Astragaloside IV alleviates neuronal ferroptosis in ischemic stroke by regulating fat mass and obesity-associated-N6-methyladenosine-acyl-CoA synthetase long-chain family member 4 axis.

Ischemic stroke (IS) is a detrimental neurological disease with limited treatment options. Astragaloside IV (As-IV) was a promising bioactive constituent in the treatment of IS. However, the functional mechanism remains unclear. Here, IS cell and mouse models were established by oxygen glucose deprivation/re-oxygenation (OGD/R) and middle cerebral artery occlusion (MCAO). Quantitative reverse transcription PCR (RT-qPCR), Western blotting, or Immunofluorescence staining measured related gene and protein expression of cells or mice brain tissues, and the results revealed altered expression of acyl-CoA synthetase long-chain family member 4 (Acsl4), fat mass and obesity-associated (Fto), and activation transcription factor 3 (Atf3) after treatment with As-IV. Then, increased N6 -methyladenosine (m6 A) levels caused OGD/R or MCAO were reduced by As-IV according to the data from methylated RNA immunoprecipitation (MeRIP)-qPCR and dot blot assays. Moreover, through a series of functional experiments such as observing mitochondrial changes under transmission electron microscopy (TEM), evaluating cell viability by cell counting kit-8 (CCK-8), analyzing infract area of brain tissues by 2,3,5-triphenyltetrazolium chloride (TTC) staining, measuring levels of malondialdehyde (MDA), lactate dehydrogenase (LDH), Fe2+ , solute carrier family 7 member 11 (Slc7a11) and glutathione peroxidase 4 (Gpx4) and concentration of glutathione (GSH), we found that Fto knockdown, Acsl4 overexpression or Atf3 knockdown promoted the viability of OGD/R cells, inhibited cell ferroptosis, reduced infract size, while As-IV treatment or Fto overexpression reversed these changes. In mechanism, the interplays of YTH N6 -methyladenosine RNA-binding protein 3 (Ythdf3)/Acsl4 and Atf3/Fto were analyzed by RNA-pull down, RNA immunoprecipitation (RIP), chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assay. Fto regulated the m6 A levels of Acsl4. Ythdf3 bound to Acsl4, and modulated its levels through m6 A modification. Atf3 bound to Fto and positively regulated its levels. Overall, As-IV promoted the transcription of Fto by upregulating Atf3, resulting in decreased m6 A levels of Acsl4, thus, improving neuronal injury in IS by inhibiting ferroptosis.

Jin Z ，Gao W ，Guo F ，Liao S ，Hu M ，Yu T ，Yu S ，Shi Q ... -  《-》

Ring Finger Protein 146-mediated Long-chain Fatty-acid-Coenzyme a Ligase 4 Ubiquitination Regulates Ferroptosis-induced Neuronal Damage in Ischemic Stroke.

Ischemic stroke (IS) is one of the leading causes of disability and death worldwide. Long-chain fatty-acid-coenzyme A ligase 4 (ACSL4) is a critical isozyme for ferroptosis that participates in the progression of IS. RING finger protein 146 (RNF146) is an E3 ligase predicted to interact with ACSL4 and regulated by activating transcription factor 3 (ATF3). The molecular mechanism of the RNF146/ACSL4 axis in IS is still unclear. Oxygen-glucose deprivation/reperfusion (OGD/R) treatment was used as the in vitro model, and middle cerebral artery occlusion (MCAO) mice were established for the in vivo model for IS. The protein level of ACSL4 was monitored by Western blot during ischemic injury. RNF146 was overexpressed in vitro and in vivo. The interaction of RNF146 and ACSL4 was determined by co-immunoprecipitation (Co-IP) assay. Chromatin immunoprecipitation (ChIP) assay and luciferase assay were utilized to determine the regulation of ATF3 on RNF146. Ferroptosis was evaluated by the levels of lactate dehydrogenase (LDH), malondialdehyde (MDA), Fe2+, and protein levels of related genes including ACSL4, SLC7A11, and GPX4. ACSL4 was downregulated upon OGD treatment and then increased by re-oxygenation. RNF146 was responsible for the ubiquitination and degradation of ACSL4 protein. RNF146 overexpression could prevent the stimulation of OGD/R-induced LDH, MDA, and Fe2+ levels and ferroptosis-related gene expression. ATF3 could activate the transcription and expression of RNF146, leading to the inhibition of OGD/R-induced neuron ferroptosis. The ATF3-mediated RNF146 could alleviate neuronal damage in IS by regulating ACSL4 ubiquitination and ferroptosis, providing a novel theoretical basis for exploring therapeutic targets and strategies.

Jin ZL ，Gao WY ，Guo F ，Liao SJ ，Hu MZ ，Yu T ，Yu SZ ，Shi Q ... -  《-》

Inhibition of Acyl-CoA Synthetase Long-Chain Family Member 4 Facilitates Neurological Recovery After Stroke by Regulation Ferroptosis.

