acyl-CoA Synthetase Long-chain Family Member Research Articles

Ketone Body β-Hydroxybutyric Acid Ameliorates Dopaminergic Neuron Injury Through Modulating Zinc Finger Protein 36/Acyl-CoA Synthetase Long-Chain Family Member Four Signaling Axis-Mediated Ferroptosis

β-hydroxybutyrate (BHB) is one of main component of ketone body, which plays an important protective role in various tissues and organs. Whereas, its exact regulatory roles and mechanisms in Parkinson's disease (PD) have not been full elucidated. In this study, SN4741 cells and C57BL/6 mice were treated with 1-methyl-4-phenylpyridinium ion (MPP+)/1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to establish the PD model in vitro and in vivo. Cell viability and damage to dopaminergic neurons were measured by cell counting kit 8, Calcein-AM/PI staining, terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling and hematoxylin & eosin staining. Corresponding assay kits and BODIPY 581/591 C11 probe evaluated oxidative stress and intracellular iron levels. Western blot examined the ferroptosis-related proteins. MPTP/MPP+-treatment reduced cell viability but triggered oxidative stress and ferroptosis in SNA4741 cells and brain tissues of mice. However, these effects were dramatically reversed by BHB and Fer-1 treatment. Mechanistically, Zinc finger protein 36 (ZFP36) was a target of BHB, and its depletion could reverse the anti-oxidative stress and anti-ferroptosis roles of BHB. Moreover, ZFP36 could directly bound to acyl-CoA synthetase long-chain family member 4 (ACSL4) mRNA to decay its expression, thus negatively modulating ACSL4-mediated oxidative stress and ferroptosis. Summary, BHB alleviated oxidative stress and ferroptosis of dopaminergic neurons in PD via modulating ZFP36/ACSL4 axis, which provided some new understanding for PD prevention and treatment.

HJ11 decoction restrains development of myocardial ischemia-reperfusion injury in rats by suppressing ACSL4-mediated ferroptosis.

HJ11 is a novel traditional Chinese medicine developed from the appropriate addition and reduction of Si-Miao-Yong-An decoction, which has been commonly used to treat ischemia-reperfusion (I/R) injury in the clinical setting. However, the mechanism of action of HJ11 components remains unclear. Ferroptosis is a critical factor that promotes myocardial I/R injury, and the pathophysiological ferroptosis-mediated lipid peroxidation causes I/R injury. Therefore, this study explored whether HJ11 decoction ameliorates myocardial I/R injury by attenuating ACSL4-mediated ferroptosis. This study also explored the effect of ACSL4 expression on iron-dependent programmed cell death by preparing a rat model of myocardial I/R injury and oxygen glucose deprivation/reperfusion (OGD/R)-induced H9c2 cells. The results showed that HJ11 decoction improved cardiac function; attenuated I/R injury, apoptosis, oxidative stress, mitochondrial damage, and iron accumulation; and reduced infarct size in the myocardial I/R injury rat model. Additionally, HJ11 decoction suppressed the expression of ferroptosis-promoting proteins [Acyl-CoA synthetase long-chain family member 4 (ACSL4) and cyclooxygenase-2 (COX2)] but promoted the expression of ferroptosis-inhibiting proteins [ferritin heavy chain 1 (FTH1) and glutathione-dependent lipid hydroperoxidase glutathione peroxidase 4 (GPX4)] in the myocardial tissues of the I/R injury rat model. Similar results were found with the OGD/R-induced H9c2 cells. Interestingly, ACSL4 knockdown attenuated iron accumulation, oxidative stress, and ferroptosis in the OGD/R-treated H9c2 cells. However, ACSL4 overexpression counteracted the inhibitory effect of the HJ11 decoction on OGD/R-triggered oxidative stress and ferroptosis in H9c2 cells. These findings suggest that HJ11 decoction restrained the development of myocardial I/R injury by regulating ACSL4-mediated ferroptosis. Thus, HJ11 decoction may be an effective medication to treat myocardial I/R injury.

SARS-CoV-2 infection induces activation of ferroptosis in human placenta.

