Disulfiram downregulates ferredoxin 1 to maintain copper homeostasis and inhibit inflammation in cerebral ischemia/reperfusion injury.

Disulfiram downregulates ferredoxin 1 to maintain copper homeostasis and inhibit inflammation in cerebral ischemia/reperfusion injury. | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Disulfiram downregulates ferredoxin 1 to maintain copper homeostasis and inhibit inflammation in cerebral ischemia/reperfusion injury. Shuai Yang, Xudong Li, Jinhong Yan, Fangchao Jiang, Xuehui Fan, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4052488/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract In the current study, we aimed to investigate whether disulfiram (DSF) plays a neuroprotective role in cerebral ischemia-reperfusion (CI-RI) injury by regulating ferredoxin 1 (FDX1) by modulating copper ion (Cu) levels and inhibiting the inflammatory response. To simulate CI-RI, a transient middle cerebral artery occlusion (tMCAO) model in C57/BL6 mice was employed. Mice were administered with or without DSF before and after tMCAO. Changes in infarct volume after tMCAO were observed using TTC staining. Nissl staining and hematoxylin-eosin (he) staining were used to observe the morphological changes of nerve cells at the microscopic level. FDX1 is the main regulatory protein of copper death, and the occurrence of copper death will lead to the increase of HSP70 stress and inflammatory response. Cuproptosis-related proteins and downstream inflammatory factors were detected by western blotting, immunofluorescence staining, and immunohistochemistry. The content of copper ions was detected using a specific kit, while electron microscopy was employed to examine mitochondrial changes. We found that DSF reduced the cerebral infarction volume, regulated the expression of cuproptosis-related proteins, and reduced FDX1 expression without inducing Cu accumulation. Moreover, DSF inhibited the HSP70/TLR-4/NLRP3 signaling pathway. Collectively, DSF could regulate Cu homeostasis by inhibiting FDX1, acting on the HSP70/TLR4/NLRP3 pathway to alleviate CI/RI. Accordingly, DSF could mitigate inflammatory responses and safeguard mitochondrial integrity, yielding novel therapeutic targets and mechanisms for the clinical management of ischemia-reperfusion injury. Health sciences/Diseases/Neurological disorders/Cerebrovascular disorders Health sciences/Diseases/Neurological disorders/Hypoxic ischaemic encephalopathy Health sciences/Diseases/Neurological disorders/Stroke Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction The incidence and mortality rates of cerebral ischemic disease are gradually increasing worldwide. Moreover, cerebral ischemic disease is the most common cause of permanent disability in adults. 1 Thrombolysis/thrombolysis is the most effective treatment for ischemic stroke. Since 2015, substantial advancements have been made in research exploring the treatment of acute ischemic stroke, leading to an expanded time window for emergency endovascular intervention, from the initial 6 h to a more extended period of 24 h. 2 Following cerebral ischemia, reperfusion of blood flow induces a cascade of injurious effects, commonly referred to as cerebral ischemia/reperfusion injury (CI/RI). This process involves various types of regulated cell death (RCD) and neuroinflammatory responses, considerably contributing to its progression. 3 , 4 Mitigating the adverse effects of reperfusion has been a persistent focal point for clinicians striving for breakthroughs. In living organisms, cells exposed to extreme physicochemical or mechanical stressors may undergo an immediate and uncontrolled structural collapse, a phenomenon known as accidental cell death. Conversely, RCD involves specific signaling cascades and molecular-defined effector mechanisms, encompassing fundamental processes, such as organogenesis and tissue remodeling, eliminating unnecessary structures or cells, and controlling cell numbers. Additionally, RCD can be triggered by exogenous perturbations in the intracellular or extracellular microenvironments. 5 , 6 Currently, more than 10 types of RCDs have been identified, encompassing a diverse range of non-apoptotic RCD modalities, such as autophagy, pyroptosis, ferroptosis, and endogenous cell demise, in addition to the conventional apoptotic pathway. 7 , 8 In contrast to apoptosis, these RCD pathways elicit inflammatory responses within the body and have been increasingly associated with cancer. 9 CI/RI induces a robust inflammatory response initiated by damage-associated molecular patterns (DAMPs) released from injured cells, including signaling molecules, such as adenosine, heat shock proteins (HSPs), high-mobility group protein B1 (HMGB1), and interleukin (IL). 10 – 12 In a healthy central nervous system, various types of DAMPs are expressed and released following injury to activate inflammatory signaling pathways. 11 The aforementioned DAMPs use pattern recognition receptors (PRRs) to initiate and augment immune responses. 13 Extracellular HSP70 is a typical DAMP. 14 PRRs are predominantly expressed in astrocytes and microglia, enabling the detection of pathogen-derived or endogenous ligand release. Toll-like receptors (TLRs) of the PRR family are an example of this phenomenon. 15 TLR4 plays a pivotal role and serves as the primary receptor for HMGB1. 16 In addition, HSP70 regulates TLR4. 17 , 18 TLR4 is most commonly associated with the expression and release of IL-1 and tumor necrosis factor-α (TNF-α), and this association typically occurs in a ligand-dependent manner. Nuclear factor (NF)-κB, positioned downstream of the TLR4 signaling pathway, plays a pivotal role in orchestrating immune responses, cellular proliferation, and differentiation. Upon activation, TLR4 induces nuclear translocation of NF-κB(p65), thereby facilitating the expression of diverse inflammatory cytokines, such as IL-1β and TNF-α. 19 The activation of the TLR4/NF-κB signaling pathway facilitates the assembly of a complex between NLRP3 and apoptosis-associated speck-like protein with a CARD (ASC), which subsequently interacts with the cysteine protease caspase-1 to form inflammasomes. 20 , 21 The classic pyroptotic pathway, mediated by the NLRP3 inflammasome, is pivotal in determining functional outcomes following stroke. 22 Recently, a novel RCD pattern, distinct from the well-known RCD pattern, has been identified and designated as "cuproptosis.” This unique mode of cell death relies on the copper (Cu)-mediated targeting of lipoacylated tricarboxylic acid (TCA) cycle proteins and is strongly associated with mitochondrial respiration. 23 FDX1 converts Cu (II) to the highly toxic Cu (I), resulting in the aggregation of fatty acylated proteins, exhaustion of iron-sulfur cluster proteins, HSP70 activation, and induction of intracellular toxic oxidative stress, ultimately causing cell death. Importantly, this process is associated with mitochondrial respiration. A meta-analysis has revealed that serum Cu levels are substantially elevated during the acute phase of stroke. 24 Recent evidence suggests that Cu ions participate in various transformation mechanisms that damage brain tissue during fusion injury. 25 The induced cuproptosis activates HSP70 to initiate an immune inflammatory response. Disulfiram (DSF) is a well-established anti-alcoholic medication scientifically validated for its safety and potential in targeted tumor therapy. 26 , 27 Recent research has demonstrated the potential of DSF in inhibiting multiple inflammatory reactions and regulating inflammation-related targets, highlighting its potential as an anti-inflammatory agent. 28 – 30 The DSF-Cu complexes were found to be safe and efficacious in the prevention and treatment of various types of cancers. 31 The inclusion of DSF in the Cu carrier category aimed to enhance the induction of apoptosis in tumor cells. 23 Recently, Cu (II) bis(diethyldithiocarbamate), a potent anticancer agent, has been identified as a bioactive metabolite of alcohol abuse. 32 The administration of this drug was shown to reduce the expression of an upstream regulator involved in mitochondrial proteolipid acylation during cuproptosis. 