Method for inhibiting ferroptosis and applying ferroptosis to cerebral hemorrhage by using alliin

Abstract

The invention provides a method for inhibiting ferroptosis by using alliin and treating cerebral hemorrhage, and application of alliin in inhibiting ALOX15-mediated ferroptosis and treating cerebral hemorrhage. According to the method for inhibiting ferroptosis and the application of alliin in cerebral hemorrhage provided by the invention, alliin inhibits neuronal cell ferroptosis, definitely improves dyskinesia caused by cerebral hemorrhage, inhibits occurrence of ferroptosis after cerebral hemorrhage, relieves secondary injury after cerebral hemorrhage and plays a role in neuroprotection; the used alliin is natural active small molecules from garlic, and has the characteristics of natural source and small toxic and side effects compared with chemical synthetic drugs, the action mechanism of the alliin on nerve cell ferroptosis and the application of the alliin in cerebral hemorrhage are verified for the first time in the mode, and the alliin has a drug development prospect.

Description

Technical Field

[0001] The invention relates to a new application field of alliin, and in particular to a method for alliin to inhibit ferroptosis and treat cerebral hemorrhage. Background Art

[0002] Garlic is widely used in traditional medicine and alternative medicine because of its advantage of "medicine and food being of the same origin". China, as the main producer and exporter of garlic, has very rich garlic resources. The health benefits of garlic are mainly attributed to its rich minerals, vitamins, essential amino acids, phenolic compounds, and especially organosulfur compounds. Alliin is the precursor of allicin. When raw garlic bulbs are crushed, cut or ground, the alliinase in garlic is released into the cytoplasm, catalyzing the conversion of alliin into diallyl thiosulfinate. Allicin has cardiovascular and cerebrovascular protection, anti-cancer and antibacterial effects.

[0003] Ferroptosis is an iron-dependent form of non-apoptotic cell death that has become a promising target for the treatment of cerebral hemorrhage. Biochemical hallmarks of ferroptosis include dysfunction of the glutathione antioxidant system, depletion of glutathione peroxidase 4, accumulation of lipid peroxides, and mitochondrial membrane shrinkage. When lipid peroxides overwhelm the cell's antioxidant defenses, the accumulation of cytotoxic compounds may accelerate protein breakdown, lipid degradation, and neuronal death. The iron-rich and heme-free fatty acid dioxygenase, arachidonic acid 15-lipoxygenase, has the ability to metabolize a variety of polyunsaturated fatty acids. ALOX15 is a member of the lipoxygenase family that is specifically responsible for oxidative stress-induced cell death. ALOX15 produces ferroptosis signals by directly oxidizing arachidonic acid and adrenal acyl-phosphatidylethanolamine.

[0004] Cerebral hemorrhage refers to non-traumatic spontaneous bleeding in the brain parenchyma. It is a form of stroke with a high mortality and morbidity rate. Blood leaks into the brain parenchyma, leading to hematoma formation, which compresses surrounding tissues and is the main mechanism of injury in cerebral hemorrhage. Hemoglobin is the main protein in the blood. It is released from ruptured red blood cells after cerebral hemorrhage and is considered to be a potential neurotoxin. Microglia and invading macrophages in the area around the hematoma can phagocytize it and metabolize it into ferrous / ferric. After cerebral hemorrhage, the lysis of red blood cells leads to the release of a large amount of potentially neurotoxic iron into the brain parenchyma, triggering neurotoxic effects and subsequent neuronal death. Neuronal ferroptosis plays a key role in secondary brain injury caused by cerebral hemorrhage. Iron is the main degradation product of hemoglobin decomposition. It causes secondary brain injury after cerebral hemorrhage by promoting the production of free radicals and triggering inflammatory responses. Secondary brain injury is closely related to the prognosis of cerebral hemorrhage and is considered a potential target for the treatment of cerebral hemorrhage.

[0005] Although allicin has attracted much attention for its significant pharmacological activity, relatively few studies have been conducted on its active precursor, small molecule alliin. The specific mechanism of action of alliin in ferroptosis is still unclear, and whether it can protect the brain from secondary injury induced by cerebral hemorrhage is still unclear. Currently, the treatment of cerebral hemorrhage in clinical practice mainly includes conservative medical treatment and surgical hematoma removal, but there is still a lack of drugs with high-level evidence-based medicine.

