Gut microbiota metabolite Urolithin B inhibits chondrocyte ferroptosis by rewriting iron homeostasis via FGFR3/NCOA4/FTH1 axis, alleviating osteoarthritis.

To explore the effect of acupuncture treatment on cerebral ischaemia-reperfusion injury (CIRI) and reveal the underlying mechanism of the effect based on nuclear receptor coactivator 4 (NCOA4) mediated ferritinophagy. Sprague-Dawley male rats were divided into four groups: the sham group, model group, acupuncture group, and sham acupuncture group. After 2 h of middle cerebral artery occlusion (MCAO), reperfusion was performed for 24 h to induce CIRI. The rats were treated with acupuncture at the Neiguan (PC6) and Shuigou (GV26) acupoints. Their neurological function was evaluated by taking their Bederson scores at 2 h after ischaemia and 24 h after reperfusion. Triphenyltetrazolium chloride staining was applied to assess the cerebral infarct volume at 24 h after reperfusion. The malondialdehyde (MDA) and ferrous iron (Fe2+) levels were observed after 24 h of reperfusion using an assay kit. Western blotting was performed to detect the expression of NCOA4 and ferritin heavy chain 1 (FTH1) at 24 h after reperfusion. Moreover, the colocalization of ferritin with neurons, NCOA4 with microtubule-associated protein 1 light chain 3 (LC3), and NCOA4 with ferritin was visualized using immunofluorescence staining. Acupuncture significantly improved neurological function and decreased cerebral infarct volume in the acupuncture group. Following CIRI, the expression of NCOA4, LC3 and FTH1 was increased, which enhanced ferritinophagy and induced an inappropriate accumulation of Fe2+ and MDA in the ischaemic brain. However, acupuncture dramatically downregulated the expression of NCOA4, LC3 and FTH1, inhibited the overactivation of ferritinophagy, and decreased the levels of MDA and Fe2+. Acupuncture can inhibit NCOA4-mediated ferritinophagy and protect neurons against CIRI in a rat model.

Ferritinophagy-derived iron causes protein nitration and mitochondrial dysfunction in acetaminophen-induced liver injury.

Static magnetic field (SMF) can alter cell fate decisions in many ways. However, the effects of SMF on cancer stem cells (CSCs) are little-known. In this particular study, we evaluate the biological effect of moderate-intensity SMF on osteosarcoma stem cells (OSCs) and try to clarify the underlying mechanisms of action. First, we demonstrated that prolonged exposure to SMF induced the proliferation and tumorsphere formation in K7M2 and MG63 OSCs. Moreover, SMF promoted the release of ferrous iron (Fe2+ ) and provoked reactive oxygen species (ROS) in OSCs. Interestingly, SMF evidently triggered the autophagic degradation of ferritin, which is characterized by the activation of microtubule-associated protein 1 light chain 3 (LC3) and nuclear receptor co-activator 4 (NCOA4), and downregulation of ferritin heavy chain 1 (FTH1) in OSCs. Particularly, the colony-forming ability of K7M2 OSCs promoted by SMF was obviously abolished by using a small interfering RNA (siRNA) against NCOA4. Finally, treatment of the tumor-bearing mice with SMF did not affect the tumor volume or tumor mass, nor pulmonary metastasis of K7M2 OSCs, but the SMF-treated K7M2 OSCs caused a preference of pulmonary metastasis in a mouse model, which suggested that SMF might induce the metastatic characteristic of OSCs. Consequently, this paper demonstrates for the first time that the cumulative SMF exposure promoted the self-renewal ability of OSCs via autophagic degradation of ferritin, implying that ferritinophagy may be a potential molecular target for cancer. © 2021 Bioelectromagnetics Society.

Severe acute pancreatitis (SAP) is an acute abdominal disease with extremely high mortality; autophagy-dependent ferroptosis plays a crucial role in acute pancreatitis. However, the specific underlying mechanism remains unclear. To investigate the role of nuclear receptor coactivator 4 (NCOA4) in SAP and the mechanism by which tetrandrine influences it. Experimental SAP models were established using L-arginine (L-Arg) induction to observe changes in NCOA4 expression. Knockout and overexpression experiments of NCOA4 were conducted to assess the impact on SAP. Additionally, invitro cell experiments were performed to verify these findings. Furthermore, the impact of N-glycosylation of NCOA4 on its function, particularly its binding ability with ferritin heavy chain 1 (FTH1), was studied. Finally, the effects of tetrandrine on N-glycosylation of NCOA4, the binding between NCOA4 and FTH1, and the progression of SAP were analyzed. NCOA4 expression was significantly upregulated in SAP. Knockout of NCOA4 improved the phenotype of SAP, whereas its overexpression exacerbated SAP. This was also confirmed invitro. N-glycosylation of NCOA4 is crucial for its binding with FTH1, which in turn affects ferroptosis. Tetrandrine targets the N-glycosylation of NCOA4, weakening the interaction between NCOA4 and FTH1, thereby inhibiting the progression of SAP. This study demonstrates that tetrandrine targets the N-glycosylation of NCOA4, inhibiting autophagy-dependent ferroptosis mediated by its binding to FTH1 and thus ameliorates SAP. This finding provides us with a novel therapeutic approach for SAP and offers a new perspective on understanding the mechanism of action of tetrandrine in SAP.

