acyl-CoA Synthetase Long-chain Family Research Articles

Sulforaphane, an NRF2 agonist, alleviates ferroptosis in acute liver failure by regulating HDAC6 activity

ObjectiveAcute liver failure (ALF) is characterized by severe liver dysfunction, rapid progression and high mortality and is difficult to treat. Studies have found that sulforaphane (SFN), a nuclear factor E2-related factor 2 (NRF2) agonist, has anti-inflammatory, antioxidant and anticancer effects, and has certain protective effects on neurodegenerative diseases, cancer and liver fibrosis. This paper aimed to explore the protective effect of SFN in ALF and it possible mechanisms of action. MethodsLipopolysaccharide and D-galactosamine were used to induce liver injury in vitro and in vivo. NRF2 agonist SFN and histone deacetylase 6 (HDAC6) inhibitor ACY1215 were used to observe the protective effect and possible mechanisms of SFN in ALF, respectively. Cell viability, lactate dehydrogenase (LDH), Fe2+, glutathione (GSH) and malondialdehyde (MDA) were detected. The expression of HDAC6, NRF2, glutathione peroxidase 4 (GPX4), acyl-CoA synthetase long-chain family member 4 (ACSL4) and solute carrier family 7 member 11 (SLC7A11) were detected by Western blotting and immunofluorescence. ResultsOur results show that NRF2 was activated by SFN. LDH, Fe2+, MDA and ACSL4 were downregulated, while GSH, GPX4 and SLC7A11 were upregulated by SFN in vitro and in vivo, indicating the inhibitory effect of SFN on ferroptosis. Additionally, HDAC6 expression was decreased in the SFN group, indicating that SFN could downregulate the expression of HDAC6 in ALF. After using the HDAC6 inhibitor, ACY1215, SFN further reduced HDAC6 expression and inhibited ferroptosis, indicating that SFN may inhibit ferroptosis by regulating HDAC6 activity. ConclusionSFN has a protective effect on ALF, and the mechanism may include reduction of ferroptosis through the regulation of HDAC6.Please cite this article as: Zhang YQ, Shi CX, Zhang DM, Zhang LY, Wang LW, Gong ZJ. Sulforaphane, an NRF2 agonist, alleviates ferroptosis in acute liver failure by regulating HDAC6 activity. J Integr Med. 2023; 21(5): 464–473.

Icariin Supplementation Suppresses the Markers of Ferroptosis and Attenuates the Progression of Nonalcoholic Steatohepatitis in Mice Fed a Methionine Choline-Deficient Diet.

Icariin, a flavonoid abundant in the herb Epimedium, exhibits anti-ferroptotic activity. However, its impact on nonalcoholic steatohepatitis (NASH) development remains unclear. This study aimed to investigate the potential role of icariin in mitigating methionine choline-deficient (MCD) diet-induced NASH in C57BL/6J mice. The results showed that icariin treatment significantly reduced serum alanine aminotrasferase and aspartate aminotransferase activities while improving steatosis, inflammation, ballooning, and fibrosis in the liver tissues of mice fed the MCD diet. These improvements were accompanied by a substantial reduction in the hepatic iron contents and levels of malondialdehyde and 4-hydroxynonenal, as well as an increase in the activities of catalase and superoxide dismutase. Notably, icariin treatment suppressed the hepatic protein levels of ferroptosis markers such as acyl-CoA synthetase long-chain family member 4 and arachidonate 12-lipoxygenase, which were induced by the MCD diet. Furthermore, transmission electron microscopy confirmed the restoration of morphological changes in the mitochondria, a hallmark characteristic of ferroptosis, by icariin. Additionally, icariin treatment significantly increased the protein levels of Nrf2, a cystine/glutamate transporter (xCT), and glutathione peroxidase 4 (GPX4). In conclusion, our study suggests that icariin has the potential to attenuate NASH, possibly by suppressing ferroptosis via the Nrf2-xCT/GPX4 pathway.

Low androgen levels induce ferroptosis of rat penile cavernous endothelial cells.