[0006] Therefore, it is necessary to provide a method for alliin to inhibit ferroptosis and its use in cerebral hemorrhage to solve the above technical problems. Summary of the invention

[0007] The present invention provides a method for inhibiting ferroptosis by alliin and its use in cerebral hemorrhage, which solves the problem that the treatment of cerebral hemorrhage in clinic mainly includes conservative medical treatment and surgical hematoma removal, but lacks drugs with high-level evidence-based medicine.

[0008] In order to solve the above technical problems, the present invention provides alliin for inhibiting ALOX15-mediated ferroptosis and its application in the treatment of cerebral hemorrhage

[0009]

[0010] Preferably, there are 98 intersection targets between the potential targets of garlic components in the alliin and the potential therapeutic targets for cerebral hemorrhage, and the intersection target enrichment analysis shows that ferroptosis is a related action pathway.

[0011] Preferably, the alliin can stably bind to ALOX15 to inhibit its expression.

[0012] Preferably, the alliin inhibits the occurrence of erastin-induced ferroptosis in HT22 cells.

[0013] Preferably, the alliin inhibits the formation of hematoma and neuronal damage after cerebral hemorrhage.

[0014] Preferably, the alliin inhibits brain tissue damage after cerebral hemorrhage.

[0015] Preferably, the alliin inhibits the occurrence of ferroptosis after cerebral hemorrhage.

[0016] Compared with the related art, the method of alliin inhibiting ferroptosis and its use in cerebral hemorrhage provided by the present invention has the following beneficial effects:

[0017] The invention provides a method for inhibiting ferroptosis by alliin and its use in cerebral hemorrhage. Alliin inhibits ferroptosis of neuronal cells, clearly improves motor dysfunction caused by cerebral hemorrhage, inhibits the occurrence of ferroptosis after cerebral hemorrhage, alleviates secondary damage after cerebral hemorrhage, and plays a neuroprotective role. The alliin used is a natural active small molecule derived from garlic, which has the characteristics of being derived from nature and having less toxic and side effects compared with chemically synthesized drugs. This method verifies for the first time the mechanism of action of alliin on ferroptosis of neuronal cells and its application in cerebral hemorrhage, and has the prospect of developing drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The Venn diagram is the intersection of the potential targets of garlic and the potential targets of cerebral hemorrhage;

[0019] Figure 2 KEGG pathway enrichment results for intersection targets;

[0020] Figure 3 Searching for the compound-target-signaling pathway-disease network diagram of garlic's effect on cerebral hemorrhage for network pharmacology;

[0021] Figure 4 Molecular docking for the binding ability of alliin to ALOX15;

[0022] Figure 5 The figure shows that alliin can enhance the viability of HT22 cells induced by erastin;

[0023] Figure 6 Schematic diagram of alliin alleviating mitochondrial damage during ferroptosis in HT22 cells;

[0024] Figure 7 Schematic diagram of alliin alleviating the accumulation of mitochondrial ROS and the damage of mitochondrial membrane potential in HT22 cells induced by iron death;

[0025] Figure 8 Alliin reduces the occurrence of ferroptosis in HT22 cells 2+ Schematic diagram of the level;

[0026] Fig. 9 Schematic diagram of alliin inhibiting the accumulation of lipid peroxidation in HT22 cells during ferroptosis;

[0027] Fig.10 Schematic diagram of the expression of ALOX15 and GPX4 when alliin inhibits ferroptosis in HT22 cells;

[0028] Fig.11 Schematic diagram of transcriptomic data validation of the effect of alliin on ferroptosis after ICH.

[0029] Fig.12This is a schematic diagram showing that alliin can alleviate brain tissue damage after cerebral hemorrhage in mice, and alleviate movement disorders and coordination in mice with cerebral hemorrhage;

[0030] Fig.13 Schematic diagram of the inhibitory effect of alliin on brain edema and hematoma in mice with cerebral hemorrhage;

[0031] Fig.14 Schematic diagram of alliin alleviating neuronal damage in brain tissue after cerebral hemorrhage;

[0032] Fig.15 This is a schematic diagram showing that alliin inhibits brain tissue damage after cerebral hemorrhage, inhibits the expression of ALOX15, promotes the expression of GPX4, and thus inhibits the occurrence of ferroptosis after cerebral hemorrhage;

[0033] Fig.16 Schematic diagram of how alliin alleviates the occurrence of ferroptosis in brain tissue of mice after cerebral hemorrhage. DETAILED DESCRIPTION

[0034] The present invention will be further described below in conjunction with the accompanying drawings and implementation modes.

