Application of exogenous recombinant protein ANXA5 in preparation of medicine for treating cerebral hemorrhage

Abstract

The invention belongs to the technical field of biomedicine, and particularly relates to application of an exogenous recombinant protein ANXA5 in preparation of a medicine for treating cerebral hemorrhage. The invention reveals that the expression level of ANXA5 after cerebral hemorrhage is increased; by adding the recombinant ANXA5 protein, the expression level of ferroptosis related protein can be reduced; meanwhile, the expression level of apoptotic protein is reversed, the oxidative stress level is reduced, and the MDA and active oxygen levels are obviously reduced, which proves that the recombinant protein ANXA5 plays an inhibiting role in neuronal ferroptosis after cerebral hemorrhage. The invention provides a basis for using the recombinant protein ANXA5 as a novel medicine for treating cerebral hemorrhage, especially ferroptosis caused by cerebral hemorrhage.

Description

Technical Field

[0001] The invention belongs to the field of biomedical technology, and particularly relates to application of exogenous recombinant protein ANXA5 in preparing a drug for treating cerebral hemorrhage. Background Art

[0002] Intracerebral hemorrhage (ICH) refers to primary non-traumatic intracerebral parenchymal hemorrhage, which accounts for 20%-30% of acute cerebrovascular diseases. About 80% of the bleeding sites occur in the cerebral hemispheres, and about 20% of the bleeding sites occur in the brainstem and cerebellum. Compared with ischemic stroke, intracerebral hemorrhage has a higher mortality and disability rate, which imposes a heavy burden on society and families. Intracerebral hemorrhage is divided into primary intracerebral hemorrhage and secondary intracerebral hemorrhage. Primary intracerebral hemorrhage refers to the formation of a huge hematoma after the sudden rupture of cerebral blood vessels, which mechanically compresses the surrounding brain tissue, leading to intracranial hypertension and deterioration of neurological function. Secondary intracerebral hemorrhage refers to a series of pathological processes triggered by red blood cell fragments and degradation products, such as inflammatory response, oxidative stress and blood-brain barrier destruction. In recent decades, new therapeutic targets have been focused on exploring the mechanism of ICH-induced secondary intracerebral hemorrhage. The pathophysiological mechanisms of secondary ICH include: (1) After ICH, hemoglobin, iron ions, free radicals and other factors lead to apoptosis of neurons around the hematoma, which ultimately leads to symptoms such as memory loss, cognitive dysfunction, and poor language expression ability in ICH patients; (2) Reactively activated astrocytes play protective and harmful functions, thereby regulating pathological processes such as neuronal and synaptic development, blood-brain barrier destruction and angiogenesis, cytotoxic edema and neuroinflammation; (3) After ICH, microglia activated by hematoma components and their degradation products play pro-inflammatory or anti-inflammatory functions, promote neuroinflammatory processes, and ultimately lead to cell function changes such as astrocyte proliferation and neuronal apoptosis; (4) After ICH, endothelial cell apoptosis, tight junction changes and capillary leakage lead to the destruction of the blood-brain barrier, thereby causing brain edema and deterioration of neurological function.

[0003] Ferroptosis was first proposed by Dr. Brent R. Stockwell of Columbia University in 2012. It is an iron-dependent process that mainly relies on iron-mediated oxidative damage and subsequent cell membrane damage. It is a new type of programmed cell death that is different from apoptosis, necrosis, and autophagy. The essence of ferroptosis is the depletion of glutathione (GSH), the decrease in the activity of glutathione peroxidase (GPX4), and the inability of lipid oxides to be metabolized by GPX4-catalyzed glutathione reductase reactions. After that, divalent iron ions oxidize lipids to produce reactive oxygen species (ROS), thereby promoting the occurrence of ferroptosis. Ferroptosis is mainly divided into three aspects: System Xc, GSH and GPX4, and ROS. Secondary brain damage after ICH is mainly due to irreversible damage to neurons caused by hemoglobin lysates, iron ions, etc.

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

[0005] The latest research reports that in addition to its anticoagulant function, ANXA5 is also involved in multiple processes such as inflammatory response and cell apoptosis, and plays an important role in tumors, immune diseases, and neurodegenerative diseases. However, whether ANXA5 is involved in regulating the process of neuronal ferroptosis after cerebral hemorrhage remains unknown. Summary of the invention

[0006] To solve the above problems, the present invention analyzes the expression change trend of ANXA5 protein after cerebral hemorrhage, and further explores whether exogenous recombinant protein ANXA5 can inhibit the occurrence of ferroptosis after cerebral hemorrhage.

