Ginkgolide B attenuates cerebral ischemia-reperfusion injury via inhibition of ferroptosis through disrupting NCOA4-FTH1 interaction.

ETHNOPHARMACOLOGICAL RELEVANCE: Cerebral ischemia/reperfusion (I/R) injury is a major cause of neuronal damage and death. Ginkgolide B (GB) has been shown to exhibit neuroprotective effects in various brain injury models. AIM OF STUDY: The aim of study was to investigate the potential role of GB in protecting against cerebral I/R injury and explore the underlying mechanisms. MATERIALS AND METHODS: Adult male Sprague-Dawley rats were exposed to transient middle cerebral artery occlusion (tMCAO) followed by reperfusion in order to trigger cerebral I/R injury. The rats were treated with different doses of GB, vehicle control or positive drug. Neurological function, infarct volume, and levels of ferroptosis markers were evaluated. In vitro experiments were performed using OGD/R-induced PC12 cells to further investigate the effects of GB on ferroptosis and its mechanisms. In addition, molecular docking, and microscale thermophoresis (MST) assay were conducted to explore the combination of GB and NCOA4. RESULTS: Reduced infarct volume and enhanced neurological function were signs of dose-dependent protection from cerebral I/R injury by GB therapy. Additionally, GB treatment had an impact on the levels of oxidative stress and ferroptosis markers, including reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD), and Fe2+ in the cerebral environment during IR injury. Moreover, relevant ferroptosis key factors such as ACSL4, GPX4, FTH1, and NCOA4 can be regulated by GB. In OGD/R-induced PC12 cells, GB protected against ferroptosis by inhibiting autophagy and disrupting the interaction of NCOA4-FTH1. CONCLUSION: Our findings suggest that GB may protect against cerebral I/R injury by inhibiting ferroptosis through disrupting NCOA4-FTH1 interaction. GB has potential therapeutic applications for cerebral I/R injury, and further investigation of the underlying mechanisms and clinical trials are warranted.

Tuberostemonine may enhance the function of the SLC7A11/glutamate antiporter to restrain the ferroptosis to alleviate pulmonary fibrosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Stemona is a medicinal plant that has been used in China for thousands of years to treat respiratory diseases such as cough and tuberculosis. The tuberostemonine is the component of the Stemona tuberosa Lour., Stemona sessilifolia (Miq.) Miq. or Stemona japonica (Blume) Miq. (The plant name has been checked with http://www.theplantlist.org), of which multiple biological activities has been verified. However, whether it may alleviate pulmonary fibrosis via regulating ferroptosis mechanism has not been confirmed. AIM OF THE STUDY: The aim of this study is to observe whether tuberostemonine alleviates pulmonary fibrosis by enhancing the function of the SLC7A11/glutamate antiporter to restrain the ferroptosis. MATERIALS AND METHODS: We validated the effects of tuberostemonine and ferroptosis on TGF-ß1-induced proliferation of human lung fibroblast and bleomycin-induced pulmonary fibrosis in mice. In vitro, the ferroptosis effect of TGF-ß1 on human lung fibroblast were examined and the activity of ɑ-SMA, collagen, hydroxyproline and ferrous ions in cells were also examined. In vivo, ferroptosis impacts respiratory function. Inflammatory manifestations, hydroxyproline, collagen activity and ferrous ions in the lung or blood were subject to evaluation. RESULTS: Tuberostemonine significantly improved respiratory function in mice with bleomycin-induced pulmonary fibrosis, decreased cellular and lung hydroxyproline content, reduced inflammation and collagen deposition in cells and lung, and promoted an increase in the SLC7A11 and GPX4 proteins. Tuberostemonine inhibits the ferroptosis phenomenon, up-regulates SLC7A11, GPX4 and GSH, and down-regulates the accumulation of iron and ROS. CONCLUSIONS: Tuberostemonine significantly inhibited ferroptosis and improved pulmonary fibrosis both in vivo and vitro.

Buddlejasaponin IVb ameliorates ferroptosis of dopaminergic neuron by suppressing IRP2-mediated iron overload in Parkinson's disease.

