Inhibition of Nrf2/HO-1 signaling pathway exacerbates Copper homeostasis in cerebral ischemia

Inhibition of Nrf2/HO-1 signaling pathway exacerbates Copper homeostasis in cerebral ischemia | 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 Research Article Inhibition of Nrf2/HO-1 signaling pathway exacerbates Copper homeostasis in cerebral ischemia Ting Lang, Yanhao Liao, Bingbin Fan, Huixian Chen, Junjie Hu, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6650982/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Ischemic stroke is the third leading cause of death in the world, and oxidative stress is the key pathological mechanism of cerebral ischemia. The Nrf2/HO-1 signaling pathway is an endogenous defense system that regulates oxidative stress in the brain, and is involved in the imbalance regulation of copper homeostasis. The brain is one of the main target organs for copper accumulation, and copper is involved in the formation of myelin sheaths and the regulation of synaptic activity and signaling cascades. Imbalance of copper homeostasis leads to metabolic abnormalities and toxic effects on cells, and the mechanisms involved in cerebral ischemic injury are unknown. In this study, we found for the first time that the expression of copper homeostatic imbalance markers was elevated after cerebral ischemia, and the silencing of Nrf2 promoted the further elevation of copper homeostatic imbalance-related markers, which suggests that copper homeostatic imbalance is involved in the pathological process of cerebral ischemic neurological injury and that Nrf2/HO-1 signaling pathway is closely related to copper homeostatic imbalance in cerebral ischemia. Our study provides an experimental basis for further investigation of cuproptosis in cerebral ischemia. cerebral ischemia copper homeostatic imbalance Nrf2/HO-1 signaling pathway oxidative stress Nrf2 FDX1 LIAS HO-1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Highlights MCAO promotes brain damage with increased expression of copper homeostatic imbalance markers. OGD promotes primary neuronal cell injury and increases expression of copper homeostatic imbalance markers. Elesclomol exacerbates neuronal cell damage and copper homeostatic imbalance through Nrf2/HO-1 signaling pathway. Silencing Nrf2 exacerbates oxidative stress and inhibits the Nrf2/HO-1 signaling pathway in OGD primary neuronal cells. Silencing Nrf2 exacerbates copper homeostatic imbalance in OGD neuronal cells. 1. Introduction With the aging of the population, stroke has become a heavy global health and economic burden, with ischemic stroke as the main type of stroke[ 1 ]. Copper is involved in important physiological processes in the body, such as energy metabolism, mitochondrial respiration, enzyme synthesis and oxidative stress. Under physiological conditions, copper metabolism in the body is tightly regulated to keep copper concentration in dynamic balance, a process known as copper homeostasis[ 2 , 3 ]. The brain is a target organ for copper accumulation, and copper ions can be distributed to different brain regions through the blood-brain barrier[ 4 , 5 ], and the physiological state and function of neurons are regulated by copper. It is found that the imbalance of copper homeostasis is closely related to the development of neurological diseases[ 6 ], such as cerebral ischemia[ 7 , 8 ], Alzheimer's disease[ 9 ], and Parkinson's disease. Bioinformatics software such as R software, Gene Ontology (GO) and Genes and Genomes (KEGG) were used to identify 10 copper metabolism-related genes in cerebral ischemia, including key genes such as FDX1 and LIAS[ 10 ]. Another study showed that the neuroprotective effect of disulfiram (DSF) in the treatment of cerebral ischemia is closely related to FDX1, a key protein in the regulation of copper homeostatic imbalance[ 11 ]. It has been shown that co-culturing neuron-like differentiated cells with copper ions for 48 hours induced an accumulation of intracellular phosphorylated alpha-synuclein, similar to that which occurs in the pathogenesis of Parkinson’s disease(PD)[ 12 ]. In addition, autopsy studies have shown that copper levels are significantly elevated in the brains of patients with Alzheimer's disease (AD)[ 13 ]. It was found that intra-striatal injection of Cu 2+ in the brain leads to increased oxidative stress in the striatum and substantia nigra, and that copper does not cause neurodegeneration, but may be involved in the Parkinson's disease pathogenesis through enhanced oxidative stress[ 14 ]. Copper overload can generate large amounts of ROS through the Fenton reaction, which reduces intracellular reduced glutathione (GSH) content and leads to increased cellular sensitivity to noxious stimuli[ 15 ]. The Nrf2/HO-1 signaling pathway is an important intracellular antioxidant signaling pathway. Cerebral ischemia and hypoxia stimulate the release of excessive ROS, prompting Nrf2 to enter the cell nucleus, and through the activation of intracellular detoxification enzymes and antioxidant proteins, thus exerting antioxidant, anti-inflammatory, and maintenance of mitochondrial homeostasis and other effects[ 16 , 17 ]. It was found that copper induced oxidative stress and activated Nrf2 signaling pathway[ 18 , 19 ]. In this project, we first clarified whether copper homeostatic imbalance in cerebral ischemic nerve cells exists in the pathological process of cerebral ischemic nerve injury, and further explored the relationship between the Nrf2/HO-1 signaling pathway and cerebral ischemic copper homeostatic imbalance. We aimed to provide new directions and new ideas for clinical treatment of ischemic stroke. 2. Materials and methods 2.1 Animals Hunan University of Chinese Medicine (SCXK2019-0004) provided SD rats weighing 260–300 g,10–12 weeks old. The rats were provided with sufficient water and normal food. Animal experiments were conducted in strict accordance with the regulations of the Animal Ethics Committee (Certificate No. ZYFY20230920005)(All programs meet the ARRIVE guideline 2.0). 2.2 Experimental designs (a)Animal experiment I : Twenty four healthy SD rats were randomly divided into 4 groups. (a) Sham operation group (Sham group): normal SD rats, only the common carotid artery was isolated without bolus insertion. (b) Middle cerebral artery ischemia 6h group (MCAO 6h): ischemia was performed for 6h according to the above cerebral ischemia model. (c) Middle cerebral artery ischemia 12h group (MCAO 12h): ischemia was performed for 12h according to the above cerebral ischemia model. (d) Middle cerebral artery ischemia 24h group (MCAO 24h): ischemia was performed for 24h according to the above cerebral ischemia model. (b)Cell experiment I : The effect of different OGD time periods on cuproptosis of primary nerve cells was affected by oxygen glucose deprivation of primary nerve cells, and the same group of cells was divided into four groups according to different oxygen and sugar deprivation times: control group; oxygen glucose deprivation 1h (OGD 1h) group; oxygen glucose deprivation 2h (OGD 2h) group; oxygen glucose deprivation 3h (OGD 3h) group. Each group was given the following treatments: Control group: same treatment as OGD steps except no oxygen glucose deprivation incubation; OGD1h, 2h and 3h groups had the same steps except different treatment of oxygen glucose deprivation time. Three parallel control wells were set up in each group, and the experiment was repeated three times. (c) Cell experiment II : the intervention dose of Ele. The same batch of cells was divided into 6 groups: control group; oxygen glucose deprivation (OGD) group; oxygen glucose deprivation + Ele6.25 nmol/mL (OGD + Ele6.25 nmol/mL) group; oxygen glucose deprivation + Ele12.5 nmol/mL (OGD + Ele12.5 nmol/mL) group; oxygen glucose deprivation + Ele18.75nmol/mL (OGD + Ele18.75nmol/mL) group; oxygen glucose deprivation + Ele25nmol/mL (OGD + Ele25nmol/mL). Each group was given the following treatments: Control group: same steps as OGD except no oxygen sugar deprivation incubation; OGD group: oxygen sugar deprivation for 2h; OGD + Ele6.25, 12.5, 18.75, and 25nmol/mL groups were given the same steps as the OGD group except different concentrations of Ele were added during the first 24h of OGD. Three parallel control wells were set up in each group and the experiment was repeated three times. (d) Cell experiment III : Effect of Nrf2/HO-1 signaling pathway on cuproptosis of primary neuronal cells after OGD injury. The same batch of cells was divided into 4 groups: control; oxygen glucose deprivation (OGD) group; oxygen glucose deprivation + Ele6.25nmol/mL (OGD + Ele6.25nmol/mL) group; oxygen glucose deprivation + Ele12.5nmol/mL (OGD + Ele12.5nmol/mL) group. Each group was given the following treatments: Control group: same steps as OGD except no oxygen sugar deprivation incubation; OGD group: oxygen sugar deprivation for 2h; OGD + Ele6.25 and 12.5nmol/mL groups were the same as the OGD group except different concentrations of Ele were added at 24h before OGD. Three parallel control wells were set up in each group and the experiment was repeated three times. (e) Cell experiment IV : the effect of silencing Nrf2 on cuproptosis after OGD injury in primary neuronal cells. The same batch of cells was divided into 5 groups: control + negative control (Control + NC) group; oxygen sugar deprivation + negative control (OGD + NC) group; oxygen sugar deprivation + negative control + Ele12.5nmol/mL (OGD + NC + Ele12.512nmol/mL ) group; oxygen glucose deprivation + Si-Nrf2 (OGD + Si-Nrf2) group; oxygen glucose deprivation + Si-Nrf2 + Ele12.5nmol/mL (OGD + Si-Nrf2 + Ele12.5nmol/mL) group. Each group was given the following treatments: Control + NC group: same treatments as OGD steps except no oxygen sugar deprivation incubation; OGD + NC group: negative control vector transfected 72h before oxygen sugar deprivation, then oxygen sugar deprivation for 2h; OGD + NC + Ele12.5nmol/mL group: same treatments as OGD steps except 12.5nmol/mL Ele added at 24h before OGD treatment was the same as the OGD step; OGD + Si-Nrf2 group: the rest of the treatment was the same as the OGD group step, except for the transfer of Si-Nrf2 72h before OGD; OGD + Si-Nrf2 + Ele12.5nmol/mL group: the rest of the treatment was the same as the OGD steps. Three parallel control wells were set up in each group and the experiment was repeated three times . All the experimental plans above were approved by the Center for Medical Innovation and Experimentation of Hunan University of Traditional Chinese Medicine. 2.3 MCAO rat model Rats were anesthetized by taking an intraperitoneal injection of sodium pentobarbital (60mg·kg − 1 ). The right carotid artery was isolated.4 − 0 medical silk nonabsorbable sutures were used to ligate the external carotid and common carotid arteries. The internal carotid artery was inserted about 17–20 mm using a bolus wire of the corresponding size. The neck wound was sutured and disinfected with an iodophor. Ischemia was performed for 6h, 12h, and 24h. Sham-operated animals were only operated without bolus wire insertion. 