Ferroptosis, a regulated non-apoptotic form of cell death, has been implicated in the response to varied types of infectious agents including virus. In this study, we sought to determine whether SARS-CoV-2 infection can induce activation of ferroptosis in the human placenta. We collected placentas from 23 pregnant females with laboratory-confirmed SARS-CoV-2 following delivery and then used RNA in situ hybridization assay for detection of viral positive-sense strand (PSS) to confirm that these placentas have been infected. We also used immunohistochemistry assay to assess expression levels of acyl-CoA synthetase long-chain family member 4 (ACSL4), an essential executioner of ferroptosis in the same specimens. Our results showed that ACSL4 expression was significantly increased in the group with positive positive-sense strand staining compared to their negative counterparts (p = 0.00022). Furthermore, we found that there was a positive trend for increased PSS staining along with increased ACSL4 expression. Our study supports that ferroptosis is activated in the response to SARS-CoV-2 infection in the human placenta, highlighting a molecular mechanism potentially linking this coronavirus infection and pathogenesis of adverse pregnancy outcomes.

Apolipoprotein H: a novel regulator of fat accumulation in duck myoblasts

Apolipoprotein H (APOH) primarily engages in fat metabolism and inflammatory disease response. This study aimed to investigate the effects of APOH on fat synthesis in duck myoblasts (CS2s) by APOH overexpression and knockdown. CS2s overexpressing APOH showed enhanced triglyceride (TG) and cholesterol (CHOL) contents and elevated the mRNA and protein expression of AKT serine/threonine kinase 1 (AKT1), ELOVL fatty acid elongase 6 (ELOVL6), and acetyl-CoA carboxylase 1 (ACC1) while reducing the expression of protein kinase AMP-activated catalytic subunit alpha 1 (AMPK), peroxisome proliferator activated receptor gamma (PPARG), acyl-CoA synthetase long chain family member 1 (ACSL1), and lipoprotein lipase (LPL). The results showed that knockdown of APOH in CS2s reduced the content of TG and CHOL, reduced the expression of ACC1, ELOVL6, and AKT1, and increased the gene and protein expression of PPARG, LPL, ACSL1, and AMPK. Our results showed that APOH affected lipid deposition in myoblasts by inhibiting fatty acid beta-oxidation and promoting fatty acid biosynthesis by regulating the expression of the AKT/AMPK pathway. This study provides the necessary basic information for the role of APOH in fat accumulation in duck myoblasts for the first time and enables researchers to study the genes related to fat deposition in meat ducks in a new direction.

Genome-Wide Association Study of Exercise-Induced Fat Loss Efficiency

There is a wide range of individual variability in the change of body weight in response to exercise, and this variability partly depends on genetic factors. The study aimed to determine DNA polymorphisms associated with fat loss efficiency in untrained women with normal weight in response to a 12-week aerobic training program using the GWAS approach, followed by a cross-sectional study in athletes. The study involved 126 untrained young Polish women (age 21.4 ± 1.7 years; body mass index (BMI): 21.7 (2.4) kg/m2) and 550 Russian athletes (229 women, age 23.0 ± 4.1; 321 men, age 23.9 ± 4.7). We identified one genome-wide significant polymorphism (rs116143768) located in the ACSL1 gene (acyl-CoA synthetase long-chain family member 1, implicated in fatty acid oxidation), with a rare T allele associated with higher fat loss efficiency in Polish women (fat mass decrease: CC genotype (n = 122) -3.8%; CT genotype (n = 4) -31.4%; p = 1.18 × 10-9). Furthermore, male athletes with the T allele (n = 7) had significantly lower BMI (22.1 (3.1) vs. 25.3 (4.2) kg/m2, p = 0.046) than subjects with the CC genotype (n = 314). In conclusion, we have shown that the rs116143768 T allele of the ACSL1 gene is associated with higher fat loss efficiency in response to aerobic training in untrained women and lower BMI in physically active men.

Comparative Transcriptome Analysis Reveals the Key Genes Involved in Lipid Deposition in Pekin Ducks (Anas platyrhynchos domesticus)