[0007] A first aspect of the present invention provides a use of an exogenous recombinant protein ANXA5 in the preparation of a drug for treating cerebral hemorrhage.

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

[0009] Preferably, the drug for inhibiting neuronal ferroptosis reduces the expression level of ferroptosis-related gene protein.

[0010] Preferably, the drug for inhibiting neuronal ferroptosis is a drug for reducing the level of oxidative stress.

[0011] Preferably, the drug for inhibiting neuronal ferroptosis is a drug for reducing the accumulation of lipid peroxides.

[0012] Preferably, the drug for inhibiting neuronal ferroptosis is a drug for slowing down neuronal apoptosis.

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

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

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

[0016] The beneficial effects of the present invention are:

[0017] 1. The present invention reveals the increased expression level and cellular distribution of ANXA5 after cerebral hemorrhage.

[0018] 2. The present invention proves that ANXA5 can significantly reduce neuronal apoptosis by inhibiting ferroptosis.

[0019] 3. The present invention can effectively inhibit neuronal ferroptosis by adding recombinant protein ANXA5, which proves that the recombinant protein ANXA5 has potential neuroprotective effects.

[0020] 4. The present invention provides a new type of intervention method, which is expected to play an important role in the treatment of cerebral hemorrhage and has broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0022] Figure 2 ANXA5 and astrocyte marker GFAP ( Figure 2A) IBA-1, a marker of microglia ( Figure 2 B) Neuronal marker NEUN ( Figure 2 C) Co-localization staining analysis; Figure 2 D is the co-localization result of ANXA5 and neuronal marker NEUN.

[0023] Figure 3 This is a diagram showing the expression of related proteins in the hemin-stimulated neuron HT22 cell model, where: Figure 3 A is the protein expression result diagram of related proteins. Figure 3 BF is the statistical result graph of membrane-attached protein ANXA5, ferroptosis-related protein GPX4, oxidative stress-related protein SOD-2, inflammatory factor IL-1β, and apoptosis protein Caspase-3.

[0024] Figure 4 This is a diagram showing the expression of membrane-attached protein ANXA5 after overexpression intervention and Hemin stimulation, among which, Figure 4 A protein expression results of related proteins, Figure 4 BG is the statistical result diagram of ANXA5, ferroptosis-related proteins GPX4 and SLC7A11, apoptosis proteins Bax and Bcl-2, and oxidative stress-related molecule NRF2.

[0025] Figure 5 This is a diagram showing the expression of membrane-linked protein ANXA5 after overexpression intervention and Hemin stimulation. Figure 5 A is the change of MDA level, Figure 5 BC is the result diagram and statistical result diagram of active oxygen generation. DETAILED DESCRIPTION

[0026] The following is a detailed description of an embodiment of the present invention in conjunction with the accompanying drawings: This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and process are given, which are intended to be used to explain the present invention, but the implementation does not constitute a limitation to the present invention.

[0027] Example 1: Construction of mouse ICH model

[0028] 70 SPF male C57BL / 6 mice, 8 weeks old, were selected and randomly divided into 7 groups, including 1 control group and 6 experimental groups (6h, 12h, 1d, 3d, 7d, 14d). The mice were anesthetized with 4% chloral hydrate and placed on a stereotaxic frame to expose the skull and bregma. A bone window with a diameter of 0.05mm was drilled on the surface of the skull with a dental drill at 0.2mm behind the bregma and 2.3mm to the right of the midline. 15μl of blood was collected from the tail vein of the mouse using a micro-syringe for later use. The needle was inserted vertically along the bone window for 3.5mm and injected into the right basal ganglia of the mouse at a constant speed of 2μl / min. After stopping the needle for 10min, the needle was slowly withdrawn, the scalp was sutured, and the mouse was placed on a 37℃ constant temperature pad and returned to the cage after the body temperature returned to normal. No special treatment was given to the sham group.

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

[0030] After establishing the cerebral hemorrhage model in mice at different time points and in the Sham group, the mice were anesthetized and the areas around the hematoma in the mouse brain tissue were separated.

[0031] 1. Extract protein:

[0032] The separated and extracted brain tissue was added to the strong RIPA lysis solution (Biyuntian / P0013B) for low-temperature homogenization and lysis by a homogenizer, rotated at 4°C for 15 minutes, and centrifuged at 4°C, 15000rpm for 20 minutes to extract the supernatant, measure the protein concentration, and add 5X loading buffer after balancing to prepare a protein sample.