Osteoarthritis (OA) is a common joint disease with high morbidity, but there is still no definitive treatment for it. Long noncoding RNAs (lncRNAs) have been confirmed to play key roles in OA progression. This work was done to investigate the roles and action mechanism of lncRNA TNFSF10 in OA. The messenger RNA levels of TNFSF10 in articular cartilage samples from patients or chondrocytes were detected by Quantitative real-time PCR assay (qRT-PCR). The effects of TNFSF10 on chondrocytes were evaluated on the basis of cell growth, apoptosis, and inflammation. Then, the interaction between TNFSF10 and miR-376-3p was explored by dual-luciferase reporter test, RNA-binding protein immunoprecipitation, and RNA pull-down assay. Finally, various cell experiments, Western blot analysis, and qRT-PCR were performed to study the interaction among TNFSF10, miR-376-3p, and fibroblast growth factor receptor 1 (FGFR1). It was found that TNFSF10 was upregulated in OA cartilages and stimulated cell proliferation, antiapoptosis, and inflammation for chondrocytes. In addition, TNFSF10 acted as a competing endogenous RNA to downregulate miR-376-3p, and the influence of TNFSF10 on chondrocytes was partly reversed by miR-376-3p. Moreover, FGFR1, as a target of miR-376-3p, had reversal functions on the outcomes mediated by miR-376-3p. The further analysis displayed that there was a negative relationship between TNFSF10 and miR-376-3p as well as miR-376-3p and FGFR1, while FGFR1 was positively related with TNFSF10. Altogether, TNFSF10 overexpression probably stimulated proliferation and inflammation, and inhibited apoptosis by regulating the miR-376-3p/FGFR1 axis, implying that its increase contributed to OA progression. Our study provided a new potential biomarker or therapeutic target-TNFSF10, which was helpful to develop an efficient approach to cure OA.

Iron homeostasis plays a crucial role in the combat against pathogen invasion. Ferrous iron can trigger generous production of reactive oxygen species (ROS) by Fenton reaction. Nuclear receptor coactivator 4 (NCOA4), a selectivecargoreceptor to deliver ferritin to lysosome, may trigger release of ferritin-bound iron into the cytosol. The aim of the present study was to explore whether NCOA4-mediated ferritinophagy participated in the pathogenesis of periodontitis, and its role in promoting the periodontal inflammation. Inflamed and healthy periodontal tissues were harvested for immunobiological staining of ferritinophagy-related genes in the periodontal tissues, while real-time quantitative PCR (qPCR) was utilized to detect mRNA transcription. Periodontal ligament fibroblasts (PDLFs) were isolated and infected with Porphyromonas gingivalis. The mRNA transcription and protein expression of genes involved in the iron metabolism, including NCOA4, transferrin receptor 1 (TFR1), and ferroportin (SLC40A1) were detected by qPCR and western blot. Levels of labile iron pool and ROS production were detected by flow cytometry and confocal endoscopy. Small interference RNA was utilized to knock down NCOA4. Elevated expression of NCOA4, ferritin heavy chain, and light chain were observed in the diseased periodontal tissues. P. gingivalis infection promoted expression of TFR1, NCOA4, and microtubule-associated protein 1-light chain 3 B (LC3B), enhanced levels of intracellular labile iron pool and ROS production. NCOA4 knockdown reduced ROS generation in PDLFs in response to P. gingivalis and mitigated production of pro-inflammatory monocyte chemoattractant protein-1 and interleukin 6. P. gingivalis triggered activation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase signaling pathway. In addition, inhibitors of JNK, SP600125, and inhibitors of p38, SB203580 blocked NCOA4 transcription. NCOA4-ferritinophagy participated in the progress of periodontitis progression. P. gingvalis-triggered ferritinophagy aggravated production of ROS and inflammatory responses in PDLFS. These findings suggest iron homeostasis plays an important role in the pathogenesis of periodontitis.