33 Further comprehensive investigations are warranted to ascertain the potential of DSF in ameliorating CI/RI damage and to elucidate the underlying mechanisms. Therefore, in the present study, we aimed to validate the effect of DSF on CI/RI and elucidate the underlying molecular mechanisms. We hypothesized that DSF could modulate Fdx1-induced CI-RI damage and inhibit inflammatory responses. We believe that the findings of the current study would further unravel the crosstalk relationship between each death mechanism and provide new targets for drug application and treatment of CI-RI. 2. Experimental methods 2.1 Animals and tMCAO models Male C57BL/6 mice (6–8 weeks old, weight 20 ± 3 g) were procured from Liaoning Changsheng Biotechnology Co., Ltd and housed in a hygienic and comfortable habitat under a 12-h natural light-dark cycle to simulate their circadian rhythm. The Animal Experiment Center of Harbin Medical University provided adequate food and water. The animals were randomly divided into four groups (n = 4/group): sham group, 24h-tMCAO group, DSF + 24h-tMCAO group, and vehicle + 24h-tMCAO (dimethyl sulfoxide < 2%). The DSF + 24h-tMCAO group was intraperitoneally administered DSF (50 mg/kg, twice daily), starting from the day before the surgery and continuing postoperatively. The mice were anesthetized with 2% isoflurane, and body temperature was maintained at ~ 37°C throughout the surgical and postoperative periods using a servo-controlled heating blanket. Focal cerebral ischemia was achieved by occluding the middle cerebral artery (MCA). After performing a midline incision in the neck, a doccol suture (602256PK5Re; US) was inserted into the right external carotid artery and then reversed into the right internal carotid artery via the common carotid artery to occlude the blood supply to the MCA. The sutures were then removed after 1 h.Following suture removal, the mice exhibited recovery times of 6 h, 24 h, 3 d, 5 d and 7 d.Mice died of cervical dislocation after anesthesia at the above time points. This experiment was reviewed and approved by the ethical review committee of Harbin Medical University. (IACUU NO.2022133).The management and use of mice are consistent with the relevant guidelines of US National Institutes of Health. 2.2 2,3,5-Triphenyltetrazolium chloride (TTC) staining Briefly, the harvest mouse brain was sliced into seven pieces (1 mm thick) from the rostral end of the frontal lobe. Cells were then incubated in TTC (Solarbio, Beijing, China), shielded from light for 30 min, and fixed in 4% paraformaldehyde. Planar infarct volume measurements were performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA). 2.3 Hematoxylin-eosin and Nissl stainings The specimens were fixed in a 4% paraformaldehyde solution and subsequently embedded in paraffin. Histological sections (4 µm thick) were prepared and stained with hematoxylin-eosin. For Nissl staining, a 1% toluidine blue solution was heated to 60°C, followed by the immersion of all sections for 40 min. Subsequently, sections were washed with 70% ethanol and rapidly differentiated using 95% ethanol. The tissue sections were examined and imaged under a light microscope (Nikon, Tokyo, Japan). 2.4 Immunofluorescent staining Briefly, the prepared brain tissues were sequentially immersed in a 4% paraformaldehyde solution and 30% sucrose solution for 48 h. After embedding in optimal cutting temperature compound and freezing, frozen blocks were sectioned (7-µm thick slices) using a freezing microtome. Sections were treated with a blocking solution (Beyotime, China) containing Triton X-100 for 20 min and incubated overnight at 4°C with the following primary antibodies: ASC (sc-514414, 1:50 dilution, Santa Cruz, USA), Caspase-1 (sc-56036, 1:50 dilution, Santa Cruz, USA), GSDMD (sc-393581, 1:50 dilution, Santa Cruz, USA), HSP70 (T55496, 1:50 dilution, Abmart, China). Following hydrations with PBST (3×), the samples were incubated with the appropriate fluorophore-conjugated secondary antibody (Boster, China) for 1 h. Nuclei were stained with 30µL DAPI (Abcam, UK) to ensure no bubbles. After 10 min, the slides were examined using light microscopy. 2.5 Immunohistochemical staining The fixed brain tissues were embedded in paraffin, sectioned, deparaffinized, and subjected to antigen retrieval using a citrate solution after blocking with 3% hydrogen peroxide. The sections were then blocked with fetal bovine serum for 1 h. Subsequently, the sections were incubated overnight at 4°C with primary antibodies against FDX1 (T510671,1:200 dilution, Abmart, China), DLST (TD13671,1:200 dilution, Abmart, China), ATP7A (PA7106, 1:200 dilution, Abmart, China), ATP7B (TA0410, 1:200 dilution, Abmart, China), Caspase-1 ( 22915-1-AP, 1:200 dilution, Proteintech, USA), and IL-18 (10663-1-AP, 1:200 dilution, Proteintech, USA), followed by subsequent incubation with secondary antibodies (Boster, China) for 1 h at room temperature. Color development was achieved using diaminobenzidine. Then, sections were stained with hematoxylin. Subsequently, the sections were dehydrated and sealed before imaging under a light microscope at 200× magnification. The acquired images were analyzed using ImageJ software to determine the average density within the observation area. 2.6 Cu level detection Cu levels were detected using colorimetric quantitative kits provided by Nanjing Jiancheng BioEngineering Institute (Jiangsu, China). The right brain tissue was measured, followed by the addition of double-distilled water at a ratio of 1:9 and supernatant collection by ultrasonic centrifugation. For protein quantification, the absorbance (580 nm) was measured using a microplate reader (MD, Shanghai, China) after the sequential addition of reagents. 2.7 Western blot analysis Brain tissue from the right hemisphere was lysed (RIPA: protease inhibitor: phosphatase inhibitor, 100:1:1; lipoacylated protein was added in an equal proportion of TCEP). To determine the sample protein concentration, the supernatant was denatured by boiling, using the BCA protein assay kit (Beyotime, China). Proteins were subjected to sodium dodecyl-sulfate polyacrylamide gel electrophoresis and subsequently transferred onto polyvinylidene fluoride membranes using the sandwich method. Then, the membranes were blocked with 5% skim milk for 1 h at room temperature, followed by overnight incubation with primary antibodies at 4°C in a refrigerator. The membranes were incubated with goat anti-mouse and anti-rabbit secondary antibodies (Abmart, China) at room temperature for 60 min. Next, the membranes were washed with Tris-buffered saline with 0.1% Tween® 20 detergent and incubated with an enhanced chemiluminescence reagent (Biosharp, China) for detection. The primary antibodies employed were as follows: GSDMD (EPR20859, 1:1000, Abcam, UK); Caspase-1(22915-1-AP, 1:1000 dilution, Proteintech, USA), ASC (sc-514414, 1:500, Santa Cruz, USA),IL-1β(RM1009, 1:1000, Abcam, UK), IL-18 (10663-1-AP, 1:1000 dilution, Proteintech, USA), IL-17(26163-1-AP, 1:1000, Proteintech,USA),TLR4(66350-1-Ig, 1:1000dilution, Proteintech, USA),NLRP3(EPR23073-96, 1:1000, Abcam, UK),TNFα(60291-1-Ig, 1:1000 dilution, Proteintech, USA), LIAS(67298-1-Ig, 1:1000 dilution, Proteintech, USA), NF-κB(66535-1-Ig, 1:1000 dilution, Proteintech, USA) and SLC31A1 (CTR1), ATP7B (TA0410, 1:1000 dilution, Abmart, China), HSP70(T55496, 1:1000 dilution, Abmart, China), FDX1(T510671, 1:1000 dilution, Abmart, China), SDHB(EPR10880, 1:50000, Abcam, UK), DLAT(T58125, 1:1000 dilution, Abmart, China), and DLST (TD13671, 1:1000 dilution, Abmart, China). For protein bands, gray values were quantified using ImageJ software. 2.8 Transmission electron microscopy Brain tissues were cut into cubes (≤ 1 mm 3 ) in 2.5% glutaraldehyde solution. After fixation with osmium tetroxide, tissue samples were subjected to repeated dehydration using acetone, followed by the addition of resin overnight at room temperature. The resulting tissue sections (70 nm) were stained with uranyl acetate and lead citrate and dried for transmission electron microscopy. 2.9 Statistical analyses All results are presented as the mean ± standard error of the mean. Data were analyzed using analysis of variance, followed by Tukey's post-hoc test. Statistical analyses were performed using GraphPad Prism version 9.5 (GraphPad Software, Inc., Boston, MA, USA). Statistical significance was set at P < 0.05. 3. Results 3.1FDX1 expression is up-regulated in the cerebral infarction area after tMCAO. To explore the relationship between FDX1 and CI/RI, FDX1 protein was determined at different time points after tMCAO. WB showed that FDX1 protein expression reached the peak at 24h and then gradually decreased compared with Sham group.(Fig. 1A) 3.2 DSF attenuates the cerebral infarction volume and mitigates neuronal damage following tMCAO TTC staining confirmed the DSF-mediated neuroprotective effect, reducing CI/RI. Compared with the 24h-tMCAO group, the DSF + 24h-tMCAO group exhibited a significant decrease in the infarct volume. (P < 0.05) (Fig. 2A-B) The microstructural characteristics of the brain tissue were observed following tMCAO. Hematoxylin and eosin staining revealed that tMCAO damaged the area of cerebral infarction and the surrounding tissues. The nerve cell stroma appeared loose and edematous, whereas neuronal nuclei displayed pyknosis and hyperchromatism. Additionally, vacuolation was observed in the neurodermal membrane, along with gliosis. In the DSF + 24 h-tMCAO group, pyknosis and hyperchromatism of neurons were observed in the infarct area, while neuropil vacuolation in the infarct focus was alleviated, with a reduction in the number of unidentified structures and glial cells surrounding the infarct (Fig. 2C). Based on Nissl staining, Nissl bodies disappeared in neurons after CI/RI, and a large number of damaged neurons with karyopyknosis and dark staining were observed in and around the infarct area. DSF administration preserved normal neuronal morphology in most mice, with most cells displaying visible Nissl bodies. Only a small number of injured neurons with pyknosis and dark staining were observed around the infarction (Fig. 2D). 