[0035] Please refer to Figure 1 , Figure 2 , Figure 3 , Figure 4 , Figure 5 , Figure 6 , Figure 7 , Figure 8 , Fig. 9 , Fig.10 , Fig.11 , Fig.12 , Fig.13 , Fig.14 , Fig.15 and Fig.16 ,in, Figure 1 The Venn diagram is the intersection of the potential targets of garlic and the potential targets of cerebral hemorrhage; Figure 2 KEGG pathway enrichment results for intersection targets; Figure 3 Searching for the compound-target-signaling pathway-disease network diagram of garlic's effect on cerebral hemorrhage for network pharmacology; Figure 4 Molecular docking for the binding ability of alliin to ALOX15; Figure 5 The figure shows that alliin can enhance the viability of HT22 cells induced by erastin; Figure 6 Schematic diagram of alliin alleviating mitochondrial damage during ferroptosis in HT22 cells; Figure 7 Schematic diagram of alliin alleviating the accumulation of mitochondrial ROS and the damage of mitochondrial membrane potential in HT22 cells induced by iron death; Figure 8 Schematic diagram of how alliin reduces the level of Fe2+ in HT22 cells when ferroptosis occurs; Fig. 9Schematic diagram of alliin inhibiting the accumulation of lipid peroxidation in HT22 cells during ferroptosis; Fig.10 Schematic diagram of the expression of ALOX15 and GPX4 when alliin inhibits ferroptosis in HT22 cells; Fig.11 Schematic diagram of transcriptomic data validation of the effect of alliin on ferroptosis after ICH. Fig.12 This is a schematic diagram showing that alliin can alleviate brain tissue damage after cerebral hemorrhage in mice, and alleviate movement disorders and coordination in mice with cerebral hemorrhage; Fig.13 Schematic diagram of the inhibitory effect of alliin on brain edema and hematoma in mice with cerebral hemorrhage; Fig.14 Schematic diagram of alliin alleviating neuronal damage in brain tissue after cerebral hemorrhage; Fig.15 This is a schematic diagram showing that alliin inhibits brain tissue damage after cerebral hemorrhage, inhibits the expression of ALOX15, promotes the expression of GPX4, and thus inhibits the occurrence of ferroptosis after cerebral hemorrhage; Fig.16 Schematic diagram of alliin alleviating ferroptosis in brain tissue of mice after cerebral hemorrhage. Alliin inhibits ALOX15-mediated ferroptosis and its application in the treatment of cerebral hemorrhage.

[0036] Preferably, the potential targets of the garlic component in the alliin and the potential therapeutic targets for cerebral hemorrhage have 98 intersection targets, and the intersection target enrichment analysis shows that ferroptosis is a related action pathway.

[0037]

[0038] The alliin can stably bind to ALOX15 to inhibit its expression.

[0039] The alliin inhibits the occurrence of ferroptosis of HT22 cells induced by erastin.

[0040] The alliin inhibits the formation of hematoma and neuron damage after cerebral hemorrhage.

[0041] The alliin inhibits brain tissue damage after cerebral hemorrhage.

[0042] The alliin inhibits the occurrence of ferroptosis after cerebral hemorrhage.

[0043] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in combination with specific embodiments and comparative examples:

[0044] S1. Network pharmacology to find potential targets and pathways after intracerebral hemorrhage;

[0045] S11. Target screening of garlic active ingredients;

[0046] S12, Screening of targets related to cerebral hemorrhage;

[0047] S13, construct component target-disease target network and screen core targets;

[0048] S14, construction of target-biological process / signaling pathway network;

[0049] S15, compound-target-pathway disease network diagram establishment;

[0050] S2, interaction between alliin and ALOX15, a target of ferroptosis;

[0051] S21, molecular docking analysis;

[0052] S3, alliin inhibits erastin-induced ferroptosis in HT22 cells;

[0053] S31, cell culture and viability experiments;

[0054] S32, the effect of inhibiting ferroptosis in HT22 cells;

[0055] S4. Application of alliin in the treatment of ICH;

[0056] S41. 6-8 week old C57BL / 6J mice were randomly divided into 3 groups: control group, cerebral hemorrhage model group, and alliin group;

