Use of exogenous recombinant protein ANXA5 in preparation of drugs for treating cerebral hemorrhage

Abstract

The application belongs to the technical field of biomedicine, and particularly relates to application of exogenous recombinant protein ANXA5 in preparation of a medicine for treating cerebral hemorrhage. The application discloses that the expression level of ANXA5 is increased after cerebral hemorrhage; by adding the recombinant ANXA5 protein, the expression level of an iron death related protein can be reduced; meanwhile, the expression level of an apoptosis protein is reversed, the oxidative stress level is slightly reduced, and the levels of MDA and active oxygen water are obviously decreased, which proves that the recombinant protein ANXA5 plays an inhibiting role on neuron iron death after cerebral hemorrhage. The application provides a new type of medicine basis for using the recombinant protein ANXA5 as a treatment for cerebral hemorrhage, especially iron death caused after cerebral hemorrhage.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of exogenous recombinant protein ANXA5 in the preparation of drugs for treating cerebral hemorrhage. Background Technology

[0002] Intracranial hemorrhage (ICH) refers to primary, non-traumatic bleeding within the brain parenchyma, accounting for 20%-30% of acute cerebrovascular diseases. Approximately 80% of hemorrhages occur in the cerebral hemispheres, and about 20% occur in the brainstem and cerebellum. Compared to ischemic stroke, ICH has a higher mortality and disability rate, imposing a heavy burden on society and families. ICH is classified into primary and secondary ICH. Primary ICH occurs when a blood vessel ruptures suddenly, forming a large hematoma that mechanically compresses surrounding brain tissue, leading to increased intracranial pressure and neurological deterioration. Secondary ICH involves a series of pathological processes triggered by red blood cell fragments and degradation products, such as inflammatory responses, oxidative stress, and blood-brain barrier disruption. In recent decades, new therapeutic targets have focused on exploring the mechanisms of ICH-induced secondary ICH. The pathophysiological mechanisms of secondary cerebral hemorrhage include: (1) after cerebral hemorrhage, factors such as heme, iron ions, and free radicals lead to apoptosis of neurons around the hematoma, which ultimately causes symptoms such as memory loss, cognitive impairment, and poor language expression in ICH patients; (2) reactive activated astrocytes play protective and harmful functions, thereby regulating pathological processes such as neurogenesis and synapsis, destruction of the blood-brain barrier and angiogenesis, cytotoxic edema, and neuroinflammation; (3) after cerebral hemorrhage, microglia activated by hematoma components and their degradation products play a pro-inflammatory or anti-inflammatory function, promoting the neuroinflammation process, which ultimately leads to changes in cell function such as astrocyte proliferation and neuronal apoptosis; (4) after cerebral hemorrhage, endothelial cell apoptosis, changes in tight junctions, and capillary leakage lead to destruction of the blood-brain barrier, thereby causing cerebral edema and deterioration of neurological function.

[0003] Ferroptosis, first proposed by Dr. Brent R. Stockwell of Columbia University in 2012, is an iron-dependent process primarily dependent on iron-mediated oxidative damage and subsequent cell membrane injury. It is a novel form of programmed cell death, distinct from apoptosis, necrosis, and autophagy. The essence of ferroptosis is the depletion of glutathione (GSH), leading to a decrease in glutathione peroxidase (GPX4) activity. Lipid oxides cannot be metabolized by GPX4-catalyzed glutathione reductase, resulting in the oxidation of lipids by divalent iron ions, producing reactive oxygen species (ROS), thus promoting ferroptosis. Ferroptosis mainly involves three aspects: System Xc, GSH, and GPX4 / ROS. Secondary brain injury following ICH is primarily due to irreversible neuronal damage caused by hemoglobin lysates and iron ions.

[0004] Annexin A5 (Annexin V) is a protein widely distributed in animal cells with diverse biological functions. It is a calcium-dependent phospholipid-binding protein that regulates processes such as anticoagulation, apoptosis, and signal transduction, and participates in the occurrence and development of various diseases. ANXA5 can recognize phosphatidylserine (PS) residues exposed on the surface of apoptotic cells due to loss of membrane asymmetry, and is therefore considered an early marker of apoptosis.