[0033] Prepare electrophoresis buffer (Tris 3.02g, glycine 18.8g, SDS1g, ddH2O to 1000ml), transfer buffer (Tris 3.03g, glycine 14.4g, methanol 200ml, ddH2O to 1000ml), TBST (Tris 2.42g, NaCl 8g, Tween20 0.5ml, ddH2O to 1000ml) and blocking buffer (5% skim milk powder + TBST) in advance, and pre-cool the transfer buffer in a 4°C refrigerator.

[0034] 2. Electrophoresis:

[0035] The protein sample was centrifuged for later use. The glass plate was placed on the electrophoresis device, electrophoresis solution was added, and the comb was removed. A 10 μl pipette was used to blow each loading well to disperse the residual gel in the well. After adding equal amounts of sample to each well, the voltage was set to 80 V and the gel was started. When the sample was run to the separation gel, the voltage was increased to 120 V. The electrophoresis was terminated when the bromophenol blue just ran out.

[0036] 3. Transfer membrane:

[0037] Remove the glass plate, rinse the electrophoresis solution with ddH2O, put it into the transfer solution, and cut off the unwanted parts. Put the PVDF membrane in methanol for about 1 minute to activate it, and then put it in ddH2O to rinse and depolarize. Assemble the sandwich from bottom to top in the order of sponge-three-layer filter paper-gel-PVDF membrane-three-layer filter paper-sponge, and put it into the transfer tank. Set the transfer conditions: constant current 300mA, 90 minutes.

[0038] 4. Blocking and antibody incubation:

[0039] Remove the PVDF membrane, wash off the methanol on the membrane with ddH2O, put it in the blocking solution, place it on a shaker, and block it at room temperature for 1-2 hours. Wash the blocking solution on the membrane with TBST, make necessary cuts, apply the primary antibody, and place it on a shaker in a 4°C refrigerator overnight. The next day, recover the primary antibody and wash the membrane with TBST for 5 minutes / time, wash 3 times. Apply the secondary antibody, incubate at room temperature for 2 hours, wash the membrane with TBST, 5 minutes / time, wash 3 times. Prepare an appropriate amount of developer according to the instructions of the ultrasensitive ECL chemiluminescence detection kit. Finally, scan the membrane for imaging and analyze the results.

[0040] In order to explore the expression changes of ANXA5 in ICH mice, a mouse intracerebral hemorrhage model was first established. Figure 1 As shown in Figure 2, the expression levels of related proteins in mice after intracerebral hemorrhage ( Figure 1 A), the intracerebral hemorrhage model was successfully established. The expression of membrane protein ANXA5 increased after ICH, and the expression level was the highest on the first day ( Figure 1 B). ICH is a devastating form of stroke with high mortality and morbidity. It can induce cell apoptosis or necrosis, resulting in neurological deficits after injury. The apoptosis-inhibiting molecule Bcl-2 shows a continuous downward trend after ICH, indicating that apoptosis after ICH is aggravated ( Figure 1 C). The level of Bax, a molecule that promotes apoptosis, increased after ICH, which was inversely proportional to the level of Bcl-2 protein. The combined detection of the two indicated that the level of apoptosis increased significantly after ICH ( Figure 1 D). The expression levels of ferroptosis-related molecules GPX4 and SLC7A11 showed a downward trend after cerebral hemorrhage, and the expression level was the lowest around the first day, indicating that the brain tissue produced too much lipid peroxide, the redox reaction was unbalanced, and ferroptosis occurred ( Figure 1 E, F). The apoptosis-promoting molecule Caspase-3 showed a continuous upward trend after ICH, with the highest expression level on the seventh day, which also indicated that apoptosis was aggravated ( Figure 1 G). The inflammatory factor iNOS showed an upward trend after ICH, with the highest expression on the fifth day, indicating that the level of inflammation continued to rise ( Figure 1H). The oxidative stress-related molecule NOX4 showed an upward trend after ICH, and its expression level was the highest on the third day, indicating that the degree of oxidative stress was aggravated ( Figure 1 I). Studies have shown that ferroptosis plays an important role in the regulation of oxidative stress and inflammatory responses. Therefore, after cerebral hemorrhage in mice, the expression levels of oxidative stress and inflammation-related proteins were changed.

[0041] The above results show that the cerebral hemorrhage model was successfully established, the levels of apoptosis and inflammation increased, and ferroptosis occurred after ICH. The research results are consistent with previous reports. In addition, the expression of ANXA5 protein level gradually increased after ICH, and the highest expression level was 7 days after hemorrhage.