[0057] S42, forearm placement test;

[0058] S43, balance beam test;

[0059] S44, rotarod test;

[0060] S5. Changes in transcriptome of mice with ICH;

[0061] S51. On the 7th day after the establishment of the mouse ICH model, the mouse brain tissue was obtained, quickly frozen in liquid nitrogen, and stored in an EP tube in a -80°C refrigerator;

[0062] S52. After alliin treatment, the genes in the mouse brain tissue changed significantly. Compared with the cerebral hemorrhage group, the differentially expressed genes in the alliin group were mainly enriched in hemoglobin complex, haptoglobin-hemoglobin complex, HFE-transferrin receptor complex, haptoglobin binding, peroxidase activity, hemoglobin α binding and other functions, involving proteins involved in the binding and transport of Fe3+ on the membrane;

[0063] S6. Alliin protects against brain damage caused by cerebral hemorrhage through the ferroptosis pathway;

[0064] S61. The modeling method for cerebral hemorrhage is the same as that in Example 4.1. 48 hours after the occurrence of cerebral hemorrhage, the brain is removed and divided into left and right parts;

[0065] S62. Brain tissues from the control group, model group, and alliin group were taken for brain sectioning, and the effectiveness of alliin in reducing hematoma volume was evaluated by brain slice imaging.

[0066] S63. On the 7th day of modeling administration, mice were deeply anesthetized and perfused with 40 mL of pre-cooled phosphate buffer solution.

[0067] S64, remove the brain and store it in 4% PFA solution at 4°C for 24-48 hours, then immerse it in 30% sucrose solution for 72 hours to completely dehydrate;

[0068] S65. Take fresh brain tissue, extract protein from it, and determine the protein content. According to the operating instructions of the kit, determine the contents of MDA, SOD, LPO, and GSH-Px in the three groups of brain tissue samples;

[0069] S66. Take fresh brain tissue, extract the protein therein, and then determine the protein content.

[0070] Screening of garlic medicinal ingredient targets in S11: predicting target proteins of compounds of characteristic ingredients in garlic in SymMap, TCMSP database and Traditional Chinese Medicine Encyclopedia database, and using the universal protein resource UniProt database to obtain gene names of these targets after removing duplicates;

[0071] Screening of cerebral hemorrhage-related targets in S12: searching for cerebral hemorrhage-related targets in the GeneCards database, removing duplicates and merging to obtain cerebral hemorrhage-related targets;

[0072] The component target-disease target network and core target screening in S13 are constructed: the compound targets and disease targets are screened for intersection targets using Venny 2.1 to obtain potential pharmacological compound therapeutic targets for cerebral hemorrhage in garlic, the intersection targets are imported into the Metascape database, the species definition is set as "Homo sapiens", and the gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis is performed on the intersection targets;

[0073] Construction of the target-biological process / signaling pathway network in S14: GO and KEGG enrichment analysis results were screened, with P<0.05 as the screening condition, the top 15 of GO and the top 20 of KEGG were screened respectively, and a visualization graph of "target-biological process / signaling pathway" was constructed;

[0074] The compound-target-pathway-disease network diagram in S15 is established by merging the compound target and the gene-pathway network to establish a compound-target-pathway-disease network, and using Cytoscape software to construct a pharmacodynamic compound-target-pathway network diagram to screen out pathways and targets that garlic has a potential therapeutic effect on cerebral hemorrhage.

[0075] Molecular docking analysis in the described S21: To study the binding affinity and interaction between the active ingredient and its potential target, the three-dimensional structure of ALOX15 was obtained from the protein database. After removing water molecules and adding polar hydrogen atoms to the protein structure, molecular docking was performed using Autodock Vina 1.2.2. The binding energy was used to evaluate the binding interaction between the ligand and the receptor protein. The receptor-ligand interaction was visualized using Discovery software;

[0076] The cell culture and viability experiment in S31: HT22 cells were cultured at 37°C and 5% CO2 using DMEM medium containing 10% fetal bovine serum, and the medium contained 100U / mL penicillin and 100g / mL streptomycin. When the cell growth reached 90-95%, 2.5% trypsin was used to digest and separate the cells. The experiment studied the effect of alliin on ferroptosis of HT22 cells induced by ellastin.

