Ferroptosis Suppressor Protein Research Articles

Edaravone Alleviates Traumatic Brain Injury by Inhibition of Ferroptosis via FSP1 Pathway.

Traumatic brain injury (TBI) is a highly severe form of trauma with complex series of reactions in brain tissue which ultimately results in neuronal damage. Previous studies proved that neuronal ferroptosis, which was induced by intracranial haemorrhage and other reasons, was one of the most primary causes of neuronal damage following TBI. However, the association between neuronal mechanical injury and ferroptosis in TBI and relevant treatments remain unclear. In the present study, we first demonstrated the occurrence of neuronal ferroptosis in the early stage of TBI and preliminarily elucidated that edaravone (EDA), a cerebroprotective agent that eliminates oxygen radicals, was able to inhibit ferroptosis induced by TBI. A cell scratching model was established in PC12 cells, and it was confirmed that mechanical injury induced ferroptosis in neurons at the early stage of TBI. Ferroptosis suppressor protein 1 (FSP1) plays a significant role in inhibiting ferroptosis, and we found that iFSP, a ferroptosis agonist which is capable to inhibit FSP1 pathway, attenuated the anti-ferroptosis effect of EDA. In conclusion, our results suggested that EDA inhibited neuronal ferroptosis induced by mechanical injury in the early phase of TBI by activating FSP1 pathway, which could provide evidence for future research on prevention and treatment of TBI.

SRSF2 promotes glioblastoma cell proliferation by inducing alternative splicing of FSP1 and inhibiting ferroptosis

Objective: To investigate the effect of serine/arginine-rich splicing factor 2 (SRSF2) on ferroptosis and its possible mechanism in glioblastoma cells. Methods: The online database of gene expression profiling interactive analysis 2 (GEPIA 2) and Chinese Glioma Genome Atlas were used to analyze the expression of SRSF2 in glioblastoma tissue and its association with patients prognosis. To validate the findings of the online databases, the pathological sections of glioblastoma and non-tumor brain tissues from Tianjin Medical University General Hospital, Tianjin, China were collected and analyzed by using immunohistochemistry. Silencing SRSF2 gene expression in glioblastoma cells by siRNA was analyzed with Western blot. The proliferation index was detected by using CCK8 assay. The rescued experiment was conducted by using expression plasmid of pcDNA3.1(+)-SRSF2. The activity of ferroptosis was assessed by using the levels of iron ions and malondialdehyde in glioblastoma cells and the changes in the ratio of glutathione to oxidized glutathione. The changes of gene expression and differential pre-mRNA alternative splicing (PMAS) induced by SRSF2 were monitored by using the third-generation sequencing technology analysis, namely Oxford nanopore technologies (ONT) sequencing analysis. Results: SRSF2 expression was higher in glioblastoma tissues than non-tumor brain tissues. Immunohistochemistry also showed a positive rate of 88.48%±4.60% in glioblastoma tissue which was much higher than the 9.97%±4.57% in non-tumor brain tissue. The expression of SRSF2 was inversely correlated with overall and disease-free disease survivals (P<0.01). The proliferation index of glioblastoma cells was significantly reduced by silencing with SRSF2 siRNA (P<0.01) and could be reversed with transfection of exogenous SRSF2. The levels of intracellulariron ions and malondialdehyde increased (P<0.05), but the glutathione/oxidized glutathione ratio and the expression of key proteins in the glutathione pathway remained unchanged (P>0.05). ONT sequencing results showed that silencing SRSF2 in glioblastoma cells could induce a significant alternative 3' splice site change on ferroptosis suppressor protein 1 (FSP1). Conclusion: SRSF2 inhibits the ferroptosis in glioblastoma cells and promotes their proliferation, which may be achieved by regulating FSP1 PMAS.

Protopanaxadiol prevents cisplatin-induced acute kidney injury by regulating ferroptosis.