2.4 Nerve cells Primary neuronal cells were extracted based on the method of the previous study of our group. Briefly, anesthetize 24-hour-old SD rats. The cerebral cortex tissue was isolated and cut into 1–2 mm 2 pieces, and then 2 mL of 0.25% trypsin was added to digest the tissue. The supernatant was discarded after centrifugation at 1000 rpm/min for 5 min. Subsequently, 3 mL DMEM-F12 solution was added and the cells were filtered using a 70µm filter after resuspension and mixing. This filtration process was repeated twice to adjust the cell density to 5×10 5 cells/mL. The neural cells were distributed in culture plates or culture flasks coated with poly-D-lysine and cultured in a 5% CO 2 , 37℃cell culture incubator for 4h before being switched to neural cell culture medium. Neural cells were cultured for 7d and then used for subsequent experiments. 2.5 OGD nerve cell modeling The neuronal cells were placed in a three-gas incubator containing 5% CO 2 , 1% O 2 and 94% N 2 , and different oxygen sugar deprivation times were set according to the experimental requirements. 2.6 Drug treatment Elesclomol (Ele) (22345, MCE, USA) was dissolved in DMSO (D8371, solarbio, China), and different concentrations of Ele were added 24 h before cell modeling and treated with the same concentration of DMSO as control. 2.7 Cell transfection Nrf2 adenovirus (Si-Nrf2) and control empty vector lentivirus (NC-Nrf2), constructed by Shanghai Jikai Gene. When the primary neuronal cells reached 40%-60% confluence, transfection was performed according to the transfection experimental manual, and the adenovirus expressing only green fluorescent protein (Ad-GFP) viral stock solution was diluted to a titer of 1.5×10 8 PFU/mL, the original medium was discarded, and the viral dilution and neuronal cell medium were added sequentially and mixed thoroughly, and the subsequent experiments were carried out after 72h of transfection. 2.8 Cell viability The cells were inoculated in 96-well cell culture plates at a density of 1×10 4 neuronal cells per well. After cell treatment, each well was rinsed twice with 100µLPBS. The CCK-8 working solution was prepared by mixing the neuronal cell culture medium with CCK-8 at a ratio of 9:1. 100µL of CCK-8 working solution was added to each well and incubated at 37℃for 2h. The absorbance at 450 nm was measured by an enzyme meter. Cell viability was the OD value of experimental wells divided by the OD value of normal control wells. 2.9 LDH release test The cells were inoculated in 96-well cell culture plates at a density of 1×10 4 neuronal cells per well. After incubation, 100µL of each cell culture solution was taken into 0.5 mLEP tubes. 1000 r/min centrifugation was performed for 10 min, and the supernatant was collected. Subsequently, at room temperature and protected from light, 60µL of LDH assay working solution was added to the EP tubes and incubated for 30 min. After incubation, the OD value was detected at 490 nm. 2.10 malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD), and Cu content MDA, GSH, SOD, and Cu content were detected in rat ischemic lateral cerebral cortex and neuronal cells using appropriate kits (E-BC-K028-M, E-BC-K030-M, E-BC-K020-M, E-EL-0128, E-BC-K300-M/E-BC-K775-M, Elabscience, China) according to the manufacturer's instructions. 2.11 Cellular reactive oxygen species (ROS) measurement The treated neuronal cells were incubated with dihydroethidium (DHE, C1300-2, Applygen, China) probe working solution in a 5% CO 2 , 37℃cell culture incubator for 1h. The cells were washed with PBS for 3 times, and the images were observed and captured under fluorescence microscope. The images were analyzed using ImageJ software. 2.12 Immunofluorescence staining Rat brain tissue was preserved at room temperature using 4% paraformaldehyde fixative. Subsequently, paraffin-embedded brain tissue sections with a thickness of 5 mm were produced. Paraffin sections were routinely deparaffinized, followed by auto fixation in citrate buffer for 15 min, and phosphate buffer solution (PBS) was used as fixative. The sections were permeabilized with 0.2% TritonX-100 (AWH0299a, abiowell, China) for 10 min and incubated with 5% BSA containing 5% BSA for 1 h at room temperature, followed by dropwise addition of anti-Neun antibody (66836-1-lg, Proteintech, 1:200) and anti-FDX1 antibody (A20895. ABclonal,1:100) were incubated overnight at 4℃. After washing with PBS, 488-labeled and 594-labeled secondary antibodies (ab150077, ab150078, Abcam, 1:1000) were added dropwise. The samples were incubated at 37℃under light protection for 1.5h. After DAPI staining, the samples were sealed with an anti-fluorescence quenching sealer. On the ischemic side of the brain, we observed the fluorescence intensity and localization by fluorescence microscopy. The positive signal was Neun (green) + FDX1 (red) fluorescence, and DAPI (blue) was the nuclear staining signal. Neuronal cells were washed three times with PBS (5 min per wash) and fixed in 4% PFA for 15 min. After three washes with PBS, neuronal cells were permeabilized with 0.2% TritonX-100 for 10 min, followed by closure with 5% BSA at room temperature for 1 h. Neuronal cells were then incubated overnight at 4℃. Primary antibodies included FDX1 (A20895, ABclonal. 1:100) and Nrf2 (A21508, ABclonal, 1:100). 488-labeled and 594-labeled secondary antibodies were incubated for 1.5h at room temperature, and the nuclei were labeled by DAPI staining. Finally, images were acquired using fluorescence microscopy. 2.13 Western blotting Ischemic cortical and neuronal cell proteins were extracted with protease inhibitor-containing RIPA. Protein concentrations were calculated based on the results of the Bicinchoninic acid (BCA) method. Protein samples were separated on 12% or 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and transferred to polyvinylidene fluoride membranes (PVDF) at 200 mA for 1.5h. Subsequently, they were incubated with 5% skimmed milk powder for 2 h at room temperature to prevent nonspecific binding. FDX1(A20895, ABclonal, 1:500), LIAS(11577-1-AP, Proteintech, 1:1000), Nrf2(A21508, ABclonal,1:1000), HO-1(10701-1-AP, Proteintech, 1:2000), the β-tubulin (66240-1-lg, Proteintech, 1:20000) antibodies were incubated with the samples overnight at 4℃. The samples were washed three times with Tris-buffered saline with Tween (TBST), each time lasting for 10 min. HRP-labeled secondary antibodies were diluted with TBST as goat anti-mouse IgG (H + L) secondary antibodies against HRP (AWS0001, 1:10000) and goat anti-rabbit IgG(H + L) secondary antibody HRP (AWS0002, 1:10000). The diluted secondary antibodies were co-incubated with the membranes at room temperature for 1.5 h. Subsequently, the membranes were washed three times with TBST, each lasting for 10 min. After incubation, the membranes were treated with ECL chemiluminescent solution for 1 min, followed by development of the membranes with a gel imaging system. The intensity of protein bands was analyzed using ImageJ software. 2.14 Statistical analysis Data were analyzed using GraphPad Prism 9.5.1 (GraphPad Software, San Diego, CA, USA, https://www.graphpad-prism.cn/ ), and the mean and variability of the data were expressed as mean ± standard deviation (mean ± standard deviation, SD). One-way analysis of variance (ANOVA) and Dunnett's test were used to determine differences between groups. 3. Results 3.1 MCAO promotes neurological damage in cortical areas on the ischemic side of the rat brain with increased expression of copper homeostatic imbalance markers. The neuronal cells in the sham-operated group were structurally intact, neatly arranged, with mostly round nuclei, deeply stained cytoplasm, and a high number of Nissl bodies. The morphology of neuronal cells in the cortical area of the ischemic side of the brain in the MCAO group was mostly vacuolated, with inconspicuous nuclei, and the number of Nissl bodies was further decreased with the prolongation of the MCAO time (Fig. 1 A, B). The cortical area on the ischemic side of the brain copper content increased, with the most significant increase at 24h of MCAO (Fig. 1 C). We further observed that the immunofluorescence intensity of FDX1, a key protein for copper homeostatic imbalance in the cortical region on the ischemic side of the brain, increased at MCAO 24h (Fig. 1 D, E); the protein expression of FDX1, a key protein for copper homeostatic imbalance in the brain tissue on the ischemic side of the brain, was increased at MCAO 24h, and the expression of LIAS was significantly expressed at MCAO 12 h and MCAO 24h (Fig. 1 F-H). 3.2 OGD promotes primary neuronal cell injury and increases expression of copper homeostatic imbalance markers. We used immunofluorescence to characterize Microtubule-associated protein 2 (MAP2), a marker for primary neuronal cells, and the results showed that the extracted primary neuronal cells were of high purity (> 95%) (Fig. 2 A). The neuronal reticulation was lost, most of the neuronal cells were rounded, the number of cells was decreased, the cell viability was significantly reduced, and the LDH level was significantly increased at all time points after OGD injury (Fig. 2 B-D). We further observed a significant increase in intracellular Cu content at OGD 2h (Fig. 2 E), a significant increase in the fluorescence intensity of FDX1, a key protein for cuproptosis, at OGD 1h and OGD 2h (Fig. 2 F-G), a significant increase in FDX1, a key protein for Cu homeostatic imbalance, at OGD 2h, and a significant increase in LIAS expression at OGD 2h and OGD 3h (Fig. 2 H -J). 3.3 Elesclomol exacerbates neuronal cell damage and copper homeostatic imbalance We intervened different concentrations of Elesclomol (6.25 nmol/mL, 12.5 nmol/mL, 18.75 nmol/mL and 25 nmol/mL) in primary neuronal OGD models. It was found that with Elesclomol prompted a significant decrease in cell viability and a significant increase in cell damage (Fig. 3 A, B). It was further observed that the intracellular Cu content was elevated after the administration of Ele interventions at concentrations of 6.25 nmol/mL and 12.5 nmol/mL, but the Cu content was decreasing with increasing concentration. (Fig. 3 C). OGD induced an increase in the expression of the key proteins of copper homeostatic imbalance, FDX1 and LIAS, and the expression of intracellular FDX1 protein and LIAS protein was significantly elevated after the administration of Ele intervention at a concentration of 12.5 nmol/mL (Fig. 3 D-F). 3.4 Elesclomol promotes oxidative stress in nerve cells The relationship between Elesclomol and oxidative stress was explored through four aspects: ROS fluorescence expression, intracellular MDA, GSH and SOD content. We observed that the relative fluorescence intensity of intracellular ROS was enhanced in the OGD group, which was significantly enhanced after the intervention of 6.25 nmol/mL and 12.5 nmol/mL Elesclomol was given (Fig. 4 A, B).The intracellular MDA content in the OGD group was significantly increased, the GSH content was significantly decreased, and the SOD content was significantly increased by the intervention of 6.25 nmol/mL and 12.5 nmol/mL Elesclomol was given. 6.25 nmol/mL and 12.5 nmol/mL Ele intervention, the intracellular MDA content was significantly increased, the intracellular GSH content was decreased, and the intracellular SOD content was increased (Fig. 4 C, D). 