There are differences in lipid deposition in fatty-type (FT) and lean-type (LT) ducks. Fatty ducks have a higher rate of sebum and abdominal fat, lower meat yield and hepatic lipid contents than LT ducks. However, the underlying changes in gene expression profiles regarding the lipid deposition between FT and LT ducks have not yet been clarified. To identify the differentially expressed genes in the liver, sebum, and abdominal fat between both ducks, we identified the gene expression profiles in the liver, sebum, and abdominal fat derived from FT and LT ducks by comparing the multistage transcriptomes. Our results showed that there were 622, 1536, and 224 differentially expressed genes (DEGs) in the liver, sebum, and abdominal fat between the FT and LT ducks, respectively. KEGG enrichment showed that the DEGs related to lipid metabolism were enriched in the biosynthesis of unsaturated fatty acid, glycerolipid and fatty acid metabolism in the liver; and were enriched in the fatty acid metabolism, fatty acid biosynthesis, glycerolipid metabolism, linoleic acid metabolism, and the PPAR signaling pathway in the sebum. There was no pathway related to a lipid metabolism enriched in abdominal fat. A gene functional analysis showed that the DEGs involved in adipogenesis were found to be upregulated. In contrast, those involved in lipolysis were downregulated in the liver and serum of FT ducks. The DEGs showed that ATP-binding cassette sub-family G member 8 (ABCG8), fatty acid synthase (FASN), and phospholipid transfer protein (PLTP) were highly expressed in the liver of FT ducks, and acyl-CoA synthetase long-chain family member3 (ACSL3), ACSL5, ACSL6, 1-acyl-sn-glycerol-3-phosphate acyltransferase alpha (AGPAT1), AGPAT9, ELOVL fatty acid elongase 6 (ELVOL6), fatty acid desaturase 1 (FADS1), FADS2, monoacylglycerol O-acyltransferase 1 (MOGAT1), serine/threonine kinase 17a (STK17A), and serine/threonine kinase 39 (STK39) were highly expressed in the sebum of FT ducks. A weighted correlation network analysis (WGCNA) of the DEGs showed ABCG8, FADS2, ACSL5, and ELOVL6 positively correlated with hepatic fatty acid synthesis, and AGPAT1, STK17A, STK32A, FADS1, and ACSL3 positively correlated with lipid deposition in the sebum. In summary, ABCG8 might be the key gene for the reduced hepatic lipid deposition in FT Pekin ducks, and FADS2, ACSL5, ELOVL6, AGPAT1, STK17A, STK32A, FADS1, and ACSL3 were the key genes for lipid deposition in the sebum of FT Pekin ducks. Our results provide new insights into the transcriptome regulation in lipid deposition of Pekin ducks and will be helpful for duck breeding.

Mori fructus aqueous extracts attenuate carbon tetrachloride-induced renal injury via the Nrf2 pathway and intestinal flora

Mori fructus aqueous extracts (MFAEs) have been used as a traditional Chinese medicine for thousands of years with the function of strengthening the liver and tonifying the kidney. However, its inner mechanism to alleviative renal injury is unclear. To investigate the attenuation of MFAEs on nephrotoxicity and uncover its potential molecular mechanism, we established a nephrotoxicity model induced by carbon tetrachloride (CCl4). The mice were randomly divided into control group, CCl4 model group (10% CCl4), CCl4 + low and high MFAEs groups (10% CCl4 + 100 mg/kg and 200 mg/kg MFAEs). We found that MFAEs decreased the kidney index of mice, restored the pathological changes of renal structure induced by CCl4, reduced cystatin C, neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule 1 (Kim-1) blood urea nitrogen and creatinine contents in serum, promoted the nuclear transportation of Nrf2 (nuclear factor erythroid derived 2 like 2), elevated the expression of HO-1 (heme oxygenase 1), GPX4 (glutathione peroxidase 4), SLC7A11 (solute carrier family 7 member 11), ZO-1 (zonula occludens-1) and Occludin, suppressed the expression of Keap1 (kelch-like ECH-associated protein 1), HMGB1 (High Mobility Group Protein 1), ACSL4 (acyl-CoA synthetase long chain family member 4) and TXNIP (thioredoxin interacting protein), upregulated the flora of Akkermansia, Anaerotruncus, Clostridium_sensu_stricto, Ihubacter, Alcaligenes, Dysosmobacter, and downregulated the flora of Clostridium_XlVa, Helicobacter, Paramuribaculum. Overlapped with Disbiome database, Clostridium_XlVa, Akkermansia and Anaerotruncus may be the potential genera treated with renal injury. It indicated that MFAEs could ameliorate kidney injury caused by CCl4 via Nrf2 signaling.

Cooperation effects of radiation and ferroptosis on tumor suppression and radiation injury.

Ferroptosis is a kind of oxidative stress-dependent cell death characterized by iron accumulation and lipid peroxidation. It can work in conjunction with radiation to increase reactive oxygen species (ROS) generation and disrupt the antioxidant system, suppressing tumor progression. Radiation can induce ferroptosis by creating ROS, depleting glutathione, activating genes linked to DNA damage and increasing the expression of acyl-CoA synthetase long-chain family member 4 (ACSL4) in tumor cells. Furthermore, ferroptosis can enhance radiosensitivity by causing an iron overload, destruction of the antioxidant system, and lipid peroxidation. Radiation can also cause ferroptosis in normal cells, resulting in radiation injury. The role of ferroptosis in radiation-induced lung, intestinal, skin, and hematological injuries have been studied. In this review, we summarize the potential mechanisms linking ferroptosis, oxidative stress and radiation; analyze the function of ferroptosis in tumor suppression and radiation injury; and discuss the potential of ferroptosis regulation to improve radiotherapy efficacy and reduce adverse effects.