Acute kidney injury (AKI) caused by cisplatin (CDDP) is a complex, critical illness with no effective or specific treatment. The purpose of the study was to assess the protective effect of protopanaxadiol (PPD) on the kidneys in CDDP-induced AKI models and its possible mechanisms. In vitro, the protection of PPD was assessed in HK-2. KM mice were injected with CDDP to induce AKI models in vivo. The determination of blood urea nitrogen and serum creatinine (SCr) was performed, and pathological changes were examined by histopathological examination. Immunostaining and western blot analyses were used to analyze the expression levels of proteins. PPD can increase the viability of HK-2 cells damaged by CDDP, improve cell morphology, and alleviate the symptoms of AKI in mice. In addition, PPD can down-regulate the protein expression of TRF and up-regulate the protein expression of Ferritin heavy chain, Glutathione peroxidase 4, and ferroptosis suppressor protein 1 reduce the iron content in cells and kidney tissues, and restore the antioxidant defense system. PPD has an inhibitory effect on cisplatin-induced nephrotoxicity, which may be related to the inhibition of ferroptosis by regulating iron metabolism and lipid peroxidation.

Ginsenoside Rg1 Suppresses Ferroptosis of Renal Tubular Epithelial Cells in Sepsis-induced Acute Kidney Injury via the FSP1-CoQ10- NAD(P)H Pathway.

Sepsis-induced acute kidney injury is related to an increased mortality rate by modulating ferroptosis through ginsenoside Rg1. In this study, we explored the specific mechanism of it. Human renal tubular epithelial cells (HK-2) were transfected with oe-ferroptosis suppressor protein 1 and treated with lipopolysaccharide for ferroptosis induction, and they were then treated with ginsenoside Rg1 and ferroptosis suppressor protein 1 inhibitor. Ferroptosis suppressor protein 1, CoQ10, CoQ10H2, and intracellular NADH levels in HK-2 cells were assessed by Western blot, ELISA kit, and NAD/NADH kit. NAD+/NADH ratio was also calculated, and 4-Hydroxynonal fluorescence intensity was assessed by immunofluorescence. HK-2 cell viability and death were assessed by CCK-8 and propidium iodide staining. Ferroptosis, lipid peroxidation, and reactive oxygen species accumulation were assessed by Western blot, kits, flow cytometry, and C11 BODIPY 581/591 molecular probe. Sepsis rat models were established by cecal ligation and perforation to investigate whether ginsenoside Rg1 regulated the ferroptosis suppressor protein 1-CoQ10-NAD(P)H pathway in vivo. LPS treatment diminished ferroptosis suppressor protein 1, CoQ10, CoQ10H2, and NADH contents in HK-2 cells, while facilitating NAD+/NADH ratio and relative 4- Hydroxynonal fluorescence intensity. FSP1 overexpression inhibited lipopolysaccharideinduced lipid peroxidation in HK-2 cells via the ferroptosis suppressor protein 1-CoQ10- NAD(P)H pathway. The ferroptosis suppressor protein 1-CoQ10-NAD(P)H pathway suppressed lipopolysaccharide-induced ferroptosis in HK-2 cells. Ginsenoside Rg1 alleviated ferroptosis in HK-2 cells by regulating the ferroptosis suppressor protein 1-CoQ10- NAD(P)H pathway. Moreover, ginsenoside Rg1 regulated the ferroptosis suppressor protein 1-CoQ10-NAD(P)H pathway in vivo. Ginsenoside Rg1 alleviated sepsis-induced acute kidney injury by blocking renal tubular epithelial cell ferroptosis via the ferroptosis suppressor protein 1-CoQ10- NAD(P)H pathway.

The suppression of FSP1 expression via NRF2 promotes ferroptosis induced by reactive oxygen species in vascular smooth muscle cells

Death of vascular smooth muscle cells (VSMC) induced by reactive oxygen species (ROS) may occur as ferroptosis, contributing to atherosclerotic plaque instability and rupture. Although it is involved in ROS-induced VSMC death, the role of nuclear transcription factors erythroid 2 related factor 2 (NRF2) in ferroptosis remains unclear. This study was designed to determine the coupling between NRF2 and antioxidant ferroptosis suppressor protein 1 (FSP1) in ROS-induced VSMC ferroptosis. We exposed mouse aortic VSMCs to hydrogen peroxide (H2O2), and cell death was characterized by increased NRF2 expression and inhibition of FSP1 expression. The results demonstrated that VSMC exposure to H2O2 emerged as the characteristic of ferroptosis. H2O2 promoted the expression and nuclear translocation of NRF2 and downregulated FSP1 expression. These data imply a novel and important role for NRF2/FSP1 in ferroptosis for treating VSMCs ferroptosis-associated diseases.