[0005] Recent research reports that ANXA5, in addition to its anticoagulant function, participates in various processes such as inflammatory responses and apoptosis, playing an important role in tumors, immune diseases, and neurodegenerative diseases. However, whether ANXA5 participates in regulating neuronal ferroptosis after cerebral hemorrhage remains unknown. Summary of the Invention

[0006] To address the aforementioned issues, this invention analyzes the expression trends of ANXA5 protein after cerebral hemorrhage and further explores whether exogenous recombinant protein ANXA5 can inhibit ferroptosis after cerebral hemorrhage.

[0007] The first aspect of the present invention provides the use of exogenous recombinant protein ANXA5 in the preparation of drugs for treating cerebral hemorrhage.

[0008] Preferably, the drug for treating cerebral hemorrhage is a drug that inhibits neuronal ferroptosis.

[0009] Preferably, the drug that inhibits neuronal ferroptosis is one that reduces the expression level of ferroptosis-related gene proteins.

[0010] Preferably, the drug that inhibits neuronal ferroptosis is a drug that reduces oxidative stress levels.

[0011] Preferably, the drug that inhibits neuronal ferroptosis is a drug that reduces the accumulation of lipid peroxides.

[0012] Preferably, the drug that inhibits neuronal ferroptosis is a drug that slows down neuronal apoptosis.

[0013] Preferably, the dosage form of the above-mentioned drug is any pharmacologically acceptable dosage form.

[0014] Preferably, the dosage of the above-mentioned drug is any pharmacologically acceptable dosage.

[0015] A second aspect of the present invention provides a pharmaceutical composition for treating neuronal ferroptosis induced after cerebral hemorrhage, wherein the active ingredient of the pharmaceutical composition comprises exogenous recombinant protein ANXA5.

[0016] The beneficial effects of this invention are:

[0017] 1. This invention reveals the increased expression level and cell distribution of ANXA5 after cerebral hemorrhage.

[0018] 2. This invention demonstrates that ANXA5 can significantly reduce neuronal apoptosis by inhibiting ferroptosis.

[0019] 3. This invention demonstrates that the addition of recombinant protein ANXA5 can effectively inhibit neuronal ferroptosis, proving that recombinant protein ANXA5 has potential neuroprotective effects.

[0020] 4. This invention provides a novel intervention method that is expected to play an important role in the treatment of cerebral hemorrhage and has broad clinical application prospects. Attached Figure Description

[0021] Figure 1 This diagram shows the expression of proteins related to cerebral hemorrhage in mice. Figure 1 Figure A shows the protein expression levels of the relevant proteins. Figure 1 Figure B shows the statistical results of ANXA5 expression after cerebral hemorrhage in mice. Figure 1 CD is a graph showing the statistical results of the expression of apoptosis-related proteins Bcl-2 and Bax. Figure 1 EF is a graph showing the statistical results of the expression of ferroptosis-related molecules GPX4 and SLC7A11. Figure 1 G represents the statistical results of the expression of the pro-apoptotic protein Caspase-3. Figure 1 H represents the statistical results of the expression of the pro-inflammatory factor iNOS. Figure 1 Figure I shows the statistical results of NOX4 expression, a molecule related to oxidative stress.

[0022] Figure 2 ANXA5 and GFAP (a marker of astrocytes) were compared after intracranial hemorrhage in mice. Figure 2A) Microglial cell marker IBA-1 ( Figure 2 B) Neuronal markers NEUN Figure 2 C) colocalization staining analysis; Figure 2 D shows the co-localization results of ANXA5 and the neuronal marker NEUN.

[0023] Figure 3 This diagram illustrates the expression of related proteins in a heme-stimulated neuronal HT22 cell model. Figure 3 Figure A shows the protein expression levels of the relevant proteins. Figure 3 BF is a statistical result graph of connexin ANXA5, ferroptosis-associated protein GPX4, oxidative stress-associated protein SOD-2, pro-inflammatory cytokine IL-1β, and apoptosis-inducing protein Caspase-3.

[0024] Figure 4 The figure shows the expression of ANXA5 after intervention with overexpression and stimulation with Hemin. Figure 4 The graph shows the protein expression levels of protein A. Figure 4 BG is a statistical result figure for ANXA5, ferroptosis-related proteins GPX4 and SLC7A11, apoptosis proteins Bax and Bcl-2, and oxidative stress-related molecule NRF2.