Repositioning of FDA-Approved antifungal agents to interrogate Acyl-CoA synthetase long chain family member 4 (ACSL4) in ferroptosis

Ferroptosis, first coined in 2012, is an iron-dependent regulated cell death (RCD) characterized by the accumulation of lipid peroxides to toxic levels. This mechanism is currently being evaluated as a target for a variety of diseases offering new opportunities for drug design and development. Recent reports uncovered acyl-CoA synthetase long-chain 4 (ACSL4) as a critical contributor to ferroptosis execution. Therefore, ACSL4 inhibitors are emerging as attractive anti-ferroptotic agents. Herein, we developed a robust screening cascade with orthogonal biophysical and biochemical techniques to identify original human ACSL4 inhibitors. By screening an FDA-approved drug library, we were able to identify and validate new inhibitors with micromolar-range activities against ACSL4. With an IC50 of 280 nM against hACSL4, antifungal agent sertaconazole is to our knowledge, the most potent ACSL4 inhibitor identified so far. In addition, sertaconazole significantly reduced lipid peroxidation and ferroptosis in human differentiated dopaminergic neurons (Lund human mesencephalic LUHMES cells), demonstrating that it is a valuable chemical tool for further investigating the role of ACSL4 in ferroptosis. This study highlights the phenethyl-imidazole scaffold as a novel and promising starting point for the development of anti-ferroptotic agents targeting ACSL4.

Dihydromyricetin Attenuates Cerebral Ischemia Reperfusion Injury by Inhibiting SPHK1/mTOR Signaling and Targeting Ferroptosis.

BackgroundDihydromyricetin (DHM) exerts protective effects in various brain diseases. The aim of this research was to investigate the biological role of DHM in cerebral ischemia reperfusion (I/R) injury.MethodsWe generated a rat model of cerebral I/R injury by performing middle cerebral artery occlusion/reperfusion (MCAO/R). The neurological score and brain water content of the experimental rats was then evaluated. The infarct volume and extent of apoptosis in brain tissues was then assessed by 2,3,5-triphenyltetrazolium (TTC) and TdT-mediated dUTP nick end labeling (TUNEL) staining. Hippocampal neuronal cells (HT22) were subjected to oxygen-glucose deprivation/reperfusion (OGD/R) and cell counting kit-8 (CCK-8) assays and flow cytometry were performed to detect cell viability and apoptosis. The levels of lipid reactive oxygen species (ROS) and iron were detected and the expression levels of key proteins were assessed by Western blotting.ResultsDHM obviously reduced neurological deficits, brain water content, infarct volume and cell apoptosis in the brain tissues of MCAO/R rats. DHM repressed ferroptosis and inhibited the sphingosine kinase 1 (SPHK1)/mammalian target of rapamycin (mTOR) pathway in MCAO/R rats. In addition, DHM promoted cell viability and repressed apoptosis in OGD/R-treated HT22 cells. DHM also suppressed the levels of lipid ROS and intracellular iron in OGD/R-treated HT22 cells. The expression levels of glutathione peroxidase 4 (GPX4) was enhanced while the levels of acyl-CoA synthetase long-chain family member 4 (ACSL4) and phosphatidylethanolamine binding protein 1 (PEBP1) were reduced in OGD/R-treated HT22 cells in the presence of DHM. Moreover, the influence conferred by DHM was abrogated by the overexpression of SPHK1 or treatment with MHY1485 (an activator of mTOR).ConclusionThis research demonstrated that DHM repressed ferroptosis by inhibiting the SPHK1/mTOR signaling pathway, thereby alleviating cerebral I/R injury. Our findings suggest that DHM may be a candidate drug for cerebral I/R injury treatment.

LncRNA ZFAS1 Positively Facilitates Endothelial Ferroptosis via miR-7-5p/ACSL4 Axis in Diabetic Retinopathy.