[0077] The effect of S32 in inhibiting ferroptosis of HT22 cells: HT22 cells were seeded in 6-well plates at a density of 1×10^6 cells / well and treated with erastin, alliin and Fer-1 as described above, and then JC-1, 2,7-dichlorofluorescein diacetate and C11 BODIPY 581 / 591 were added to the wells and incubated in the dark at 37°C for 20 minutes. After the incubation, the cells were observed under a fluorescence microscope after washing twice with PBS, and then the ratio of JC-1 monomers to polymers was calculated. In order to analyze lipid peroxidation, the oxidized form of the dye was compared with its normal form, and FerroOrange was used to visualize the Fe2+ in the cells, and the fluorescence intensity was measured by a fluorescent digital microscope. The morphology of the cell mitochondria was observed using a transmission electron microscope.

[0078] The S31 cells were treated with alliin (0, 100, 200, 400, 600 μM) for 24 hours in the presence of elastin (1 μM), and Fer-1 (5 μM) was used as a positive control. MTT was used to evaluate cell viability.

[0079] The dosage of the alliin group in the S41 is based on the dosage of allicin for treating cerebral hemorrhage, according to a 12-hour light / 12-hour dark cycle, the environmental humidity is maintained at 55-60%, and the temperature is controlled at 22°C. Since alliin is easily eliminated by the first-pass effect when taken orally and the blood-brain barrier has limited permeability, the experimental plan selects tail vein injection to ensure that the drug is effectively delivered to the target site to achieve the maximum therapeutic effect. The cerebral hemorrhage model is established by injecting collagenase into the basal ganglia. Mice are anesthetized intraperitoneally with 2% sodium pentobarbital (25 mg / kg). The mice are kept in a prone position in a stereotaxic frame, and a 1 mm deep hole is drilled with a dental drill, located 2.0 mm to the right of Brima and 3.5 mm below the surface of the skull. 0.1U type IV collagenase dissolved in 1 μL of normal saline is injected to a depth of 3.5 mm at a rate of 0.2 μL per minute. After the injection is completed, the needle is retained for 5-10 minutes before the injection needle is removed to avoid reflux. After modeling, behavioral evaluation and animal weight records are performed on all groups of animals;

[0080] The forearm placement experiment in S42: The forearm placement experiment is also called the whisker forearm placement experiment. During the test, pinch the mouse neck skin with one hand and pull the tail with the other hand to stretch the body. Slowly move the whisker on one side of the mouse close to the edge of the table. When the whisker touches the edge of the table, the normal reaction is to place the forelimb on the same side at the edge of the table, while the cerebral hemorrhage model group loses this reflex. In the experiment, each mouse is tested 10 times on each side of the forearm, and the number of times the forearm on that side is correctly placed to the corner of the table after the whisker is touched is recorded. Before the formal experiment, the experimental animal is moved up and down several times with gentle manipulation to relax the animal's muscles and reduce resistance activities;

[0081] The balance beam test in S43: The test lasts for three days, with the first two days being training and the last day being testing. The performance is quantified by measuring the time required for mice to walk across the balance beam and the number of times they slip during the process. Five narrow wooden bars of different widths are used to evaluate the motor coordination and balance ability of mice. A 1-meter-long wooden bar is placed horizontally, 0.5 meters above the ground, with one end fixed to a narrow support and the other end connected to a cage to which the mice can escape. The mice are trained three times on the training wooden bar, each time starting from a different starting point: the starting point close to the home cage, the center of the balance beam, and one end of the light. After the training, the mice will undergo five consecutive tests, each time walking on a different wooden bar with increasing difficulty. The test process will be recorded and subsequently evaluated. The mice are required to walk across a circular wooden bar with a diameter of 1.5 cm, a length of 70 cm, and a height of 30 cm from the ground. After walking, they will receive a corresponding score. The test is repeated three times, and the score is determined based on the walking distance and gait.