Regulation of FSP1 myristoylation by NADPH: A novel mechanism for ferroptosis inhibition

Excitotoxicity is a prevalent pathological event in neurodegenerative diseases. The involvement of ferroptosis in the pathogenesis of excitotoxicity remains elusive. Transcriptome analysis has revealed that cytoplasmic reduced nicotinamide adenine dinucleotide phosphate (NADPH) levels are associated with susceptibility to ferroptosis-inducing compounds. Here we show that exogenous NADPH, besides being reductant, interacts with N-myristoyltransferase 2 (NMT2) and upregulates the N-myristoylated ferroptosis suppressor protein 1 (FSP1). NADPH increases membrane-localized FSP1 and strengthens resistance to ferroptosis. Arg-291 of NMT2 is critical for the NADPH-NMT2-FSP1 axis-mediated suppression of ferroptosis. This study suggests that NMT2 plays a pivotal role by bridging NADPH levels and neuronal susceptibility to ferroptosis. We propose a mechanism by which the NADPH regulates N-myristoylation, which has important implications for ferroptosis and disease treatment.

Ferroptosis Biology.

Ferroptosis is a regulated cell death modality triggered by iron-dependent lipid peroxidation. Ferroptosis plays a causal role in the pathophysiology of various diseases, making it a promising therapeutic target. Unlike all other cell death modalities dependent on distinct signaling cues, ferroptosis occurs when cellular antioxidative defense mechanisms fail to suppress the oxidative destruction of cellular membranes, eventually leading to cell membrane rupture. Physiologically, only two such surveillance systems are known to efficiently prevent the lipid peroxidation chain reaction by reducing (phospho)lipid hydroperoxides to their corresponding alcohols or by reducing radicals in phospholipid bilayers, thus maintaining the integrity of lipid membranes. Mechanistically, these two systems are linked to the reducing capacity of glutathione peroxidase 4 (GPX4) by consuming glutathione (GSH) on the one and ferroptosis suppressor protein 1 (FSP1, formerly AIFM2) on the other hand. Notably, the importance of ferroptosis suppression in physiological contexts has been linked to a particular vulnerability of renal tissue. In fact, early work has shown that mice genetically lacking Gpx4 rapidly succumb to acute renal failure with pathohistological features of acute tubular necrosis. Promising research attempting to implicate ferroptosis in various renal disease entities, particularly those with proximal tubular involvement, has generated a wealth of knowledge with widespread potential for clinical translation. Here, we provide a brief overview of the involvement of ferroptosis in nephrology. Our goal is to introduce this expanding field for clinically versed nephrologists in the hope of spurring future efforts to prevent ferroptosis in the pathophysiological processes of the kidney.

Osteopontin-driven partial epithelial-mesenchymal transition governs the development of middle ear cholesteatoma

ABSTRACT Cholesteatoma is a common disease of the middle ear. Currently, surgical removal is the only treatment option and patients face a high risk of relapse. The molecular basis of cholesteatoma remains largely unknown. Here, we show that Osteopontin (OPN), a predominantly secreted protein, plays a crucial role in the development of middle ear cholesteatoma. Global transcriptome analysis revealed the loss of epithelial features and an enhanced immune response in human cholesteatoma tissues. Quantitative RT-PCR and immunohistochemical staining of middle ear cholesteatoma validated the reduced expression of epithelial markers, as well as the elevated expression of mesenchymal markers including Vimentin and Fibronectin, but not N-Cadherin, α-smooth muscle actin (α-SMA) or ferroptosis suppressor protein 1 (FSP1), indicating a partial epithelial-mesenchymal transition (EMT) state. Besides, the expression of OPN was significantly elevated in human cholesteatoma tissues. Treatment with OPN promoted cell proliferation, survival and migration and led to a partial EMT in immortalized human keratinocyte cells. Importantly, blockade of OPN signaling could remarkably improve the cholesteatoma-like symptoms in SD rats. Our mechanistic study demonstrated that the AKT-zinc finger E-box binding homeobox 2 (ZEB2) axis mediated the effects of OPN. Overall, these findings suggest that targeting the OPN signaling represents a promising strategy for the treatment of middle ear cholesteatoma.