[0025] Figure 5 The graph shows the expression of ANXA5 after intervention with overexpression and stimulation with Hemin. Figure 5 A represents the change in MDA level. Figure 5 BC represents the results and statistical results of reactive oxygen species production. Detailed Implementation

[0026] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings. These embodiments are implemented based on the technical solution of the present invention and provide detailed implementation methods and processes, which are intended to explain the present invention. However, the implementation does not constitute a limitation on the present invention.

[0027] Example 1: Construction of a mouse ICH model

[0028] Seventy SPF-grade male C57BL / 6 mice, 8 weeks old, were randomly divided into 7 groups, including one control group and six experimental groups (6h, 12h, 1d, 3d, 7d, and 14d). Mice were anesthetized with 4% chloral hydrate and placed on a stereotaxic apparatus frame. The skull and anterior fontanelle were exposed. A 0.05mm diameter bone window was drilled on the skull surface 0.2mm posterior to the anterior fontanelle and 2.3mm to the right of the midline using a dental drill. 15μl of blood was collected from the mouse's tail vein using a micro-syringe. The needle was inserted vertically 3.5mm along the bone window and injected into the mouse's right basal ganglia at a constant rate of 2μl / min. After 10 minutes of needle withdrawal, the needle was slowly withdrawn, the scalp was sutured, and the mice were placed on a 37℃ constant-temperature pad until their body temperature returned to normal before being returned to their cages. The Sham group received no special treatment.

[0029] Example 2: Changes in ANXA5 levels after cerebral hemorrhage

[0030] After establishing a brain hemorrhage model in mice at different time points and in the Sham group, the mice were anesthetized, and the area surrounding the hematoma in the mouse brain tissue was separated.

[0031] 1. Protein extraction:

[0032] The isolated brain tissue was added to high-efficiency ripa lysis buffer (Beyotime / P0013B) and homogenized at low temperature using a homogenizer. After 15 minutes of rotation at 4°C and centrifugation at 15,000 rpm for 20 minutes at 4°C, the supernatant was extracted, the protein concentration was measured, and after balancing, 5X loading buffer was added to prepare the protein sample.

[0033] Prepare the following in advance: electrophoresis buffer (Tris 3.02g, glycine 18.8g, SDS 1g, ddH2O to 1000ml), transfer buffer (Tris 3.03g, glycine 14.4g, methanol 200ml, ddH2O to 1000ml), TBST (Tris 2.42g, NaCl 8g, Tween 20 0.5ml, ddH2O to 1000ml), and blocking buffer (5% skim milk powder + TBST). Pre-cool the transfer buffer in a 4°C refrigerator.

[0034] 2. Electrophoresis:

[0035] Centrifuge the protein samples and set aside. Mount the glass plate onto the electrophoresis apparatus, add the electrophoresis buffer, and remove the comb. Use a 10 μl pipette to blow away any residual gel in each well. After adding an equal amount of sample to each well, set the voltage to 80V and begin gel electrophoresis. Once the sample reaches the separating gel, increase the voltage to 120V and stop electrophoresis when the bromophenol blue just appears.

[0036] 3. Transfer:

[0037] Remove the glass plate, rinse off the electrophoresis buffer with ddH2O, place it in transfer buffer, and cut off any unwanted parts. Activate the PVDF membrane by immersing it in methanol for about 1 minute, then rinse and depolarize it in ddH2O. Assemble the sandwich clamp from bottom to top in the following order: sponge - three layers of filter paper - gel - PVDF membrane - three layers of filter paper - sponge, and place it in the transfer tank. Set the transfer conditions: constant current 300mA, 90 minutes.

[0038] 4. Blocking and antibody incubation:

[0039] The PVDF membrane was retrieved, washed with ddH2O to remove methanol, and placed in blocking buffer. It was then placed on a shaker and blocked at room temperature for 1-2 hours. The blocking buffer was washed off with TBST, and the membrane was trimmed as necessary. Primary antibody was then applied, and the membrane was incubated overnight on a shaker at 4°C. The next day, the primary antibody was recovered, and the membrane was washed with TBST for 5 minutes at a time, for a total of 3 washes. Secondary antibody was applied, and the membrane was incubated at room temperature for 2 hours. TBST was then applied for 5 minutes at a time, for a total of 3 washes. An appropriate amount of developing solution was prepared according to the instructions of the ultrasensitive ECL chemiluminescence detection kit. Finally, the membrane was scanned for imaging, and the results were analyzed.