Endothelial dysfunction caused by low androgen levels in penile tissue can lead to erectile dysfunction. The exact mechanism of endothelial dysfunction has not been thoroughly studied. The study sought to verify whether low androgen levels induce ferroptosis of endothelial cells in rat penile tissue. Rat penile cavernous endothelial cells (CP-R133) were divided into a no-androgen group (Dihydrotestosterone (DHT): 0nmol/L), very low-androgen group (DHT: 0.1nmol/L), low-androgen group (DHT: 1nmol/L), DHT = 10nmol/L group, DHT (0nmol/L) + ferrostatin-1 (Fer-1) group, DHT (0.1nmol/L) + Fer-1 group, DHT (1nmol/L) + Fer-1 group, DHT (10nmol/L) + Fer-1 group. Cell viability, intracellular ferrous ion (Fe2+), malondialdehyde (MDA), GSH into oxidized glutathione (GSSG), reactive oxygen species (ROS), nitric oxide (NO), transferrin receptor 1 protein (TfR1), solute carrier family 7 member 11 (SLC7A11), glutathione peroxidase 4 (GPX4), acyl-CoA synthetase long-chain family member 4 (ACSL4), endothelial nitric oxide synthase (eNOS), and phospho-eNOS (p-eNOS) were detected. Low androgen levels could induce ferroptosis of rat penile cavernous endothelial cells in vivo by upregulating the expressions of TfR1 and ACSL4 and downregulating the expressions of SLC7A11 and GPX4. Cell viability, the levels of glutathione (GSH), NO, SLC7A11, GPX4, and p-eNOS/eNOS in the DHT = 0nmol/L group were lower than those in the other groups (P < .05). The levels of Fe2+, ROS, MDA, GSSG, TfR1, and ACSL4 in the DHT = 0nmol/L group were higher than those in the other groups (P < .05). Cell viability and the levels of GSH, NO, SLC7A11, GPX4, and p-eNOS/eNOS in the DHT = 1nmol/L group were lower than those in the DHT (1nmol/L) + Fer-1 group, DHT = 10nmol/L group, and DHT (10nmol/L) + Fer-1 group (P < .05). The levels of Fe2+, ROS, MDA, GSSG, TfR1, and ACSL4 in the DHT = 1nmol/L group were higher than those in the DHT (1nmol/L) + Fer-1 group, DHT = 10nmol/L group, and DHT (10nmol/L) + Fer-1 group (P < .05). A ferroptosis inhibitor might be a novel drug for treating erectile dysfunction caused by low androgen level. The results of this study need to be further confirmed in in vitro and in human studies. Meanwhile, further investigation is needed to clarify whether low androgen levels affect ferroptosis of rat penile cavernous smooth muscle and nerve cells. Low androgen levels can induce ferroptosis of endothelial cells in rat penile tissue. Inhibition of ferroptosis can reverse endothelial dysfunction caused by low androgen levels.

Signal transducer and activator of transcription 6 mediates skeletal muscle cell injury in septic mice by regulating ferroptosis