FSP1-mediated ferroptosis in cancer: from mechanisms to therapeutic applications.

Ferroptosis is a new discovered regulated cell death triggered by the ferrous ion (Fe2+)-dependent accumulation of lipid peroxides associated with cancer and many other diseases. The mechanism of ferroptosis includes oxidation systems (such as enzymatic oxidation and free radical oxidation) and antioxidant systems (such as GSH/GPX4, CoQ10/FSP1, BH4/GCH1 and VKORC1L1/VK). Among them, ferroptosis suppressor protein 1 (FSP1), as a crucial regulatory factor in the antioxidant system, has shown a crucial role in ferroptosis. FSP1 has been well validated to ferroptosis in three ways, and a variety of intracellular factors and drug molecules can alleviate ferroptosis via FSP1, which has been demonstrated to alter the sensitivity and effectiveness of cancer therapies, including chemotherapy, radiotherapy, targeted therapy and immunotherapy. This review aims to provide important frameworks that, bring the regulation of FSP1 mediated ferroptosis into cancer therapies on the basis of existing studies.

RNF126-mediated ubiquitination of FSP1 affects its subcellular localization and ferroptosis.

Medulloblastoma (MB) is a prevalent malignant brain tumor among children, which can be classified into four primary molecular subgroups. Group 3 MB (G3-MB) is known to be highly aggressive and associated with a poor prognosis, necessitating the development of novel and effective therapeutic interventions. Ferroptosis, a regulated form of cell death induced by lipid peroxidation, has been identified as a natural tumor suppression mechanism in various cancers. Nevertheless, the potential role of ferroptosis in the treatment of G3-MB remains unexplored. In this study, we demonstrate that RNF126 acts as an anti-ferroptotic gene by interacting with ferroptosis suppressor protein 1 (FSP1, also known as AIFM2) and ubiquitinating FSP1 at the 4KR-2 sites. Additionally, the deletion of RNF126 reduces the subcellular localization of FSP1 in the plasma membrane, resulting in an increase in the CoQ/CoQH2 ratio in G3-MB. The RNF126-FSP1-CoQ10 pathway plays a pivotal role in suppressing phospholipid peroxidation and ferroptosis both in vivo and in vitro. Clinically, RNF126 exhibited elevated expression in G3-MB and its overexpression was significantly associated with reduced patient survival. Our findings indicate that RNF126 regulates G3-MB sensitivity to ferroptosis by ubiquitinating FSP1, which provides new evidence for the potential G3-MB therapy.

Sensing of mitochondrial DNA by ZBP1 promotes RIPK3-mediated necroptosis and ferroptosis in response to diquat poisoning.

Diquat (DQ) poisoning is a severe medical condition associated with life-threatening implications and multiorgan dysfunction. Despite its clinical significance, the precise underlying mechanism remains inadequately understood. This study elucidates that DQ induces instability in the mitochondrial genome of endothelial cells, resulting in the accumulation of Z-form DNA. This process activates Z-DNA binding protein 1 (ZBP1), which then interacts with receptor-interacting protein kinase 3 (RIPK3), ultimately leading to RIPK3-dependent necroptotic and ferroptotic signaling cascades. Specific deletion of either Zbp1 or Ripk3 in endothelial cells simultaneously inhibits both necroptosis and ferroptosis. This dual inhibition significantly reduces organ damage and lowers mortality rate. Notably, our investigation reveals that RIPK3 has a dual role. It not only phosphorylates MLKL to induce necroptosis but also phosphorylates FSP1 to inhibit its enzymatic activity, promoting ferroptosis. The study further shows that deletion of mixed lineage kinase domain-like (Mlkl) and the augmentation of ferroptosis suppressor protein 1 (FSP1)-dependent non-canonical vitamin K cycling can provide partial protection against DQ-induced organ damage. Combining Mlkl deletion with vitamin K treatment demonstrates a heightened efficacy in ameliorating multiorgan damage and lethality induced by DQ. Taken together, this study identifies ZBP1 as a crucial sensor for DQ-induced mitochondrial Z-form DNA, initiating RIPK3-dependent necroptosis and ferroptosis. These findings suggest that targeting the ZBP1/RIPK3-dependent necroptotic and ferroptotic pathways could be a promising approach for drug interventions aimed at mitigating the adverse consequences of DQ poisoning.