Cellular iron, at the physiological level, is essential to maintain several metabolic pathways, while an excess of free iron may cause oxidative damage and/or provoke cell death. Consequently, iron homeostasis has to be tightly controlled. Under hypoxia these regulatory mechanisms for human macrophages are not well understood. Hypoxic primary human macrophages reduced intracellular free iron and increased ferritin expression, including mitochondrial ferritin (FTMT), to store iron. In parallel, nuclear receptor coactivator 4 (NCOA4), a master regulator of ferritinophagy, decreased and was proven to directly regulate FTMT expression. Reduced NCOA4 expression resulted from a lower rate of hypoxic NCOA4 transcription combined with a micro RNA 6862-5p-dependent degradation of NCOA4 mRNA, the latter being regulated by c-jun N-terminal kinase (JNK). Pharmacological inhibition of JNK under hypoxia increased NCOA4 and prevented FTMT induction. FTMT and ferritin heavy chain (FTH) cooperated to protect macrophages from RSL-3-induced ferroptosis under hypoxia as this form of cell death is linked to iron metabolism. In contrast, in HT1080 fibrosarcome cells, which are sensitive to ferroptosis, NCOA4 and FTMT are not regulated. Our study helps to understand mechanisms of hypoxic FTMT regulation and to link ferritinophagy and macrophage sensitivity to ferroptosis.

Dimethyl malonate preserves brain and neurobehavioral phenotype following neonatal hypoxia–ischemia by inhibiting FTH1-mediated ferritinophagy

Osteoarthritis (OA) is one of the most prevalent arthritis types globally, with the knee being particularly susceptible due to its frequent and strenuous use. Urolithin B (UB) exhibits various biological properties, with meniscal repair playing an important role in preventing knee OA. This study aimed to explore the impact of UB on meniscal regeneration and OA progression. Initially, we explored the effect of UB on meniscal cells. Utilizing the cell counting kit (CCK)-8 assay, we determined the optimum concentration of UB treatment. Enzyme-linked immunosorbent assay (ELISA) was used for detecting inflammation-related interleukin-1beta (IL-1β). Real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) was used for measuring the expression of extracellular matrix (ECM)-related proteins, ECM-degrading enzymes, and genes associated with joint formation in meniscal cells. Furthermore, 5-Bromo-2'-deoxyuridine (BrdU) staining was used to evaluate the proliferation of meniscal cells. Meniscal tissues were cultured in vitro, and western blot analysis was used to detect levels of proliferation-related markers such as proliferating cell nuclear antigen (PCNA) and vascular endothelial growth factor (VEGF), as well as ECM protein collagen-1 (COL-1) and ECM degradation-related matrix metallopeptidase-13 (MMP-13). Mice were subjected to meniscus injury to establish a knee joint model of meniscus injury-induced osteoarthritis (MIOA) and to verify the effect of UB on meniscal cells in vivo. Pathological changes in knee joints were observed using hematoxylin-eosin (H&E) staining. Additionally, western blot was used to assess PCNA, VEGF, COL-1, and MMP-13 levels, while ELISA was used to detect inflammation-related tumor necrosis factor-alpha (TNF-α), IL-1β, IL-6, and interferon-gamma (IFN-γ) in mouse menisci. At concentrations up to 100 μM, UB exhibited non-toxicity and concomitantly decreased IL-1β in meniscal cells (p < 0.001). Moreover, UB increased the expression of ECM-related proteins (p < 0.001) and genes associated with joint formation (p < 0.001), while concurrently decreasing the expression of ECM-degrading enzymes (p < 0.001) in meniscal cells. UB promoted meniscal cell proliferation (p < 0.001). Additionally, UB increased PCNA, VEGF, and COL-1 while suppressing MMP-13 in menisci cultured in vitro (p < 0.001). Moreover, UB mitigated the pathological alterations observed in knee joints affected by meniscus injury. In murine models, MIOA led to decreased PCNA, VEGF, and COL-1 levels, alongside increased MMP-13, TNF-α, IL-1β, IL-6, and IFN-γ levels (p < 0.001), all of which were effectively reversed by UB treatment (p < 0.001). UB effectively promotes meniscal regeneration and repair, while protecting against knee OA in mice, suggesting its potential role in clinical OA treatment.