To explore the effect of signal transducer and activator of transcription 6 (STAT6) on ferroptosis in skeletal muscle cells in sepsis model and its potential mechanism. Twenty-four 8-week-old male specific pathogen free Kunming mice were divided into normal control group, sham group, sepsis model group and STAT6 inhibitor pretreatment group according to random number table method with 6 mice in each group. A mouse sepsis model was reproduced by cecal ligation and perforation (CLP). In the sham group, the skin of mice was sutured after exposing the cecum tissue. In the STAT6 inhibitor pretreatment group, 10 mg/kg AS1517499 was injected intraperitoneally 1 hour before model reproduction. The sham group and the model group were intraperitoneally injected with the same volume of normal saline. Mice in the normal control group did not receive any operation or drug intervention. The mice were sacrificed 24 hours after model reproduction, and the muscle tissue of hind limb was obtained under sterile condition. Hematoxylin-eosin (HE) staining was used to observe the histopathology with optical microscope, and mitochondrial morphological changes were observed by transmission electron microscopy after double staining with uranium acetate lead citrate. The ferroptosis marker proteins expressions of chitinase-3-like protein 1 (CHI3L1), cyclooxygenase-2 (COX-2), acyl-CoA synthetase long-chain family member 4 (ACSL4), ferritin heavy chain 1 (FTH1), and glutathione peroxidase 4 (GPx4) were detected by Western blotting. Under the optical microscope, the morphology and structure of skeletal muscle tissues in the normal control and sham groups were normal. In the model group, the structure of skeletal muscle tissues was loose, the muscle fiber became smaller and atrophic, inflammatory cell infiltration and even muscle fiber loss were found. Compared with the model group, the structure of skeletal muscle tissues was tight and skeletal muscle atrophy was improved in the STAT6 inhibitor pretreatment group. The ultrastructure of skeletal muscle cell in the normal control and sham groups was normal under transmission electron microscope. The ultrastructure characteristics of skeletal muscle in the model group showed that cell membrane was broken and blister, mitochondria became smaller and membrane density increased, the mitochondrial crista decreased or disappeared, the mitochondrial outer membrane was broken, and the nucleus was normal in size but lacked chromatin condensation. Compared with the model group, the STAT6 inhibitor pretreatment group had a significant improvement in the ultrastructure of muscle cells. Compared with the normal control and sham groups, the protein expressions of CHI3L1, COX-2, ACSL4 and FTH1 in the muscle of the model group were significantly increased, while the protein expression of GPx4 was decreased significantly, indicating that the skeletal muscle cells in the mouse sepsis model showed characteristic mitochondrial injury and abnormal expression of ferroptosis markers. Compared with the model group, the protein expressions of CHI3LI, COX-2, ACSL4 and FTH1 in the STAT6 inhibitor pretreatment group were significantly decreased [CHI3L1 protein (CHI3L1/GAPDH): 0.70±0.08 vs. 0.97±0.09, COX-2 protein (COX-2/GAPDH): 0.61±0.03 vs. 0.83±0.03, ACSL4 protein (ACSL4/GAPDH): 0.75±0.04 vs. 1.02±0.16, FTH1 protein (FTH1/GAPDH): 0.49±0.06 vs. 0.76±0.13, all P < 0.05], while the protein expression of GPx4 was significantly increased (GPx4/GAPDH: 1.14±0.29 vs. 0.53±0.03, P < 0.05). Sepsis can induce ferroptosis in skeletal muscle cells of mice. STAT6 may mediate ferroptosis in mouse skeletal muscle cells by regulating the expressions of COX-2, ACSL4, FTH1 and GPx4, thereby inducing skeletal muscle cell injury in sepsis.

FLRT2 suppresses bladder cancer progression through inducing ferroptosis.

Bladder cancer is a common tumour worldwide and exhibits a poor prognosis. Fibronectin leucine rich transmembrane protein 2 (FLRT2) is associated with the regulation of multiple tumours; however, its function in human bladder cancer remain unclear. Herein, we found that FLRT2 level was reduced in human bladder cancer and that higher FLRT2 level predicted lower survival rate. FLRT2 overexpression inhibited, while FLRT2 silence facilitated tumour cell growth, migration and invasion. Mechanistic studies revealed that FLRT2 elevated acyl-CoA synthetase long-chain family member 4 (ACSL4) expression, increased lipid peroxidation and subsequently facilitated ferroptosis of human bladder cancer cells. In summary, we demonstrate that FLRT2 elevates ACSL4 expression to facilitate lipid peroxidation and subsequently triggers ferroptosis, thereby inhibiting the malignant phenotype of human bladder cancer cells. Overall, we identify FLRT2 as a tumour suppressor gene.

Etomidate Improves the Antidepressant Effect of Electroconvulsive Therapy by Suppressing Hippocampal Neuronal Ferroptosis via Upregulating BDNF/Nrf2.

Electroconvulsive therapy (ECT) performed under general anesthesia is an effective treatment for severe depression. Etomidate is an intravenous anesthetic that shows beneficial effects on ECT. However, the potential mechanisms have rarely been reported. In this study, male rats were exposed to chronic unpredictable mild stress for 4weeks, followed by ECT for 10days, with or without intervention with ferrostatin-1 (2mg/kg) or all-trans retinoic acid (ATRA, 5mg/kg). Rats subjected to etomidate (20mg/kg) or propofol (120mg/kg) treatment were administered with designated anesthetic before ECT. Compared to depressive rats without ECT, those who received ECT showed increased numbers of hippocampal neurons, increased expression of negative regulators of ferroptosis including glutathione peroxidase 4, ferritin heavy chain 1, and ferroptosis suppressor protein 1, upregulation of brain-derived neurotrophic factor and nuclear factor erythroid 2-related factor, and downregulation of acyl-CoA synthetase long-chain family member 4, a positive regulator of ferroptosis in the hippocampus. Additionally, compared with propofol, etomidate used in ECT resulted in higher upregulation of BDNF/Nrf2 and inhibited neuronal ferroptosis in hippocampus. These results showed etomidate may enhance the antidepressant effect of ECT by protecting hippocampal neurons against ferroptosis.