Tanshinone Ⅱ_A inhibits ferroptosis via Nrf2 signaling pathway to protect liver in rats of non-alcoholic fatty liver disease

This study investigated the protective effect of tanshinone Ⅱ_A(TSⅡ_A) on the liver in the rat model of non-alcoholic fatty liver disease(NAFLD) and the mechanism of TSⅡ_A in regulating ferroptosis via the nuclear factor E2-related factor 2(Nrf2) signaling pathway. The rat model of NAFLD was established with a high-fat diet for 12 weeks. The successfully modeled rats were assigned into model group, low-and high-dose TSⅡ_A groups, and inhibitor group, and normal control group was set. Enzyme-linked immunosorbent assay was employed to determine the content of superoxide dismutase(SOD) and malondialdehyde(MDA) in the serum of rats in each group. A biochemical analyzer was used to measure the content of aspartate aminotransferase(AST), alaninl aminotransferase(ALT), total cholesterol(TC), and triglycerides(TG). Hematoxylin-eosin(HE) staining was used to detect pathological damage in liver tissue. Terminal-deoxynucleoitidyl transferase-mediated nick end labeling(TUNEL) was employed to examine the apoptosis of the liver tissue. Oil red O staining, MitoSOX staining, and Prussian blue staining were conducted to reveal lipid deposition, the content of reactive oxygen species(ROS), and iron deposition in liver tissue. Western blot was employed to determine the expression of Nrf2, heme oxygenase-1(HO-1), glutathione peroxidase 4(GPX4), ferroptosis suppressor protein 1(FSP1), B cell lymphoma-2(Bcl-2), and Bcl-2 associated X protein(Bax) in the liver tissue. The result showed that TSⅡ_A significantly reduced the content of MDA, AST, ALT, TC, and TG in the serum, increased the activity of SOD, decreased the apoptosis rate, lipid deposition, ROS, and iron deposition in the liver tissue, up-regulated the expression of Nrf2, HO-1, FSP1, GPX, and Bcl-2, and inhibited the expression of Bax in the liver tissue of NAFLD rats. However, ML385 partially reversed the protective effect of TSⅡ_A on the liver tissue. In conclusion, TSⅡ_A could inhibit ferroptosis in the hepatocytes and decrease the ROS and lipid accumulation in the liver tissue of NAFLD rats by activating the Nrf2 signaling pathway.

Pitavastatin induces autophagy-dependent ferroptosis in MDA-MB-231 cells via the mevalonate pathway

Triple-negative breast cancer (TNBC) is more prone to recurrence and metastasis relative to other subtypes of breast cancer, leading to an extremely poor prognosis. The increasing potential chemoresistance of TNBC patients is mainly due to that tumor cells escape from apoptosis. In recent years, statins have demonstrated extensive anti-tumor effects. It is worth noting that statins have more effective anti-tumor effects on TNBC cells and drug-resistant breast cancer cells. Therefore, this study examines the superior cytotoxic effects of statins on TNBC cell lines and further explores their potential therapeutic mechanisms. We detected different cell phenotypes and found that statins significantly reduced the cell viability of TNBC cells. Specifically, pitavastatin showed an obvious induction in cell death, cell cycle arrest and oxidative stress in TNBC MDA-MB-231 cells. The reversal effect of iron chelator desferrioxamine (DFO) on the morphological and molecular biological changes induced by pitavastatin has revealed a new mode of cell death induced by pitavastatin: ferroptosis. This ferroptotic effect was strengthened by the decreased expression of glutathione peroxidase 4 (GPx4) as well as newly discovered ferroptosis suppressor protein 1 (FSP1). The data showed that ferroptotic death of MDA-MB-231 cells is autophagy-dependent and mediated by the mevalonate pathway. Finally, we found that therapeutic oral doses of statins can inhibit the growth of transplanted tumors, which establishes statins as a potential treatment for TNBC patients. In conclusion, we found pitavastatin could induce autophagy-dependent ferroptosis in TNBC cells via the mevalonate pathway which may become a potential adjuvant treatment option for TNBC patients.