Accumulating evidence has suggested the significant role of long noncoding RNAs (lncRNA) in regulating ferroptosis, while its regulatory mechanism in diabetic retinopathy (DR) remains unelucidated. In this work, we first demonstrated that lncRNA zinc finger antisense 1 (ZFAS1) is upregulated in high glucose-cultured human retinal endothelial cells (hRECs) and ZFAS1 inhibition attenuated high glucose- (HG-) induced ferroptosis, which was evidenced by cell viability, total iron and ferrous iron levels, reactive oxygen species (ROS) level, and Glutathione Peroxidase 4 (GPX4) expression detection. Mechanistically, we validated that ZFAS1 may act as a competing endogenous RNA by competitively binding with microRNA-7-5p (miR-7-5p) and modulating the expression of its downstream molecule acyl-CoA synthetase long-chain family member 4 (ACSL4), which is now identified as a classic driver gene of ferroptosis process. In conclusion, our results demonstrate that HG-induced ZFAS1 elevation activates ferroptosis in hRECs and the ZFAS1/miR-7-5p/ACSL4 axis may serve as a therapeutic target for endothelial dysfunction in DR.

Perfluorooctane Sulfonate Induces Dysfunction of Human Umbilical Vein Endothelial Cells via Ferroptosis Pathway.

(1) Background: Perfluorooctane sulfonate (PFOS) is a persistent organic pollutant, and it is receiving increasing attention regarding its human health risks due to its extensive use. Endothelial dysfunction is a mark of cardiovascular disease, but the basic mechanism of PFOS-induced endothelial dysfunction is still not fully understood. Ferroptosis is a newly defined regulatory cell death driven by cellular metabolism and iron-dependent lipid peroxidation. Although ferroptosis has been shown to be involved in the pathogenesis of cardiovascular diseases, the involvement of ferroptosis in the pathogenesis of endothelial dysfunction caused by PFOS remains unclear. (2) Purpose: To explore the role of ferroptosis in the dysfunction of endothelial cells and underlying mechanisms. (3) Methods: Human umbilical vein endothelial cells (HUVECs) were exposed to PFOS or PFOS and Fer-1. The viability, morphology change under electronic microscope, lipid-reactive oxygen species (lipid-ROS), and production of nitric oxide (NO) were determined. The expression of glutathione peroxidase 4(GPX4), ferritin heavy chain protein 1 (FTH1), heme oxygenase 1 (HO-1) and Acyl-CoA synthetase long-chain family member 4 (ACSL4) were analyzed via Western blot analysis. (4) Results: PFOS was shown to cause a decrease in viability and morphological changes of mitochondria, and well as an increase in lipid droplets. The expression of GPX4, FTH1 and HO-1 was decreased, and that of ACSL4 was increased after exposure to PFOS. In addition to the above-mentioned ferroptosis-related manifestations, there was also a reduction in NO content. (5) Conclusions: PFOS induces ferroptosis by regulating the GPX4 and ACSL4 pathways, which leads to HUVEC dysfunction.

Neratinib inhibits proliferation and promotes apoptosis of acute myeloid leukemia cells by activating autophagy-dependent ferroptosis.

Acute myeloid leukemia (AML) is a hematologic malignancy with increased lethality. We focused on elucidating the role of Neratinib, a tyrosine kinase inhibitor, in the progression of AML and identify the potential mechanisms. Upon the treatment of Neratinib, autophagy suppressor 3-methyladenine (3-MA) and ferroptosis stimulator Erastin, the viability and proliferation of HL-60 cells were evaluated by cell counting kit-8 and 5-Ethynyl-20-Deoxyuridinestaining assays. A flow cytometer was to observe cell cycle and apoptosis. Production of reactive oxygen species (ROS) was tested via 2,7-dichlorodihydrofluorescein diacetate assay. Additionally, malondialdehyde (MDA) content and Fe2+ activity were examined with commercial kits. LC3-II expression was examined by using immunofluoresencestaining. Western blot analysis ascertained the expression of proliferation, apoptosis, ferroptosis and autophagy-associated proteins. It was noted that Neratinib notably mitigated cell viability and proliferation, cut down Ki67 and proliferating cell nuclear antigen expression. Moreover, Neratinib hindered cell cycle at G0/G1 phase whereas exacerbated apoptosis. ROS, MDA and Fe2+ activities were elevated by Neratinib, coupled with the reduced glutathione peroxidase 4, ferritin heavy chain 1 expression and enhanced acyl-CoA synthetase long-chain family member 4expression. Furthermore, Neratinib promoted autophagy of HL-60 cells, evidenced by raised LC3-II, ATG5, Beclin1 expression and lessened p62 expression. Importantly, 3-MA eased the impacts of Neratinib on cell ferroptosis, proliferation and apoptosis, which were offset by further administration of Erastin. To conclude, Neratinib could suppress proliferation and promote apoptosis of HL-60 cells through autophagy-dependent ferroptosis.