ETHNOPHARMACOLOGICAL RELEVANCE: Parkinson's disease (PD) is the second neurodegenerative disease that lacks effective treatments. Buddlejasaponin IVb (BJP-IVb) is the main bioactive component of herbs in genus Clinopodium which display antioxidative, anti-inflammatory and neuroprotective activities. However, the role of BJP-IVb in PD still remains unknown. AIM OF THE STUDY: This study aimed to evaluate the effect of BJP-IVb on dopaminergic neurodegeneration in PD and clarified the underlying mechanisms from the aspect of iron overload-mediated ferroptosis. MATERIALS AND METHODS: One-methyl-4-phenylpyridinium (MPP+) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD models were established in this study. Behavioral tests, cell cytotoxicity assay, tyrosine hydroxylase (TH) and Nissl staining were performed to evaluate the antiparkinsonian effect of BJP-IVb. Cellular ultrastructure, iron content and lipid peroxidation were detected to evaluate iron overload-mediated dopaminergic neuron ferroptosis. Iron regulatory protein 2 (IRP2) and iron transport-related proteins were detected by immunofluorescence and Western blot to evaluated iron transport. Finally, plasmid vector-mediated IRP2 overexpression were performed to further clarify the molecular mechanism. RESULTS: BJP-IVb alleviated MPP+-induced neurotoxicity in vitro and improved MPTP-induced dopaminergic neuron loss and motor dysfunctions of PD mice, confirming an effect of BJP-IVb against dopaminergic neurodegeneration of PD. Further results revealed that BJP-IVb protected against PD by suppressing iron overload-mediated dopaminergic neuron ferroptosis, as evidenced by the attenuated lipid peroxidation, decreased iron content and changes in cellular ultrastructure. Finally, the decreased iron regulatory protein (IRP2) was confirmed to be responsible for BJP-IVb-mediated ferroptosis suppression by modulating iron transport-related proteins and alleviating iron overload. CONCLUSION: BJP-IVb suppressed iron overload-mediated dopaminergic neuron ferroptosis and improved motor dysfunctions in PD, which was achieved by inhibiting IRP2-mediated iron overload. This study provided a potential drug candidate for the treatment of PD.

Visualizing intracellular membrane interactions and cell type-specific differentiation in ferroptosis and apoptosis with Boranil-Carbazole derivative.

Intracellular lipid systems play essential roles in various physiological functions and cell growth processes. However, our understanding of the intricate interactions within this system, especially between mitochondria and lipid droplets, is limited, particularly in the context of cancer cells' altered lipid metabolism. To address this, our study introduces an N-B-O BODIPY-hexylcarbazole derivative, named Cz-Boranil, that sets a new benchmark in visualizing these critical interactions. Cz-Boranil's unique capability lies in its ability to display distinct intracellular distribution patterns in both normal and cancer cells, offering nuanced cell type-specific differentiation. More impressively, this probe tracks the coordinated interactions of lipid droplets and mitochondria during the critical processes of ferroptosis and apoptosis. We believe that the innovative capabilities of Cz-Boranil will revolutionize our understanding of intracellular lipid interactions and prove pivotal in identifying and studying cancerous cells.

Niujiao Dihuang Jiedu decoction promotes SLC7A11 m5C methylation modification against ferroptosis in acute-on-chronic liver failure.

BACKGROUND: Acute-on-chronic liver failure (ACLF) constitutes a prevalent manifestation of liver failure within clinical settings. This condition manifests swiftly and is characterized by an exceedingly elevated fatality rate. OBJECTIVE: While numerous investigations have delved into the role of RNA methylation in ferroptosis, the impact of such methylation on ACLF-associated ferroptosis remains notably underexplored. This study aimed to elucidate the molecular mechanism underlying the efficacy of Niujiao Dihuang Jiedu decoction (NDD) in mitigating ferroptosis in ACLF, with a specific focus on RNA 5-methylcytosine (m5C) methylation. MATERIALS AND METHODS: An ACLF rat model was established alongside an erastin-induced ferroptosis model in LO2 cells. Both in vitro and in vivo experiments were conducted to substantiate NDD's influence on ferroptosis. The modifying influence of methylase NOL1/NOP2/sun domain (NSUN5) upon SLC7A11, a key ferroptosis-associated gene, was probed through dot blot, immunofluorescence co-localization, and RNA binding protein immunoprecipitation (RIP) experiments. RESULTS: Serological and hepatic histopathological findings indicated NDD's discernible therapeutic impact on ACLF. Furthermore, ferroptosis phenotype experiments revealed NDD's proficiency in effectively impeding the occurrence and development of ferroptosis. Dot blot assays demonstrated a reduction in the overall RNA m5C levels during cellular ferroptosis. Furthermore, through immunofluorescence co-localization and RIP techniques, we found that the propensity of methylase NSUN5 to associate with SLC7A11 mRNA, thereby enhancing its protein translation and conferring resistance against ferroptosis. CONCLUSION: RNA methylation is involved in the process of ACLF-associated ferroptosis, and NDD can inhibit ACLF-associated ferroptosis by fostering SLC7A11 m5C methylation.

Salvianolic acid A attenuates arsenic-induced ferroptosis and kidney injury via HIF-2α/DUOX1/GPX4 and iron homeostasis.