A tangible method to assess native ferroptosis suppressor activity

Ferroptosis, a regulated cell death hallmarked by unrestrained lipid peroxidation, plays a pivotal role in the pathophysiology of various diseases, making it a promising therapeutic target. Glutathione peroxidase 4 (GPX4) prevents ferroptosis by reducing (phospho)lipid hydroperoxides, yet evaluation of its actual activity has remained arduous. Here, we present a tangible method using affinity-purified GPX4 to capture a snapshot of its native activity. Next to measuring GPX4 activity, this improved method allows for the investigation of mutational GPX4 activity, exemplified by the GPX4U46C mutant lacking selenocysteine at its active site, as well as the evaluation of GPX4 inhibitors, such as RSL3, as a showcase. Furthermore, we apply this method to the second ferroptosis guardian, ferroptosis suppressor protein 1, to validate the newly identified ferroptosis inhibitor WIN62577. Together, these methods open up opportunities for evaluating alternative ferroptosis suppression mechanisms.

FSP1 inhibition enhances olaparib sensitivity in BRCA-proficient ovarian cancer patients via a nonferroptosis mechanism

Poly ADP-ribose polymerase inhibitors (PARPis) exhibit promising efficacy in patients with BRCA mutations or homologous repair deficiency (HRD) in ovarian cancer (OC). However, less than 40% of patients have HRD, it is vital to expand the indications for PARPis in BRCA-proficient patients. Ferroptosis suppressor protein 1 (FSP1) is a key protein in a newly identified ferroptosis-protective mechanism that occurs in parallel with the GPX4-mediated pathway and is associated with chemoresistance in several cancers. Herein, FSP1 is reported to be negatively correlated with the prognosis in OC patients. Combination therapy comprising olaparib and iFSP1 (a FSP1 inhibitor) strongly inhibited tumour proliferation in BRCA-proficient OC cell lines, patient-derived organoids (PDOs) and xenograft mouse models. Surprisingly, the synergistic killing effect could not be reversed by ferroptosis inhibitors, indicating that mechanisms other than ferroptosis were responsible for the synergistic lethality. In addition, cotreatment was shown to induce increased γH2A.X foci and to impair nonhomologous end joining (NHEJ) activity to a greater extent than did any single drug. Mass spectrometry and immunoprecipitation analyses revealed that FSP1 interacted with Ku70, a classical component recruited to and occupying the end of double-strand breaks (DSBs) in the NHEJ process. FSP1 inhibition decreased Ku70 PARylation, impaired subsequent DNA-PKcs recruitment to the Ku complex at DSB sites and was rescued by restoring PARylation. These findings unprecedentedly reveal a novel role of FSP1 in DNA damage repair and provide new insights into how to sensitize OC patients to PARPi treatment.

FSP1 is a predictive biomarker of osteosarcoma cells’ susceptibility to ferroptotic cell death and a potential therapeutic target

Human osteosarcoma (OS) is a relatively rare malignancy preferentially affecting long body bones which prognosis is often poor also due to the lack of effective therapies. Clinical management of this cancer basically relies on surgical removal of primary tumor coupled with radio/chemotherapy. Unfortunately, most osteosarcoma cells are resistant to conventional therapy, with the undergoing epithelial-mesenchymal transition (EMT) giving rise to gene expression reprogramming, thus increasing cancer cell invasiveness and metastatic potential. Alternative clinical approaches are thus urgently needed. In this context, the recently described ferroptotic cell death represents an attractive new strategy to efficiently kill cancer cells, since most chemoresistant and mesenchymal-shaped tumors display high susceptibility to pro-ferroptotic compounds. However, cancer cells have also evolved anti-ferroptotic strategies, which somehow sustain their survival upon ferroptosis induction. Indeed, here we show that osteosarcoma cell lines display heterogeneous sensitivity to ferroptosis execution, correlating with the mesenchymal phenotype, which is consistently affected by the expression of the well-known anti-ferroptotic factor ferroptosis suppressor protein 1 (FSP1). Interestingly, inhibiting the activity or expression of FSP1 restores cancer cell sensitivity to ferroptosis. Moreover, we also found that: i) AKRs might also contribute to resistance; ii) NRF2 enhances FSP1 expression upon ferroptosis induction; while iii) p53 contributes to the regulation of FSP1 basal expression in OS cells.In conclusion, FSP1 expression can potentially be used as a valuable predictive marker of OS sensitivity to ferroptosis and as a new potential therapeutic target.