Clinical and Biological Significances of a Ferroptosis-Related Gene Signature in Lung Cancer Based on Deep Learning.

Acyl-CoA synthetase long-chain family member 4 (ACSL4) has been linked to the occurrence of tumors and is implicated in the ferroptosis process. Deep learning has been applied to many areas in health care, including imaging diagnosis, digital pathology, classification of cancer, and prediction of metastasis. Nonetheless, neither the level of ACSL4 expression nor its predictive significance in non-small-cell lung cancer (NSCLC) is well understood at this time. Predictions of the ACSL4 mRNA expressions in NSCLC and its link to NSCLC prognosis were made with the aid of the Oncomine and TCGA databases. By performing real-time PCR, we detected the levels of ACSL4 expression that were present in human NSCLC samples. Analyses of the diagnostic, as well as the prognostic significance of ACSL4 in NSCLC, were performed with the use of Kaplan-Meier curves. To assess the influence of ACSL4 on ferroptosis in NSCLC cell lines, an inducer of ferroptosis, namely, erastin, was utilized in this study. In NSCLC tissues, there was a substantial decrease in the level of ACSL4 expression (p < 0.001), and this was in line with the findings of the inquiry into the Oncomine and TCGA databases. After that, the findings of the immunohistochemistry analysis revealed that the ACSL4 staining was weakened in NSCLC samples in contrast with the normal samples. It was shown that the differential expression of ACSL4 was substantially linked to the stages of cancer, smoking behaviors, and the status of nodal metastases (all p < 0.001). According to the findings of the survival analysis, both RFS and OS were favorable among NSCLC patients who had elevated expression of ACSL4. The ferroptosis sensitization in cancer cells may be reestablished with upregulation of ACSL4 through gene transfection. Mechanistically, protein ubiquitination could perform a remarkable function in ACSL4-induced ferroptosis. ACSL4, which has a function in ferroptosis as both a contributor and monitor, was shown to be downregulated in NSCLC. This finding suggests that ACSL4 might function as a helpful diagnostic and prognostic biological marker and might also be considered a novel possible treatment target for NSCLC.

Shenmai Injection Attenuates Myocardial Ischemia/Reperfusion Injury by Targeting Nrf2/GPX4 Signalling-Mediated Ferroptosis.

To examine the effect of Shenmai Injection (SMJ) on ferroptosis during myocardial ischemia reperfusion (I/R) injury in rats and the underlying mechanism. A total of 120 SPF-grade adult male SD rats, weighing 220-250 g were randomly divided into different groups according to a random number table. Myocardial I/R model was established by occluding the left anterior descending artery for 30 min followed by 120 min of reperfusion. SMJ was injected intraperitoneally at the onset of 120 min of reperfusion, and erastin (an agonist of ferroptosis), ferrostatin-1 (Fer-1, an inhibitor of ferroptosis) and ML385 (an inhibitor of nuclear factor erythroid-2 related factor 2 (Nrf2)) were administered intraperitoneally separately 30 min before myocardial ischemia as different pretreatments. Cardiac function before ischemia, after ischemia and after reperfusion was analysed. Pathological changes in the myocardium and the ultrastructure of cardiomyocytes were observed, and the myocardial infarction area was measured. Additionally, the concentration of Fe2+ in heart tissues and the levels of creatine kinase-MB (CK-MB), troponin I (cTnl), malondialdehyde (MDA) and superoxide dismutase (SOD) in serum were measured using assay kits, and the expressions of Nrf2, glutathione peroxidase 4 (GPX4) and acyl-CoA synthetase long-chain family member 4 (ACSL4) were examined by Western blot. Compared with the sham group, I/R significantly injured heart tissues, as evidenced by the disordered, ruptured and oedematous myocardial fibres; the increases in infarct size, serum CK-MB, cTnI and MDA levels, and myocardial Fe2+ concentrations; and the decreases in SOD activity (P<0.05). These results were accompanied by ultrastructural alterations to the mitochondria, increased expression of ACSL4 and inhibited the activation of Nrf2/GPX4 signalling (P<0.05). Compared with I/R group, pretreatment with 9 mL/kg SMJ and 2 mg/kg Fer-1 significantly reduced myocardial I/R injury, Fe2+ concentrations and ACSL4 expression and attenuated mitochondrial impairment, while 14 mg/kg erastin exacerbated myocardial I/R injury (P<0.05). In addition, cardioprotection provided by 9 mL/kg SMJ was completely reversed by ML385, as evidenced by the increased myocardial infarct size, CK-MB, cTnI, MDA and Fe2+ concentrations, and the decreased SOD activity (P<0.05). Ferroptosis is involved in myocardial I/R injury. Pretreatment with SMJ alleviated myocardial I/R injury by activating Nrf2/GPX4 signalling-mediated ferroptosis, thereby providing a strategy for the prevention and treatment of ischemic heart diseases.