Arsenic (As) is a prevalent pollutant in the environment and causes a high frequency of kidney disease in areas of high arsenic contamination, but its pathogenic mechanisms have yet to be completely clarified. Ferroptosis is a new form of cell death mainly dependent on lipid peroxidation and iron accumulation. Several reports have suggested that ferroptosis is operative in a spectrum of diseases caused by arsenic exposure, including in the lungs, pancreas, and testis. However, the mechanism and exact role of ferroptosis in arsenic-induced kidney injury is not known. Firstly, by constructing in vivo and in vitro arsenic exposure models, we confirmed the occurrence of ferroptosis based on the identification of the ability of NaASO2 to cause kidney injury. In addition, we found that arsenic exposure could upregulate DUOX1 expression in kidney and HK-2 cells, and after knocking down DUOX1 using siRNA was able to significantly upregulate GPX4 expression and attenuate ferroptosis. Subsequently using bioinformatics, we identified and proved the involvement of HIF-2α in the course of ferroptosis, and further confirmed by dual luciferase assay that HIF-2α promotes DUOX1 transcription to increase its expression. Finally, intervention with the natural ingredient SAA significantly attenuated arsenic-induced ferroptosis and kidney injury. These results showed that arsenic could cause ferroptosis and kidney injury by affecting HIF-2α/DUOX1/GPX4 and iron homeostasis and that SAA was an effective intervention component. This study not only discovered the molecular mechanism of sodium arsenite-induced kidney injury but also explored an active ingredient with intervention potential, providing a scientific basis for the prevention and treatment of sodium arsenite-induced kidney injury.

In silico prediction and experimental validation to reveal the protective mechanism of Puerarin against excessive extracellular matrix accumulation through inhibiting ferroptosis in diabetic nephropathy.

ETHNOPHARMACOLOGICAL RELEVANCE: Puerarin (PUR) isolated from the root of Pueraria lobata (Willd.) Ohwi is considered as one of the main medicines to alleviate asthenic splenonephro-yang of diabetic nephropathy (DN). Whereas, the exact mechanism of Puerarin on diabetic nephropathy is still unclear. AIM OF STUDY: In this study, we aimed to investigate the protective effects of PUR on type 2 diabetic nephropathy in vivo, in silico and in vitro, as well as unveil the underlying mechanism through inhibiting ferroptosis. MATERIALS AND METHODS: In vivo, blood glucose and lipid, renal function, kidney histology and immunohistochemistry analysis were used to vindicate the protective effects of PUR on diabetic nephropathy in type 2 DN rat model. In silico, pharmacophore matching and enrichment analysis were adopted to predict the potential mechanism of PUR on DN. In vitro, we utilized high glucose stress to induce impairment in glomerular mesangial cells (GMCs) as diabetic nephropathy cell model. Cell count kit-8 (CCK-8) was used to observe cell viability. qPCR, Western blot, immunofluorescence staining and flow cytometry were used to evaluate the effect of PUR on the generation of extracellular matrix (ECM), ferroptosis and iron homeostasis in vitro and in vivo. RESULTS: PUR markedly improved glucose and lipid metabolism, as well as alleviated renal dysfunction in diabetic nephropathy rats. Pharmacophore matching and enrichment analysis predicted the anti-DN effect of PUR may correlate with ECM. Experimental validation suggested that PUR treatment could inhibit the generation of ECM to alleviate high-glucose-induced cell impairments, suppressing ROS production and excessive collagen fiber accumulation in GMSs, and reduce mesangial matrix expansion and renal fibrosis in type 2 DN rats. Further study suggested that PUR protected GMCs against ferroptosis via reducing LDH release and GSH disruption, suppressing key regulators of two pathways for ferroptosis execution. Moreover, PUR also maintained iron metabolism hemostasis by regulating iron transportation proteins, iron exporter proteins, and iron storage proteins and reducing intracellular iron in type 2 DN rats. CONCLUSION: PUR inhibited excessive ECM accumulation to protect against type 2 diabetic nephropathy, which meditated by regulating iron homeostasis and mitigating ferroptosis. This study provides promising therapeutics for diabetic nephropathy treatment.