[0082] The rotarod test in S44: the mice were placed on the rotarod in the compartment and the rotarod was controlled to rotate for 5 minutes to allow the mice to adapt to the new environment and movement mode. After every 5 mice test, the test compartment was wiped with 75% alcohol to avoid odor interference. The operation method of the test phase was the same as that of the habituation phase. The rotarod motor function test was performed for 5 minutes. At 0, 9, and 16 o'clock, the mice were placed on the fatigue rod respectively, and the speed of the rotating rod was adjusted to 40 cycles / min. The time for the mice to fall or passively rotate continuously for 2 weeks was measured. Each experimental animal was repeated 3 times, with an interval of 5 minutes each time, and the average value was taken as the result. The bottom of the box was a copper fence with a spacing of 1 cm, and a 10V current was passed to prevent the mice from actively jumping off the rotating rod. During the experiment, the mice were placed on the rotating rod, and the residence time of the mice on the rod was tested at 18r / min. The total distance and average speed of the mice crawling on the rotating rod were recorded. Each mouse was tested 3 times at each speed, and the average value was taken. If the mouse stayed on the rod for more than 120s, it was counted as 120s. At the same time, the time it jumped on the rotating rod after falling off the rotating rod due to electrical stimulation was recorded. If the mouse still did not jump on the rotating rod after 120s, it was counted as 120s.

[0083] S51 On the 7th day after the establishment of the mouse ICH model, the mouse brain tissue was obtained, quickly frozen in liquid nitrogen, and stored in an EP tube at -80°C. It was taken out before use, and total RNA was extracted using TRIzol reagent according to the instructions. The RNA purity and quantification were identified using a NanoDrop2000 spectrophotometer (Thermo Scientific, USA), and the RNA integrity was assessed using an Agilent2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The library was sequenced using the llumina Novaseq 6000 sequencing platform, and differentially expressed genes were analyzed. Genes that met the threshold of q value <0.05 and foldchange>2 or foldchange <0.5 were defined as differentially expressed genes (DEGs). The differentially expressed genes were analyzed using GO, KEGG, and chromatin. Pathway, Reactome and WikiPathways enrichment analysis were used to screen for significantly enriched functional entries. The results showed that genes in brain tissue changed significantly after ICH. GO functional enrichment analysis of up-regulated genes showed that differentially expressed genes in the ICH group were mainly enriched in functions such as arachidonic acid epoxidase activity, iron ion binding, heme binding and oxidoreductase activity. KEGG pathway analysis showed that these up-regulated genes were enriched in 20 signaling pathways, mainly including 8 metabolic pathways and 3 lipid metabolic pathways, including arachidonic acid metabolism, linoleic acid metabolism, oxidative phosphorylation and steroid hormone synthesis. Transcriptomics showed that ferroptosis occurred in the brain tissue of mice after ICH.

[0084] After S52 alliin treatment, the genes in the mouse brain tissue changed significantly. Compared with the cerebral hemorrhage group, the differentially expressed genes in the alliin group were mainly enriched in hemoglobin complex, haptoglobin-hemoglobin complex, HFE-transferrin receptor complex, haptoglobin binding, peroxidase activity, hemoglobin α binding and other functions, involving Fe 3+ Proteins that bind and transport across membranes, Fig.11 The top 20 enriched pathways are shown, such as eicosanoid metabolism and iron homeostasis via lipoxygenase (LOX). The analysis of enriched pathways covers three databases, Figure 7 Some significantly enriched related pathways were shown, such as arachidonic acid metabolism, lipids, and atherosclerosis. Transcriptomics results once again confirmed the role of alliin in inhibiting lipid peroxidation and ferroptosis in brain tissue after ICH. Ferroptosis is involved in neuronal cells after intracerebral hemorrhage, and lipid peroxidation and lipoxygenase (LOXs) synthesis are key signaling pathways related to this process, indicating that alliin has the potential to regulate the synthesis of lipoxygenases (such as ALOX15), affecting arachidonic acid metabolism and the production of its eicosatetraenoic acid derivatives. This event cascade affects the production of lipid peroxides and glutathione (GSH) metabolism, while maintaining the homeostasis of intracellular iron.

[0085] In order to determine the dry weight of each part in S61, we used an electronic analytical balance to weigh it immediately after taking it out of the oven, brain water content (%) = [(wet weight-dry weight) / wet weight] × 100%. The results showed that alliin reduced brain edema in mice with intracerebral hemorrhage;

[0086] The brain tissue was removed from the S63, fixed with 4% paraformaldehyde, and embedded in paraffin. According to the manufacturer's instructions, the brain tissue sections were stained with hematoxylin / eosin after dewaxing. HE staining showed that the neurons in the cerebral hemorrhage group were irregularly arranged, with vacuolar degeneration and nuclear condensation, and the administration of alliin significantly improved these effects caused by cerebral hemorrhage.