3.3 DSF modulates key factors associated with cuproptosis Functional proteins associated with cuproptosis were determined by western blotting. Induction of tMCAO increased the expression levels of SLC31A1/CTR1, ATP7B, FDX1, and HSP70. Additionally, tMCAO enhanced the levels of crucial proteins associated with the TCA cycle (LIAS, DLAT, DLST, and PDHB). Notably, administration of DSF mitigated the fluctuations observed in these protein levels, SDHB fluctuations are not significant. (Fig. 3A-B). Immunohistochemical staining with FDX1, DLST, ATP7A, and ATP7B antibodies revealed that the positive rate of antigen and antibody reaction in DSF + 24h-tMCAO group was lower than that in 24h-tMCAO group. (Fig. 4A) Based on the results of the semi-quantitative analysis, the expression of the aforementioned proteins was statistically significant. (Fig. 4B). Immunofluorescence staining revealed the upregulation of HSP70 expression following tMCAO; however, treatment with DSF mitigated the IR-induced elevation in protein expression DSF regulates the expression cuproptosis-related proteins. (Fig. 5A-B) 3.4 DSF inhibits the activation of the HSP70/TLR-4 /NLRP3 signaling pathway and reduces inflammatory cytokines after tMCAO DAMPs can activate TLR4 to trigger downstream signaling pathways, and HSP70 is an important endogenous TLR4 agonist. Based on the western blotting analysis, the 24h-tMCAO group showed increased protein expression levels of TLR-4, NF-κB, NLRP3, TNF-α, ASC, Pro-caspase-1, Cleaved-caspase-1, GASDMD-N, IL-18, IL-1β, and IL-17 when compared with the sham group. However, the DSF-treated group exhibited downregulated protein expression when compared with the control group(Fig. 5A-B). Moreover, immunofluorescence staining revealed that the expression of ASC, caspase-1, and GASDMD were upregulated following tMCAO; treatment with DSF could alleviate the reperfusion injury-induced increase in protein expression (Fig. 6A-B). Immunohistochemical staining with IL-18 antibody showed that treatment with DSF decreased positive staining in the 24h-tMCAO group compared with that in the 24h-tMCAO group. Based on the results of the semi-quantitative analysis, the expression of the aforementioned proteins was statistically significant in the 24h-tMCAO group compared with that in the DSF + 24h-tMCAO group (Fig. 4A-B). 3.5 DSF did not induce Cu retention and conferred mitochondrial protection following tMCAO The impact of DSF on Cu levels during CI/RI was assessed by measuring Cu ions. Following CI/RI, the total amount of Cu ions was increased in the 24h-tMCAO group when compared with that in the sham group, with no further increase observed following DSF administration (Fig. 7B). The instability of Cu metabolism leads to mitochondrial morphological damage and dysfunction. Transmission electron microscopy was performed to examine morphological alterations in mitochondria in the brain tissue. The control group exhibited intact mitochondrial membranes and aligned cristae. However, the induction of tMCAO resulted in substantial mitochondrial swelling; most matrixes in the mitochondrial membrane appeared less intense, with a broken crest, which disappeared. The rough endoplasmic reticulum exhibited dilation, degranulation, and vacuolation. The mitochondria in the DSF + 24h-tMCAO group exhibited intact morphology, suggesting that DSF could suppress mitochondrial fission and preserve mitochondrial structural integrity (Fig. 7A). 4. Discussion Herein, our findings suggest that DSF can exert inhibitory effects on the inflammatory response and cell death induced by CI/RI via the modulation of the TLR4/NLRP3/NF-κB pathway and FDX1/TCA cycle, thereby affording a neuroprotective role. Treatment with DSF alone could suppress the expression of FDX1/TCA cycle-related proteins and the HSP70/TLR4/NLRP3 pathway during CI-RI to protect nerves and inhibit inflammation. Cu plays an indispensable role in the physiological processes of the central nervous system. The disruption of Cu homeostasis can exert a multitude of detrimental effects on brain development and function. Compared with other organs, the brain exhibits the highest Cu concentration, second only to the liver. Notably, the average Cu content in the globus pallidus surpasses that in the liver. 34 As a cofactor, Cu plays an indispensable role as a reducing agent in the catalytic activity of peptidyl-glycine alpha-amide monooxygenase, dopamine β-monooxygenase, Cu-zinc (Zn) superoxide dismutase, and ceruloplasmin. 35 The presence of Cu is crucial for catecholamine biosynthesis, neuropeptide activation, and the regulation of mitochondrial function. 36 However, excess Cu induces lipid deposition through oxidative stress, mitochondrial dysfunction, 37 promotes neurodegenerative changes, and cell death. 38 Cu transporters SLC31A1 (CTR1), ATP7A, and ATP7B are known to regulate the Cu content in cellular compartments and maintain Cu homeostasis. CTR1, a master regulator of Cu uptake, acts on the plasma membrane, and the genetic inactivation of CTR1 results in Cu deficiency in cells. 39 In the choroid plexus (ChPl), Cu flows from the epithelial cells of the ChPl into the cerebrospinal fluid (CSF). In patients with Menkes disease, ATP7A inactivation is known to cause Cu deficiency in the brain, numerous metabolic abnormalities (including catecholamine imbalance), delayed neurodevelopment, and death during early childhood. 40 Generally, ATP7B transports Cu from the cytosol into the lumen of the secretory pathway for incorporation into Cu-dependent enzymes and traps excess Cu in vesicles for further export from the cell. The precise impact of these activities on cellular metabolism depends on the specific cell type and can exhibit significant variation across different tissues. 41 Therefore, the negative effects of ATP7B inactivation on brain metabolism are evident in Wilson’s disease. Copper-induced protein transport has been described as a key feature of copper-ATPase, and the central link of this mechanism is the transfer of ATP7A and ATP7B from TGN to peripheral or cytoplasmic vesicles, respectively. The displacement of these proteins is specifically triggered by elevated levels of their own ligand, Cu (I), not Cu (II), and is intended to efflux to restore intracellular copper levels. 42 – 44 FDX1 helps convert Cu (II) to the more toxic Cu (I), and in our study, DSF inhibited FDX1 expression, reduced Cu(I) levels, and reduced transport of ATP7A and ATP7B. Interestingly, dysregulation of the Cu transport mechanism in the choroid plexus of Atp7b -/- mice has been demonstrated in recent studies; these mice exhibited low Cu levels in the brain at 4 weeks postnatally, emphasizing the crucial role of ATP7B in Cu accumulation. 45 In summary, the inhibition of CTR1 could regulate the cuproptosis pathway by affecting Cu uptake. It is yet to be established whether the upregulation of ATP7A and ATP7B expression following CI-RI serves as a protective mechanism by promoting Cu efflux or accumulation. However, inhibition of the dual-flow ring can explain the lack of decrease in total Cu levels after DSF administration, and this mechanism aligns with the anticancer effects of Cu ionophores. This discrepancy can be partly attributed to the limited understanding of the complex mechanisms of action of ATP7A and ATP7B in the brain.(Fig. 8) The immune system relies on Cu to execute certain functions. Recent studies have demonstrated that even in the presence of a marginal deficiency, Cu contributes to a decrease in IL concentrations. Neutrophils accumulate Cu as they differentiate into more mature cell populations, and this accumulation is not reflected by an increase in Cu/Zn superoxide dismutase or cytochrome c oxidase activity. 46 HSP70 is induced by Cu exposure. 47 TLR4 is a PRR that recognizes specific DAMPs such as HSP70, which, in turn, triggers TLR4-mediated inflammatory responses. Thus, HSP70 in the CSF may function as a neuroinflammatory mediator. 17 The inflammasome protein complex has recently emerged as a pivotal component of the innate immune response during ischemic stroke. 48 NLRP3 has been extensively studied in central nervous system diseases. 49 The activation of the NLRP3 inflammasome necessitates TLR4-mediated activation of the p65 subunit within the downstream NFκB pathway. 50 Subsequently, NLRP3 binds to ASC to form a complex, and Caspase-1 specifically cleaves GSDMD, releasing the N-terminus from its self-inhibitory C-terminus. GSDMD-N binds to lipids to form non-selective pores, resulting in cell membrane rupture and promoting the release of a large number of inflammatory factors, ultimately triggering pyroptosis. 51 With the maturation and efflux of IL-18 and IL-1β, Th17 cells and γδT cells release interleukin-17 (IL-17), an important factor in the adaptive immune system. 52 Several studies have shown that IL-17 is associated with the pathogenesis of cerebral ischemia-reperfusion. Loss of γδT cells alleviates brain tissue damage after ischemia-reperfusion, and IL-17 positive lymphocytes are also detected in brain tissue validated in the field of stroke patients. 