Canine osteosarcomas (COS) are the most common bone tumors in dogs, characterized by high metastatic rates, poor prognosis, and poor responsiveness to routine therapies, which highlights the need for new treatment targets. In this context, the metabolism of neoplastic cells represents an increasingly studied element, as cancer cells depend on particular metabolic pathways that are also elements of vulnerability. Among these, tumor cells (TCs) show higher iron requirements to sustain proliferation (so-called iron addiction), which are achieved by increasing iron uptake and/or by activating ferritinophagy, a process mediated by the Nuclear receptor Co-Activator 4 (NCOA4) leading to iron mobilization from ferritin (Ft) deposits. Previous studies have shown that COS cells overexpress Transferrin Receptor 1 (TfR1) to increase iron uptake. In this study we evaluated the immunohistochemical expression of ferritinophagy-related proteins, namely Ferritin Heavy chain (FTH1) and NCOA4, and proliferating cell nuclear antigen (PCNA) in canine normal bone and canine osteoblastic osteosarcoma (COOS) samples. Normal samples revealed negative/weak immunoreactivity for FTH1, NCOA4 and PCNA in <10% of osteocytes. In COOS samples the majority of neoplastic cells showed immunoreactivity to FTH1, NCOA4 and PCNA. Our data suggest that the activation of ferritinophagy by COOS cells responds to the need for feed their "iron addiction." These data, though preliminary, further suggest that targeting iron metabolism represents a new potential strategy worthy of further study to be transferred into clinical practice.

JNK-JUN-NCOA4 axis contributes to chondrocyte ferroptosis and aggravates osteoarthritis via ferritinophagy.

Osteoarthritis (OA) is a common joint disease, and chondrocyte extracellular matrix (ECM) degradation was closely associated with its progression. This study investigated the regulatory mechanisms of ECM degradation during OA development. A rat model of OA was established by anterior cruciate ligament transection (ACL-T) and interleukin-1 beta (IL-1β)-stimulated rat chondrocytes were used to simulate OA invitro. Cartilage damage was evaluated by hematoxylin-eosin (HE) and safranin O-fast green staining. The ECM content in chondrocytes was assessed by alcian blue staining. Real-time quantitative PCR (RT-qPCR), Western Blotting, immunohistochemical staining, and immunofluorescent staining were adopted to analyze associated molecule expression. Molecular mechanisms were elucidated by Co-immunoprecipitation (Co-IP) and GST pull-down assay. We found that Aurora kinase A (AURKA), p-eukaryotic translation initiation factor 4E (eIF4E), and ADAM metallopeptidase with thrombospondin type 1 motif 12 (ADAMTS12) levels were elevated in the human and rat cartilage tissues of OA, as well as IL-1β-exposed chondrocytes. AURKA inhibition restrained ECM degradation to relieve OA via down-regulation of ADAMTS12. AURKA overexpression phosphorylated eIF4E, which promoted cap-dependent translation of ADAMTS12. Moreover, DNA methyltransferase 1 (DNMT1)-mediated methylation down-regulated HECT domain E3 ubiquitin protein ligase 1 (HECTD1) in the OA model and consequently enhanced AURKA expression via inhibiting its ubiquitination. HECTD1 knockdown or ubiquitination repression intensified ECM degradation in IL-1β-stimulated chondrocytes. Taken together, low expression of HECTD1 repressed AURKA ubiquitination to elevate AURKA protein level and subsequently facilitated eIF4E-mediated cap-dependent translation of ADAMTS12, thus resulting in ECM degradation during OA progression.

The aim of this study is to explore the mechanism of ligustilide, the main active constituent of essential oils of traditional Chinese medicine Angelicae Sinensis Radix, on alleviating oxygen-glucose deprivation/reperfusion(OGD/R) injury in PC12 cells from the perspective of ferroptosis. OGD/R was induced in vitro, and 12 h after ligustilide addition during reperfusion, cell viability was detected by cell counting kit-8(CCK-8) assay. DCFH-DA staining was used to detect the level of intracellular reactive oxygen species(ROS). Western blot was employed to detect the expression of ferroptosis-related proteins, glutathione peroxidase 4(GPX4), transferrin receptor 1(TFR1), and solute carrier family 7 member 11(SLC7A11), and ferritinophagy-related proteins, nuclear receptor coactivator 4(NCOA4), ferritin heavy chain 1(FTH1), and microtubule-associated protein 1 light chain 3(LC3). The fluorescence intensity of LC3 protein was analyzed by immunofluorescence staining. The content of glutathione(GSH), malondialdehyde(MDA), and Fe was detected by chemiluminescent immunoassay. The effect of ligustilide on ferroptosis was observed by overexpression of NCOA4 gene. The results showed that ligustilide increased the viability of PC12 cells damaged by OGD/R, inhibited the release of ROS, reduced the content of Fe and MDA and the expression of TFR1, NCOA4, and LC3, and improved the content of GSH and the expression of GPX4, SLC7A11, and FTH1 compared with OGD/R group. After overexpression of the key protein NCOA4 in ferritinophagy, the inhibitory effect of ligustilide on ferroptosis was partially reversed, indicating that ligustilide may alleviate OGD/R injury of PC12 cells by blocking ferritinophagy and then inhibiting ferroptosis. The mechanism by which ligustilide reduced OGD/R injury in PC12 cells is that it suppressed the ferroptosis involved in ferritinophagy.