Gastrodin alleviates cisplatin nephrotoxicity by inhibiting ferroptosis via the SIRT1/FOXO3A/GPX4 signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Cisplatin (CP) results in acute kidney injury (AKI) and negatively affects patients' therapy and survival. The dried rhizome of Gastrodia elata Blume has been used to treat clinical kidney diseases. Gastrodin (GAS) is an active ingredient of the G. elata tuber. It is unknown whether GAS can alleviate CP-induced AKI. AIM OF THE STUDY: This study aimed to investigate whether GAS, an active ingredient of G. elata Blume, can alleviate CP-induced AKI and to explore its underlying mechanisms. MATERIALS AND METHODS: Experiments were conducted with a CP-induced AKI mouse model and an immortalized human renal tubular epithelial cell line (HK-2). Serum creatinine, Periodic acid-Schiff staining, tissue iron, glutathione, malondialdehyde, and 4-Hydroxynonenal were detected in serum and kidney samples to observe whether GAS inhibits CP-induced tubule ferroptosis. The drug target was verified by detecting the effects of GAS on sirtuin-1 (SIRT1) activity in vitro. Transcriptional regulation of glutathione peroxidase 4 (GPX4) by forkhead box O3A (FOXO3A) was verified by siRNA knockdown, overexpression, and chromatin immunoprecipitation. The effects of FOXO3A, SIRT1, and GAS on CP-induced ferroptosis were measured with propidium iodide, dihydroethidium, monobromobimane, and dipyrromethene boron difluoride staining in HK-2 cells. The relationship between GAS and the SIRT1/FOXO3A/GPX4 pathway was studied using Western blotting. RESULTS: GAS treatment inhibited CP-induced reactive oxygen species, lipid peroxidation, and tubule death in the cell and animal models. GAS activated SIRT1 in vitro. The SIRT1 inhibitor blocked the protective role of GAS in reducing lipid peroxidation in HK-2 cells. FOXO3A transcriptionally regulated GPX4 expression and inhibited CP-induced cell ferroptosis. Compared to CP-damaged mouse kidneys, GAS-treated mice demonstrated significantly increased SIRT1 and GPX4 expression levels, decreased CP-induced acetylation of FOXO3A, and inhibited lipid peroxidation and cell death. CONCLUSIONS: GAS alleviated CP-induced AKI by inhibiting ferroptosis via the SIRT1/FOXO3A/GPX4 signaling pathway. The results offer new insights into the development of new anti-AKI drugs from traditional Chinese medicine.

Ambra1 in exosomes secreted by HK-2 cells damaged by supersaturated oxalate induce mitophagy and autophagy-ferroptosis in normal HK-2 cells to participate in the occurrence of kidney stones.

Injury to the renal tubular epithelium has emerged as a leading factor underlying the formation of kidney stones. Indeed, epithelial cell damage contributes to the adherence and aggregation of crystals, thereby accelerating the formation of renal stones. Meanwhile, exosomes play an instrumental role in cellular communication, including DNA, RNA, mRNA, etc. In this study, homogenous cells were treated with exosomes derived from damaged cells in an attempt to establish "positive feedback" of cell damage, and the desired results were achieved. To begin, a serum-free medium and supersaturated concentrations of oxalate were added to the HK-2 cell line, and then exosomes were isolated from the two groups for analysis and comparison, and the autophagy-related gene Ambra1 (autophagy and beclin-1 regulator 1) was detected. Subsequently, normal HK-2 cells were treated with exosomes, and the related indexes of autophagy, ferroptosis and mitophagy were determined. Thereafter, Ambra1 was knocked down in exosome-derived HK-2 cells, resulting in the down-regulation of Ambra1 expression in exosomes produced by HK-2 cells following oxalate intervention. Thereafter, the ability of exosomes to stimulate autophagy, mitophagy and ferroptosis was re-evaluated in HK-2 cells after Ambra1 knockdown. The results corroborated that exosomes secreted by oxalate-treated HK-2 can directly elevate autophagy, ferroptosis and mitophagy levels in normal cells, and this effect was significantly mitigated following Ambra1 knockdown within exosomes. Meanwhile, exosomes-induced autophagy and ferroptosis were alleviated after knockdown of beclin-1 in recipient HK-2 cells. These results further suggest that beclin-1 plays a critical role in the process of exosome-induced autophagy-ferroptosis.

Modified Shoutai Pill inhibited ferroptosis to alleviate recurrent pregnancy loss.

ETHNOPHARMACOLOGICAL RELEVANCE: Modified Shoutai Pill, also called Jianwei Shoutai Pill (JSP), is a traditional Chinese medicine prescription that has been used as an effective agent for the treatment of miscarriage. AIM OF THE STUDY: To explore the potential molecular mechanism of JSP against recurrent pregnancy loss (RPL). MATERIALS AND METHODS: In vivo, CBA/J mated DBA/2 mice were used to conduct RPL model, while CBA/J mated BALB/c mice were seen as the control group. Mice were orally administered with JSP, Fer-1 (a ferroptosis inhibitor) or distilled water from day 0.5-12.5 of gestation (GD 0.5-12.5). Pregnancy outcomes were analyzed and ferroptosis related indexes of the whole implantation sites were measured on GD 12.5. In vitro, human trophoblast cell line HTR-8/SVneo was cultured and treated with RAS-selective lethal small molecule 3 (RSL3) (a ferroptosis agonist) or different concentrations of JSP. Then, ferroptosis related indexes were tested to analyze whether JSP could inhibit ferroptosis in HTR-8/SVneo cells. RESULTS: In vivo consequences demonstrated that JSP or Fer-1 alleviated pregnancy outcomes including lower resorption rate and abortion rate. In addition, excessive iron accumulation and MDA level were inhibited, while GSH and GPX content were raised under JSP or Fer-1 exposure. Also, JSP or Fer-1 enhanced protein expressions of GPX4 and SLC7A11 which suppress ferroptosis, and lightened protein expression of ACSL4 which boosts ferroptosis. In vitro, JSP rescued HTR-8/SVneo cell death and migration ability that were injured by RSL3. Furthermore, JSP inhibited RSL3-induced intracellular reactive oxygen species (ROS), lipid ROS and iron deposition. CONCLUSIONS: Collectively, our findings illustrated that the mechanism of JSP in treating RPL might be related to inhibiting ferroptosis, which provided a novel insight into the application of JSP in RPL intervention.