53 The activated NLRP3 inflammasome promotes the activation of IL-17 in CI/RI. 54 The anti-inflammatory efficacy of DSF has been substantiated in numerous studies. However, its role in CI-RI remains elusive. In the current study, we found that the association between CI/RI and Cu-induced cell death was accompanied by an enhanced inflammatory response (classic pyroptosis), which was mediated via the HSP70/TLR4/NLRP3 pathway. Importantly, the administration of DSF effectively inhibited this pathway to alleviate CI/RI. Our findings suggest that DSF exhibits robust safety as a well-established pharmaceutical employed in clinical settings for an extensive duration. This finding challenges the conventional belief that DSF, in conjunction with Cu ionophores, forms a complex that effectively treats cancer and induces apoptosis, specifically in cancer cells. However, conventional copper chelators hamper angiogenesis, impeding brain tissue recovery following ischemia. Hence, further investigations into drug applications are warranted. Collectively, the findings of the current study further suggest the presence of potential crosstalk among multiple RCDs in ischemic stroke-induced neuroinflammation, such as pan-apoptosis, potentially mediated via multiple mechanisms. 5. Conclusions The CI/RI model in C57BL/6 mice revealed that the cuproptosis-induced inflammatory pathway contributes to CI/RI and that DSF exerts a protective effect on mitochondria while reducing the cerebral infarct size by inhibiting FDX1-mediated protein esterification and HSP70-mediated inflammatory response. The findings of the current study present a novel concept for enhanced mitigation of cerebral blood flow recanalization-induced damage. Declarations Conflict of Interest Declarations of interest: none. Ethics Statement The animal experiment was approved by the Animal Management and Use Committee of the First Affiliated Hospital of Harbin Medical University(NO.2022133) and the management and use of mice are consistent with the relevant guidelines of US National Institutes of Health.the study is reported in accordance with ARRIVE guidelines Funding This work was supported by the Heilongjiang Province Key R&D Program (Grant no. JD22C002 and GA21C005). Author Contribution SY conceived the idea of the study,performed experiments and wrote the manuscript. XL designed the experiments. JY, FJ, XF, JJ and WZ analyzed the data. GL and DZ revised the final manuscript. All authors contributed to the article and approved the submitted version. Data availability statement All data generated or analysed during this study are included in this published article [and its supplementary information files]. References Donnan, G. A., Fisher, M., Macleod, M., & Davis, S. M. Stroke. 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J Mol Neurosci 70, 475–480 (2020). Guo, Q., Ma, M., Yu, H., Han, Y., & Zhang, D. Dexmedetomidine enables copper homeostasis in cerebral ischemia/reperfusion via ferredoxin 1. Annals of medicine, 55(1), 2209735 (2023). Lu, C., Li, X., Ren, Y. & Zhang, X. Disulfiram: a novel repurposed drug for cancer therapy. Cancer Chemother Pharmacol 87, 159–172 (2021). Wright, C., & Moore, R. D.Disulfiram treatment of alcoholism. The American journal of medicine, 88(6), 647–655 (1990). Huang Qiu-yang, Chen Xiao-zhong, Dai Chen, Yu Xiu-Yan, Shen Yan & Lin Zhi-hua. Research progress on the role of disulfiram in inflammation-related diseases. West China Journal of Pharmacy (01),117–122 (2023). Deng, W., Yang, Z., Yue, H., Ou, Y., Hu, W., & Sun, P.Disulfiram suppresses NLRP3 inflammasome activation to treat peritoneal and gouty inflammation. Free radical biology & medicine, 152, 8–17(2020). Bai, Y. et al. Disulfiram blocks inflammatory TLR4 signaling by targeting MD-2. 120, (2023). Li, H., Wang, J., Wu, C., Wang, L., Chen, Z. S., & Cui, W.The combination of disulfiram and copper for cancer treatment. Drug discovery today, 25(6), 1099–1108 (2020). Skrott, Zdenek et al. “Alcohol-abuse drug disulfiram targets cancer via p97 segregase adaptor NPL4.” Nature vol. 552,7684: 194–199 (2017). Lu, Y. Reversal of cisplatin chemotherapy resistance by glutathione-resistant copper-based nanomedicine via cuproptosis. J. Mater. Chem. B (2022). Cumings, J. N. THE COPPER AND IRON CONTENT OF BRAIN AND LIVER IN THE NORMAL AND IN HEPATO-LENTICULAR DEGENERATION. Rongzhu, L. et al. Zinc, Copper, Iron, and Selenium Levels in Brain and Liver of Mice Exposed to Acrylonitrile. Biol Trace Elem Res 130, 39–47 (2009). Lutsenko, S., Bhattacharjee, A. & Hubbard, A. L. Copper handling machinery of the brain. Metallomics 2, 596 (2010). Zhong, C.-C. et al. Copper (Cu) induced changes of lipid metabolism through oxidative stress-mediated autophagy and Nrf2/PPARγ pathways. The Journal of Nutritional Biochemistry 100, 108883 (2022). Zhang Y, Zhou Q, Lu L, et al. Copper Induces Cognitive Impairment in Mice via Modulation of Cuproptosis and CREB Signaling. Nutrients. 15(4):972(2023). Lee, J., Petris, M. J. & Thiele, D. J. Characterization of Mouse Embryonic Cells Deficient in the Ctr1 High Affinity Copper Transporter. Journal of Biological Chemistry 277, 40253–40259 (2002). Tümer, Z. & Møller, L. B. Menkes disease. Eur J Hum Genet 18, 511–518 (2010). Lutsenko S. Dynamic and cell-specific transport networks for intracellular copper ions. Journal of cell science, 134(21), jcs240523 (2021). Petris, M. J. et al. Ligand-regulated transport of the Menkes copper P-type ATPase efflux pump from the Golgi apparatus to the plasma membrane: a novel mechanism of regulated trafficking. The EMBO Journal 15, 6084–6095 (1996). Hung, I. H. et al. Biochemical Characterization of the Wilson Disease Protein and Functional Expression in the Yeast Saccharomyces cerevisiae. Journal of Biological Chemistry 272, 21461–21466 (1997). La Fontaine, S. & Mercer, J. F. B. Trafficking of the copper-ATPases, ATP7A and ATP7B: Role in copper homeostasis. Archives of Biochemistry and Biophysics 463, 149–167 (2007). Washington-Hughes, C. L. et al. Atp7b-dependent choroid plexus dysfunction causes transient copper deficit and metabolic changes in the developing mouse brain. PLoS Genet 19, e1010558 (2023). Percival S. S. Copper and immunity. The American journal of clinical nutrition, 67(5 Suppl), 1064S–1068S(1998). Urani, C., Melchioretto, P., Morazzoni, F., Canevali, C., & Camatini, M. Copper and zinc uptake and hsp70 expression in HepG2 cells. Toxicology in vitro: an international journal published in association with BIBRA, 15(4–5), 497–502(2001). Walsh, J. G., Muruve, D. A. & Power, C. Inflammasomes in the CNS. Nat Rev Neurosci 15, 84–97 (2014). Wang H, Zhong D, Chen H, Jin J, Liu Q, Li G. NLRP3 inflammasome activates interleukin-23/interleukin-17 axis during ischaemia-reperfusion injury in cerebral ischaemia in mice. Life Sciences 227: 101–113(2019). Luo L, Liu M, Fan Y, et al. Intermittent theta-burst stimulation improves motor function by inhibiting neuronal pyroptosis and regulating microglial polarization via TLR4/NFκB/NLRP3 signaling pathway in cerebral ischemic mice. J Neuroinflammation. 19(1):141(2022). Coll, R. C., Schroder, K. & Pelegrín, P. NLRP3 and pyroptosis blockers for treating inflammatory diseases. Trends in Pharmacological Sciences 43, 653–668 (2022). Korn, T., Bettelli, E., Oukka, M. & Kuchroo, V. K. IL-17 and Th17 Cells. Annu. Rev. Immunol. 27, 485–517 (2009). Zhang, J., Mao, X., Zhou, T., Cheng, X. & Lin, Y. IL-17A contributes to brain ischemia reperfusion injury through calpain-TRPC6 pathway in mice. Neuroscience 274, 419–428 (2014). Wang, H. et al. NLRP3 inflammasome activates interleukin-23/interleukin-17 axis during ischaemia-reperfusion injury in cerebral ischaemia in mice. Life Sciences 227, 101–113 (2019). Additional Declarations No competing interests reported. Supplementary Files Exampleoforiginalwesternblot.pdf WB.pdf Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 19 Apr, 2024 Reviews received at journal 13 Apr, 2024 Reviews received at journal 12 Apr, 2024 Reviewers agreed at journal 04 Apr, 2024 Reviewers agreed at journal 03 Apr, 2024 Reviewers invited by journal 03 Apr, 2024 Editor assigned by journal 03 Apr, 2024 Editor invited by journal 02 Apr, 2024 Submission checks completed at journal 26 Mar, 2024 First submitted to journal 09 Mar, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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04:24:14","extension":"pdf","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":995470,"visible":true,"origin":"","legend":"","description":"","filename":"Exampleoforiginalwesternblot.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4052488/v1/dc574ade59f7e327bede38ee.pdf"},{"id":53697897,"identity":"aadcd659-7653-4004-9d40-aaaed09138ae","added_by":"auto","created_at":"2024-03-29 04:24:14","extension":"pdf","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":791333,"visible":true,"origin":"","legend":"","description":"","filename":"WB.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4052488/v1/02768b9abb5d9e95b95373d7.