Exploring the anti-ferroptosis mechanism of Kai-Xin-San against Alzheimer's disease through integrating network pharmacology, bioinformatics, and experimental validation strategy in vivo and in vitro

Ethnopharmacological relevanceKai Xin San (KXS), first proposed by Sun Simiao during the Tang Dynasty, has been utilized to treat dementia by tonifying qi and dispersing phlegm. Aim of the studyThis study aimed to elucidate the mechanism by which KXS exerts its therapeutic effects on Alzheimer's disease (AD) by targeting ferroptosis, using a combination of network pharmacology, bioinformatics, and experimental validation strategies. Materials and methodsThe active target sites and the further potential mechanisms of KXS in protecting against AD were investigated through molecular docking, molecular dynamics simulation, and network pharmacology, and combined with the validation of animal experiments. ResultsComputational and experimental findings provide the first indication that KXS significantly improves learning and memory defects and inhibits neuronal ferroptosis by repairing mitochondria damage and upregulating the protein expression of ferroptosis suppressor protein 1 (FSP1) in vivo APP/PS1 mice AD model. According to bioinformatics analysis, the mechanism by which KXS inhibits ferroptosis may involve SIRT1. KXS notably upregulated the mRNA and protein expression of SIRT1 in both vivo APP/PS1 mice and in vitro APP-overexpressed HT22 cells. Additionally, KXS inhibited ferroptosis induced by APP-overexpression in HT22 cells through activating the SIRT1-FSP1 signal pathway. ConclusionsCollectively, our findings suggest that KXS may inhibit neuronal ferroptosis through activating the SIRT1/FSP1 signaling pathway. This study reveals the scientific basis and underlying modern theory of replenishing qi and eliminating phlegm, which involves the inhibition of ferroptosis. Moreover, it highlights the potential application of SIRT1 or FSP1 activators in the treatment of AD and other ferroptosis-related diseases.

Mediation of Ferroptosis Suppressor Protein 1 Expression via 4-Hydroxy-2-Nonenal Accumulation Contributes to Acquisition of Resistance to Apoptosis and Ferroptosis in Diffuse Large B-Cell Lymphoma

Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin lymphoma. New therapeutic strategies are needed for the treatment of refractory DLBCL. 4-Hydroxy-2-nonenal (4-HNE) is a cytotoxic lipid peroxidation marker, which alters intracellular signaling and induces genetic mutations. Lipid peroxidation is associated with nonapoptotic cell death, called ferroptosis. However, the relationship between 4-HNE accumulation and feroptotic regulators in DLBCL has not been fully evaluated. Here, we aimed to evaluate the accumulation of lipid peroxide and the expression of ferroptosis suppressor protein 1 (FSP1) in DLBCL using immunohistochemistry. We found a significant increase in the expression of FSP1 in cases with nuclear 4-HNE accumulation (P = .021). Both nuclear and cytoplasmic 4-HNE accumulation and FSP1 positivity were independent predictors of worse prognosis. In vitro exposure to 4-HNE resulted in its concentration- and time-dependent intracellular accumulation and increased expression of FSP1. Furthermore, short-term (0.25 and 1.0 μM) or long-term (0.25 μM) exposure to 4-HNE induced resistance to not only apoptosis but also ferroptosis. Taken together, regulation of FSP1 through 4-HNE accumulation may attenuate resistance to cell death in treatment-resistant DLBCL and might help develop novel therapeutic strategies for refractory DLBCL.