3.5 Elesclomol activates the Nrf2/HO-1 signaling pathway in neuronal cells We evaluated the relationship between copper homeostatic imbalance and Nrf2/HO-1 signaling pathway through the expression of key proteins of Nrf2/HO-1 signaling pathway. It was found that Nrf2 activated by intranuclear was significantly elevated in the OGD group, and there was a trend of increased intranuclear activation of Nrf2 after the administration of 6.25 nmol/mL Elesclomol intervention, whereas the intranuclear activation of Nrf2 was significantly decreased by the administration of 12.5 nmol/mL Elesclomol intervention (Fig. 5 A, B). Further protein assay revealed that the expression of Nrf2 and HO-1 proteins related to Nrf2/HO-1 signaling pathway was significantly increased in the OGD group, and Nrf2 protein expression of the Nrf2/HO-1 signaling pathway was significantly increased, and HO-1 protein expression was increased after the administration of 6.25 nmol/mL Elesclomol intervention; the Nrf2/HO-1 signaling pathway protein expression was significantly decreased in the OGD + 6.25 nmol/mL group when compared with that in the OGD + 6.25 nmol/mL Nrf2/HO-1 signaling pathway Nrf2 and HO-1 protein expression was significantly decreased in the OGD + 12.5 nmol/mL group compared with the OGD + 6.25 nmol/mL Elesclomol group (Fig. 5 C-E). 3.6 Silencing Nrf2 exacerbates oxidative stress and inhibits the Nrf2/HO-1 signaling pathway in OGD primary neuronal cells We explored the effect of cuproptosis inducer on oxidative stress in silencing Nrf2 primary neuronal cells OGD by ROS fluorescence expression and SOD content and expression of key proteins of Nrf2/HO-1 signaling pathway. It was found that silencing Nrf2 promoted the increase of ROS and SOD levels after OGD in neuronal cells, and Elesclomol induced the oxidative stress response and further increased the ROS and SOD levels after OGD in silenced Nrf2 neuronal cells (Fig. 6 A-C). The expression of Nrf2/HO-1 pathway-associated proteins Nrf2 and HO-1 was down-regulated after silencing Nrf2 neuronal cells OGD, and Nrf2 and HO-1 were in an inhibitory state after Elesclomol intervention in neuronal cells OGD (Fig. 6 D-F). 3.7 Silencing Nrf2 exacerbates copper homeostatic imbalance in OGD neuronal cells We further constructed a silenced Nrf2 neuronal OGD model and administered Elesclomol to synchronize the intervention, and detected primary neuronal cell damage using cell morphology observation method, CCK8 method and LDH release assay. We found that silencing Nrf2 promoted cytosolic rounding, decreased cell number, cell viability, and cell leakage in OGD neuronal cells, and Elesclomol induced OGD neuronal cell injury and further aggravated OGD injury in silenced Nrf2 neuronal cells (Fig. 7 A-C). We found that silencing Nrf2 neuronal cells with OGD significantly increased Cu content and further increased the promotion of Cu content by Elesclomol (Fig. 7 D). The expression of FDX1 and LIAS, key proteins for copper homeostatic imbalance, was up-regulated after silencing OGD in Nrf2 neuronal cells, and the promotion of FDX1 and LIAS, key proteins for copper homeostatic imbalance, by Ele was inhibited (Fig. 7 E-G). 4. Discussion Copper is a key component in the normal metabolism of brain cells and in the formation of neurotransmitters, and it has been found that imbalance in copper homeostasis is involved in the development of central nervous system diseases, and that excess copper interacts with aggregated proteins and participates in cellular oxidative stress leading to nerve cell necrosis[ 20 , 21 ]. Several clinical studies have found that baseline plasma copper levels are approximately linearly and positively correlated with the risk of first ischemic stroke, and that toxic/co-toxic effects of copper ions are involved in the development of several cerebrovascular diseases, including stroke. It is now clear that copper ions are widely involved in several pathological processes in stroke, such as serum copper, copper/zinc, and copper/selenium ratios reflecting the level of oxidative stress and the trophic status of brain cells in acute ischemic stroke[ 22 , 23 ]. A genome-wide CRISPR/Cas9 screen identified seven genes associated with cuproptosis, among which the Fe-S cluster proteins FDX1 and LIAS are markers of cuproptosis[ 24 ]. Firstly, as key regulatory factors of protein lipid acylation, FDX1 and LIAS are involved in the regulation of protein lipid acylation including dihydrolipoamide acyltransferase (DLAT), and the lipid acylated proteins can directly bind to copper ions to form oligomers. Secondly, FDX1 reduces Cu 2+ to the more cytotoxic Cu + , leading to the destabilization of iron-sulfur cluster proteins[ 14 , 15 ]. In our rat MCAO model, we found that with the prolongation of the MCAO time in rats, the neurological impairment increased, and a simultaneous increase in the copper concentration in the ischemic side of the brain tissue, the fluorescence intensity of FDX1, a key marker of copper homeostatic imbalance, and the expression of FDX1 and LIAS proteins were observed. This result suggests that ischemic-hypoxic stimulation induces a high copper state, which exacerbates cellular damage. In the present study, we isolated and cultured primary neural cells in vitro, identified the purity of the extracted primary neural cells by morphological observation and immunofluorescence staining with MAP2, a neural cell-specific marker, and established an OGD model to simulate cerebral ischemia in vivo. We found that cellular copper concentration, the key protein of copper homeostatic imbalance, FDX1, immunofluorescence intensity, FDX1, and LIAS, and the expression of LIAS, all of which are key proteins of the Cu homeostatic imbalance, reached a peak at 2 h of OGD, which may be a critical time point for the occurrence of ischemia-hypoxia-induced copper homeostatic imbalance. In conclusion, our animal and cellular experiments have clearly demonstrated that copper homeostatic imbalance is involved in the pathological process of cerebral ischemic neuronal cell injury. Elesclomol is a copper carrier that targets mitochondria, binds to copper in the extracellular environment, generates a membrane-permeable complex, and transports copper to mitochondria, thereby inducing an intracellular high copper state[ 25 , 26 ]. We firstly intervened primary neuronal cells with OGD using different concentrations (6.25 nmol/mL, 12.5 nmol/mL, and 18.75 nmol/mL) of Ele, and found that Ele promoted primary neuronal cell damage in OGD without a dose-dependent characteristic. Ele at concentrations of 6.25 nmol/mL and 12.5 nmol/mL promoted increased intracellular copper concentrations ( P < 0.01, P < 0.05), which was hypothesized to be a possible effector concentration for Ele-induced copper homeostatic imbalance. Related studies have found that intracerebral striatal injection of Cu 2+ leads to increased oxidative stress in the striatum and substantia nigra[ 27 ]. Other studies have shown that excess copper can induce oxidative stress through activation of the ROS/HO-1/NQO1 pathway, and inhibition of HO-1 may attenuate copper-induced oxidative stress[ 28 ]. We further observed that 6.25 nmol/mL and 12.5 nmol/mL of Ele promoted higher ROS fluorescence expression, increased MDA content, and decreased GSH content. This result suggests that copper homeostatic imbalance induces oxidative stress, which may be a key pathological pathway leading to neuronal cell injury. When oxidative stress occurs, the imbalance of intracellular oxidative and antioxidant systems can lead to the activation of multiple signaling pathways, and the Nrf2/HO1 signaling pathway is one of the key signaling pathways for sensing the environment and regulating endogenous oxidative stress, which maintains cellular redox homeostasis by transcriptionally inducing protective genes. Excess ROS prompts Nrf2 to dissociate from Keap1, and Nrf2 enters the nucleus and activates gene expression of intracellular detoxifying enzymes and antioxidant proteins through ARE (antioxidant response element). HO-1 belongs to the class of metabolizing enzymes, and is a member of the heme oxygenase (HO) family, which is regulated at the transcriptional level by the transcription factor Nrf2. The activation of the Nrf2/HO-1 signaling pathway has a variety of effects such as antioxidant, anti-inflammatory, maintenance of mitochondrial homeostasis, inhibition of apoptosis, and regulation of pyroptosis[ 29 , 30 ]. In HT22 mouse hippocampal neuronal cells exposed to copper ions, copper-induced oxidative damage reduced CREB phosphorylation, decreased Bcl-2 expression, increased Bax expression, activated the Nrf2 signaling pathway, facilitated the dissociation of the keap1-Nrf2 complex, enhanced the nuclear translocation of Nrf2, and stimulated the expression of the antioxidant molecules HO-1 and NQO1[ 19 ]. In CD-1 rats treated with copper ions, higher levels of copper treatment activated the Keap1/Nrf2 signaling pathway and significantly increased the expression of genes related to redox status[ 31 ]. Notably, our study observed a trend of increased Nrf2 activation by Ele given 6.25 nmol/mL and a significant decrease in Nrf2 activation by Ele given 12.5 nmol/mL (P < 0.01) and increased expression of copper homeostatic marker proteins, FDX1 and LIAS, in primary neuronal cells given 12.5 nmol/mL of Ele. This result suggests that Ele promotes oxidative stress and activates the Nrf2/HO-1 signaling pathway, and that low-dose Ele promotes Nrf2 expression and inhibits copper homeostatic imbalance, whereas high-dose Ele inhibits Nrf2 expression and promotes copper homeostatic imbalance. Subsequently, we used adenoviral transfection to knock down Nrf2, a key regulator of the Nrf2/HO-1 signaling pathway, and showed that silencing of Nrf2 resulted in cellular damage, oxidative stress, and copper homeostatic imbalance in heavy OGD, and further exacerbated the Ele-induced cellular damage and copper homeostatic imbalance. This result demonstrates for the first time that the Nrf2/HO-1 signaling pathway has an effect on regulating copper homeostatic imbalance. 5. Conclusion Our study in this work firstly found that copper homeostatic imbalance occurs after cerebral ischemia and hypoxia, further observed the relationship between copper homeostatic imbalance and oxidative stress, and for the first time found that the Nrf2/HO-1 signaling pathway is involved in the pathological process of copper homeostatic imbalance under cerebral ischemia and hypoxia, and Nrf2 is a key factor regulating the imbalance of copper homeostatic imbalance. Abbreviations OGD, Oxygen-Glucose Deprivation; MACO, Medication Adherence Context and Outcomes; Ele, Elesclomol; MDA, malondialdehyde; SOD, superoxide dismutase; ROS, Reactive Oxygen Species; HO-1, heme oxygenase-1; MAP2,Microtubule-associated protein 2; Si-Nrf2,Nrf2 adenovirus; NC-Nrf2,control empty vector lentivirus; FDX1, ferredoxin 1; LIAS, lipoic acid synthase. Declarations A uthor contribution s Ting Lang and Yanhao Liao : Formulation and implementation of experimental research, Writing of original manuscript. Bingbin Fan and Huixian Chen : Research work of animal experiment in vivo. Junjie Hu and Lijin g Xie : Research work of cell experiment in vitro. Keyi Qin and Zhenlin Zhong : Analyze experimental data using data analysis software. Keyan Jiao , Yan She and Le Shao : Guidance and supervision of experimental research, revision of manuscript. All data were generated in-house, and no paper mill was used. All authors agree to be accountable for all aspects of work ensuring integrity and accuracy. Clinical trial number: not applicable Acknowledgements / Funding Declaration All the images were drawn by Yanhao Liao and Binbing Fan (Using Adobe illustrator 2021, https://www.adobe.com/cn/creativecloud/roc/business.html). This research was supported by the National Natural Science Foundation of China (No: 81603415,82174176), Education Department of Hunan Province of China (No: 23A0288). Availability of materials and data The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. Consent to Participate Informed consent was obtained from all individual participants included in the studyCompeting Interest All authors of this research declare that there are no conflicts of interest. Ethics approval Animal experiments were conducted in strict accordance with the regulations of the Animal Ethics Committee (Certificate No. ZYFY20230920005) (All programs meet the ARRIVE guideline 2.0). 