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Disulfiram downregulates ferredoxin 1 to maintain copper homeostasis and inhibit inflammation in cerebral ischemia/reperfusion injury.","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe incidence and mortality rates of cerebral ischemic disease are gradually increasing worldwide. Moreover, cerebral ischemic disease is the most common cause of permanent disability in adults.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e Thrombolysis/thrombolysis is the most effective treatment for ischemic stroke. Since 2015, substantial advancements have been made in research exploring the treatment of acute ischemic stroke, leading to an expanded time window for emergency endovascular intervention, from the initial 6 h to a more extended period of 24 h.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e Following cerebral ischemia, reperfusion of blood flow induces a cascade of injurious effects, commonly referred to as cerebral ischemia/reperfusion injury (CI/RI). This process involves various types of regulated cell death (RCD) and neuroinflammatory responses, considerably contributing to its progression.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e Mitigating the adverse effects of reperfusion has been a persistent focal point for clinicians striving for breakthroughs.\u003c/p\u003e \u003cp\u003eIn living organisms, cells exposed to extreme physicochemical or mechanical stressors may undergo an immediate and uncontrolled structural collapse, a phenomenon known as accidental cell death. Conversely, RCD involves specific signaling cascades and molecular-defined effector mechanisms, encompassing fundamental processes, such as organogenesis and tissue remodeling, eliminating unnecessary structures or cells, and controlling cell numbers. Additionally, RCD can be triggered by exogenous perturbations in the intracellular or extracellular microenvironments.\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e Currently, more than 10 types of RCDs have been identified, encompassing a diverse range of non-apoptotic RCD modalities, such as autophagy, pyroptosis, ferroptosis, and endogenous cell demise, in addition to the conventional apoptotic pathway.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e In contrast to apoptosis, these RCD pathways elicit inflammatory responses within the body and have been increasingly associated with cancer.\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eCI/RI induces a robust inflammatory response initiated by damage-associated molecular patterns (DAMPs) released from injured cells, including signaling molecules, such as adenosine, heat shock proteins (HSPs), high-mobility group protein B1 (HMGB1), and interleukin (IL).\u003csup\u003e\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e In a healthy central nervous system, various types of DAMPs are expressed and released following injury to activate inflammatory signaling pathways.\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e The aforementioned DAMPs use pattern recognition receptors (PRRs) to initiate and augment immune responses.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e Extracellular HSP70 is a typical DAMP.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e PRRs are predominantly expressed in astrocytes and microglia, enabling the detection of pathogen-derived or endogenous ligand release. Toll-like receptors (TLRs) of the PRR family are an example of this phenomenon.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e TLR4 plays a pivotal role and serves as the primary receptor for HMGB1.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e In addition, HSP70 regulates TLR4.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e TLR4 is most commonly associated with the expression and release of IL-1 and tumor necrosis factor-α (TNF-α), and this association typically occurs in a ligand-dependent manner. Nuclear factor (NF)-κB, positioned downstream of the TLR4 signaling pathway, plays a pivotal role in orchestrating immune responses, cellular proliferation, and differentiation. Upon activation, TLR4 induces nuclear translocation of NF-κB(p65), thereby facilitating the expression of diverse inflammatory cytokines, such as IL-1β and TNF-α.\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e The activation of the TLR4/NF-κB signaling pathway facilitates the assembly of a complex between NLRP3 and apoptosis-associated speck-like protein with a CARD (ASC), which subsequently interacts with the cysteine protease caspase-1 to form inflammasomes.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e The classic pyroptotic pathway, mediated by the NLRP3 inflammasome, is pivotal in determining functional outcomes following stroke.\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eRecently, a novel RCD pattern, distinct from the well-known RCD pattern, has been identified and designated as \"cuproptosis.\u0026rdquo; This unique mode of cell death relies on the copper (Cu)-mediated targeting of lipoacylated tricarboxylic acid (TCA) cycle proteins and is strongly associated with mitochondrial respiration.\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e FDX1 converts Cu (II) to the highly toxic Cu (I), resulting in the aggregation of fatty acylated proteins, exhaustion of iron-sulfur cluster proteins, HSP70 activation, and induction of intracellular toxic oxidative stress, ultimately causing cell death. Importantly, this process is associated with mitochondrial respiration. A meta-analysis has revealed that serum Cu levels are substantially elevated during the acute phase of stroke.\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e Recent evidence suggests that Cu ions participate in various transformation mechanisms that damage brain tissue during fusion injury.\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e The induced cuproptosis activates HSP70 to initiate an immune inflammatory response.\u003c/p\u003e \u003cp\u003eDisulfiram (DSF) is a well-established anti-alcoholic medication scientifically validated for its safety and potential in targeted tumor therapy.\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e Recent research has demonstrated the potential of DSF in inhibiting multiple inflammatory reactions and regulating inflammation-related targets, highlighting its potential as an anti-inflammatory agent.\u003csup\u003e\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e The DSF-Cu complexes were found to be safe and efficacious in the prevention and treatment of various types of cancers.\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e The inclusion of DSF in the Cu carrier category aimed to enhance the induction of apoptosis in tumor cells.\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e Recently, Cu (II) bis(diethyldithiocarbamate), a potent anticancer agent, has been identified as a bioactive metabolite of alcohol abuse.\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e The administration of this drug was shown to reduce the expression of an upstream regulator involved in mitochondrial proteolipid acylation during cuproptosis.\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e Further comprehensive investigations are warranted to ascertain the potential of DSF in ameliorating CI/RI damage and to elucidate the underlying mechanisms. Therefore, in the present study, we aimed to validate the effect of DSF on CI/RI and elucidate the underlying molecular mechanisms. We hypothesized that DSF could modulate Fdx1-induced CI-RI damage and inhibit inflammatory responses. We believe that the findings of the current study would further unravel the crosstalk relationship between each death mechanism and provide new targets for drug application and treatment of CI-RI.\u003c/p\u003e"},{"header":"2. Experimental methods","content":" \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Animals and tMCAO models\u003c/h2\u003e \u003cp\u003eMale C57BL/6 mice (6\u0026ndash;8 weeks old, weight 20\u0026thinsp;\u0026plusmn;\u0026thinsp;3 g) were procured from Liaoning Changsheng Biotechnology Co., Ltd and housed in a hygienic and comfortable habitat under a 12-h natural light-dark cycle to simulate their circadian rhythm. The Animal Experiment Center of Harbin Medical University provided adequate food and water. The animals were randomly divided into four groups (n\u0026thinsp;=\u0026thinsp;4/group): sham group, 24h-tMCAO group, DSF\u0026thinsp;+\u0026thinsp;24h-tMCAO group, and vehicle\u0026thinsp;+\u0026thinsp;24h-tMCAO (dimethyl sulfoxide\u0026thinsp;\u0026lt;\u0026thinsp;2%). The DSF\u0026thinsp;+\u0026thinsp;24h-tMCAO group was intraperitoneally administered DSF (50 mg/kg, twice daily), starting from the day before the surgery and continuing postoperatively. The mice were anesthetized with 2% isoflurane, and body temperature was maintained at ~\u0026thinsp;37\u0026deg;C throughout the surgical and postoperative periods using a servo-controlled heating blanket. Focal cerebral ischemia was achieved by occluding the middle cerebral artery (MCA). After performing a midline incision in the neck, a doccol suture (602256PK5Re; US) was inserted into the right external carotid artery and then reversed into the right internal carotid artery via the common carotid artery to occlude the blood supply to the MCA. The sutures were then removed after 1 h.Following suture removal, the mice exhibited recovery times of 6 h, 24 h, 3 d, 5 d and 7 d.Mice died of cervical dislocation after anesthesia at the above time points. This experiment was reviewed and approved by the ethical review committee of Harbin Medical University. (IACUU NO.2022133).The management and use of mice are consistent with the relevant guidelines of US National Institutes of Health.