TMT quantitative proteomics analysis reveals molecular mechanism of ferroptosis during beef refrigeration.

Ferroptosis is a recently identified cell death process in refrigerated beef, and its mediated protein oxidation and cell death may reduce muscle quality, but the mechanism of ferroptosis is unclear. In the study, free iron accumulation reached 19.670 ± 0.482 µg/g after 6 days refrigeration, the levels of apoptosis, ROS, and lipid peroxidation increased significantly (P < 0.05), and muscle tissue cells exhibited typical ferroptosis characteristics. A total of 377 differentially expressed proteins (DEPs) were identified by TMT quantitative proteomics. 15 DEPs, including transferrin, ferritin, glutathione peroxidase (GPX) 4, and heme oxygenase 1, were involved in lipid peroxidation, Fe2+ and Fe3+ conversion, iron ion accumulation, and mitochondrial oxidative stress to induce ferroptosis. In addition, signalling pathways, such as chemical carcinogenesis-ROS, glutathione metabolism, HIF-1, and PPAR may promote ferroptosis by affecting free iron overload and GPX4 inactivation.

Synergistic effect of PS-MPs and Cd on male reproductive toxicity: Ferroptosis via Keap1-Nrf2 pathway.

It has been wildly reported that microplastics (MPs) can adsorb heavy metals and act as carriers for their transport into organisms. However, the combined toxicity of MPs and heavy metals remains poorly studied. In this study, we established single or co-exposure (i.e. complex/combined exposure) mice models to investigate the combined toxicity of MPs and cadmium (Cd) on male reproduction. The complexation of MPs and Cd enhanced the bioavailability of Cd, while the combination of MPs and Cd exerted synergistic effect. Ultimately, the co-exposure was reported to enhance the reproduction toxicity by single exposure, which reflected in testicular structure, spermatogenesis and sex hormone synthesis. More in-depth mechanistic investigation suggested that MPs and Cd synergistically inhibited the Keap1-Nrf2 pathway and its downstream genes, induced lipid peroxidation and ferroptosis, ultimately caused damage to reproductive structures and functions. Our results highlighted the synergistic effect of MPs and Cd on the reproductive toxicity in male mammals for the first time, which also provided valuable insights into the combined toxicity mechanisms of MPs and other pollutants.

NRF2 connects Src tyrosine kinase to ferroptosis resistance in glioblastoma.

Glioblastoma is a severe brain tumor characterized by an extremely poor survival rate of patients. Glioblastoma cancer cells escape to standard therapeutic protocols consisting of a combination of ionizing radiation and temozolomide alkylating drugs that trigger DNA damage by rewiring of signaling pathways. In recent years, the up-regulation of factors that counteract ferroptosis has been highlighted as a major driver of cancer resistance to ionizing radiation, although the molecular connection between the activation of oncogenic signaling and the modulation of ferroptosis has not been clarified yet. Here, we provide the first evidence for a molecular connection between the constitutive activation of tyrosine kinases and resistance to ferroptosis. Src tyrosine kinase, a central hub on which deregulated receptor tyrosine kinase signaling converge in cancer, leads to the stabilization and activation of NRF2 pathway, thus promoting resistance to ionizing radiation-induced ferroptosis. These data suggest that the up-regulation of the Src-NRF2 axis may represent a vulnerability for combined strategies that, by targeting ferroptosis resistance, enhance radiation sensitivity in glioblastoma.

Inhibition of Apoc1 reverses resistance of sorafenib by promoting ferroptosis in esophageal cancers.