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Proc Natl Acad Sci U S A 120(10):e2216722120 Cruces-Sande A, Rodríguez-Pérez AI, Herbello-Hermelo P, et al. (2019) Copper Increases Brain Oxidative Stress and Enhances the Ability of 6-Hydroxydopamine to Cause Dopaminergic Degeneration in a Rat Model of Parkinson's Disease. Mol Neurobiol 56(4):2845-2854 Fang Y, Xing C, Wang X, et al. (2021) Activation of the ROS/HO-1/NQO1 signaling pathway contributes to the copper-induced oxidative stress and autophagy in duck renal tubular epithelial cells. Sci Total Environ 757:143753 Telkoparan-Akillilar P, Panieri E, Cevik D, Suzen S, Saso L (2021) Therapeutic Targeting of the NRF2 Signaling Pathway in Cancer. Molecules 26(5):1417 Liu S, Pi J, Zhang Q (2022) Signal amplification in the KEAP1-NRF2-ARE antioxidant response pathway. Redox Biol 54:102389 Zhong G, He Y, Wan F, et al. (2021) Effects of Long-Term Exposure to Copper on the Keap1/Nrf2 Signaling Pathway and Msr-Related Redox Status in the Kidneys of Rats. Biol Trace Elem Res 199(11):4205-4217 Additional Declarations No competing interests reported. Supplementary Files westernblot.pptx Cite Share Download PDF Status: Posted Version 1 posted 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6650982","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":466166539,"identity":"a280da7d-80e5-466c-b726-fa8f3d174448","order_by":0,"name":"Ting Lang","email":"","orcid":"","institution":"Hunan University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Ting","middleName":"","lastName":"Lang","suffix":""},{"id":466166540,"identity":"8fd33f02-f90f-421f-a410-d9e2dba4ae12","order_by":1,"name":"Yanhao Liao","email":"","orcid":"","institution":"Hunan University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yanhao","middleName":"","lastName":"Liao","suffix":""},{"id":466166541,"identity":"99ba7781-c348-44c3-b844-2cb064ed607b","order_by":2,"name":"Bingbin Fan","email":"","orcid":"","institution":"Hunan University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Bingbin","middleName":"","lastName":"Fan","suffix":""},{"id":466166542,"identity":"95195ec4-7033-41ba-9a80-33115cea7cb8","order_by":3,"name":"Huixian Chen","email":"","orcid":"","institution":"Hunan University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Huixian","middleName":"","lastName":"Chen","suffix":""},{"id":466166543,"identity":"f24fa583-c38d-430a-99fc-89efb2418ca8","order_by":4,"name":"Junjie Hu","email":"","orcid":"","institution":"Hunan University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Junjie","middleName":"","lastName":"Hu","suffix":""},{"id":466166544,"identity":"658f8565-2b65-4708-80aa-70245b922816","order_by":5,"name":"Lijing Xie","email":"","orcid":"","institution":"Hunan University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Lijing","middleName":"","lastName":"Xie","suffix":""},{"id":466166545,"identity":"d18e1a52-610c-4147-9efb-0af0bfb314de","order_by":6,"name":"Keyi Qin","email":"","orcid":"","institution":"Hunan University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Keyi","middleName":"","lastName":"Qin","suffix":""},{"id":466166546,"identity":"48b6e953-c891-4973-b42f-064b4c62ae3e","order_by":7,"name":"Zhenlin Zhong","email":"","orcid":"","institution":"Hunan University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Zhenlin","middleName":"","lastName":"Zhong","suffix":""},{"id":466166548,"identity":"8b88033e-d0ec-4aea-ae1a-a445daa7d774","order_by":8,"name":"Keyan Jiao","email":"","orcid":"","institution":"Hunan University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Keyan","middleName":"","lastName":"Jiao","suffix":""},{"id":466166549,"identity":"d00c2b74-dd68-4325-ae5e-a94687ccde1a","order_by":9,"name":"Le Shao","email":"","orcid":"","institution":"The First Hospital of Hunan University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Le","middleName":"","lastName":"Shao","suffix":""},{"id":466166552,"identity":"ea63d565-35e4-4cd6-a3bf-f644b825a0e8","order_by":10,"name":"Yan She","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0ElEQVRIiWNgGAWjYBACNmb2Awc+VNgkQPnMhLXws/ckHpxxJo0ELZI9B4wP87YdJkGLwY2EBKCW83nmEslPNzBUWCc2sJ89QEBL4oGDc87dLrackWZ2g+FMemIDT14CAS0JCQfelN1O3HAjh+0GY9vhxAYJHgNCWgwO8LCdg2r5R4QWoPcNDvK0HYBqaSBCCzCQE4CBnJy44cwzsxsJx9KN23hy8GsBRuXhDx8q7BI3HE9+duNDjbVsP/sZ/FpQQQLIEBLUj4JRMApGwSjAAQD5QFLQBTmR1AAAAABJRU5ErkJggg==","orcid":"","institution":"Hunan University of Chinese Medicine","correspondingAuthor":true,"prefix":"","firstName":"Yan","middleName":"","lastName":"She","suffix":""}],"badges":[],"createdAt":"2025-05-13 03:38:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6650982/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6650982/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":84058168,"identity":"ca1f163a-96bd-43c1-bac4-1df3c170c640","added_by":"auto","created_at":"2025-06-06 09:42:28","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":339945,"visible":true,"origin":"","legend":"\u003cp\u003eMCAO promotes neurological injury and increases expression of copper homeostatic imbalance markers in cortical areas on the ischemic side of the brain in rats (A-B) Representative and statistical graphs of Nissl bodies in cortical areas on the ischemic side of the brain (* \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01.); (C) Statistical graphs of copper content (* \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01.); (D-E) Immunofluorescent expression of FDX1 in cortical areas on the ischemic side of the brain, along with representative images and statistical graphs. (*\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01.); (F-H) FDX1 and LIAS protein expression in the cortical region on the ischemic side of the brain (* \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.01.).\u003c/p\u003e","description":"","filename":"image1.tiff.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6650982/v1/b906d8d7a35ef5828bd48b94.jpg"},{"id":84058387,"identity":"16c39e3c-65ed-4d56-91ef-9ef7100d2324","added_by":"auto","created_at":"2025-06-06 09:50:28","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":364326,"visible":true,"origin":"","legend":"\u003cp\u003eOGD promotes primary neuronal cell injury and increases expression of copper homeostatic imbalance markers. (A) Fluorescence expression of the primary neuronal marker MAP2; (B) Primary neuronal cell morphology; (C) Cell viability statistic (* \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01.); (D) Cell LDH content statistic (* \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.01.); (E) Cell Cu content statistic (* \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01.); (F-G) Representative and statistical graphs of cellular FDX1 immunofluorescence expression (*\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.05, ** p \u0026lt; 0.01.); (H-J) Representative and statistical graphs of cellular FDX1 and LIAS protein expression (* \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01.).\u003c/p\u003e","description":"","filename":"image2.tiff.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6650982/v1/b9a8cc98fce425f95003f1e3.jpg"},{"id":84058166,"identity":"9c1705df-42bf-4887-9c1b-e06d3ae2ffb3","added_by":"auto","created_at":"2025-06-06 09:42:28","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":125769,"visible":true,"origin":"","legend":"\u003cp\u003eElesclomol aggravates neuronal cell damage and copper homeostatic imbalance (A) Statistical graphs of cell viability (* \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01.); (B) Statistical graphs of cellular LDH content (* \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01.); (C) Statistical graphs of cellular Cu content (* \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01.); (D-F) Representative and statistical graphs of FDX1 and LIAS protein expression in cells (* \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01.).\u003c/p\u003e","description":"","filename":"image3.tiff.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6650982/v1/7d838e57e0b8a9be6e1247cb.jpg"},{"id":84058389,"identity":"1fe06d34-7508-4de1-b788-e7eb64182fef","added_by":"auto","created_at":"2025-06-06 09:50:28","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":157630,"visible":true,"origin":"","legend":"\u003cp\u003eElesclomol prompts oxidative stress response in neuronal cells. (A-B) Statistical graph of ROS fluorescence expression (* \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01.); (C) Statistical graph of cellular MDA content (* \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01.); (D) Statistical graph of cellular GSH content (* \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01.).\u003c/p\u003e","description":"","filename":"image4.tiff.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6650982/v1/267c54ae6f7bda7c9de9f42c.jpg"},{"id":84058173,"identity":"bde09633-5c2e-47ac-b7a8-bdc28fd4ebca","added_by":"auto","created_at":"2025-06-06 09:42:29","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":204734,"visible":true,"origin":"","legend":"\u003cp\u003eElesclomol activation of the Nrf2/HO-1 signaling pathway in neuronal cells (A-B) Nrf2 fluorescence expression and statistical graphs (* \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.01.); (C-E) Representative graphs of Nrf2 and HO-1 protein expression and statistical graphs (* \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01.).\u003c/p\u003e","description":"","filename":"image5.tiff.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6650982/v1/21b8377b21e3a97e4d81e2e6.jpg"},{"id":84058172,"identity":"c286c026-d32d-4bcd-af57-20b1b59b678c","added_by":"auto","created_at":"2025-06-06 09:42:28","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":159698,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of silencing Nrf2 on oxidative stress and Nrf2/HO-1 signaling pathway in OGD primary neuronal cells. (A-B) Representative and statistical plots of ROS fluorescence expression (* \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01.); (C) Statistical plots of SOD content (* \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01.); (D-F) Representative and statistical plots of Nrf2 and HO-1 protein expression (* \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01.).\u003c/p\u003e","description":"","filename":"image6.tiff.