TNFα-reliant FSP1 up-regulation promotes intervertebral disc degeneration via caspase 3-dependent apoptosis

Intervertebral disc degeneration (IDD) is a common chronic inflammatory degenerative disease that causes lower back pain. However, the underlying mechanisms of IDD remain unclear. Ferroptosis suppressor protein 1 (FSP1) is a newly identified suppressor for ferroptosis. This study aims to investigate the role of FSP1 in IDD. Nucleus pulposus (NP) tissues in humans were collected and NP cells from rats were isolated to detect FSP1 expression pattern. The relationship between FSP1-mediated ferroptosis and apoptosis was identified using FSP1 inhibitor iFSP1. RNA sequencing was utilized to seek downstream molecules and related signaling pathways. Moreover, both exogenous recombinant FSP1 protein and endogenous small interfering RNA were implemented in this study to clarify the role of FSP1 in tumor necrosis factor-alpha (TNFα)-mediated NP cell apoptosis. Ultimately, the underlying mechanisms of FSP1-related signaling pathway in IDD were uncovered both in vitro and in vivo. As a result, FSP1 was up-regulated in human degenerative NP tissues and after TNFα stimulation. FSP1 inhibition by iFSP1 fails to trigger ferroptosis in NP cells while inhibiting TNFα-mediated apoptosis. Further experiments demonstrated that FSP1 was closely related to TNFα-reliant caspase 3 activation and mitochondrial damage. However, the exogenous addition of recombinant protein FSP1 does not induce cell death or intensify the efficacy of TNFα. Mechanically, FSP1 is involved in TNFα-mediated NF-κB signaling activation to accelerate the development of IDD. This study demonstrated that FSP1 promotes IDD through TNFα-reliant NF-κB signaling activation and caspase 3-dependent apoptosis. These findings suggested a novel therapeutic target for the treatment of IDD.

Targeting of FSP1 regulates iron homeostasis in drug-tolerant persister head and neck cancer cells via lipid-metabolism-driven ferroptosis.

Research has demonstrated that some tumor cells can transform into drug-tolerant persisters (DTPs), which serve as a reservoir for the recurrence of the disease. The persister state in cancer cells arises due to temporary molecular reprogramming, and exploring the genetic composition and microenvironment during the development of head and neck squamous cell carcinoma (HNSCC) can enhance our comprehension of the types of cell death that HNSCC, thus identifying potential targets for innovative therapies. This project investigated lipid-metabolism-driven ferroptosis and its role in drug resistance and DTP generation in HNSCC. High levels of FSP1 were discovered in the tissues of patients who experienced relapse after cisplatin treatment. RNA sequencing indicated that a series of genes related to lipid metabolism were also highly expressed in tissues from these patients. Consistent results were obtained in primary DTP cells isolated from patients who experienced relapse. The Cancer Genome Atlas database confirmed this finding. This revealed that the activation of drug resistance in cancer cells is influenced by FSP1, intracellular iron homeostasis, and lipid metabolism. The regulatory roles of ferroptosis suppressor protein 1 (FSP1) in HNSCC metabolic regulation were investigated. We generated human oral squamous cell carcinoma DTP cells (HNSCC cell line) to cisplatin and observed higher expression of FSP1 and lipid-metabolism-related targets in vitro. The shFSP1 blockade attenuated HNSCC-DTP cell stemness and downregulated tumor invasion and the metastatic rate. We found that cisplatin induced FSP1/ACSL4 axis expression in HNSC-DTPC cells. Finally, we evaluated the HNSCC CSC-inhibitory functions of iFSP1 (a metabolic drug and ferroptosis inducer) used for neo-adjuvant chemotherapy; this was achieved by inducing ferroptosis in a patient-derived xenograft mouse model. The present findings elucidate the link between iron homeostasis, ferroptosis, and cancer metabolism in HNSCC-DTP generation and acquisition of chemoresistance. The findings may serve as a suitable model for cancer treatment testing and prediction of precision treatment outcomes. In conclusion, this study provides clinically oriented platforms for evaluating metabolism-modulating drugs (FSP1 inhibitors) and new drug candidates of drug resistance and ferroptotic biomarkers.