Novel aspect of neprilysin in kidney fibrosis via ACSL4-mediated ferroptosis of tubular epithelial cells.

Although inhibition of neprilysin (NEP) might be a therapeutic strategy with the potential to improve the outcome of chronic kidney disease (CKD), the versatile function of NEP with its mechanism remains obscure in kidney fibrosis. In the study, we found that NEP was abnormally increased in tubular epithelial cells of CKD patients, as well as unilateral ureteral obstruction and adenine diet-induced mice. Treatment with a United States Food and Drug Administration-approved NEP inhibitor Sacubitrilat (LBQ657) could alleviate ferroptosis, tubular injury, and delay the progression of kidney fibrosis in experimental mice. Similarly, genetic knockdown of NEP also inhibited tubular injury and fibrosis in transforming growth factor (TGF)-β1 -induced tubular cells. Mechanically, NEP overexpression aggravated the ferroptotic and fibrotic phenotype, which was restored by acyl-CoA synthetase long-chain family member 4 (ACSL4) knockdown. The NEP silencing attenuated TGF-β1-induced tubular cell ferroptosis and was exacerbated by ACSL4 overexpression. Collectively, for the first time, a novel aspect of NEP was explored in kidney fibrosis through ACSL4-mediated tubular epithelial cell ferroptosis. Our data further confirmed that NEP inhibition exerted a promising therapeutic against fibrotic kidney diseases.

ACSL4 promotes ferroptosis and M1 macrophage polarization to regulate the tumorigenesis of nasopharyngeal carcinoma

BackgroundNasopharyngeal carcinoma (NPC) is a head and neck malignant tumor with a high incidence and recurrence rate. The crosstalk between ferroptosis and tumor-associated macrophages (TAMs) is thought to have major implications in interfering with cancers. We intended to explore the effect of acyl-CoA synthetase long-chain family member 4 (ACSL4) on the pathogenesis of NPC via ferroptosis and TAMs. MethodsDifferential genes in NPC patients were analyzed using publicly available databases, and the ferroptosis-related gene ACSL4 was identified. Expression of ACSL4 in NPC cell lines and xenografted mice was examined. Colony formation, cell proliferation, migration, and invasion were assessed. The abundance of epithelial-mesenchymal transition (EMT) markers (E-cadherin, N-cadherin, and Vimentin) was confirmed. Lipid peroxidation levels and related markers were measured. Clophosome was administered to determine the role of TAMs in NPC mice. ResultsLow levels of ACSL4 were observed in NPC patients and CNE-2 and 5-8F cells. Erastin (a ferroptosis inducer) and ACSL4 increased lipid peroxidation, decreased cell viability, colony formation, cell proliferation, migration and invasion, and inhibited EMT. Moreover, Erastin and ACSL4 promoted M2 to M1 macrophage polarization. The effects of erastin and ACSL4 were additive. Ferrostatin-1, an inhibitor of ferroptosis, exerted the opposite effect and reversed the beneficial effects of ACSL4 overexpression. In xenograft mice, ACSL4 and clophosome hindered the growth of NPC, and extra clophosome slightly enhanced the antitumor effect of ACSL4. ConclusionOur findings indicated that ACSL4 inhibited the pathogenesis of NPC, at least through crosstalk between ferroptosis and macrophages, providing potential direction for NPC therapy.

Inhibition of CDK1 Overcomes Oxaliplatin Resistance by Regulating ACSL4-mediated Ferroptosis in Colorectal Cancer.

