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 ... -  《-》

Silencing of lncRNA NEAT1 alleviates acute myocardial infarction by suppressing miR-450-5p/ACSL4-mediated ferroptosis.

Ferroptosis is principally initiated by dysregulation of iron metabolism and excessive accumulation of ROS, which exacerbates myocardial injury during acute myocardial infarction (AMI). Previous studies have indeed demonstrated the significant involvement of long non-coding RNA (lncRNA) nuclear paraspeckle assembly transcript 1 (NEAT1) exerts its pleiotropic effects in the pathophysiology of myocardial infarction, heart failure and atherosclerosis by modulating inflammation, apoptosis, and oxidative stress. However, whether and how NEAT1 mediates myocardial ferroptosis remain unknown. In this study, we found that NEAT1 expression was significantly elevated in hypoxic HL-1 cells and AMI mice, while silencing of NEAT1 alleviated lipid peroxidation and myocardial ferroptosis both in vitro and in vivo. Mechanistically, NEAT1 directly sponged miR-450b-5p and negatively regulated its expression. In addition, miR-450b-5p directly targeted Acyl-CoA synthase long-chain family member 4 (ACSL4). Notably, inhibition of miR-450b-5p reversed the role of NEAT1 in AMI mice. Collectively, these findings newly illustrated that NEAT1 acts as a competitive endogenous RNA (ceRNA) of miR-450-5p in AMI. Especially, silencing of NEAT1 effectively ameliorated myocardium ischemia by suppression of ferroptosis via miR-450-5p/ACSL4 pathway, which providing a brand-new therapeutic strategy for myocardial ischemia injury.

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

Plasma exosomes from patients with acute myocardial infarction alleviate myocardial injury by inhibiting ferroptosis through miR-26b-5p/SLC7A11 axis.

Ferroptosis promotes myocardial injury in acute myocardial infarction (AMI). Increasing evidence suggests the crucial role of exosomes in post-AMI pathophysiological regulation. We aimed to investigate the effects and underlying mechanisms of plasma exosomes derived from patients with AMI in inhibiting ferroptosis after AMI. Plasma exosomes were isolated from controls (Con-Exo) and patients with AMI (MI-Exo). These exosomes were incubated with hypoxic cardiomyocytes or intramyocardially injected into the AMI mice. Histopathological changes, cell viability, and cell death were measured to evaluate the myocardial injury. For the ferroptosis evaluation, iron particle deposition, Fe2+, ROS, MDA, GSH, and GPX4 levels were detected. Exosomal miR-26b-5p expression was detected by qRT-PCR, and the targeting relationship between miR-26b-5p and SLC7A11 was confirmed by dual luciferase reporter gene assay. The role of the miR-26b-5p/SLC7A11 axis in the regulation of ferroptosis was validated by rescue experiments in cardiomyocytes. Hypoxia-treatment induced ferroptosis and injury in H9C2 cells and primary cardiomyocytes. MI-Exo performed better than Con-Exo in inhibiting hypoxia-induced ferroptosis. miR-26b-5p expression was downregulated in MI-Exo, and miR-26b-5p overexpression significantly eliminated the inhibitory effect of MI-Exo on ferroptosis. Mechanistically, knockdown of miR-26b-5p upregulated SLC7A11/GSH/GPX4 expressions by directly targeting SLC7A11. Moreover, SLC7A11 silencing also reversed the inhibitory effect of MI-Exo on hypoxia-induced ferroptosis. In vivo, MI-Exo significantly inhibited ferroptosis, reduced myocardial injury, and improved the cardiac function of AMI mice. Our findings revealed a novel mechanism of myocardial protection that downregulation of miR-26b-5p in MI-Exo notably upregulated SLC7A11 expression, thereby inhibiting post-AMI ferroptosis and alleviating myocardial injury.

Li H ，Ding J ，Liu W ，Wang X ，Feng Y ，Guan H ，Chen Z ... -  《-》

MicroRNA-384-5p/Beclin-1 As Potential Indicators For Epigallocatechin Gallate Against Cardiomyocytes Ischemia Reperfusion Injury By Inhibiting Autophagy Via PI3K/Akt Pathway.