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6650982/v1/6e83851c9f08c06e6d38a9ef.jpg"},{"id":84058171,"identity":"716ea83f-4ad6-4e71-89d6-e7462633b5b5","added_by":"auto","created_at":"2025-06-06 09:42:28","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":262513,"visible":true,"origin":"","legend":"\u003cp\u003eSilencing of Nrf2 exacerbates copper homeostatic imbalance in OGD neuronal cells. (A) Primary cell morphology, (B) Cell viability (* \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01.), (C) Cellular LDH content (* \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01.), (D) Intracellular Cu content (* p \u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01.). (E-G) Representative and statistical plots of FDX1 and LIAS protein expression (* \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01.).\u003c/p\u003e","description":"","filename":"image7.tiff.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6650982/v1/ac1cf648daa8fe7ae33e607b.jpg"},{"id":84475324,"identity":"a9d29604-304f-4038-a002-2ac96ad83318","added_by":"auto","created_at":"2025-06-12 11:17:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2594529,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6650982/v1/c04a08fb-b280-4878-8c47-baf570201436.pdf"},{"id":84058176,"identity":"26c6eb9d-439f-41de-ac67-40268d2d4b74","added_by":"auto","created_at":"2025-06-06 09:42:29","extension":"pptx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":7888870,"visible":true,"origin":"","legend":"","description":"","filename":"westernblot.pptx","url":"https://assets-eu.researchsquare.com/files/rs-6650982/v1/9c98d2bba1a8474b3932d1d0.pptx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Inhibition of Nrf2/HO-1 signaling pathway exacerbates Copper homeostasis in cerebral ischemia","fulltext":[{"header":"Highlights","content":"\u003cul\u003e\n \u003cli\u003eMCAO promotes brain damage with increased expression of copper homeostatic imbalance markers.\u003c/li\u003e\n \u003cli\u003eOGD promotes primary neuronal cell injury and increases expression of copper homeostatic imbalance markers.\u003c/li\u003e\n \u003cli\u003eElesclomol exacerbates neuronal cell damage and copper homeostatic imbalance through Nrf2/HO-1 signaling pathway.\u003c/li\u003e\n \u003cli\u003eSilencing Nrf2 exacerbates oxidative stress and inhibits the Nrf2/HO-1 signaling pathway in OGD primary neuronal cells.\u003c/li\u003e\n \u003cli\u003eSilencing Nrf2 exacerbates copper homeostatic imbalance in OGD neuronal cells.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"1. Introduction","content":"\u003cp\u003eWith the aging of the population, stroke has become a heavy global health and economic burden, with ischemic stroke as the main type of stroke[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Copper is involved in important physiological processes in the body, such as energy metabolism, mitochondrial respiration, enzyme synthesis and oxidative stress. Under physiological conditions, copper metabolism in the body is tightly regulated to keep copper concentration in dynamic balance, a process known as copper homeostasis[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The brain is a target organ for copper accumulation, and copper ions can be distributed to different brain regions through the blood-brain barrier[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], and the physiological state and function of neurons are regulated by copper. It is found that the imbalance of copper homeostasis is closely related to the development of neurological diseases[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], such as cerebral ischemia[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], Alzheimer's disease[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], and Parkinson's disease. Bioinformatics software such as R software, Gene Ontology (GO) and Genes and Genomes (KEGG) were used to identify 10 copper metabolism-related genes in cerebral ischemia, including key genes such as FDX1 and LIAS[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Another study showed that the neuroprotective effect of disulfiram (DSF) in the treatment of cerebral ischemia is closely related to FDX1, a key protein in the regulation of copper homeostatic imbalance[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. It has been shown that co-culturing neuron-like differentiated cells with copper ions for 48 hours induced an accumulation of intracellular phosphorylated alpha-synuclein, similar to that which occurs in the pathogenesis of Parkinson\u0026rsquo;s disease(PD)[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In addition, autopsy studies have shown that copper levels are significantly elevated in the brains of patients with Alzheimer's disease (AD)[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. It was found that intra-striatal injection of Cu\u003csup\u003e2+\u003c/sup\u003e in the brain leads to increased oxidative stress in the striatum and substantia nigra, and that copper does not cause neurodegeneration, but may be involved in the Parkinson's disease pathogenesis through enhanced oxidative stress[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCopper overload can generate large amounts of ROS through the Fenton reaction, which reduces intracellular reduced glutathione (GSH) content and leads to increased cellular sensitivity to noxious stimuli[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The Nrf2/HO-1 signaling pathway is an important intracellular antioxidant signaling pathway. Cerebral ischemia and hypoxia stimulate the release of excessive ROS, prompting Nrf2 to enter the cell nucleus, and through the activation of intracellular detoxification enzymes and antioxidant proteins, thus exerting antioxidant, anti-inflammatory, and maintenance of mitochondrial homeostasis and other effects[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. It was found that copper induced oxidative stress and activated Nrf2 signaling pathway[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this project, we first clarified whether copper homeostatic imbalance in cerebral ischemic nerve cells exists in the pathological process of cerebral ischemic nerve injury, and further explored the relationship between the Nrf2/HO-1 signaling pathway and cerebral ischemic copper homeostatic imbalance. We aimed to provide new directions and new ideas for clinical treatment of ischemic stroke.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Animals\u003c/h2\u003e \u003cp\u003eHunan University of Chinese Medicine (SCXK2019-0004) provided SD rats weighing 260\u0026ndash;300 g,10\u0026ndash;12 weeks old. The rats were provided with sufficient water and normal food. Animal experiments were conducted in strict accordance with the regulations of the Animal Ethics Committee (Certificate No. ZYFY20230920005)(All programs meet the ARRIVE guideline 2.0).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Experimental designs\u003c/h2\u003e \u003cp\u003e \u003cb\u003e(a)Animal experiment I\u003c/b\u003e:\u003c/p\u003e \u003cp\u003eTwenty four healthy SD rats were randomly divided into 4 groups. (a) Sham operation group (Sham group): normal SD rats, only the common carotid artery was isolated without bolus insertion. (b) Middle cerebral artery ischemia 6h group (MCAO 6h): ischemia was performed for 6h according to the above cerebral ischemia model. (c) Middle cerebral artery ischemia 12h group (MCAO 12h): ischemia was performed for 12h according to the above cerebral ischemia model. (d) Middle cerebral artery ischemia 24h group (MCAO 24h): ischemia was performed for 24h according to the above cerebral ischemia model.\u003c/p\u003e \u003cp\u003e \u003cb\u003e(b)Cell experiment I\u003c/b\u003e: The effect of different OGD time periods on cuproptosis of primary nerve cells was affected by oxygen glucose deprivation of primary nerve cells, and the same group of cells was divided into four groups according to different oxygen and sugar deprivation times: control group; oxygen glucose deprivation 1h (OGD 1h) group; oxygen glucose deprivation 2h (OGD 2h) group; oxygen glucose deprivation 3h (OGD 3h) group. Each group was given the following treatments: Control group: same treatment as OGD steps except no oxygen glucose deprivation incubation; OGD1h, 2h and 3h groups had the same steps except different treatment of oxygen glucose deprivation time. Three parallel control wells were set up in each group, and the experiment was repeated three times.\u003c/p\u003e \u003cp\u003e \u003cb\u003e(c) Cell experiment II\u003c/b\u003e: the intervention dose of Ele. The same batch of cells was divided into 6 groups: control group; oxygen glucose deprivation (OGD) group; oxygen glucose deprivation\u0026thinsp;+\u0026thinsp;Ele6.25 nmol/mL (OGD\u0026thinsp;+\u0026thinsp;Ele6.25 nmol/mL) group; oxygen glucose deprivation\u0026thinsp;+\u0026thinsp;Ele12.5 nmol/mL (OGD\u0026thinsp;+\u0026thinsp;Ele12.5 nmol/mL) group; oxygen glucose deprivation\u0026thinsp;+\u0026thinsp;Ele18.75nmol/mL (OGD\u0026thinsp;+\u0026thinsp;Ele18.75nmol/mL) group; oxygen glucose deprivation\u0026thinsp;+\u0026thinsp;Ele25nmol/mL (OGD\u0026thinsp;+\u0026thinsp;Ele25nmol/mL). Each group was given the following treatments: Control group: same steps as OGD except no oxygen sugar deprivation incubation; OGD group: oxygen sugar deprivation for 2h; OGD\u0026thinsp;+\u0026thinsp;Ele6.25, 12.5, 18.75, and 25nmol/mL groups were given the same steps as the OGD group except different concentrations of Ele were added during the first 24h of OGD. Three parallel control wells were set up in each group and the experiment was repeated three times.\u003c/p\u003e\u003cp\u003e \u003cb\u003e(d) Cell experiment III\u003c/b\u003e: Effect of Nrf2/HO-1 signaling pathway on cuproptosis of primary neuronal cells after OGD injury. The same batch of cells was divided into 4 groups: control; oxygen glucose deprivation (OGD) group; oxygen glucose deprivation\u0026thinsp;+\u0026thinsp;Ele6.25nmol/mL (OGD\u0026thinsp;+\u0026thinsp;Ele6.25nmol/mL) group; oxygen glucose deprivation\u0026thinsp;+\u0026thinsp;Ele12.5nmol/mL (OGD\u0026thinsp;+\u0026thinsp;Ele12.5nmol/mL) group. Each group was given the following treatments: Control group: same steps as OGD except no oxygen sugar deprivation incubation; OGD group: oxygen sugar deprivation for 2h; OGD\u0026thinsp;+\u0026thinsp;Ele6.25 and 12.5nmol/mL groups were the same as the OGD group except different concentrations of Ele were added at 24h before OGD. Three parallel control wells were set up in each group and the experiment was repeated three times.