[0040] To investigate the expression changes of ANXA5 in ICH mice, a mouse model of brain hemorrhage was first constructed. The results are as follows: Figure 1 As shown, the expression levels of various related proteins changed after cerebral hemorrhage in mice. Figure 1 A) A successful model of cerebral hemorrhage was established. The expression level of connexin ANXA5 increased after ICH, reaching its highest level on day 1. Figure 1 B). ICH is a devastating form of stroke with high mortality and morbidity. It can induce events such as apoptosis or necrosis of cells, resulting in post-traumatic neurological deficits. The anti-apoptotic molecule Bcl-2 shows a continuous decreasing trend after ICH, indicating that apoptosis is aggravated after ICH. Figure 1 C). The apoptosis-promoting molecule Bax showed an increasing trend after ICH, which was opposite to the trend of Bcl-2 protein levels. Combined detection of both indicated a significant increase in apoptosis levels after intracerebral hemorrhage. Figure 1 D). The expression levels of ferroptosis-related molecules GPX4 and SLC7A11 showed a decreasing trend after cerebral hemorrhage, with the lowest expression levels around the first day, indicating excessive lipid peroxide production in brain tissue, redox imbalance, and the occurrence of ferroptosis. Figure 1 E, F). The apoptosis-promoting molecule Caspase-3 showed a continuous increasing trend after ICH, with the highest expression level on day 7, also indicating aggravated apoptosis. Figure 1 G). The pro-inflammatory cytokine iNOS showed an increasing trend after ICH, with the highest expression level on day 5, indicating a sustained increase in inflammation levels. Figure 1H). The oxidative stress-related molecule NOX4 showed an increasing trend after ICH, with the highest expression level on day 3, indicating a worsening of oxidative stress. Figure 1 I). Studies have shown that ferroptosis plays an important role in the regulation of oxidative stress and inflammatory responses; therefore, the expression levels of oxidative stress and inflammation-related proteins are altered after cerebral hemorrhage in mice.

[0041] The above results demonstrate that a brain hemorrhage model was successfully established, with increased levels of apoptosis and inflammation, and ferroptosis occurring after ICH, consistent with previous reports. Furthermore, ANXA5 protein expression gradually increased after ICH, reaching its highest level 7 days post-hemorrhage.

[0042] Example 3: Distribution of ANXA5 in neurons after cerebral hemorrhage

[0043] To further observe the localization of ANXA5 in different cell types after ICH, this invention uses double immunofluorescence staining with cell-specific markers to identify cell types. Frozen sections of mouse brain tissue from the sham-operated group and 1 day after ICH were stained with anti-ANXA5 antibodies for fluorescence staining.

[0044] Immunofluorescence staining: After anesthetizing mice, blood vessels were flushed with physiological saline, and the tissues were perfused with 4% paraformaldehyde. The brain tissue was isolated and fixed overnight in 4% paraformaldehyde. After dehydration with a 20% and 30% sucrose gradient, brain tissue was embedded in OCT and sectioned into 10 μm sections using a cryostat. The sections were washed with PBS for 15 minutes to remove the embedding agent, then blocked with blocking buffer at room temperature for 2 hours. After washing with PBS for 15 seconds to remove the blocking buffer, the sections were incubated overnight at 4°C with rabbit anti-ANXA5 and mouse anti-NEUN. The primary antibody was washed away, and the sections were incubated with secondary antibody at room temperature for 2 hours. The secondary antibody was then washed away, and the nuclei were stained with DAPI for 10 minutes. The sections were then mounted. Images were observed using a fluorescence microscope.

[0045] like Figure 2 As shown, among the three marker molecules—astrocyte marker GFAP, microglia marker IBA-1, and neuronal marker NEUN—the positive signals of ANXA5 and NEUN (neuronal activation marker) increased after ICH compared with the sham-operated group. In addition, there was an overlap between the positive signals of ANXA5 and NEUN, indicating that ANXA5 may be a potential molecule involved in neuronal activation after ICH. Therefore, HT22 neuronal cells were selected for subsequent in vitro cell experiments.

[0046] Example 4: Construction of a heme-stimulated neuronal HT22 cell model

[0047] The complete culture medium formula used for cell culture was: DMEM (4.5 g / L D-Glucose) + 10% fetal bovine serum + 1% penicillin-streptomycin (100 mg / ml). Culture environment: 37℃ incubator (containing 5% CO2).