Oxaliplatin is a widely used chemotherapy drug for patients with advanced colorectal cancer (CRC); however, frequent drug resistance limits its therapeutic efficacy in patients. Here, this work identifies cyclin-dependent kinase 1 (CDK1) as a critical contributor to oxaliplatin resistance via in vitro and in vivo CRISPR/Cas9 screening. CDK1 is highly expressed in oxaliplatin-resistant cells and tissues due to the loss of N6-methyladenosine modification. Genetic and pharmacological blockade of CDK1 restore the susceptibility of CRC cells to oxaliplatin in vitro and in cell/patient-derived xenograft models. Mechanistically,CDK1 directly binds to and phosphorylates Acyl-CoA synthetase long-chain family 4 (ACSL4) at S447, followed by recruitment of E3 ubiquitin ligase UBR5 and polyubiquitination of ACSL4 at K388, K498, and K690, which leads to ACSL4 protein degradation. Reduced ACSL4 subsequently blocks the biosynthesis of polyunsaturated fatty acid containing lipids, thereby inhibiting lipid peroxidation and ferroptosis, a unique iron-dependent form of oxidative cell death. Moreover, treatment with a ferroptosisinhibitor nullifies the enhancement of CRC cell sensitivity to oxaliplatin by CDK1 blockade in vitro and in vivo. Collectively, the findings indicate that CDK1 confers oxaliplatin resistance to cells by suppressing ferroptosis. Therefore, administration of a CDK1 inhibitor may be an attractive strategy to treat patients with oxaliplatin-resistant CRC.

Protective benefits of salvianic acid A against retinal iron overload by inhibition of ferroptosis

BackgroundBoth the accumulation of reactive oxygen species (ROS) and iron overload are significant variables that enhance the incidence of photoreceptor cell death and retinal degeneration. The discovery of ferroptosis, which is characterized by iron-dependent lipid peroxidation, has led to a new perspective on how retinal degeneration develops. As a natural phenolic acid, salvianic acid A (SAA) from Salvia miltiorrhiza has promise in treating eye diseases. The purpose of this research was to learn more about SAA and its function in the development of iron-overload-induced retinal degeneration. MethodsModels of iron overload in Kunming mice and the murine photoreceptor cell line 661 W were established, then the protective and antiferroptotic properties of SAA were assessed in vivo and in vitro. ResultsBiochemical and histopathological findings on the retina confirmed that SAA successfully alleviated retinal injury. In photoreceptor cells, iron overload caused cell death, mitochondrial dysfunction, ROS generation, and iron deposition. Salvianic acid A relieved lipid peroxidation and decreased iron accumulation by modulating Acyl-CoA synthetase long-chain family member 4, glutathione peroxidase 4, solute carrier family 7 member 11, and iron-metabolism-related proteins. The mitochondrial morphology suggests that the retinal protective effect of SAA is mediated via antiferroptotic action. ConclusionFerroptosis plays an important role in the pathogenesis of iron-overload-induced retinal degeneration. New roles of SAA in ferroptosis prevention via iron deposit inhibition, lipid peroxidation inhibition, and mitochondrial dysfunction reduction, were identified.

Glycyrrhizic acid glycosides reduces extensive tripterygium glycosides-induced lipid deposition in hepatocytes

AimTripterygium glycosides (TG) extracted from the plant Tripterygium wilfordii Hook F has been used to treat chronic kidney diseases for many years. However, hepatotoxicity limits its clinical application. Glycyrrhizic acid glycosides (GA) can reduce TG hepatotoxicity, however, further investigation into the underlying molecular mechanisms by which GA attenuates TG-induced hepatotoxicity is required. MethodsSprague‒Dawley rats were randomly divided into the control group, the TG groups (TG189 mg/kg group, TG472.5 mg/kg group), and the TG + GA groups (TG189 mg/kg + GA20.25 mg/kg group, TG472.5 mg/kg + GA20.25 mg/kg group). After 21 consecutive days of intragastric administration, structural and molecular changes in hepatocytes were detected. ResultsAfter 21 days of TG treatment, the serum level of the total bilirubin, triglyceride, total cholesterol, and low-density lipoprotein cholesterol increased in the TG189 mg/kg and TG472.5 mg/kg groups when compared to the control group. High-density lipoprotein cholesterol levels were reduced in both TG groups. The ultrastructure of hepatocytes and the structural integrity of the liver were compromised. In addition, the relevant molecular level of the peroxisome proliferators-activated receptor α (PPARα) and acyl-CoA synthetase long-chain family members (ACSLs) pathway was modulated. With the addition of 20.25 mg/kg GA, the serum biochemical indexes and liver tissue structure ultrastructure of hepatocytes were improved, and the PPARα-ACSLs pathway was corrected. ConclusionThe combined application of GA and TG improved abnormal lipid metabolism, repaired liver structure, reduced lipid deposition in hepatocytes, and reduced TG-induced hepatotoxicity.