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 2,3,5-Triphenyltetrazolium chloride (TTC) staining\u003c/h2\u003e \u003cp\u003eBriefly, the harvest mouse brain was sliced into seven pieces (1 mm thick) from the rostral end of the frontal lobe. Cells were then incubated in TTC (Solarbio, Beijing, China), shielded from light for 30 min, and fixed in 4% paraformaldehyde. Planar infarct volume measurements were performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Hematoxylin-eosin and Nissl stainings\u003c/h2\u003e \u003cp\u003eThe specimens were fixed in a 4% paraformaldehyde solution and subsequently embedded in paraffin. Histological sections (4 \u0026micro;m thick) were prepared and stained with hematoxylin-eosin. For Nissl staining, a 1% toluidine blue solution was heated to 60\u0026deg;C, followed by the immersion of all sections for 40 min. Subsequently, sections were washed with 70% ethanol and rapidly differentiated using 95% ethanol. The tissue sections were examined and imaged under a light microscope (Nikon, Tokyo, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Immunofluorescent staining\u003c/h2\u003e \u003cp\u003eBriefly, the prepared brain tissues were sequentially immersed in a 4% paraformaldehyde solution and 30% sucrose solution for 48 h. After embedding in optimal cutting temperature compound and freezing, frozen blocks were sectioned (7-\u0026micro;m thick slices) using a freezing microtome. Sections were treated with a blocking solution (Beyotime, China) containing Triton X-100 for 20 min and incubated overnight at 4\u0026deg;C with the following primary antibodies: ASC (sc-514414, 1:50 dilution, Santa Cruz, USA), Caspase-1 (sc-56036, 1:50 dilution, Santa Cruz, USA), GSDMD (sc-393581, 1:50 dilution, Santa Cruz, USA), HSP70 (T55496, 1:50 dilution, Abmart, China). Following hydrations with PBST (3\u0026times;), the samples were incubated with the appropriate fluorophore-conjugated secondary antibody (Boster, China) for 1 h. Nuclei were stained with 30\u0026micro;L DAPI (Abcam, UK) to ensure no bubbles. After 10 min, the slides were examined using light microscopy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Immunohistochemical staining\u003c/h2\u003e \u003cp\u003eThe fixed brain tissues were embedded in paraffin, sectioned, deparaffinized, and subjected to antigen retrieval using a citrate solution after blocking with 3% hydrogen peroxide. The sections were then blocked with fetal bovine serum for 1 h. Subsequently, the sections were incubated overnight at 4\u0026deg;C with primary antibodies against FDX1 (T510671,1:200 dilution, Abmart, China), DLST (TD13671,1:200 dilution, Abmart, China), ATP7A (PA7106, 1:200 dilution, Abmart, China), ATP7B (TA0410, 1:200 dilution, Abmart, China), Caspase-1 ( 22915-1-AP, 1:200 dilution, Proteintech, USA), and IL-18 (10663-1-AP, 1:200 dilution, Proteintech, USA), followed by subsequent incubation with secondary antibodies (Boster, China) for 1 h at room temperature. Color development was achieved using diaminobenzidine. Then, sections were stained with hematoxylin. Subsequently, the sections were dehydrated and sealed before imaging under a light microscope at 200\u0026times; magnification. The acquired images were analyzed using ImageJ software to determine the average density within the observation area.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e2.6 Cu level detection\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eCu levels were detected using colorimetric quantitative kits provided by Nanjing Jiancheng BioEngineering Institute (Jiangsu, China). The right brain tissue was measured, followed by the addition of double-distilled water at a ratio of 1:9 and supernatant collection by ultrasonic centrifugation. For protein quantification, the absorbance (580 nm) was measured using a microplate reader (MD, Shanghai, China) after the sequential addition of reagents.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Western blot analysis\u003c/h2\u003e \u003cp\u003eBrain tissue from the right hemisphere was lysed (RIPA: protease inhibitor: phosphatase inhibitor, 100:1:1; lipoacylated protein was added in an equal proportion of TCEP). To determine the sample protein concentration, the supernatant was denatured by boiling, using the BCA protein assay kit (Beyotime, China). Proteins were subjected to sodium dodecyl-sulfate polyacrylamide gel electrophoresis and subsequently transferred onto polyvinylidene fluoride membranes using the sandwich method. Then, the membranes were blocked with 5% skim milk for 1 h at room temperature, followed by overnight incubation with primary antibodies at 4\u0026deg;C in a refrigerator. The membranes were incubated with goat anti-mouse and anti-rabbit secondary antibodies (Abmart, China) at room temperature for 60 min. Next, the membranes were washed with Tris-buffered saline with 0.1% Tween\u0026reg; 20 detergent and incubated with an enhanced chemiluminescence reagent (Biosharp, China) for detection. The primary antibodies employed were as follows: GSDMD (EPR20859, 1:1000, Abcam, UK); Caspase-1(22915-1-AP, 1:1000 dilution, Proteintech, USA), ASC (sc-514414, 1:500, Santa Cruz, USA),IL-1β(RM1009, 1:1000, Abcam, UK), IL-18 (10663-1-AP, 1:1000 dilution, Proteintech, USA), IL-17(26163-1-AP, 1:1000, Proteintech,USA),TLR4(66350-1-Ig, 1:1000dilution, Proteintech, USA),NLRP3(EPR23073-96, 1:1000, Abcam, UK),TNFα(60291-1-Ig, 1:1000 dilution, Proteintech, USA), LIAS(67298-1-Ig, 1:1000 dilution, Proteintech, USA), NF-κB(66535-1-Ig, 1:1000 dilution, Proteintech, USA) and SLC31A1 (CTR1), ATP7B (TA0410, 1:1000 dilution, Abmart, China), HSP70(T55496, 1:1000 dilution, Abmart, China), FDX1(T510671, 1:1000 dilution, Abmart, China), SDHB(EPR10880, 1:50000, Abcam, UK), DLAT(T58125, 1:1000 dilution, Abmart, China), and DLST (TD13671, 1:1000 dilution, Abmart, China). For protein bands, gray values were quantified using ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Transmission electron microscopy\u003c/h2\u003e \u003cp\u003eBrain tissues were cut into cubes (\u0026le;\u0026thinsp;1 mm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e) in 2.5% glutaraldehyde solution. After fixation with osmium tetroxide, tissue samples were subjected to repeated dehydration using acetone, followed by the addition of resin overnight at room temperature. The resulting tissue sections (70 nm) were stained with uranyl acetate and lead citrate and dried for transmission electron microscopy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Statistical analyses\u003c/h2\u003e \u003cp\u003eAll results are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean. Data were analyzed using analysis of variance, followed by Tukey's post-hoc test. Statistical analyses were performed using GraphPad Prism version 9.5 (GraphPad Software, Inc., Boston, MA, USA). Statistical significance was set at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.1FDX1 expression is up-regulated in the cerebral infarction area after tMCAO.\u003c/h2\u003e \u003cp\u003eTo explore the relationship between FDX1 and CI/RI, FDX1 protein was determined at different time points after tMCAO. WB showed that FDX1 protein expression reached the peak at 24h and then gradually decreased compared with Sham group.(Fig.\u0026nbsp;1A)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.2 DSF attenuates the cerebral infarction volume and mitigates neuronal damage following tMCAO\u003c/h2\u003e \u003cp\u003eTTC staining confirmed the DSF-mediated neuroprotective effect, reducing CI/RI. Compared with the 24h-tMCAO group, the DSF\u0026thinsp;+\u0026thinsp;24h-tMCAO group exhibited a significant decrease in the infarct volume. (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;2A-B) The microstructural characteristics of the brain tissue were observed following tMCAO. Hematoxylin and eosin staining revealed that tMCAO damaged the area of cerebral infarction and the surrounding tissues. The nerve cell stroma appeared loose and edematous, whereas neuronal nuclei displayed pyknosis and hyperchromatism. Additionally, vacuolation was observed in the neurodermal membrane, along with gliosis. In the DSF\u0026thinsp;+\u0026thinsp;24 h-tMCAO group, pyknosis and hyperchromatism of neurons were observed in the infarct area, while neuropil vacuolation in the infarct focus was alleviated, with a reduction in the number of unidentified structures and glial cells surrounding the infarct (Fig.