Drug resistance is an obstacle in therapy of esophageal cancers (ECs), and the role of ferroptosis in progression ECs is still not clearly clarified. In the present study, we investigated the role of Apolipoprotein C1 (Apoc1) in regulating the sorafenib resistance in EC cells. Apoc1 was knock down after infection with Apoc1 shRNA lentivirus and stable cell lines for Apoc1 knockdown were screened. Cell viabilities were tested by MTT assay. ROS, MDA, and GSH tested by specific kits. In vivo experiment in nude mice were performed to test the correlation of Apoc1 and ferroptosis. The expression of Apoc1 and GPX4 was tested by western blotting. The results showed that Apoc1 was highly expressed in EC tissues and associated with poor overall survival rate of EC. Knockdown Apoc1 overcame resistance of sorafenib in EC cells and promoted erastin and sorafenib induced ferroptosis by upregulating the levels of ROS and MDA and downregulating the level of GSH in OE19/Sora and EC109/Sora cells. Rescue experiments proved that Apoc1 regulated sorafenib induced ferroptosis via GPX4. Furthermore, knockdown of Apoc1 inhibited the tumor progression by promoting ferroptosis in nude mice. In conclusion, knockdown Apoc1 overcome resistance of sorafenib in EC cells and in vivo by promoting sorafenib induced ferroptosis via GPX4. Targeting Apoc1 might be an effective way to reverse the drug resistance of sorafenib via inducing ferroptosis in EC progression.

Polyphyllin I induced ferroptosis to suppress the progression of hepatocellular carcinoma through activation of the mitochondrial dysfunction via Nrf2/HO-1/GPX4 axis.

BACKGROUND: Ferroptosis is an emerging iron-dependent programmed cell death mode characterized by lipid peroxidation and iron accumulation, closely associated with Hepatocellular Carcinoma (HCC) progression. Although the impact of Polyphyllin I (PPI), a prominent bioactive constituent derived from Paris polyphylla, on diverse malignancies has been established, the specific role and potential mechanistic pathways through which PPI modulates ferroptosis in HCC remain elusive. PURPOSE: This study aimed to elucidate the anti-cancer properties and potential mechanisms of PPI in inducing ferroptosis and triggering mitochondrial injury in HCC. METHODS: Cell viability was assessed using CCK-8 assays. EdU proliferation and colony formation assays were employed to evaluate cell proliferation. A wound-healing assay was performed to assess cell migration. Transwell assay was utilized to evaluate cell invasion. Ferroptosis was evaluated through the utilization of a FerroOrange fluorescent probe, malondialdehyde (MDA) and reduced glutathione (GSH) assay kits, DCFH-DA fluorescent probe, western blotting, and transmission electron microscopy (TEM) analysis. Molecular docking, immunofluorescence, and western blotting were employed to predict and validate the binding and interaction of PPI with Nrf2, HO-1, xCT, and GPX4. Mitochondrial structure and membrane potential changes were evaluated using JC-1 and Mito Tracker Green fluorescent probes. A nude mice xenograft model was constructed to determine the inhibitory effects and the levels of ferroptosis of PPI on HCC through hematoxylin and eosin (H&E), Prussian blue reaction, immunofluorescence staining, immunohistochemistry, and western blotting analysis, in vivo. RESULTS: PPI exhibited dose-dependent inhibitory effects on the proliferation, invasion, and metastasis of HCC cells mediated by increasing reactive oxygen species (ROS) and MDA levels, promoting Fe2+ accumulation, depleting GSH, and suppressing the expression of xCT and GPX4, thereby inducing ferroptosis in HCC. The induction of ferroptosis by PPI was associated with the binding of PPI to Nrf2, HO-1, and GPX4 proteins, modulating the Nrf2/HO-1/GPX4 antioxidant axis. PPI also induced mitochondrial structural damage and decreased mitochondrial membrane potential (MMP). Inhibition of ferroptosis by ferrostatin-1 (Fer-1) mitigated the mitochondrial disruption induced by PPI. In vivo, PPI inhibited Nrf2/HO-1/GPX4 axis-induced ferroptosis, impeding HCC growth similar to the effects of sorafenib. CONCLUSION: These results demonstrated that PPI intervention can suppress the proliferation, invasion, and metastasis of HCC cells by enhancing mitochondrial disruption and inducing ferroptosis via the Nrf2/HO-1/GPX4 axis. Consequently, our research advances the frontiers of pharmacodynamics and deepens our comprehension of the intricate mechanisms underpinning PPI. Furthermore, it has yielded an innovative treatment stratagem rooted in the tenets of Traditional Chinese Medicine (TCM), thereby furnishing a novel therapeutic avenue for addressing HCC.

Targeting ferroptosis with natural products in liver injury: new insights from molecular mechanisms to targeted therapies.