\u003c/p\u003e \u003cp\u003e \u003cb\u003e(e) Cell experiment IV\u003c/b\u003e: the effect of silencing Nrf2 on cuproptosis after OGD injury in primary neuronal cells. The same batch of cells was divided into 5 groups: control\u0026thinsp;+\u0026thinsp;negative control (Control\u0026thinsp;+\u0026thinsp;NC) group; oxygen sugar deprivation\u0026thinsp;+\u0026thinsp;negative control (OGD\u0026thinsp;+\u0026thinsp;NC) group; oxygen sugar deprivation\u0026thinsp;+\u0026thinsp;negative control\u0026thinsp;+\u0026thinsp;Ele12.5nmol/mL (OGD\u0026thinsp;+\u0026thinsp;NC\u0026thinsp;+\u0026thinsp;Ele12.512nmol/mL ) group; oxygen glucose deprivation\u0026thinsp;+\u0026thinsp;Si-Nrf2 (OGD\u0026thinsp;+\u0026thinsp;Si-Nrf2) group; oxygen glucose deprivation\u0026thinsp;+\u0026thinsp;Si-Nrf2\u0026thinsp;+\u0026thinsp;Ele12.5nmol/mL (OGD\u0026thinsp;+\u0026thinsp;Si-Nrf2\u0026thinsp;+\u0026thinsp;Ele12.5nmol/mL) group. Each group was given the following treatments: Control\u0026thinsp;+\u0026thinsp;NC group: same treatments as OGD steps except no oxygen sugar deprivation incubation; OGD\u0026thinsp;+\u0026thinsp;NC group: negative control vector transfected 72h before oxygen sugar deprivation, then oxygen sugar deprivation for 2h; OGD\u0026thinsp;+\u0026thinsp;NC\u0026thinsp;+\u0026thinsp;Ele12.5nmol/mL group: same treatments as OGD steps except 12.5nmol/mL Ele added at 24h before OGD treatment was the same as the OGD step; OGD\u0026thinsp;+\u0026thinsp;Si-Nrf2 group: the rest of the treatment was the same as the OGD group step, except for the transfer of Si-Nrf2 72h before OGD; OGD\u0026thinsp;+\u0026thinsp;Si-Nrf2\u0026thinsp;+\u0026thinsp;Ele12.5nmol/mL group: the rest of the treatment was the same as the OGD steps. Three parallel control wells were set up in each group and the experiment was repeated \u003cb\u003ethree times\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e All the experimental plans above were approved by the Center for Medical Innovation and Experimentation of Hunan University of Traditional Chinese Medicine.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 MCAO rat model\u003c/h2\u003e \u003cp\u003eRats were anesthetized by taking an intraperitoneal injection of sodium pentobarbital (60mg\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). The right carotid artery was isolated.4\u0026thinsp;\u0026minus;\u0026thinsp;0 medical silk nonabsorbable sutures were used to ligate the external carotid and common carotid arteries. The internal carotid artery was inserted about 17\u0026ndash;20 mm using a bolus wire of the corresponding size. The neck wound was sutured and disinfected with an iodophor. Ischemia was performed for 6h, 12h, and 24h. Sham-operated animals were only operated without bolus wire insertion.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Nerve cells\u003c/h2\u003e \u003cp\u003ePrimary neuronal cells were extracted based on the method of the previous study of our group. Briefly, anesthetize 24-hour-old SD rats. The cerebral cortex tissue was isolated and cut into 1\u0026ndash;2 mm\u003csup\u003e2\u003c/sup\u003e pieces, and then 2 mL of 0.25% trypsin was added to digest the tissue. The supernatant was discarded after centrifugation at 1000 rpm/min for 5 min. Subsequently, 3 mL DMEM-F12 solution was added and the cells were filtered using a 70\u0026micro;m filter after resuspension and mixing. This filtration process was repeated twice to adjust the cell density to 5\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/mL. The neural cells were distributed in culture plates or culture flasks coated with poly-D-lysine and cultured in a 5% CO\u003csub\u003e2\u003c/sub\u003e, 37℃cell culture incubator for 4h before being switched to neural cell culture medium. Neural cells were cultured for 7d and then used for subsequent experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 OGD nerve cell modeling\u003c/h2\u003e \u003cp\u003eThe neuronal cells were placed in a three-gas incubator containing 5% CO\u003csub\u003e2\u003c/sub\u003e, 1% O\u003csub\u003e2\u003c/sub\u003e and 94% N\u003csub\u003e2\u003c/sub\u003e, and different oxygen sugar deprivation times were set according to the experimental requirements.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Drug treatment\u003c/h2\u003e \u003cp\u003eElesclomol (Ele) (22345, MCE, USA) was dissolved in DMSO (D8371, solarbio, China), and different concentrations of Ele were added 24 h before cell modeling and treated with the same concentration of DMSO as control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Cell transfection\u003c/h2\u003e \u003cp\u003eNrf2 adenovirus (Si-Nrf2) and control empty vector lentivirus (NC-Nrf2), constructed by Shanghai Jikai Gene. When the primary neuronal cells reached 40%-60% confluence, transfection was performed according to the transfection experimental manual, and the adenovirus expressing only green fluorescent protein (Ad-GFP) viral stock solution was diluted to a titer of 1.5\u0026times;10\u003csup\u003e8\u003c/sup\u003e PFU/mL, the original medium was discarded, and the viral dilution and neuronal cell medium were added sequentially and mixed thoroughly, and the subsequent experiments were carried out after 72h of transfection.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Cell viability\u003c/h2\u003e \u003cp\u003eThe cells were inoculated in 96-well cell culture plates at a density of 1\u0026times;10\u003csup\u003e4\u003c/sup\u003e neuronal cells per well. After cell treatment, each well was rinsed twice with 100\u0026micro;LPBS. The CCK-8 working solution was prepared by mixing the neuronal cell culture medium with CCK-8 at a ratio of 9:1. 100\u0026micro;L of CCK-8 working solution was added to each well and incubated at 37℃for 2h. The absorbance at 450 nm was measured by an enzyme meter. Cell viability was the OD value of experimental wells divided by the OD value of normal control wells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 LDH release test\u003c/h2\u003e \u003cp\u003eThe cells were inoculated in 96-well cell culture plates at a density of 1\u0026times;10\u003csup\u003e4\u003c/sup\u003e neuronal cells per well. After incubation, 100\u0026micro;L of each cell culture solution was taken into 0.5 mLEP tubes. 1000 r/min centrifugation was performed for 10 min, and the supernatant was collected. Subsequently, at room temperature and protected from light, 60\u0026micro;L of LDH assay working solution was added to the EP tubes and incubated for 30 min. After incubation, the OD value was detected at 490 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD), and Cu content\u003c/h2\u003e \u003cp\u003e MDA, GSH, SOD, and Cu content were detected in rat ischemic lateral cerebral cortex and neuronal cells using appropriate kits (E-BC-K028-M, E-BC-K030-M, E-BC-K020-M, E-EL-0128, E-BC-K300-M/E-BC-K775-M, Elabscience, China) according to the manufacturer's instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Cellular reactive oxygen species (ROS) measurement\u003c/h2\u003e \u003cp\u003eThe treated neuronal cells were incubated with dihydroethidium (DHE, C1300-2, Applygen, China) probe working solution in a 5% CO\u003csub\u003e2\u003c/sub\u003e, 37℃cell culture incubator for 1h. The cells were washed with PBS for 3 times, and the images were observed and captured under fluorescence microscope. The images were analyzed using ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12 Immunofluorescence staining\u003c/h2\u003e \u003cp\u003eRat brain tissue was preserved at room temperature using 4% paraformaldehyde fixative. Subsequently, paraffin-embedded brain tissue sections with a thickness of 5 mm were produced. Paraffin sections were routinely deparaffinized, followed by auto fixation in citrate buffer for 15 min, and phosphate buffer solution (PBS) was used as fixative. The sections were permeabilized with 0.2% TritonX-100 (AWH0299a, abiowell, China) for 10 min and incubated with 5% BSA containing 5% BSA for 1 h at room temperature, followed by dropwise addition of anti-Neun antibody (66836-1-lg, Proteintech, 1:200) and anti-FDX1 antibody (A20895. ABclonal,1:100) were incubated overnight at 4℃. After washing with PBS, 488-labeled and 594-labeled secondary antibodies (ab150077, ab150078, Abcam, 1:1000) were added dropwise. The samples were incubated at 37℃under light protection for 1.5h. After DAPI staining, the samples were sealed with an anti-fluorescence quenching sealer. On the ischemic side of the brain, we observed the fluorescence intensity and localization by fluorescence microscopy. The positive signal was Neun (green)\u0026thinsp;+\u0026thinsp;FDX1 (red) fluorescence, and DAPI (blue) was the nuclear staining signal. Neuronal cells were washed three times with PBS (5 min per wash) and fixed in 4% PFA for 15 min. After three washes with PBS, neuronal cells were permeabilized with 0.2% TritonX-100 for 10 min, followed by closure with 5% BSA at room temperature for 1 h. Neuronal cells were then incubated overnight at 4℃. Primary antibodies included FDX1 (A20895, ABclonal. 1:100) and Nrf2 (A21508, ABclonal, 1:100). 488-labeled and 594-labeled secondary antibodies were incubated for 1.5h at room temperature, and the nuclei were labeled by DAPI staining. Finally, images were acquired using fluorescence microscopy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.13 Western blotting\u003c/h2\u003e \u003cp\u003eIschemic cortical and neuronal cell proteins were extracted with protease inhibitor-containing RIPA. Protein concentrations were calculated based on the results of the Bicinchoninic acid (BCA) method. Protein samples were separated on 12% or 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and transferred to polyvinylidene fluoride membranes (PVDF) at 200 mA for 1.5h. Subsequently, they were incubated with 5% skimmed milk powder for 2 h at room temperature to prevent nonspecific binding. FDX1(A20895, ABclonal, 1:500), LIAS(11577-1-AP, Proteintech, 1:1000), Nrf2(A21508, ABclonal,1:1000), HO-1(10701-1-AP, Proteintech, 1:2000), the β-tubulin (66240-1-lg, Proteintech, 1:20000) antibodies were incubated with the samples overnight at 4℃. The samples were washed three times with Tris-buffered saline with Tween (TBST), each time lasting for 10 min. HRP-labeled secondary antibodies were diluted with TBST as goat anti-mouse IgG (H\u0026thinsp;+\u0026thinsp;L) secondary antibodies against HRP (AWS0001, 1:10000) and goat anti-rabbit IgG(H\u0026thinsp;+\u0026thinsp;L) secondary antibody HRP (AWS0002, 1:10000). The diluted secondary antibodies were co-incubated with the membranes at room temperature for 1.5 h. Subsequently, the membranes were washed three times with TBST, each lasting for 10 min. After incubation, the membranes were treated with ECL chemiluminescent solution for 1 min, followed by development of the membranes with a gel imaging system. The intensity of protein bands was analyzed using ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.14 Statistical analysis\u003c/h2\u003e \u003cp\u003eData were analyzed using GraphPad Prism 9.5.1 (GraphPad Software, San Diego, CA, USA, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.graphpad-prism.cn/\u003c/span\u003e\u003cspan address=\"https://www.graphpad-prism.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and the mean and variability of the data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation, SD). One-way analysis of variance (ANOVA) and Dunnett's test were used to determine differences between groups.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e \u003cem\u003e3.1 MCAO promotes neurological damage in cortical areas on the ischemic side of the rat brain with increased expression of copper homeostatic imbalance markers.