Epigallocatechin gallate (EGCG) has established protective actions against myocardial ischemia/reperfusion (I/R) injury by regulating autophagy. However, little is known about the mechanisms of EGCG in posttranscriptional regulation in the process of cardioprotection. Here we studied whether microRNAs play a role in EGCG-induced cardioprotection. The myocardial I/R injury in vitro and in vivo model were made, with or without EGCG pretreatment. The upregulation and silencing of microRNA-384-5p (miR-384) and Beclin-1 in H9c2 cell lines were established. Rats were transfected with miR-384 specific shRNA. Dual-luciferase reporter gene assay was conducted to verify the relationship between miR-384 and Beclin-1. TTC staining was performed to analyze the area of myocardial infarct size. Cell viability was monitored by cell counting kit-8 (CCK-8). The release of cardiac troponin-I (cTnI) was examined by ELISA. The levels of autophagy-related genes or proteins expression were evaluated by qRT-PCR or Western blotting. Autophagosomes of myocardial cells were detected by transmission electron microscopy and laser scanning confocal microscope. I/R increased both autophagosomes and autolysosomes, thereby increasing autophagic flux both in vitro and in vivo. Pretreatment with EGCG attenuated I/R-induced autophagic flux expression, accompanied by an increase in cell viability and a decrease in the size of myocardial infarction. MiR-384 expression was down-regulated in H9c2 cell lines when subjected to I/R, while this suppression could be reversed by EGCG pretreatment. The dual-luciferase assay verified that Beclin-1 was a target of miR-384. Both overexpression of miR-384 and knocking down of Beclin-1 significantly inhibited I/R-induced autophagy, accompanied by the activation of PI3K/Akt pathway, thus enhanced the protective effect of EGCG. However, these functions were abrogated by the PI3K inhibitor, LY294002. We confirmed that EGCG has a protective role in microRNA-384-mediated autophagy by targeting Beclin-1 via activating the PI3K/Akt signaling pathway. Our results unveiled a novel role of EGCG in myocardial protection, involving posttranscriptional regulation with miRNA-384.

Zhang C ，Liang R ，Gan X ，Yang X ，Chen L ，Jian J ... -  《Drug Design Development and Therapy》

Human umbilical cord mesenchymal stem cells derived exosome shuttling mir-129-5p attenuates inflammatory bowel disease by inhibiting ferroptosis.

Ferroptosis, a unique form of non-apoptotic cell death, is dependent on iron and lipoperoxidation, and has been shown to be associated with the pathogenesis of inflammatory bowel disease (IBD). Human umbilical cord mesenchymal stem cell-derived exosomes (hucMSC-Ex) are involved in cell survival, immune conditioning, and damage repair. However, the relationship between hucMSC-Ex, IBD, and ferroptosis is unknown. This paper explores the role of hucMSC-Ex in the repair of IBD through the regulation of the ferroptosis signaling pathway. In this study, we used small RNA sequencing to find that miR-129-5p was highly expressed in hucMSC-Ex, and by predicting its targeting to ACSL4, we verified the effect of miR-129-5p on mice IBD in vitro and human colonic epithelial cells (HCoEpiC) in vivo. We found that miR-129-5p reduces ferroptosis in intestinal epithelial cells by targeting ACSL4 to repair IBD, which provides new strategies for the prevention and treatment of IBD. In conclusion, our results demonstrate that hucMSC-Ex relieves IBD by targeting ACSL4 with miR-129-5p to inhibit lipid peroxidation (LPO) and ferroptosis, reducing intestinal inflammation and repairing damages. Mechanism of hucMSC-Ex inhibiting ferroptosis in intestinal epithelial cells. System Xc- mediates the transport of extracellular cystine into the cell, which gets reduced to cysteine to participate in GSH-mediated metabolism. GPX4 strongly inhibits ferroptosis by helping scavenge reactive oxygen species. The depletion of GSH correlates with decreased GPX4, and the imbalance of the antioxidant system leads to the formation of toxic phospholipid hydroperoxide, which promotes the occurrence of ferroptosis with the participation of irons. HucMSC-Ex has the ability to relieve GSH and GPX4 depletion and repair the intracellular antioxidant system. Ferric ions enter the cytosol through DMT1 and participate in lipid peroxidation. HucMSC-Ex can reduce the expression of DMT1 and alleviate this process. HucMSC-Ex-derived miR-129-5p targets ACSL4 and reduces the expression of ACSL4, an enzyme that mediates the conversion of PUFAs into phospholipids in intestinal epithelial cells, and is a positive regulator of lipid peroxidation. GSH, glutathione; GPX4, glutathione peroxidase 4; GSSG, oxidized glutathione; DMT1, divalent metal transporter 1; ACSL4, acyl-CoA synthetase long-chain family member 4; PUFAs, polyunsaturated fatty acids; ALOXs, lipoxygenases; CoA, coenzyme A; PL, phospholipid; PLOOH, hydroperoxides, LOH, phospholipid alcohols; LPO, lipid peroxidation.

Wei Z ，Hang S ，Wiredu Ocansey DK ，Zhang Z ，Wang B ，Zhang X ，Mao F ... -  《JOURNAL OF NANOBIOTECHNOLOGY》