Our previous research has established that hydrogen sulfide (H 2 S) exerts an antagonistic effect against the hippocampal neurotoxicity induced by Rotenone (ROT). However, the underlying mechanisms are so far poorly understood. Substantial evidence corroborates the involvement of ferroptosis in ROT-induced neurotoxicity. To elucidate the protective mechanism of H 2 S against ROT-induced hippocampal neurotoxicity, this study explores its regulatory role in ferroptosis and its underlying mechanisms. We used Fluoro-Jade B staining to detect dead neurons. The levels of ferrous ions and glutathione (GSH) were measured by a kit. The ferroptosis-related proteins, including light-chain subunit (xCT), GSH peroxidase 4(GPX4), ferroptosis marker acyl-CoA synthetase long-chain family member 4(ACSL4), and ferritinophagy-related protein, including ferritin heavy chain 1 (FTH1), sequestosome 1 (p62), ferritinophagy markers autophagosome marker light-chain I/II (LC3I/II), and nuclear receptor coactivator 4 (NCOA4), were measured by Western blot. Our findings indicate that H 2 S reduces hippocampal neuron deaths in ROT-exposed rats. Meanwhile, H 2 S reverses the downregulations of xCT and GPX4, and the upregulations of ferrous ion and ACSL4 in the hippocampus induced by ROT. Furthermore, H 2 S reverses the upregulations of LC3I/II and NCOA4, and the downregulations of P62 and FTH1. Based on these findings, we concluded that the protective role of H 2 S against ROT-induced hippocampal neuronal death involves inhibiting ferroptosis triggered by ferritinophagy.

Programmed cell death (PCD) refers to a regulated cellular process involving a cascade of biochemical reactions and molecular mechanisms, commonly including apoptosis, necroptosis, and pyroptosis. Ferroptosis is a recently identified form of PCD distinguished by its dependence on iron. Emerging evidence underscores the significance of ferroptosis in viral infections; however, its role in Pseudorabies virus (PRV) infection, an enveloped double-stranded DNA virus belonging to the Alphaherpesvirinae subfamily, remains poorly understood. Here, we demonstrate that PRV infection induces multiple forms of PCD, including ferroptosis, which is characterized by mitochondrial shrinkage, lipid peroxidation, ferrous iron (Fe²+) accumulation, and elevated levels of reactive oxygen species (ROS). Ferroptosis facilitates PRV replication, with iron overload playing a crucial role. Mechanistically, we show that transferrin receptor 1 (TfR1) and ferritinophagy are involved in PRV-induced iron overload. Specifically, PRV infection upregulates TfR1 expression via hypoxia-inducible factor-1β (HIF-1β) and promotes its translocation to the cell membrane through Rab11a, thereby enhancing the cellular import of extracellular ferric iron (Fe³+). In parallel, PRV activates ferritinophagy to degrade ferritin heavy chain 1 (FTH1) via selective autophagy receptors, nuclear receptor coactivator 4 (NCOA4) and Tax1-binding protein 1 (TAX1BP1), further contributing to intracellular iron accumulation. Altogether, these findings demonstrate that PRV induces ferroptosis by disrupting iron homeostasis through TfR1 activation and ferritinophagy induction, providing novel insights into the pathogenesis of PRV and other herpesviruses.IMPORTANCEFerroptosis is an iron-dependent form of non-apoptotic cell death that primarily involves iron overload, lipid peroxidation, and suppression of antioxidant systems. Increasing evidence indicates that ferroptosis plays an important role in viral infections. In this study, we show that PRV induces ferroptosis by disrupting iron homeostasis through TfR1 activation and ferritinophagy induction. On one hand, PRV infection upregulates TfR1 expression through HIF-1β and facilitates TfR1 translocation to the cell membrane via Rab11a, leading to enhanced import of extracellular Fe3+ into cells. On the other hand, PRV exploits the selective autophagy receptors NCOA4 and TAX1BP1, which strengthens the interaction between NCOA4, TAX1BP1, and FTH1, triggering ferritinophagy and increasing intracellular Fe2+ levels. Collectively, these findings enrich the understanding of the mechanism by which PRV induces ferroptosis, shedding new light on PRV and other alpha-herpesvirus infections.