Ischemic stroke is the main cause of disability worldwide, leading to a serious socioeconomic burden. Ferroptosis is a non-apoptotic form of programmed cell death and is related to various diseases. Acyl-CoA synthetase long-chain family member 4 (ACSL4) is considered a target of ferroptosis, but its specific role in ischemic stroke remains unclear. In this study, we investigate whether the inhibition of ACSL4 promotes the recovery of neurological function in a way that prevents ferroptosis. A transient cerebral ischemia model was established for mice by middle cerebral artery occlusion (MCAO); glutathione peroxidase 4 (GPx4), ACSL4 and cyclooxygenase 2 (COX2) were detected by Western blot, and changes to mitochondria were observed by a transmission electron microscope. A kit was used to determine iron levels and lipid peroxide indicators, such as glutathione peroxidase (GPx), reduced glutathione (GSH), total glutathione/oxidized glutathione (GSH/GSSG), lipid peroxidation, reactive oxygen species, superoxide and malonaldehyde. Following MCAO, a ferroptosis inhibitor, liproxstatin-1, was administered intranasally immediately at a concentration of 10 mg/kg. Rosiglitazone was used to inhibit ACSL4 and was administered intravenously 1 h before MCAO at a concentration of 0.4 mg/kg. Brain injury was determined by neurological deficit scores, neuroscore (28-point), corner test and gait analyses, at 24 and 72 h after stroke. Brain infarct volume was determined by 2, 3, 5-Triphenyltetrazolium chloride (TTC) staining at 72 h after stroke. After MCAO, GPx4 protein expression decreased, ACSL4 and COX2 protein expression increased, GPx activity decreased and iron accumulation. Transmission electron microscopy confirmed that the outer mitochondrial membrane of neurons had ruptured and mitochondrial cristae had decreased or disappeared. Liproxstatin-1 could significantly attenuate the decrease of GPx4 and the increase of COX2 after MCAO, dramatically reducing iron accumulation and decreasing GPx activity, accompanied by a marked reduction in changes in lipid peroxidation indicators. The use of rosiglitazone to inhibit ACSL4 could significantly improve neurological function and reduce the brain infarct volume at 72 h after stroke. Importantly, inhibiting ACSL4 could significantly attenuate the decline of GPx4 after MCAO and markedly attenuate iron accumulation and a decrease in GPx activity. Additionally, changes in lipid peroxidation indicators were also significantly inhibited. This study indicates that inhibiting ACSL4 can promote the recovery of neurological function after stroke by suppression of ferroptosis.

Chen J ，Yang L ，Geng L ，He J ，Chen L ，Sun Q ，Zhao J ，Wang X ... -  《Frontiers in Cellular Neuroscience》

EGCG attenuated acute myocardial infarction by inhibiting ferroptosis via miR-450b-5p/ACSL4 axis.

Epigallocatechin gallate (EGCG) has multiple biological effects such as anti-tumor multiple drug resistance, antioxidation and anti-inflammatory properties. Ferroptosis is the main driving factor of ischemic heart injury, thus inhibiting ferroptosis may prove to be an effective treatment strategy for cardiovascular diseases. However, the role of EGCG on ferroptosis in ischemic myocardium and underlying mechanisms remain uncertain. This study was aimed to investigate the effects and potential mechanisms of EGCG on myocardial ischemic-induced ferroptosis both in vitro and in vivo. Cardiomyocyte hypoxia model and mouse acute myocardial infarction (AMI) model were established in vitro and in vivo. MiR-450b-5p and ACSL4 silencing or overexpression plasmids were transfected, with or without EGCG pretreatment. Cell viability was determined by the CCK-8 assay. Hematoxylin and eosin (HE) staining and transmission electron microscopy (TEM) were used to evaluate the morphologic alterations. TTC staining was used to observe the infarction area, and echocardiography was adopted to appraise the heart function. Using flow cytometry, the presence of reactive oxygen species (ROS) was assessed. The content of cardiac troponin I (cTn I), glutathione (GSH), malondialdehyde (MDA), divalent iron ions (Fe2+) and superoxide dismutase (SOD) were detected using reagent kits. A luciferase activity assay was performed to assess the binding ability of miR-450b-5p to ACSL4. Expressions of related genes and proteins were measured by RT-qPCR and western blotting respectively. EGCG attenuated AMI-induced ferroptosis and improved myocardial ischemia injury, which was associated with reducing iron deposition and cTn I, inhibition of lipid peroxidation, decreasing TFR1 and ACSL4, and upregulating SLC7A11, FTH1 and GPX4. Meanwhile, EGCG pretreatment increased miR-450b-5p expression in ischemic myocardium. Further researches discovered that knockdown of miR-450b-5p partially compromised EGCG-generated protective effect in hypoxia HL-1 cells, while combination with miR-450b-5p mimic could strengthen the potency of EGCG on ischemic myocardium. The dual-luciferase test demonstrated that miR-450b-5p has binding to ACSL4. Furthermore, silencing of ACSL4 synergistically increased the cardioprotective effect of EGCG. More significantly, EGCG treatment regulated the ferroptosis-related proteins expression via miR-450b-5p/ACSL4 axis. In summary, the present study evidently demonstrated that EGCG attenuates myocardial ischemia injury by targeting ferroptosis. Our work revealed the role of miR-450b-5p/ACSL4 axis in AMI for the first time. Further, it also elucidated the molecular mechanisms of EGCG on inhibiting ferroptosis greatly depend on the miR-450b-5p/ACSL4 axis, suggesting that EGCG may act as a novel anti-ferroptosis agent and exert a therapeutic role in AMI.

Yu Q ，Zhang N ，Gan X ，Chen L ，Wang R ，Liang R ，Jian J ... -  《-》

[Alda-1 alleviates brain injury after cardiopulmonary resuscitation by regulating acyl-CoA synthetase long-chain family member 4/glutathione peroxidase 4 pathway-mediated ferroptosis in swine].

Chen C ，Ma S ，Liao L ，Xiao Y ，Dai H ... -  《-》