BACKGROUND: Ferroptosis is a brand-new type of controlled cell death that is distinguished by its reliance on iron and the production of lipid peroxidation. The role of ferroptosis in damaging liver disorders has attracted a lot of attention in recent years. One effective strategy to reduce liver damage is to target ferroptosis. PURPOSE: The purpose of this review is to clarify the connection between ferroptosis and liver damage and to look into the potential contribution of natural products to the clinical management of liver damage and the discovery of novel medications. METHODS: To study the methods by which natural products operate on ferroptosis to cure liver damage and their main signaling pathways, we searched databases from the time of initial publication to August 2023 in PubMed, EMBASE, Web of Science, Ovid, ScienceDirect, and China National Knowledge Infrastructure. The liver illness that each natural product treats is categorized and summarized. It's interesting to note that several natural compounds, such Artemether, Fucoidan sulfate, Curcumin, etc., have the benefit of having many targets and multiple pathways of action. RESULTS: We saw that in human samples or animal models of liver injury, ferroptosis indicators were activated, lipid peroxidation levels were elevated, and iron inhibitors had the ability to reduce liver damage. Liver damage can be treated with natural products by regulating ferroptosis. This is mostly accomplished through the modulation of Nrf2-related pathways (e.g., Conclusions and Astaxanthin), biological enzymes like GPX4 and the SIRT family (e.g., Chrysophanol and Decursin), and transcription factors like P53 (e.g., Artemether and Zeaxanthin). CONCLUSIONS: This review proposes a promising path for the therapeutic therapy of liver damage by providing a theoretical foundation for the management of ferroptosis utilizing natural ingredients.

Matrine induces ferroptosis in cervical cancer through activation of piezo1 channel.

BACKGROUND: Cervical cancer, which is a significant public health concern in women, currently lacks effective therapeutic drugs. Matrine, a constituent of the traditional Chinese herb Sophora flavescentis Radix, is known for its anti-cervical cancer properties and ability to induce programmed cell death. The induction of cancer cell ferroptosis, which is a novel cell death pattern, can become an effective clinical therapy for tumor in the future. However, the effect of matrine on ferroptosis in cervical cancer remains to be elucidated. PURPOSE: In this study, we investigated whether matrine induces ferroptosis in cervical cancer and elucidated the underlying mechanisms. METHODS: We established an SiHa-derived tumor-bearing mouse model using CB17 severe combined immunodeficient (SCID) mice and administered a group of matrine (25, 50, and 75 mg/kg) and cisplatin (2 mg/kg). We meticulously tracked alterations in body weight and tumor size and evaluated liver and kidney health using haematoxylin and eosin (H&E) staining. Using Gene Expression Omnibus (GEO) Dataset (GSE201309), we evaluated the relationship between the effects of matrine on malignant tumor cells and ferroptosis. In vitro, tetrazolium-based colorimetric (MTT), lactate dehydrogenase (LDH) and colony formation assays were used to study the effects of matrine on SiHa cell activity and cytotoxicity. We assessed ferroptosis-related protein abundance using western blotting and ferroptosis-related indices in cells using confocal immunofluorescence microscopy. The interaction of matrine with a protein linked to ferroptosis was studied using cellular thermal shift assay (CETSA). The effects of matrine on Piezo1 expression were investigated using calcium imaging. We also used Piezo1-specific siRNA to explore the role of Piezo1 in ferroptosis. RESULTS: Matrine administration effectively inhibited tumor growth in a SiHa-derived tumor-bearing mouse model without inducing noticeable harm. The analysis results of GEO data set show matrine-induced effects in tumor cells were indeed involved in the process of ferroptosis. Treatment with matrine resulted in a significant reduction in GPX4 protein levels and a concurrent increase in lipid peroxide and Fe2+ content, suggesting matrine-induced modulation of ferroptosis. Matrine promoted SiHa cell death in vitro, as evidenced by the results of MTT and LDH assays. Cell death coincides with increases in intracellular Fe2+, reactive oxygen species (ROS), and lipid peroxides. Our study also revealed significant upregulation of Piezo1 expression through the action of matrine, whereas transferrin receptor (Tfr) and System Xc- (xCT) expression and interaction remained unaffected. We provided further evidence that matrine induces calcium influx through the Piezo1 channel, thereby potentially influencing ferroptosis. Transfection with Piezo1 siRNA reversed the effects of matrine in SiHa cell. CONCLUSIONS: Our findings indicate that matrine exerts a protective effect against cervical cancer by inducing ferroptosis through the activation of Piezo1, but not xCT or Tfr.

Timosaponin AIII induces lipid peroxidation and ferroptosis by enhancing Rab7-mediated lipophagy in colorectal cancer cells.