Ischemia/reperfusion-activated ferroptosis in the early stage triggers excessive inflammation to aggregate lung injury in rats.

Lung ischemia/reperfusion injury (LIRI) is a clinical syndrome of acute lung injury that occurs after lung transplantation or remote organ ischemia. Ferroptosis and inflammation are involved in the pathogenesis of LIRI according to the results of several studies on animal models. However, the interactive mechanisms between ferroptosis and inflammation contributing to LIRI remain unclear. HE staining and indicators of oxidative stress were used to evaluated the lung injury. The reactive oxygen species (ROS) level was examined by DHE staining. The quantitative Real-time PCR (qRT-PCR) and western blot analysis were employed to detect the level of inflammation and ferroptosis, and deferoxamine (DFO) was used to assess the importance of ferroptosis in LIRI and its effect on inflammation. In the present study, the link of ferroptosis with inflammation was evaluated at reperfusion 30-, 60- and 180-minute time points, respectively. As the results at reperfusion 30-minute point shown, the pro-ferroptotic indicators, especially cyclooxygenase (COX)-2 and acyl-CoA synthetase long-chain family member 4 (ACSL4), were upregulated while the anti-ferroptotic factors glutathione peroxidase 4 (GPX4), cystine-glumate antiporter (XCT) and ferritin heavy chain (FTH1) were downregulated. Meanwhile, the increased level of interleukin (IL)-6, tumor necrosis factor alpha (TNF-α) and IL-1β were observed beginning at reperfusion 60-minute point but mostly activated at reperfusion 180-minute point. Furthermore, deferoxamine (DFO) was employed to block ferroptosis, which can alleviate lung injury. Expectedly, the survival rate of rats was increased and the lung injury was mitigated containing the improvement of type II alveolar cells ultrastructure and ROS production. In addition, at the reperfusion 180-minute point, the inflammation was observed to be dramatically inhibited after DFO administration as verified by IL-6, TNF-α and IL-1β detection. These findings suggest that ischemia/reperfusion-activated ferroptosis plays an important role as the trigger for inflammation to further deteriorate lung damages. Inhibiting ferroptosis may have therapeutic potential for LIRI in clinical practice.

ACSL4-Mediated Ferroptosis and Its Potential Role in Central Nervous System Diseases and Injuries.

As an iron-dependent regulated form of cell death, ferroptosis is characterized by iron-dependent lipid peroxidation and has been implicated in the occurrence and development of various diseases, including nervous system diseases and injuries. Ferroptosis has become a potential target for intervention in these diseases or injuries in relevant preclinical models. As a member of the Acyl-CoA synthetase long-chain family (ACSLs) that can convert saturated and unsaturated fatty acids, Acyl-CoA synthetase long-chain familymember4 (ACSL4) is involved in the regulation of arachidonic acid and eicosapentaenoic acid, thus leading to ferroptosis. The underlying molecular mechanisms of ACSL4-mediated ferroptosis will promote additional treatment strategies for these diseases or injury conditions. Our review article provides a current view of ACSL4-mediated ferroptosis, mainly including the structure and function of ACSL4, as well as the role of ACSL4 in ferroptosis. We also summarize the latest research progress of ACSL4-mediated ferroptosis in central nervous system injuries and diseases, further proving that ACSL4-medicated ferroptosis is an important target for intervention in these diseases or injuries.

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

BackgroundFerroptosis, 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.ResultsIn 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.ConclusionIn 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.Graphic abstractMechanism 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. Abbreviations: 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.

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.

Tectorigenin relieved sepsis-induced myocardial ferroptosis by inhibiting the expression of Smad3.