\u0026nbsp;2C). Based on Nissl staining, Nissl bodies disappeared in neurons after CI/RI, and a large number of damaged neurons with karyopyknosis and dark staining were observed in and around the infarct area. DSF administration preserved normal neuronal morphology in most mice, with most cells displaying visible Nissl bodies. Only a small number of injured neurons with pyknosis and dark staining were observed around the infarction (Fig.\u0026nbsp;2D).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.3 DSF modulates key factors associated with cuproptosis\u003c/h2\u003e \u003cp\u003eFunctional proteins associated with cuproptosis were determined by western blotting. Induction of tMCAO increased the expression levels of SLC31A1/CTR1, ATP7B, FDX1, and HSP70. Additionally, tMCAO enhanced the levels of crucial proteins associated with the TCA cycle (LIAS, DLAT, DLST, and PDHB). Notably, administration of DSF mitigated the fluctuations observed in these protein levels, SDHB fluctuations are not significant. (Fig.\u0026nbsp;3A-B). Immunohistochemical staining with FDX1, DLST, ATP7A, and ATP7B antibodies revealed that the positive rate of antigen and antibody reaction in DSF\u0026thinsp;+\u0026thinsp;24h-tMCAO group was lower than that in 24h-tMCAO group. (Fig.\u0026nbsp;4A) Based on the results of the semi-quantitative analysis, the expression of the aforementioned proteins was statistically significant. (Fig.\u0026nbsp;4B). Immunofluorescence staining revealed the upregulation of HSP70 expression following tMCAO; however, treatment with DSF mitigated the IR-induced elevation in protein expression DSF regulates the expression cuproptosis-related proteins. (Fig.\u0026nbsp;5A-B)\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.4 DSF inhibits the activation of the HSP70/TLR-4 /NLRP3 signaling pathway and reduces inflammatory cytokines after tMCAO\u003c/b\u003e \u003c/p\u003e \u003cp\u003eDAMPs can activate TLR4 to trigger downstream signaling pathways, and HSP70 is an important endogenous TLR4 agonist. Based on the western blotting analysis, the 24h-tMCAO group showed increased protein expression levels of TLR-4, NF-κB, NLRP3, TNF-α, ASC, Pro-caspase-1, Cleaved-caspase-1, GASDMD-N, IL-18, IL-1β, and IL-17 when compared with the sham group. However, the DSF-treated group exhibited downregulated protein expression when compared with the control group(Fig.\u0026nbsp;5A-B). Moreover, immunofluorescence staining revealed that the expression of ASC, caspase-1, and GASDMD were upregulated following tMCAO; treatment with DSF could alleviate the reperfusion injury-induced increase in protein expression (Fig.\u0026nbsp;6A-B). Immunohistochemical staining with IL-18 antibody showed that treatment with DSF decreased positive staining in the 24h-tMCAO group compared with that in the 24h-tMCAO group. Based on the results of the semi-quantitative analysis, the expression of the aforementioned proteins was statistically significant in the 24h-tMCAO group compared with that in the DSF\u0026thinsp;+\u0026thinsp;24h-tMCAO group (Fig.\u0026nbsp;4A-B).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.5 DSF did not induce Cu retention and conferred mitochondrial protection following tMCAO\u003c/h2\u003e \u003cp\u003eThe impact of DSF on Cu levels during CI/RI was assessed by measuring Cu ions. Following CI/RI, the total amount of Cu ions was increased in the 24h-tMCAO group when compared with that in the sham group, with no further increase observed following DSF administration (Fig.\u0026nbsp;7B). The instability of Cu metabolism leads to mitochondrial morphological damage and dysfunction. Transmission electron microscopy was performed to examine morphological alterations in mitochondria in the brain tissue. The control group exhibited intact mitochondrial membranes and aligned cristae. However, the induction of tMCAO resulted in substantial mitochondrial swelling; most matrixes in the mitochondrial membrane appeared less intense, with a broken crest, which disappeared. The rough endoplasmic reticulum exhibited dilation, degranulation, and vacuolation. The mitochondria in the DSF\u0026thinsp;+\u0026thinsp;24h-tMCAO group exhibited intact morphology, suggesting that DSF could suppress mitochondrial fission and preserve mitochondrial structural integrity (Fig.\u0026nbsp;7A).\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eHerein, our findings suggest that DSF can exert inhibitory effects on the inflammatory response and cell death induced by CI/RI via the modulation of the TLR4/NLRP3/NF-κB pathway and FDX1/TCA cycle, thereby affording a neuroprotective role. Treatment with DSF alone could suppress the expression of FDX1/TCA cycle-related proteins and the HSP70/TLR4/NLRP3 pathway during CI-RI to protect nerves and inhibit inflammation.\u003c/p\u003e \u003cp\u003eCu plays an indispensable role in the physiological processes of the central nervous system. The disruption of Cu homeostasis can exert a multitude of detrimental effects on brain development and function. Compared with other organs, the brain exhibits the highest Cu concentration, second only to the liver. Notably, the average Cu content in the globus pallidus surpasses that in the liver.\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e As a cofactor, Cu plays an indispensable role as a reducing agent in the catalytic activity of peptidyl-glycine alpha-amide monooxygenase, dopamine β-monooxygenase, Cu-zinc (Zn) superoxide dismutase, and ceruloplasmin.\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e The presence of Cu is crucial for catecholamine biosynthesis, neuropeptide activation, and the regulation of mitochondrial function.\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e However, excess Cu induces lipid deposition through oxidative stress, mitochondrial dysfunction,\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e promotes neurodegenerative changes, and cell death.\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e Cu transporters SLC31A1 (CTR1), ATP7A, and ATP7B are known to regulate the Cu content in cellular compartments and maintain Cu homeostasis. CTR1, a master regulator of Cu uptake, acts on the plasma membrane, and the genetic inactivation of CTR1 results in Cu deficiency in cells.\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e In the choroid plexus (ChPl), Cu flows from the epithelial cells of the ChPl into the cerebrospinal fluid (CSF). In patients with Menkes disease, ATP7A inactivation is known to cause Cu deficiency in the brain, numerous metabolic abnormalities (including catecholamine imbalance), delayed neurodevelopment, and death during early childhood.\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e Generally, ATP7B transports Cu from the cytosol into the lumen of the secretory pathway for incorporation into Cu-dependent enzymes and traps excess Cu in vesicles for further export from the cell. The precise impact of these activities on cellular metabolism depends on the specific cell type and can exhibit significant variation across different tissues.\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e Therefore, the negative effects of ATP7B inactivation on brain metabolism are evident in Wilson\u0026rsquo;s disease. Copper-induced protein transport has been described as a key feature of copper-ATPase, and the central link of this mechanism is the transfer of ATP7A and ATP7B from TGN to peripheral or cytoplasmic vesicles, respectively. The displacement of these proteins is specifically triggered by elevated levels of their own ligand, Cu (I), not Cu (II), and is intended to efflux to restore intracellular copper levels.\u003csup\u003e\u003cspan additionalcitationids=\"CR43\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e FDX1 helps convert Cu (II) to the more toxic Cu (I), and in our study, DSF inhibited FDX1 expression, reduced Cu(I) levels, and reduced transport of ATP7A and ATP7B. Interestingly, dysregulation of the Cu transport mechanism in the choroid plexus of Atp7b -/- mice has been demonstrated in recent studies; these mice exhibited low Cu levels in the brain at 4 weeks postnatally, emphasizing the crucial role of ATP7B in Cu accumulation.\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e In summary, the inhibition of CTR1 could regulate the cuproptosis pathway by affecting Cu uptake. It is yet to be established whether the upregulation of ATP7A and ATP7B expression following CI-RI serves as a protective mechanism by promoting Cu efflux or accumulation. However, inhibition of the dual-flow ring can explain the lack of decrease in total Cu levels after DSF administration, and this mechanism aligns with the anticancer effects of Cu ionophores. This discrepancy can be partly attributed to the limited understanding of the complex mechanisms of action of ATP7A and ATP7B in the brain.(Fig.\u0026nbsp;8)\u003c/p\u003e \u003cp\u003eThe immune system relies on Cu to execute certain functions. Recent studies have demonstrated that even in the presence of a marginal deficiency, Cu contributes to a decrease in IL concentrations. Neutrophils accumulate Cu as they differentiate into more mature cell populations, and this accumulation is not reflected by an increase in Cu/Zn superoxide dismutase or cytochrome c oxidase activity.\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e HSP70 is induced by Cu exposure.