[0048] To investigate the expression changes of ANXA5 in in vitro cell experiments, ferroptosis was induced in neuronal cell lines using heme (30 μM hemin). HT22 cells were seeded in five large dishes. When the cell density reached 60%-70%, the experimental group was stimulated with 30 μM hemin. Cell proteins were collected at six time points: 3h, 6h, 12h, and 24h. Western blotting was used to analyze the expression changes of ANXA5, ferroptosis protein GPX4, oxidative stress molecule SOD-2, inflammatory cytokine IL-1β, and apoptosis protein Caspase-3 in the HT22 cell model. The results are as follows: Figure 3 As shown, the expression levels of various related proteins in the HT22 neuronal cell model changed after heme stimulation. Figure 3 A) shows that the expression trends of each protein are consistent with the in vivo experimental trends; the expression level of connexin ANXA5 showed a continuous upward trend after Hemin stimulation, and the expression level was the highest at 24 h, which is consistent with the trend of ANXA5 in the mouse cerebral hemorrhage model. Figure 3 B). Hemin stimulation of HT22 cells also triggered ferroptosis. In vivo experiments simulating ICH mice showed that the ferroptosis-related protein GPX4 decreased continuously after Hemin stimulation, indicating excessive intracellular lipid peroxide production, redox imbalance, and the occurrence of ferroptosis. Figure 3 C). SOD-2, an oxidative stress-related protein, is an important component of the antioxidant enzyme system in biological systems. Its level remains elevated after Hemin stimulation, indicating aggravated oxidative stress. Figure 3 D). The sustained increase in IL-1β levels, a inflammatory cytokine, after Hemin stimulation indicates exacerbated cellular inflammation. Figure 3 E). Neuronal apoptosis is an important mechanism for maintaining homeostasis in the nervous system. The expression trend of the pro-apoptotic protein Caspase-3 in cells was consistent with that after ICH, indicating that apoptosis was exacerbated. Figure 3 F) Changes in the levels of apoptosis-related proteins and inflammatory factors can attest to the successful construction of the cell model.

[0049] The above results indicate that a heme-stimulated neuronal HT22 cell model was successfully constructed, and ANXA5 showed a continuous upward trend in the cell model, consistent with the trend observed in in vivo experiments.

[0050] Example 5: Effects of recombinant protein ANXA5 on neuronal ferroptosis

[0051] To investigate the regulatory role of recombinant protein ANXA5 in neuronal ferroptosis, a heme-stimulated HT22 neuronal cell model was constructed in vitro, and 1 μL / mL of recombinant ANXA5 protein (abcom, ab89493) was added. The protective effect of ANXA5 was analyzed by Western blotting.

[0052] The complete culture medium formula used for cell culture was: DMEM (4.5 g / L D-Glucose) + 10% fetal bovine serum + 1% penicillin-streptomycin (100 mg / ml). Culture environment: 37℃ incubator (containing 5% CO2).

[0053] HT22 cells were seeded in four large dishes and divided into three groups: Control group, ANXA5 group, Hemin stimulation group, and Hemin+ANXA5 group. When the cell density reached 60%-70%, 2 μL of recombinant ANXA5 protein was added to the ANXA5 group and the Hemin+ANXA5 group for 6 hours. Then, 30 μM heme was added to the Hemin stimulation group and the Hemin+ANXA5 group for 24 hours. The Control group received no special treatment. Cells from all four dishes were harvested, and protein expression levels were detected by Western blot.

[0054] The results are as follows Figure 4 As shown, the first step was to verify whether the recombinant protein ANXA5 played an overexpression role. The changes in the expression levels of various related proteins are shown below. Figure 4 As shown in Figure A, the expression level of ANXA5 molecules was significantly increased under the action of the recombinant protein compared with the control group, indicating that the recombinant protein ANXA5 can significantly increase the expression level of ANXA5 molecules in cells. Figure 4 B); Compared with the Hemin stimulation group, overexpression of ANXA5 significantly increased the levels of ferroptosis-related proteins GPX4 and SLC7A11, and reduced intracellular lipid peroxide production, indicating that recombinant protein ANXA5 can effectively inhibit neuronal ferroptosis. Figure 4 CD); Compared with the Hemin stimulation group, the levels of apoptotic proteins Bax and Bcl-2 were reversed, indicating an improvement in neuronal apoptosis. As previous studies have shown, ANXA5 has an anti-apoptotic effect. Figure 4 (EF); Compared with the Hemin stimulation group, the expression level of the oxidative stress-related molecule NRF2 was reversed, and the oxidative stress level was reduced, indicating that the recombinant protein ANXA5 effectively reduced the neuronal oxidative stress level. At the cellular level, oxidative stress is likely to induce ferroptosis, and reducing oxidative stress levels helps to inhibit ferroptosis. Figure 4 G).

