA and how it induces ferroptosis to enhance immunotherapy. To evaluate cell viability, the Cell Counting Kit-8 (CCK-8) test was utilized. The transwell test and the wound healing assay were used to assess cell migration. A subcutaneous graft tumor model was constructed with C57BL/6 mice using MC38 colorectal cancer cell lines. The inhibitory effect of ICA on CRC, ferroptosis level and immunomodulatory effects were detected by serum biochemical assay, cytokine assay, hematoxylin-eosin (H&E) staining, immunofluorescence staining, CyTOF mass spectrometry flow screening and Western blotting. Western blotting, proteomics, molecular docking and microscale thermophoresis (MST) were used to forecast and confirm ICA's binding and interaction with HMGA2, STAT3, and HIF-1α. Moreover, the levels of lipid peroxidation and ferroptosis were assessed through the use of the C11-BODIPY fluorescent probe, the FerroOrange fluorescent probe, the iron level, the malondialdehyde (MDA) and reduced glutathione (GSH) assay kit, and Western blotting analysis. To assess alterations in mitochondrial structure and membrane potential, transmission electron microscopy (TEM) and JC-1 immunofluorescence were employed. It was demonstrated in the current study that ICA treatment inhibits CRC and enhances anti-PD-1 therapy efficacy by inciting ferroptosis. As shown in vitro, ICA inhibits CRC cell proliferation, migration, and apoptosis. As demonstrated in vivo, ICA has a dose-dependent tumor suppressor effect when combined with anti-PD-1, it can significantly inhibit tumor growth, increase the expression of serum TNF-α, IFN-γ, and granzyme B, and promote CD69+CD8+ T, CD69+CD8+Tem, CD69+CD8+Teff, TCRβ+CD8+ T, TCRβ+CD8+ T, TCRβ+CD8+Tem, TCRβ+CD8+Teff. The inhibitory effect of ICA on CRC was associated with the binding of HMGA2, STAT3, and HIF-1α proteins, which inhibited CRC by increasing the levels of reactive oxygen species (ROS) and malondialdehyde (MDA), promoting the accumulation of iron (Fe2+), depletion of reduced glutathione (GSH), inhibiting SLC7A11 and GPX4 expressions, thereby inducing ferroptosis in CRC. As a consequence of ICA-induced ferroptosis, mitochondria are dysfunctional, with increased ROS production, membrane potential depolarization (MMP), and ATP production reduced. This process can be efficiently reversed by the mitochondria-targeted antioxidant Mito-Q. It is noteworthy that the ferroptosis inhibitor liproxstatin-1 (lip-1), anti-CD8, and anti-IFN-γ exhibited a significant inhibitory effect on the level of ferroptosis and antitumor capacity of ICA combined with anti-PD-1. This finding suggests that the antitumor immunopotentiating effect of ICA on anti-PD-1 is dependent on the secretion of IFN-γ-induced ferroptosis of CRC cells by the CD8+ T cell. Our study represents the inaugural demonstration of the mechanism whereby ICA exerts anti-CRC effects and synergistically enhances the efficacy of anti-PD-1, inducing mitochondrial damage and leading to ferroptosis. ICA promotes ferroptosis of CRC cells by inducing mitochondrial dysfunction, and ICA combined with anti-PD-1 significantly promotes CD69, TCRβ signalling, activates effector CD8+ T cells to secrete IFN-γ, and achieves immunopotentiation by promoting ferroptosis of CRC cells, thus inhibiting CRC development. This study is built upon existing research into the pharmacodynamic mechanisms of ICA in the context of CRC, and offers a novel therapeutic approach in addressing the issue of CRC immunotherapy potentiation.

Haoyue W ，Kexiang S ，Shan TW ，Jiamin G ，Luyun Y ，Junkai W ，Wanli D ... -  《-》

Quercetin inhibits hydrogen peroxide-induced cleavage of heat shock protein 90 to prevent glutathione peroxidase 4 degradation via chaperone-mediated autophagy.

Oxidative stress is caused by the accumulation of reactive oxygen species (ROS) and the depletion of free radical scavengers, which is closely related to ferroptosis in diseases. Quercetin, as a natural flavonoid compound, has been reported to have multiple pharmacological effects on the basis of its anti-oxidative and anti-ferroptotic activities. This study was designed to explore the specific mechanism of quercetin against ferroptosis induced by hydrogen peroxide (H2O2). The HT22 cells (mouse hippocampal neuronal cells) treated with 40 μg·ml-1 H2O2 were used to investigate the role of ferroptosis in oxidative stress damage and the regulation of quercetin (7.5, 15, 30 μmol·l-1), as evidenced by assessments of cell viability, morphological damage, Fe2+ accumulation, and the expressions of ferroptotic-related proteins. The changes in the expression levels of glutathione peroxidase 4 (GPX4), heat shock cognate protein 70 (HSC70), lysosomal-associated membrane protein 2a (LAMP-2a), and heat shock protein (HSP90) were assessed by qPCR, western blotting (WB) and immunofluorescence (IF) assays. Additionally, the interactions of GPX4, HSC70, LAMP-2a, and HSP90 were examined by co-immunoprecipitation (Co-IP) assay to elucidate the impact of quercetin on the degradation pathway of GPX4 and the CMA pathway. To further explore the regulatory mechanism of quercetin, the si-LAMP-2a and HSP90 mutant cells were conducted. Pretreatment with 30 μmol·l-1 quercetin for 6 h significantly enhanced the survival rate (p < 0.05), maintained cell morphology, and inhibited Fe2+ levels in HT22 cells exposed to H2O2 (40 μg·ml-1). HT22 cells under oxidative stress showed lower expressions of GPX4 and ferritin heavy chain 1 (FTH1), and a higher level of Acyl-CoA synthetase long-chain family member 4 (ACSL4) (p < 0.05). And quercetin significantly reversed the expressions of these ferroptotic proteins (p < 0.05). Moreover, the autophagic lysosomal pathway inhibitor CQ effectively increased the expression of GPX4 in oxidative stress cell model. Further study showed that H2O2 increased the activity of macroautophagy and chaperone-mediated autophagy (CMA), while quercetin notably suppressed the levels of microtubule-associated protein light chain 3 Ⅱ (LC3 Ⅱ), LAMP-2a, and the activity of lysosomes (p < 0.01). Additionally, quercetin disrupted the interactions of GPX4, HSC70, and LAMP-2a, reduced cellular levels of CMA by decreasing the cleaved HSP90 (c-HSP90), and these effects were reversed in the R347 mutant HT22 cells. Quercetin has a significantly protective effect on oxidative stress cell model through the inhibition on ferroptosis, which is related to the degradation of GPX4 via CMA. And quercetin decreases the level of c-HSP90 induced by H2O2 to reduce the activity of CMA by binding to R347 of HSP90.

Peng C ，Li H ，Mao Q ，Tang K ，Sun M ，Ai Q ，Yang Y ，Liu F ... -  《-》