\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e TLR4 is a PRR that recognizes specific DAMPs such as HSP70, which, in turn, triggers TLR4-mediated inflammatory responses. Thus, HSP70 in the CSF may function as a neuroinflammatory mediator.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e The inflammasome protein complex has recently emerged as a pivotal component of the innate immune response during ischemic stroke.\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e NLRP3 has been extensively studied in central nervous system diseases.\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e The activation of the NLRP3 inflammasome necessitates TLR4-mediated activation of the p65 subunit within the downstream NFκB pathway.\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e Subsequently, NLRP3 binds to ASC to form a complex, and Caspase-1 specifically cleaves GSDMD, releasing the N-terminus from its self-inhibitory C-terminus. GSDMD-N binds to lipids to form non-selective pores, resulting in cell membrane rupture and promoting the release of a large number of inflammatory factors, ultimately triggering pyroptosis.\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e With the maturation and efflux of IL-18 and IL-1β, Th17 cells and γδT cells release interleukin-17 (IL-17), an important factor in the adaptive immune system.\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e Several studies have shown that IL-17 is associated with the pathogenesis of cerebral ischemia-reperfusion. Loss of γδT cells alleviates brain tissue damage after ischemia-reperfusion, and IL-17 positive lymphocytes are also detected in brain tissue validated in the field of stroke patients.\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e The activated NLRP3 inflammasome promotes the activation of IL-17 in CI/RI.\u003csup\u003e54\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThe anti-inflammatory efficacy of DSF has been substantiated in numerous studies. However, its role in CI-RI remains elusive. In the current study, we found that the association between CI/RI and Cu-induced cell death was accompanied by an enhanced inflammatory response (classic pyroptosis), which was mediated via the HSP70/TLR4/NLRP3 pathway. Importantly, the administration of DSF effectively inhibited this pathway to alleviate CI/RI. Our findings suggest that DSF exhibits robust safety as a well-established pharmaceutical employed in clinical settings for an extensive duration. This finding challenges the conventional belief that DSF, in conjunction with Cu ionophores, forms a complex that effectively treats cancer and induces apoptosis, specifically in cancer cells. However, conventional copper chelators hamper angiogenesis, impeding brain tissue recovery following ischemia. Hence, further investigations into drug applications are warranted. Collectively, the findings of the current study further suggest the presence of potential crosstalk among multiple RCDs in ischemic stroke-induced neuroinflammation, such as pan-apoptosis, potentially mediated via multiple mechanisms.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eThe CI/RI model in C57BL/6 mice revealed that the cuproptosis-induced inflammatory pathway contributes to CI/RI and that DSF exerts a protective effect on mitochondria while reducing the cerebral infarct size by inhibiting FDX1-mediated protein esterification and HSP70-mediated inflammatory response. The findings of the current study present a novel concept for enhanced mitigation of cerebral blood flow recanalization-induced damage.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of Interest\u003c/h2\u003e \u003cp\u003eDeclarations of interest: none.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eEthics Statement\u003c/h2\u003e \u003cp\u003e The animal experiment was approved by the Animal Management and Use Committee of the First Affiliated Hospital of Harbin Medical University(NO.2022133) and the management and use of mice are consistent with the relevant guidelines of US National Institutes of Health.the study is reported in accordance with ARRIVE guidelines\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by the Heilongjiang Province Key R\u0026amp;D Program (Grant no. JD22C002 and GA21C005).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eSY conceived the idea of the study,performed experiments and wrote the manuscript. XL designed the experiments. JY, FJ, XF, JJ and WZ analyzed the data. GL and DZ revised the final manuscript. All authors contributed to the article and approved the submitted version.\u003c/p\u003e\u003ch2\u003eData availability statement\u003c/h2\u003e \u003cp\u003eAll data generated or analysed during this study are included in this published article [and its supplementary information files].\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDonnan, G. A., Fisher, M., Macleod, M., \u0026amp; Davis, S. M. Stroke. Lancet (London, England), 371(9624), 1612\u0026ndash;1623 (2008).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXiaochuan Huo and Gaofeng. Chinese guidelines for endovascular treatment of acute ischemic stroke 2023. 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Neuroscience 274, 419\u0026ndash;428 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, H. \u003cem\u003eet al.\u003c/em\u003e NLRP3 inflammasome activates interleukin-23/interleukin-17 axis during ischaemia-reperfusion injury in cerebral ischaemia in mice. Life Sciences 227, 101\u0026ndash;113 (2019).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4052488/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4052488/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn the current study, we aimed to investigate whether disulfiram (DSF) plays a neuroprotective role in cerebral ischemia-reperfusion (CI-RI) injury by regulating ferredoxin 1 (FDX1) by modulating copper ion (Cu) levels and inhibiting the inflammatory response. To simulate CI-RI, a transient middle cerebral artery occlusion (tMCAO) model in C57/BL6 mice was employed. Mice were administered with or without DSF before and after tMCAO. Changes in infarct volume after tMCAO were observed using TTC staining. Nissl staining and hematoxylin-eosin (he) staining were used to observe the morphological changes of nerve cells at the microscopic level. FDX1 is the main regulatory protein of copper death, and the occurrence of copper death will lead to the increase of HSP70 stress and inflammatory response. Cuproptosis-related proteins and downstream inflammatory factors were detected by western blotting, immunofluorescence staining, and immunohistochemistry. The content of copper ions was detected using a specific kit, while electron microscopy was employed to examine mitochondrial changes. We found that DSF reduced the cerebral infarction volume, regulated the expression of cuproptosis-related proteins, and reduced FDX1 expression without inducing Cu accumulation. Moreover, DSF inhibited the HSP70/TLR-4/NLRP3 signaling pathway. Collectively, DSF could regulate Cu homeostasis by inhibiting FDX1, acting on the HSP70/TLR4/NLRP3 pathway to alleviate CI/RI. Accordingly, DSF could mitigate inflammatory responses and safeguard mitochondrial integrity, yielding novel therapeutic targets and mechanisms for the clinical management of ischemia-reperfusion injury.\u003c/p\u003e","manuscriptTitle":"Disulfiram downregulates ferredoxin 1 to maintain copper homeostasis and inhibit inflammation in cerebral ischemia/reperfusion injury.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-29 04:24:08","doi":"10.21203/rs.3.rs-4052488/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-04-19T09:59:20+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-04-13T19:07:05+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-04-12T21:03:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"b60d8bbe-564a-4702-bf74-75386b752b2c","date":"2024-04-04T13:55:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"5746bb26-91b0-4db3-8b99-96f03b1291d0_SNPRID","date":"2024-04-03T18:34:47+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-04-03T17:52:18+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-04-03T11:01:03+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-04-02T20:45:22+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-03-26T13:09:33+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-03-09T06:24:16+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a5aac3c5-558c-4702-9d83-b64f18b86931","owner":[],"postedDate":"March 29th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":29907318,"name":"Health sciences/Diseases/Neurological disorders/Cerebrovascular disorders"},{"id":29907319,"name":"Health sciences/Diseases/Neurological disorders/Hypoxic ischaemic encephalopathy"},{"id":29907320,"name":"Health sciences/Diseases/Neurological disorders/Stroke"}],"tags":[],"updatedAt":"2024-06-14T12:30:08+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-29 04:24:08","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4052488","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4052488","identity":"rs-4052488","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}