Myocardial injury is a serious consequence of sepsis that contributes to high rates of death. Currently, the pathophysiology of cardiac damage in sepsis is still unknown, and treatment approaches are limited. The sepsis mouse model was established inducing by Lipopolysaccharide (LPS) in vivo and Tectorigenin was pretreated to explore whether it contributed to alleviated myocardial injury. Hematoxylin-eosin (HE) stain was employed to evaluate the myocardial injury severity. TUNEL assay measured the number of apoptosis cells and the levels of B-cell lymphoma-2 associated X (Bax) and Cleaved Caspase-3 were assessed by western blot. The contents of iron and related ferroptosis molecules (acyl-CoA synthetase long-chain family (ACSL4), Glutathione Peroxidase 4 (GPX4)) were assessed. Then, interleukin-1β (IL-1β), IL-18, IL-6, tumor necrosis factor-α (TNF-α), and other inflammatory-related cytokines were detected by ELISA. The expression of the mother against decapentaplegic homolog 3 (Smad3) in heart tissues was evaluated by western blot and immunofluorescence. Tectorigenin alleviated myocardial dysfunction and myofibrillar disruption in LPS-related sepsis groups. Tectorigenin ameliorated cardiomyocyte apoptosis and myocardial ferroptosis in LPS-stimulated sepsis mice. Tectorigenin reduced inflammatory-relevant cytokines in the cardiac tissues of LPS stimuli mice. In addition, we further confirm that Tectorigenin relieved myocardial ferroptosis by inhibiting the expression of Smad3. Tectorigenin ameliorates myocardial damage stimulated by LPS and this effect exerts by inhibiting ferroptosis and the inflammation of the myocardium. Furthermore, the inhibitory effect of Tectorigenin on ferroptosis may deregulate Smad3 expression. Taken together, Tectorigenin may be a viable method for alleviating myocardial damage in sepsis.

The transcriptome reveals the molecular regulatory network of primordial follicle depletion in obese mice

To explore the dynamic transcriptional regulatory network of primordial follicle fate in obese mice to elucidate the potential mechanism of primordial follicle depletion. Experimental study and transcriptomic analysis. Healthy (n=15) and obese (n=15) female mice. Six-week-old CD-1 mice were divided into healthy and high-fat diet groups and fed continuously for 12 weeks. The diet of healthy mice contained 10% fat. The diet of high-fat mice contained 60% fat. Primordial to primary follicle transition rate, gene expression changes, enriched Kyoto Encyclopedia of Genes and Genomes pathways, and ferroptosis. Primordial follicle depletion was increased in the ovaries of obese mice. We found that deposited fat around primordial and primary follicles of obese mice was higher than that for healthy mice. The proliferation of granulosa cells around primary follicles was increased in obese mice. In addition, we uncovered specific gene signatures associated with the primordial to primary follicle transition (PPT) in obese mice using laser capture microdissection RNA sequencing analysis. Gene set enrichment analysis indicated that ferroptosis, cell oxidation, vascular endothelial growth factor, and mammalian target of rapamycin signaling were increased significantly in the primordial follicles of obese mice. Notably, the ferritin, acyl CoA synthetase long-chain family member 4, and solid carrier family 7 member 11 associated proteins of the ferroptosis signaling pathway were significantly increased in the PPT phase of obese mice. Our work suggests that ferroptosis is a key pathway activated within immature ovarian follicles in the context of obesity and that the process may be involved in the physiological regulation of the PPT as well.

LoxBlock-1 or Curcumin attenuates liver, pancreas and cardiac ferroptosis, oxidative stress and injury in Ischemia/reperfusion-damaged rats by facilitating ACSL/GPx4 signaling