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

[0043] In order to further observe the localization of ANXA5 in different cell types after ICH, the present invention uses double immunofluorescence staining with cell-specific markers to identify cell types. Anti-ANXA5 antibodies were used to perform fluorescent staining on frozen sections of brain tissue of mice in the sham operation group and 1 day after ICH.

[0044] Immunofluorescence staining: After the mice were anesthetized, the blood vessels were flushed with physiological saline, and 4% paraformaldehyde was perfused. After the brain was separated, it was fixed in 4% paraformaldehyde overnight. After 20% and 30% sucrose gradient dehydration, the brain tissue was embedded in OCT, and the brain tissue was cut into 10μm slices using a freezing slicer. PBS was shaken for 15 minutes, the embedding agent was washed off, and then blocked with blocking solution at room temperature for 2 hours, PBS was rinsed for 15s to remove the blocking solution, rabbit anti-ANXA5, mouse anti-NEUN were incubated at 4℃ overnight, the primary antibody was washed off, the secondary antibody was incubated at room temperature for 2 hours, the secondary antibody was washed off, DAPI was stained for 10 minutes, and the slices were sealed. Images were observed using a fluorescence microscope.

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

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

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

[0048] In order to explore the expression changes of ANXA5 in in vitro cell experiments, hemin (30μM hemin) was used to induce ferroptosis in neuronal cell lines. HT22 cells were plated in 5 large dishes. When the cell density grew to 60%-70%, 30μM hemin was added to the experimental group for stimulation. The cells were divided into 6 time points: 3h, 6h, 12h, and 24h. Cell proteins were collected at the time points, and Western Blot was performed to analyze the expression changes of ANXA5, ferroptosis protein GPX4, oxidative stress molecule SOD-2, inflammatory factor IL-1β, and apoptosis protein Caspase-3 in the HT22 cell model. The results are shown in Figure 3 As shown, the expression levels of various related proteins in the neuronal HT22 cell model changed after hemoglobin stimulation ( Figure 3 A), it can be seen that the expression trend of each protein is consistent with the in vivo experimental trend; the membrane protein ANXA5 showed a continuous upward trend after hemin stimulation, and the expression level was the highest at 24h, which is consistent with the trend of ANXA5 in the mouse cerebral hemorrhage model ( Figure 3 B). Ferroptosis also occurs after Hemin stimulation of HT22 cells. The results of the in vivo experiment simulating ICH mice showed that the expression of ferroptosis-related protein GPX4 continued to decrease after Hemin stimulation, indicating that excessive lipid peroxides were produced in the cells, the redox reaction was unbalanced, and ferroptosis occurred ( Figure 3 C). Oxidative stress-related protein SOD-2 is an important member of the antioxidant enzyme system in biological systems. Its level continues to increase after Hemin stimulation, indicating that oxidative stress is aggravated ( Figure 3 D). The level of IL-1β, a cytokine that promotes inflammation, continued to increase after Hemin stimulation, indicating that cellular inflammation has worsened ( Figure 3 E). Neuronal apoptosis is an important mechanism for the nervous system to maintain homeostasis. The expression trend of the pro-apoptotic protein Caspase-3 in cells is consistent with that after ICH, indicating that apoptosis is aggravated ( Figure 3 F), changes in the levels of apoptosis-related proteins and inflammatory factors can prove the successful construction of the cell model.

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

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

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

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

[0053] HT22 cells were plated in 4 large dishes and divided into three groups: Control group, ANXA5 group, Hemin stimulation group, and Hemin+ANXA5 group. When the cell density grew to 60%-70%, 2μL recombinant ANXA5 protein was added to the ANXA5 group and the hemin+ANXA5 group for 6 hours, and 30μM heme was added to the hemin stimulation group and the hemin+ANXA5 group for 24 hours. The control group was not treated specially. The cells in 4 dishes were collected and the protein expression was detected by western blot experiment.