Endometrial Regenerative Cell-Derived Exosomes Attenuate Experimental Colitis through Downregulation of Intestine Ferroptosis.

Background Endometrial regenerative cells (ERCs) have been identified to ameliorate colitis in mice; however, whether exosomes derived from ERCs (ERC-exos) own similar effects on colitis remains unclear. Ferroptosis, an iron-dependent cell programmed death form, has been reported to promote inflammation in UC. Thus, in this study, whether ERC-exos can treat colitis and regulate intestine ferroptosis will be explored. Methods In this study, iron, malondialdehyde (MDA) production, glutathione (GSH) synthesis, and acyl-CoA synthetase long-chain family member (ACSL) 4 and glutathione peroxidase 4 (GPX4) expressions were measured in colon samples from healthy people and UC patients to explore the effects of ferroptosis. In vitro, ERC-exos were cocultured with ferroptosis inducer erastin-treated NCM460 human intestinal epithelial cell line, and ferroptotic parameters were measured. In vivo, colitis was induced by 3% dextran sulfate sodium (DSS) in BALB/c mice, and animals were randomly assigned to normal, untreated, and ERC-exos-treated groups. The Disease Activity Index (DAI) score, histological features, tissue iron, MDA, GSH, ACSL4, and GPX4 were measured to verify the role of ERC-exos in attenuating UC. Results Compared with healthy people, UC samples exhibited higher levels of iron, MDA, and ACSL4, while less levels of GSH and GPX4. In vitro, the CCK-8 assay showed that ERC-exos rescued erastin-induced cell death, and ERC-exos treatment significantly increased the levels of GSH and expression of GPX4, while markedly decreasing the levels of iron, MDA, and expression of ACSL4. In vivo, ERC-exos treatment effectively reduced DAI score, ameliorated colon pathological damage, and improved disease symptoms. Moreover, ERC-exos treatment further enhanced the levels of GSH and the expression of GPX4 but reduced the levels of iron, MDA, and expression of ACSL4 in the colon of colitis mice. Conclusions Ferroptosis was involved in the pathogenesis of UC, and ERC-exos attenuated DSS-induced colitis through downregulating intestine ferroptosis. This study may provide a novel insight into treating UC in the future.

D-Penicillamine Reveals the Amelioration of Seizure-Induced Neuronal Injury via Inhibiting Aqp11-Dependent Ferroptosis.

Repetitive seizures, a common phenomenon in diverse neurologic conditions such as epilepsy, can undoubtedly cause neuronal injury and our prior work reveals that ferroptosis is a contributing factor of neuronal damage post seizure. However, there is no drug available in clinical practice for ameliorating seizure-induced neuronal impairment via targeting ferroptosis. Our present work aimed to explore whether D-penicillamine (DPA), an originally approved drug for treating Wilson’s disease, inhibited neuronal ferroptosis and alleviated seizure-associated brain damage. Our findings revealed that DPA remarkably improved neuronal survival in kainic acid (KA)-treated mouse model. Furthermore, ferroptosis-associated indices including acyl-coA synthetase long chain family member 4 (ACSL4), prostaglandin-endoperoxide synthase 2 (Ptgs2) gene and lipid peroxide (LPO) level were significantly decreased in KA mouse model after DPA treatment. In a ferroptotic cell death model induced by glutamate or erastin, DPA was also validated to evidently suppress neuronal ferroptosis. The results from RNA-seq analysis indicated that Aqp11, a gene coding previously reported channel protein responsible for transporting water and small solutes, was identified as a molecular target by which DPA exerted anti-ferroptotic potential in neurons. The experimental results from in vivo Aqp11 siRNA transfer into the brain also confirmed that knockdown of Aqp11 abrogated the inhibitory effect of seizure-induced ferroptosis after DPA treatment, suggesting that the effects of DPA on ferroptosis process are dependent upon Aqp11. In conclusion, DPA can be repurposed to cure seizure disorders such as epilepsy.

Identification of ACSL4 as a biomarker and contributor of ferroptosis in clear cell renal cell carcinoma.