In this study, the effects of the pretreatment of Curcumin and LoxBlock-1 on liver, pancreas, and cardiac dysfunction following Ischemia-Reperfusion-induced (IR) Acute Kidney Injury (AKI) were investigated through the mechanisms of oxidative stress and ferroptosis. Total antioxidant status (TAS), total oxidant status (TOS) and oxidative stress index (OSI) parameters in the tissue were analyzed to investigate the oxidative stress occurring in the liver, pancreas, and heart, and Acyl-Coa synthetase long-chain family member (ACSL4). Glutathione peroxidase 4 (GPx4) enzyme levels were also analyzed by ELISA to investigate the effect on ferroptosis. In addition, hematoxylin-eosin staining was performed for histopathological examination of the tissues. As a result of biochemical analyzes, it was observed that oxidative stress parameters increased significantly in the IR group. In addition, while the ACSL4 enzyme level increased in the IR group in all tissues, the GPx4 enzyme level decreased. In the histopathological examination, it was observed that IR caused serious damage to the heart, liver, and pancreas tissues. The present study shows that Curcumin and LoxBlock-1 have a protective effect on the liver, pancreas, and cardiac ferroptosis following the effect on AKI. In addition, Curcumin was found to be more effective than LoxBlock-1 in I/R injury with its antioxidant property.

ACSL4 serves as a novel prognostic biomarker correlated with immune infiltration in Cholangiocarcinoma

BackgroundCholangiocarcinoma (CHOL) is the second most common primary hepatic malignant tumor, following hepatocellular carcinoma (HCC). CHOL is highly aggressive and heterogeneous resulting in poor prognosis. The diagnosis and prognosis of CHOL has not improved in the past decade. Acyl-CoA synthetase long-chain family member 4 (ACSL4) is reported to be associated with tumors, however, its role in CHOL has not been revealed. This study is mainly for exploring the prognostic values and potential function of ACSL4 in CHOL.MethodsWe investigated the expression level and prognostic value of ACSL4 in CHOL based on The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets. TIMER2.0, TISIDB and CIBERSORT databases were utilized to assess the associations between ACSL4 and immune infiltration cells in CHOL. Single-cell sequencing data from GSE138709 was analyzed to study the expression of ACSL4 in different types of cells. ACSL4 co-expressed genes were analyzed by Linkedomics. Additionally, Western Blot, qPCR, EdU assay, CCK8 assay, transwell assay and wound healing assay were performed to further confirm the roles of ACSL4 in the pathogenesis of CHOL.ResultsWe found that the level of ACSL4 was higher in CHOL and it was correlated with the diagnosis and prognosis of CHOL patients. Then, we observed that the infiltration level of immune cells was related to the level of ACSL4 in CHOL. Moreover, ACSL4 and its co-expressed genes were mainly enriched in metabolism-related pathway and ACSL4 is also a key pro-ferroptosis gene in CHOL. Finally, knockdown of ACSL4 could reverse the tumor-promoting effect of ACSL4 in CHOL.ConclusionsThe current findings demonstrated ACSL4 may as a novel biomarker for CHOL patients, which might regulate immune microenvironment and metabolism resulting in poor prognosis.

Bone marrow mesenchymal stem cell-derived exosomes protect podocytes from HBx-induced ferroptosis.

Hepatitis B virus-associated glomerulonephritis (HBV-GN) is a common secondary kidney disease in China, the pathogenesis of which is not completely clear, and there is still a lack of effective treatment. The mechanism of exosomes derived from bone marrow mesenchymal stem cells (BMSCs) was investigated by using HBx-transfected human renal podocytes. Cell viability was detected by CCK8 assay. Iron and malondialdehyde (MDA) contents were detected by using commercial kits. Reactive oxygen species (ROS) levels were measured by flow cytometry analysis. The expression of ferroptosis related molecules was detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. The effect of miR-223-3p transferred by BMSC-derived exosomes on HBx-overexpressing podocytes was proved by using miR-223-3p inhibitor. The cell viability of podocytes reduced at 72 h or 96 h after the transfection of lentivirus overexpressing HBx protein (p < 0.05). Ferroptosis-related proteins, including glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11 (SLC7A11) were down-regulated upon HBx overexpression, while acyl-CoA synthetase long-chain family member 4 (ACSL4) was up-regulated (p < 0.05). Intracellular levels of iron, MDA, and ROS were also enhanced (p < 0.05). BMSC-derived exosomes protected against ferroptosis induced by HBx overexpression in podocytes. miR-223-3p was enriched in BMSC-derived exosomes. Application of miR-223-3p inhibitor reversed the protective effect of BMSC-derived exosomes on HBx-induced ferroptosis in podocytes. BMSC-derived exosomes inhibit HBx-induced podocyte ferroptosis by transferring miR-223-3p.