[0055] Example 6: Exploring the effects of recombinant protein ANXA5 on neuronal ferroptosis from multiple perspectives

[0056] MDA content is an important parameter reflecting the body's antioxidant potential. It can reflect the rate and intensity of lipid peroxidation and indirectly reflect the degree of cellular peroxidation damage.

[0057] Reactive oxygen species (ROS) refer to the collective term for oxygen-containing free radicals and peroxides that readily form free radicals, all of which are involved in oxygen metabolism in living organisms. Increased ROS levels can cause severe damage to cell structure, a process known as oxidative stress. Both ROS and peroxides are indicators of oxidative stress. Iron metabolism and lipid peroxidation signaling are increasingly considered central mediators of ferroptosis. Ferroptosis plays a crucial role in the regulation of oxidative stress.

[0058] To further investigate the regulatory role of recombinant protein ANXA5 in neuronal ferroptosis, a malondialdehyde (MDA) assay kit (A003-1-96T, Nanjing Jiancheng) was purchased to detect changes in MDA levels, and a reactive oxygen species (ROS) assay kit (CM-H2DCFDA, Beyotime) was purchased to detect ROS production. Cells were divided into three groups: Crtl group, Hemin group, and Hemin+ANXA5 group. The stimulation conditions were the same as in Example 5 to investigate the effect of recombinant protein ANXA5 on lipid peroxidation.

[0059] The results are as follows Figure 5 As shown, compared with the control group, the Hemin stimulation group had significantly increased MDA content and reactive oxygen species production. However, after the addition of recombinant protein ANXA5, the levels of MDA and reactive oxygen species decreased significantly, indicating that lipid peroxides were reduced and oxidative stress was alleviated. This suggests that recombinant protein ANXA5 has a significant inhibitory effect on neuronal ferroptosis.

[0060] In summary, this invention, through the construction of in vivo mouse models and in vitro cell models, revealed that the expression level of ANXA5 gradually increases after cerebral hemorrhage. In the in vitro model, the addition of recombinant ANXA5 protein reduced the expression levels of ferroptosis-related proteins, demonstrating that recombinant ANXA5 directly inhibits ferroptosis. Simultaneously, the expression levels of apoptosis proteins were reversed, and oxidative stress levels were alleviated, further indicating that recombinant ANXA5 inhibits ferroptosis. Furthermore, MDA and reactive oxygen species levels significantly decreased, suggesting that recombinant ANXA5 inhibits neuronal ferroptosis by reducing lipid peroxides and mitigating oxidative stress. This invention provides a basis for using recombinant ANXA5 as a novel drug for the treatment of cerebral hemorrhage, especially ferroptosis induced by cerebral hemorrhage.

[0061] The above is a further detailed description of the present invention and should not be considered as a limitation on the specific implementation of the present invention. For those skilled in the art, simple deductions or substitutions without departing from the concept of the present invention are all within the protection scope of the present invention.

Claims

1. The use of exogenous recombinant protein ANXA5 in the preparation of a drug for inhibiting neuronal ferroptosis after primary cerebral hemorrhage, characterized in that, The recombinant protein ANXA5 is purchased from abcam company, product number ab89493.

2. Use according to claim 1, characterized in that, The drug for inhibiting neuronal ferroptosis is a drug for reducing the expression level of ferroptosis-related gene protein.

3. Use according to claim 1, characterized in that, The drug for inhibiting neuronal ferroptosis is a drug for reducing the level of oxidative stress.

4. Use according to claim 1, characterized in that, The drug for inhibiting neuronal ferroptosis is a drug for reducing lipid peroxide accumulation.

5. The use according to claim 1, characterized in that, The drug for inhibiting neuronal ferroptosis is a drug for slowing down neuronal apoptosis.

6. Use according to any one of claims 1 to 5, characterized in that, The dosage form of the drug is any pharmaceutically acceptable dosage form.

7. The use according to any one of claims 1 to 5, characterized in that, The dosage of the drug is any pharmaceutically acceptable dosage.

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