\u003c/em\u003e \u003c/p\u003e \u003cp\u003eThe neuronal cells in the sham-operated group were structurally intact, neatly arranged, with mostly round nuclei, deeply stained cytoplasm, and a high number of Nissl bodies. The morphology of neuronal cells in the cortical area of the ischemic side of the brain in the MCAO group was mostly vacuolated, with inconspicuous nuclei, and the number of Nissl bodies was further decreased with the prolongation of the MCAO time (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B). The cortical area on the ischemic side of the brain copper content increased, with the most significant increase at 24h of MCAO (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). We further observed that the immunofluorescence intensity of FDX1, a key protein for copper homeostatic imbalance in the cortical region on the ischemic side of the brain, increased at MCAO 24h (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, E); the protein expression of FDX1, a key protein for copper homeostatic imbalance in the brain tissue on the ischemic side of the brain, was increased at MCAO 24h, and the expression of LIAS was significantly expressed at MCAO 12 h and MCAO 24h (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF-H).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.2 OGD promotes primary neuronal cell injury and increases expression of copper homeostatic imbalance markers.\u003c/h2\u003e \u003cp\u003eWe used immunofluorescence to characterize Microtubule-associated protein 2 (MAP2), a marker for primary neuronal cells, and the results showed that the extracted primary neuronal cells were of high purity (\u0026gt;\u0026thinsp;95%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eThe neuronal reticulation was lost, most of the neuronal cells were rounded, the number of cells was decreased, the cell viability was significantly reduced, and the LDH level was significantly increased at all time points after OGD injury (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-D).\u003c/p\u003e \u003cp\u003eWe further observed a significant increase in intracellular Cu content at OGD 2h (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE), a significant increase in the fluorescence intensity of FDX1, a key protein for cuproptosis, at OGD 1h and OGD 2h (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF-G), a significant increase in FDX1, a key protein for Cu homeostatic imbalance, at OGD 2h, and a significant increase in LIAS expression at OGD 2h and OGD 3h (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH -J).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Elesclomol exacerbates neuronal cell damage and copper homeostatic imbalance\u003c/h2\u003e \u003cp\u003eWe intervened different concentrations of Elesclomol (6.25 nmol/mL, 12.5 nmol/mL, 18.75 nmol/mL and 25 nmol/mL) in primary neuronal OGD models. It was found that with Elesclomol prompted a significant decrease in cell viability and a significant increase in cell damage (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B). It was further observed that the intracellular Cu content was elevated after the administration of Ele interventions at concentrations of 6.25 nmol/mL and 12.5 nmol/mL, but the Cu content was decreasing with increasing concentration. (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). OGD induced an increase in the expression of the key proteins of copper homeostatic imbalance, FDX1 and LIAS, and the expression of intracellular FDX1 protein and LIAS protein was significantly elevated after the administration of Ele intervention at a concentration of 12.5 nmol/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD-F).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Elesclomol promotes oxidative stress in nerve cells\u003c/h2\u003e \u003cp\u003eThe relationship between Elesclomol and oxidative stress was explored through four aspects: ROS fluorescence expression, intracellular MDA, GSH and SOD content. We observed that the relative fluorescence intensity of intracellular ROS was enhanced in the OGD group, which was significantly enhanced after the intervention of 6.25 nmol/mL and 12.5 nmol/mL Elesclomol was given (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B).The intracellular MDA content in the OGD group was significantly increased, the GSH content was significantly decreased, and the SOD content was significantly increased by the intervention of 6.25 nmol/mL and 12.5 nmol/mL Elesclomol was given. 6.25 nmol/mL and 12.5 nmol/mL Ele intervention, the intracellular MDA content was significantly increased, the intracellular GSH content was decreased, and the intracellular SOD content was increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Elesclomol activates the Nrf2/HO-1 signaling pathway in neuronal cells\u003c/h2\u003e \u003cp\u003eWe evaluated the relationship between copper homeostatic imbalance and Nrf2/HO-1 signaling pathway through the expression of key proteins of Nrf2/HO-1 signaling pathway. It was found that Nrf2 activated by intranuclear was significantly elevated in the OGD group, and there was a trend of increased intranuclear activation of Nrf2 after the administration of 6.25 nmol/mL Elesclomol intervention, whereas the intranuclear activation of Nrf2 was significantly decreased by the administration of 12.5 nmol/mL Elesclomol intervention (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B). Further protein assay revealed that the expression of Nrf2 and HO-1 proteins related to Nrf2/HO-1 signaling pathway was significantly increased in the OGD group, and Nrf2 protein expression of the Nrf2/HO-1 signaling pathway was significantly increased, and HO-1 protein expression was increased after the administration of 6.25 nmol/mL Elesclomol intervention; the Nrf2/HO-1 signaling pathway protein expression was significantly decreased in the OGD\u0026thinsp;+\u0026thinsp;6.25 nmol/mL group when compared with that in the OGD\u0026thinsp;+\u0026thinsp;6.25 nmol/mL Nrf2/HO-1 signaling pathway Nrf2 and HO-1 protein expression was significantly decreased in the OGD\u0026thinsp;+\u0026thinsp;12.5 nmol/mL group compared with the OGD\u0026thinsp;+\u0026thinsp;6.25 nmol/mL Elesclomol group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC-E).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003e3.6 Silencing Nrf2 exacerbates oxidative stress and inhibits the Nrf2/HO-1 signaling pathway in OGD primary neuronal cells\u003c/em\u003e \u003c/p\u003e \u003cp\u003eWe explored the effect of cuproptosis inducer on oxidative stress in silencing Nrf2 primary neuronal cells OGD by ROS fluorescence expression and SOD content and expression of key proteins of Nrf2/HO-1 signaling pathway. It was found that silencing Nrf2 promoted the increase of ROS and SOD levels after OGD in neuronal cells, and Elesclomol induced the oxidative stress response and further increased the ROS and SOD levels after OGD in silenced Nrf2 neuronal cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-C). The expression of Nrf2/HO-1 pathway-associated proteins Nrf2 and HO-1 was down-regulated after silencing Nrf2 neuronal cells OGD, and Nrf2 and HO-1 were in an inhibitory state after Elesclomol intervention in neuronal cells OGD (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD-F).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.7 Silencing Nrf2 exacerbates copper homeostatic imbalance in OGD neuronal cells\u003c/h2\u003e \u003cp\u003eWe further constructed a silenced Nrf2 neuronal OGD model and administered Elesclomol to synchronize the intervention, and detected primary neuronal cell damage using cell morphology observation method, CCK8 method and LDH release assay. We found that silencing Nrf2 promoted cytosolic rounding, decreased cell number, cell viability, and cell leakage in OGD neuronal cells, and Elesclomol induced OGD neuronal cell injury and further aggravated OGD injury in silenced Nrf2 neuronal cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-C). We found that silencing Nrf2 neuronal cells with OGD significantly increased Cu content and further increased the promotion of Cu content by Elesclomol (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). The expression of FDX1 and LIAS, key proteins for copper homeostatic imbalance, was up-regulated after silencing OGD in Nrf2 neuronal cells, and the promotion of FDX1 and LIAS, key proteins for copper homeostatic imbalance, by Ele was inhibited (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE-G).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eCopper is a key component in the normal metabolism of brain cells and in the formation of neurotransmitters, and it has been found that imbalance in copper homeostasis is involved in the development of central nervous system diseases, and that excess copper interacts with aggregated proteins and participates in cellular oxidative stress leading to nerve cell necrosis[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Several clinical studies have found that baseline plasma copper levels are approximately linearly and positively correlated with the risk of first ischemic stroke, and that toxic/co-toxic effects of copper ions are involved in the development of several cerebrovascular diseases, including stroke. It is now clear that copper ions are widely involved in several pathological processes in stroke, such as serum copper, copper/zinc, and copper/selenium ratios reflecting the level of oxidative stress and the trophic status of brain cells in acute ischemic stroke[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. A genome-wide CRISPR/Cas9 screen identified seven genes associated with cuproptosis, among which the Fe-S cluster proteins FDX1 and LIAS are markers of cuproptosis[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Firstly, as key regulatory factors of protein lipid acylation, FDX1 and LIAS are involved in the regulation of protein lipid acylation including dihydrolipoamide acyltransferase (DLAT), and the lipid acylated proteins can directly bind to copper ions to form oligomers. Secondly, FDX1 reduces Cu\u003csup\u003e2+\u003c/sup\u003e to the more cytotoxic Cu\u003csup\u003e+\u003c/sup\u003e, leading to the destabilization of iron-sulfur cluster proteins[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In our rat MCAO model, we found that with the prolongation of the MCAO time in rats, the neurological impairment increased, and a simultaneous increase in the copper concentration in the ischemic side of the brain tissue, the fluorescence intensity of FDX1, a key marker of copper homeostatic imbalance, and the expression of FDX1 and LIAS proteins were observed. This result suggests that ischemic-hypoxic stimulation induces a high copper state, which exacerbates cellular damage. In the present study, we isolated and cultured primary neural cells in vitro, identified the purity of the extracted primary neural cells by morphological observation and immunofluorescence staining with MAP2, a neural cell-specific marker, and established an OGD model to simulate cerebral ischemia in vivo. We found that cellular copper concentration, the key protein of copper homeostatic imbalance, FDX1, immunofluorescence intensity, FDX1, and LIAS, and the expression of LIAS, all of which are key proteins of the Cu homeostatic imbalance, reached a peak at 2 h of OGD, which may be a critical time point for the occurrence of ischemia-hypoxia-induced copper homeostatic imbalance. In conclusion, our animal and cellular experiments have clearly demonstrated that copper homeostatic imbalance is involved in the pathological process of cerebral ischemic neuronal cell injury.