BackgroundAcyl-CoA synthetase long-chain family member 4 (ACSL4) has been linked to the ferroptosis process and is implicated in the pathogenesis of tumors. Nevertheless, neither the expression levels of ACSL4 nor its prognostic significance in clear cell renal cell carcinoma (ccRCC) is completely understood at this time.MethodsPredictions of the ACSL4 mRNA expression in ccRCC and its link to ccRCC prognosis were made based on data from the Oncomine and The Cancer Genome Atlas (TCGA) databases. Through the use of real-time polymerase chain reaction (PCR), we ascertained the levels of ACSL4 expression in human RCC samples. Analyses of the link between ACSL4 expression and the survival of ccRCC patients were undertaken using the Kaplan-Meier (KM) plotter database. To study the function that ACSL4 plays in ferroptosis in ccRCC cell lines, either upregulation or knockdown of its expression was performed.ResultsWe found that the ACSL4 expression level was considerably down-modulated in ccRCC samples (P<0.001), which was in line with the analytical results of the Oncomine and TCGA database. The subsequent immunohistochemistry findings revealed that ACSL4 levels in ccRCC samples were much lower or not undetectable in contrast with those in normal samples. The level of differential ACSL4 expression was strongly associated with factors such as disease stages, gender, tumor grade, nodal invasion, and ccRCC subtypes (all P<0.001). According to the results of the survival analysis, the overall survival was satisfactory among ccRCC patients who had overexpressed ACSL4 (P=0.014). In cancer cells, ferroptosis sensitization may be restored by overexpressing ACSL4 via the process of transfection; however, increasing ferroptosis resistance can be achieved by suppressing ACSL4 expression through the use of shRNA. Protein ubiquitination could have a function in influencing the way that ACSL4-induced ferroptosis works mechanically.ConclusionsACSL4, which is a mediator and monitor of ferroptosis, was lowered in expression in ccRCC and acted as a valuable diagnostic and prognostic biological marker, thus representing a novel promising treatment target for ccRCC.

ACSL4 is essential for radiation-induced intestinal injury by initiating ferroptosis

Lipid peroxidation-induced ferroptosis is a newly recognized type of programmed cell death. With the method of RNA sequencing, we found that irradiation (IR) markedly increased the expression of ferroptosis promotive genes, whereas reduced the expression of ferroptosis suppressive genes in murine intestine tissues, when compared with those of liver and lung tissues. By using ferroptosis inducer RSL-3 and inhibitor liproxstatin-1, we found that ferroptosis is essential for IR-induced intestinal injury. Acyl-CoA Synthetase Long-Chain Family Member 4 (ACSL4) is an important component for ferroptosis execution, and we found that ACSL4 expression was significantly upregulated in irradiated intestine tissues, but not in liver or lung tissues. Antibacterial and antifungal regents reduced the expression of ASCL4 and protected against tissue injury in irradiated intestine tissues. Further studies showed that troglitazone, a ACSL4 inhibitor, succeeded to suppresses intestine lipid peroxidation and tissue damage after IR.

Puerarin Induces Molecular Details of Ferroptosis-Associated Anti-Inflammatory on RAW264.7 Macrophages.

Puerarin is a natural flavonoid with significant anti-inflammatory effects. Recent studies have suggested that ferroptosis may involve puerarin countering inflammation. However, the mechanism of ferroptosis mediated by the anti-inflammatory process of puerarin has not been widely explored. Herein, puerarin at a concentration of 40 μM showed an anti-inflammatory effect on lipopolysaccharide (LPS)-induced macrophages RAW264.7. The analysis of network pharmacology indicated that 51 common targets were enriched in 136 pathways, and most of the pathways were associated with ferroptosis. Subsequently, the analysis of metabolomics obtained 61 differential metabolites that were enriched in 30 metabolic pathways. Furthermore, integrated network pharmacology and metabolomics revealed that puerarin exerted an excellent effect on anti-inflammatory in RAW264.7 via regulating ferroptosis-related arachidonic acid metabolism, tryptophan metabolism, and glutathione metabolism pathways, and metabolites such as 20-hydroxyeicosatetraenoic acid (20-HETE), serotonin, kynurenine, oxidized glutathione (GSSG), gamma-glutamylcysteine and cysteinylglycine were involved. In addition, the possible active binding sites of the potential targeted proteins such as acyl-CoA synthetase long-chain family member 4 (ACSL4), prostaglandin-endoperoxide synthase 2 (PTGS2), arachidonate 15-lipoxygenase (ALOX15) and glutathione peroxidase 4 (GPX4) with puerarin were further revealed by molecular docking. Thus, we suggested that ferroptosis mediated the anti-inflammatory effects of puerarin in macrophages RAW264.7 induced by LPS.