[0054] The results are as follows Figure 4 As shown, first verify whether the recombinant protein ANXA5 plays an overexpression role. Among them, the expression level changes of various related proteins are shown in Figure 4 As shown in A. Compared with the control group, the ANXA5 molecule increased significantly under the action of the recombinant protein, indicating that the recombinant protein ANXA5 can significantly increase the expression of ANXA5 molecules in cells ( Figure 4 B); Compared with the Hemin stimulation group, the levels of ferroptosis-related proteins GPX4 and SLC7A11 increased significantly after overexpression of ANXA5, and the lipid peroxides produced in the cells decreased, indicating that the 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 that neuronal apoptosis was improved. As shown in previous studies, ANXA5 has an anti-apoptotic effect. ( Figure 4 EF); compared with the Hemin stimulation group, the expression level of oxidative stress-related molecule NRF2 was reversed, and the oxidative stress level was reduced, indicating that the recombinant protein ANXA5 effectively reduced the oxidative stress level of neurons. At the cellular level, oxidative stress is likely to induce ferroptosis, and reducing the oxidative stress level helps to inhibit the occurrence of 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 in the body, and can also indirectly reflect the degree of cell peroxidation damage.

[0057] Reactive oxygen species (ROS) are a general term for oxygen-containing free radicals and peroxides that are prone to forming free radicals and are related to oxygen metabolism in organisms. When ROS levels increase, they may cause serious damage to cell structures, a process known as oxidative stress. Both are indicators of oxidative stress. Iron metabolism and lipid peroxidation signaling are increasingly recognized as central mediators of ferroptosis. Ferroptosis plays an important role in the regulation of oxidative stress.

[0058] In order to further explore the regulatory effect of recombinant protein ANXA5 on neuronal ferroptosis in multiple aspects, a malondialdehyde (MDA) assay kit (A003-1-96T, Nanjing Jiancheng) was purchased to detect the level of MDA, and a reactive oxygen species detection kit (CM-H2DCFDA, Biyuntian) was purchased to detect the generation of reactive oxygen species. The cells were divided into three groups, Crtl group, Hemin group, and Hemin+ANXA5 group. The stimulation conditions were consistent with those in Example 5 to explore 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 MDA content and reactive oxygen production in the Hemin stimulation group increased significantly, while after the addition of recombinant protein ANXA5, the levels of MDA and reactive oxygen decreased significantly, indicating that lipid peroxides decreased and oxidative stress was alleviated, which suggests that recombinant protein ANXA5 has a significant inhibitory effect on neuronal ferroptosis.

[0060] In summary, the present invention constructs a mouse in vivo model and an in vitro cell model, and finds that the expression level of ANXA5 gradually increases after cerebral hemorrhage. In the in vitro model, by adding recombinant ANXA5 protein, the expression level of ferroptosis-related proteins can be reduced, which proves that the recombinant protein ANXA5 directly inhibits the occurrence of ferroptosis. At the same time, the expression level of apoptotic proteins is reversed and the level of oxidative stress is alleviated, which also shows that the recombinant protein ANXA5 inhibits the occurrence of ferroptosis. Further, both MDA and reactive oxygen levels are significantly reduced, which shows that the recombinant protein ANXA5 reduces oxidative stress by reducing lipid peroxides, thereby inhibiting neuronal ferroptosis. The present invention provides a basis for a new type of drug for the use of recombinant protein ANXA5 as a treatment for cerebral hemorrhage, especially ferroptosis caused after cerebral hemorrhage.

[0061] The above is a further detailed description of the present invention, which should not be regarded as a limitation on the specific implementation of the present invention. For ordinary technicians in the technical field to which the present invention belongs, simple deduction or replacement without departing from the concept of the present invention is within the protection scope of the present invention.

Claims

1. Application of exogenous recombinant protein ANXA5 in the preparation of drugs for the treatment of cerebral hemorrhage.

2. The use according to claim 1, characterized in that: The drug for treating cerebral hemorrhage is a drug for inhibiting neuronal ferroptosis.

3. The use according to claim 2, characterized in that: The drug for inhibiting neuronal ferroptosis reduces the expression level of ferroptosis-related gene proteins.

4. The use according to claim 2, characterized in that: The drug for inhibiting neuronal ferroptosis is a drug for reducing the level of oxidative stress.

5. The use according to claim 2, characterized in that: The drug for inhibiting neuronal ferroptosis is a drug for reducing the accumulation of lipid peroxides.

6. The use according to claim 2, characterized in that: The drug for inhibiting neuronal ferroptosis is a drug for slowing down neuronal apoptosis.

7. The use according to any one of claims 1 to 6, characterized in that: The dosage form of the drug is any pharmaceutically therapeutically acceptable dosage form.

8. The use according to any one of claims 1 to 6, characterized in that: The dosage of the drug is any therapeutically acceptable dosage.

9. A pharmaceutical composition for treating neuronal ferroptosis caused by cerebral hemorrhage, characterized in that: The active ingredient of the pharmaceutical composition includes exogenous recombinant protein ANXA5.

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