\u003c/p\u003e \u003cp\u003eElesclomol is a copper carrier that targets mitochondria, binds to copper in the extracellular environment, generates a membrane-permeable complex, and transports copper to mitochondria, thereby inducing an intracellular high copper state[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. We firstly intervened primary neuronal cells with OGD using different concentrations (6.25 nmol/mL, 12.5 nmol/mL, and 18.75 nmol/mL) of Ele, and found that Ele promoted primary neuronal cell damage in OGD without a dose-dependent characteristic. Ele at concentrations of 6.25 nmol/mL and 12.5 nmol/mL promoted increased intracellular copper concentrations (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), which was hypothesized to be a possible effector concentration for Ele-induced copper homeostatic imbalance. Related studies have found that intracerebral striatal injection of Cu\u003csup\u003e2+\u003c/sup\u003e leads to increased oxidative stress in the striatum and substantia nigra[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Other studies have shown that excess copper can induce oxidative stress through activation of the ROS/HO-1/NQO1 pathway, and inhibition of HO-1 may attenuate copper-induced oxidative stress[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. We further observed that 6.25 nmol/mL and 12.5 nmol/mL of Ele promoted higher ROS fluorescence expression, increased MDA content, and decreased GSH content. This result suggests that copper homeostatic imbalance induces oxidative stress, which may be a key pathological pathway leading to neuronal cell injury.\u003c/p\u003e \u003cp\u003eWhen oxidative stress occurs, the imbalance of intracellular oxidative and antioxidant systems can lead to the activation of multiple signaling pathways, and the Nrf2/HO1 signaling pathway is one of the key signaling pathways for sensing the environment and regulating endogenous oxidative stress, which maintains cellular redox homeostasis by transcriptionally inducing protective genes. Excess ROS prompts Nrf2 to dissociate from Keap1, and Nrf2 enters the nucleus and activates gene expression of intracellular detoxifying enzymes and antioxidant proteins through ARE (antioxidant response element). HO-1 belongs to the class of metabolizing enzymes, and is a member of the heme oxygenase (HO) family, which is regulated at the transcriptional level by the transcription factor Nrf2. The activation of the Nrf2/HO-1 signaling pathway has a variety of effects such as antioxidant, anti-inflammatory, maintenance of mitochondrial homeostasis, inhibition of apoptosis, and regulation of pyroptosis[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In HT22 mouse hippocampal neuronal cells exposed to copper ions, copper-induced oxidative damage reduced CREB phosphorylation, decreased Bcl-2 expression, increased Bax expression, activated the Nrf2 signaling pathway, facilitated the dissociation of the keap1-Nrf2 complex, enhanced the nuclear translocation of Nrf2, and stimulated the expression of the antioxidant molecules HO-1 and NQO1[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In CD-1 rats treated with copper ions, higher levels of copper treatment activated the Keap1/Nrf2 signaling pathway and significantly increased the expression of genes related to redox status[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Notably, our study observed a trend of increased Nrf2 activation by Ele given 6.25 nmol/mL and a significant decrease in Nrf2 activation by Ele given 12.5 nmol/mL (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and increased expression of copper homeostatic marker proteins, FDX1 and LIAS, in primary neuronal cells given 12.5 nmol/mL of Ele. This result suggests that Ele promotes oxidative stress and activates the Nrf2/HO-1 signaling pathway, and that low-dose Ele promotes Nrf2 expression and inhibits copper homeostatic imbalance, whereas high-dose Ele inhibits Nrf2 expression and promotes copper homeostatic imbalance. Subsequently, we used adenoviral transfection to knock down Nrf2, a key regulator of the Nrf2/HO-1 signaling pathway, and showed that silencing of Nrf2 resulted in cellular damage, oxidative stress, and copper homeostatic imbalance in heavy OGD, and further exacerbated the Ele-induced cellular damage and copper homeostatic imbalance. This result demonstrates for the first time that the Nrf2/HO-1 signaling pathway has an effect on regulating copper homeostatic imbalance.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eOur study in this work firstly found that copper homeostatic imbalance occurs after cerebral ischemia and hypoxia, further observed the relationship between copper homeostatic imbalance and oxidative stress, and for the first time found that the Nrf2/HO-1 signaling pathway is involved in the pathological process of copper homeostatic imbalance under cerebral ischemia and hypoxia, and Nrf2 is a key factor regulating the imbalance of copper homeostatic imbalance.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eOGD, Oxygen-Glucose Deprivation; MACO, Medication Adherence Context and Outcomes; Ele, Elesclomol; MDA, malondialdehyde; SOD, superoxide dismutase; ROS, Reactive Oxygen Species; HO-1, heme oxygenase-1; \u0026nbsp;MAP2,Microtubule-associated protein 2; Si-Nrf2,Nrf2 adenovirus; NC-Nrf2,control empty vector lentivirus; FDX1, ferredoxin 1; LIAS, lipoic acid synthase.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003cstrong\u003euthor contribution\u003c/strong\u003e\u003cstrong\u003es\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTing\u003c/strong\u003e\u003cstrong\u003eLang\u003c/strong\u003e and \u003cstrong\u003eYanhao Liao\u003c/strong\u003e: Formulation and implementation of experimental research, Writing of original manuscript. \u003cstrong\u003eBingbin Fan\u003c/strong\u003e and \u003cstrong\u003eHuixian Chen\u003c/strong\u003e: Research work of animal experiment in vivo. \u003cstrong\u003eJunjie Hu\u003c/strong\u003e and \u003cstrong\u003eLijin\u003c/strong\u003e\u003cstrong\u003eg\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Xie\u003c/strong\u003e: Research work of cell experiment in vitro.\u003cstrong\u003e\u0026nbsp;Keyi Qin\u003c/strong\u003e and \u003cstrong\u003eZhenlin Zhong\u003c/strong\u003e: Analyze experimental data using data analysis software.\u003cstrong\u003e\u0026nbsp;Keyan Jiao\u003c/strong\u003e, \u003cstrong\u003eYan She\u003c/strong\u003eand\u003cstrong\u003e\u0026nbsp;Le Shao\u003c/strong\u003e: Guidance and supervision of experimental research, revision of manuscript.\u0026nbsp;All data were generated in-house, and no paper mill was used. All authors agree to be accountable for all aspects of work ensuring integrity and accuracy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number:\u003c/strong\u003e not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003cstrong\u003e/\u003c/strong\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003cstrong\u003eDeclaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the images were drawn by Yanhao Liao and Binbing Fan (Using Adobe illustrator 2021, https://www.adobe.com/cn/creativecloud/roc/business.html).\u003c/p\u003e\n\u003cp\u003eThis research was supported by the National Natural Science Foundation of China (No: 81603415,82174176),\u0026nbsp;Education Department of Hunan Province of China\u0026nbsp;(No: 23A0288).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of materials and data\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInformed consent was obtained from all individual participants included in the studyCompeting Interest\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll authors of this research declare that there are no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnimal experiments were conducted in strict accordance with the regulations of the Animal Ethics Committee (Certificate No. ZYFY20230920005) (All programs meet the ARRIVE guideline 2.0).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGBD 2021 Diseases and Injuries Collaborators, (2024) Global incidence, prevalence, years lived with disability (YLDs), disability-adjusted life-years (DALYs), and healthy life expectancy (HALE) for 371 diseases and injuries in 204 countries and territories and 811 subnational locations, 1990-2021: a systematic analysis for the Global Burden of Disease Study 2021. 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(2021) Activation of the ROS/HO-1/NQO1 signaling pathway contributes to the copper-induced oxidative stress and autophagy in duck renal tubular epithelial cells. Sci Total Environ 757:143753\u003c/li\u003e\n\u003cli\u003eTelkoparan-Akillilar P, Panieri E, Cevik D, Suzen S, Saso L (2021) Therapeutic Targeting of the NRF2 Signaling Pathway in Cancer. Molecules 26(5):1417\u003c/li\u003e\n\u003cli\u003eLiu S, Pi J, Zhang Q (2022) Signal amplification in the KEAP1-NRF2-ARE antioxidant response pathway. Redox Biol 54:102389\u003c/li\u003e\n\u003cli\u003eZhong G, He Y, Wan F, et al. (2021) Effects of Long-Term Exposure to Copper on the Keap1/Nrf2 Signaling Pathway and Msr-Related Redox Status in the Kidneys of Rats. Biol Trace Elem Res 199(11):4205-4217\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"cerebral ischemia, copper homeostatic imbalance, Nrf2/HO-1 signaling pathway, oxidative stress, Nrf2, FDX1, LIAS, HO-1","lastPublishedDoi":"10.21203/rs.3.rs-6650982/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6650982/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIschemic stroke is the third leading cause of death in the world, and oxidative stress is the key pathological mechanism of cerebral ischemia. The Nrf2/HO-1 signaling pathway is an endogenous defense system that regulates oxidative stress in the brain, and is involved in the imbalance regulation of copper homeostasis. The brain is one of the main target organs for copper accumulation, and copper is involved in the formation of myelin sheaths and the regulation of synaptic activity and signaling cascades. Imbalance of copper homeostasis leads to metabolic abnormalities and toxic effects on cells, and the mechanisms involved in cerebral ischemic injury are unknown. In this study, we found for the first time that the expression of copper homeostatic imbalance markers was elevated after cerebral ischemia, and the silencing of Nrf2 promoted the further elevation of copper homeostatic imbalance-related markers, which suggests that copper homeostatic imbalance is involved in the pathological process of cerebral ischemic neurological injury and that Nrf2/HO-1 signaling pathway is closely related to copper homeostatic imbalance in cerebral ischemia. Our study provides an experimental basis for further investigation of cuproptosis in cerebral ischemia.\u003c/p\u003e","manuscriptTitle":"Inhibition of Nrf2/HO-1 signaling pathway exacerbates Copper homeostasis in cerebral ischemia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-06 09:42:24","doi":"10.21203/rs.3.rs-6650982/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"cc9845cd-a812-438d-81ef-37dc84c93db2","owner":[],"postedDate":"June 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-06-12T11:09:01+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-06 09:42:24","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6650982","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6650982","identity":"rs-6650982","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}