BACKGROUND: Colorectal cancer (CRC) is a common digestive system malignancy, and despite significant therapeutic advancements, more effective treatments are needed. Timosaponin AIII (TA-III), a major steroidal saponin derived from Anemarrhena asphodeloides Bge, is a potential anticancer agent. Ferroptosis plays an important role in cancer treatment. PURPOSE: To investigate the molecular mechanism of TA-III as a novel ferroptosis inducer in suppressing CRC through lipophagy. Ferroptosis, an autophagy-dependent mode of cell death, has been implicated in CRC. METHODS: CRC cells were treated with TA-III, and lipophagy levels were evaluated via BODIPY493/503 staining and western blotting. Autophagy turnover was tracked using GFP-RFP-LC3B. Lipid peroxidation was quantified using an malondialdehyde kit and C11-BODIPY flow assay. Mitochondrial morphology was observed using transmission electron microscopy. GC-MS/MS was used to detect lipid metabolism changes. The role of ras related protein Rab 7a (Rab7) was assessed by western blotting and glutathione S-transferase pull-down assays. In vivo, the anticancer efficacy of TA-III was tested using a xenograft model. RESULTS: RNA-seq analysis unveiled the potential of TA-III as an anticancer agent through ferroptosis. In vivo experiments revealed how TA-III treatment triggered degradation of lipid droplets in CRC cells, resulting in an accumulation of FFAs, heightened unsaturated free fatty acids, and increased lipid peroxidation. These events ultimately lead to mitochondrial shrinkage and downregulation of ferroptosis markers (FSP1 and GPX4). Intriguingly, the Rab7 protein emerged as a crucial bridge between lipophagy and ferroptosis, underlining its significance in the anticancer mechanism of TA-III. Moreover, TA-III treatment in a xenograft tumour model substantially reduced tumour volume via ferroptosis, underscoring its therapeutic efficacy. CONLUSION: Our study is the first to establish that TA-III triggers lipophagy in CRC cells via the Rab7 gene, subsequently promoting ferroptosis. This suggests its potential use as an antitumour agent.

Integrated network pharmacology and experimental validation to explore the potential pharmacological mechanism of Qihuang Granule and its main ingredients in regulating ferroptosis in AMD.

BACKGROUND: Qihuang Granule (QHG) is a traditional prescription  that has exhibited potential in safeguarding against age-related maculopathy (AMD). Salvia miltiorrhiza (SM) and Fructus lycii (FL) are the main components of QHG. Ferroptosis, a newly discovered, iron-dependent, regulated cell death pathway, have been implicated in the pathogenesis of AMD. This study delves into the intricate mechanism by which SM/FL and QHG confer protection against AMD by modulating the ferroptosis pathway, employing a combination of network pharmacology and experimental validation. METHODS: Bioactive compounds and potential targets of SM and FL were gathered from databases such as TCMSP, GeneCard, OMIM, and FerrDb, along with AMD-related genes and key genes responsible for ferroptosis regulation. Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and protein-protein interaction (PPI) network were performed to discover the potential mechanism. The construction of an interaction network involving AMD, ferroptosis, SM/FL potential target genes was facilitated by the STRING database and realized using Cytoscape software. Subsequent validation was accomplished through molecular docking and in vitro cell experiments. RESULTS: Noteworthy active compounds including quercetin, tanshinone IIA, luteolin, cryptotanshinone, and hub targets such as HIF-1α, EGFR, IL6, and VEGFA were identified. KEGG enrichment unveiled the HIF-1 signalling pathway as profoundly enriched, and IL6 and VEGF were involved. The molecular docking revealed the significant active compounds with hub genes and quercetin showed good binding to HIF-1α, which is involved in inflammation and angiogenesis. Experimental results verified that both herbs and QHG could regulate key ferroptosis-related targets in the retinal pigment epithelium and inhibit the expression of HIF-1α, VEGFA, and IL-6, subsequently increase cell viability and decrease the ROS content induced by H2O2. CONCLUSION: This study demonstrates the molecular mechanism through which SM/FL and QHG protect against AMD and emerges as a plausible mechanism underlying this protection.

Puerarin ameliorates metabolic dysfunction-associated fatty liver disease by inhibiting ferroptosis and inflammation.

Metabolic dysfunction-associated fatty liver disease (MAFLD) is frequently linked to type 2 diabetes mellitus (T2DM), and both conditions exacerbate the progression of the other. However, there is currently no standardized treatment or drug for MAFLD. In this study, A MAFLD animal model through a high-fat diet (HFD) along with administration of streptozotocin (STZ), and palmitic acid (PA)-induced AML12 cells were treated by puerarin. The objective of this study was to assess the therapeutic effect of puerarin, a flavonoid substance that possesses various pharmacological properties, on MAFLD. The results showed that puerarin administration enhanced glucose tolerance and insulin sensitivity, while also mitigating liver dysfunction and hyperlipidemia in MAFLD mice. Moreover, puerarin attenuated oxidative stress levels and inflammation in the liver. Transmission electron microscopy and Western blot analysis indicated that puerarin inhibited ferroptosis in vivo. Further mechanistic investigations revealed that puerarin upregulated SIRT1 expression, increased nuclear factor erythroid 2-related factor 2 (Nrf2) protein levels, and facilitated translocation into the nucleus. The protective effect of puerarin on PA-induced AML12 cells was diminished by the utilization of EX-527 (a SIRT1 inhibitor) and Nrf2 siRNA. Overall, the results demonstrate that puerarin ameliorates MAFLD by suppressing ferroptosis and inflammation via the SIRT1/Nrf2 signaling pathway. The results emphasize the possible medicinal application of puerarin for managing MAFLD.