[0087] In the S64, the brain was cut into 8 μm slices for immunofluorescence staining, and the immunofluorescence staining was performed after heating at room temperature for 30 minutes, followed by rinsing three times with PBS, and blocking with QuickBlotTM blocking buffer at room temperature for 60 minutes. After incubation with the primary antibody, anti-NeuN and anti-ALOX15 antibodies were applied, and then the slices were rinsed again and incubated with a fluorescently labeled secondary antibody corresponding to the primary antibody at 25°C for 2 hours. Finally, images were taken using a fluorescence microscope. The results of immunofluorescence staining showed that the co-localization of ALOX15 and neuronal cells was significantly reduced after alliin treatment compared with the simple cerebral hemorrhage group.

[0088] The results in S65 demonstrated that the activity of glutathione peroxidase decreased significantly on the third day after cerebral hemorrhage, but the activity was significantly restored by daily administration of 50 mg / kg alliin. Alliin treatment reduced the LPO level in the brain of mice with cerebral hemorrhage. After cerebral hemorrhage, the malondialdehyde level in the damaged hemisphere increased significantly. Administration of 50 mg / kg alliin effectively alleviated the increase in MDA caused by cerebral hemorrhage, while the superoxide dismutase level increased.

[0089] The data obtained by Western blot analysis in S66 showed that the increase in ALOX15 levels after ICH can be effectively inhibited by alliin treatment, thereby promoting the expression of GPX4

[0090] When treating cerebral hemorrhage, the alliin of the present invention can be used alone, or used in combination with other drugs, or prepared into a compound preparation with other drugs for use, so as to achieve the purpose of treating cerebral hemorrhage.

[0091] The present invention finds that alliin inhibits ferroptosis and secondary injury after cerebral hemorrhage by regulating ALOX15, searches for potential targets of cerebral hemorrhage through network pharmacology, proves that alliin can directly bind to ALOX15 through molecular docking and cell thermal migration experiments, proves that alliin can significantly reduce erastin-induced ferroptosis based on ALOX15, promotes the expression of GPX4, alleviates mitochondrial damage and lipid peroxidation, and verifies that alliin can alleviate neurological dysfunction and pathological injury of cerebral hemorrhage mice in vivo using a cerebral hemorrhage mouse model, inhibits the expression of ALOX15 and promotes the expression of GPX4 at the same time, inhibits the accumulation of lipid peroxides, and alleviates secondary injury. Therefore, both in vivo and in vitro studies confirm that alliin can effectively inhibit ferroptosis and alleviate secondary injury after cerebral hemorrhage.

[0092] Compared with the related art, the method of alliin inhibiting ferroptosis and its use in cerebral hemorrhage provided by the present invention has the following beneficial effects:

[0093] The invention provides a method for inhibiting ferroptosis by alliin and its use in cerebral hemorrhage. Alliin inhibits ferroptosis of neuronal cells, clearly improves motor dysfunction caused by cerebral hemorrhage, inhibits the occurrence of ferroptosis after cerebral hemorrhage, alleviates secondary damage after cerebral hemorrhage, and plays a neuroprotective role. The alliin used is a natural active small molecule derived from garlic, which has the characteristics of being derived from nature and having less toxic and side effects compared with chemically synthesized drugs. This method verifies for the first time the mechanism of action of alliin on ferroptosis of neuronal cells and its application in cerebral hemorrhage, and has the prospect of developing drugs.

[0094] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. Alliin inhibits ALOX15-mediated ferroptosis and its application in the treatment of cerebral hemorrhage.

2. The use according to claim 1, characterized in that: There are 98 intersection targets between the potential targets of the garlic component in alliin and the potential therapeutic targets for cerebral hemorrhage, and the intersection target enrichment analysis shows that ferroptosis is a related action pathway.

3. The use according to claim 1, characterized in that: The alliin can stably bind to ALOX15 to inhibit its expression.

4. The use according to claim 1, characterized in that: The alliin inhibits the occurrence of ferroptosis of HT22 cells induced by erastin.

5. The use according to claim 1, characterized in that: The alliin inhibits the formation of hematoma and neuron damage after cerebral hemorrhage.

6. The use according to claim 1, characterized in that: The alliin inhibits brain tissue damage after cerebral hemorrhage.

7. The use according to claim 1, characterized in that: The alliin inhibits the occurrence of ferroptosis after cerebral hemorrhage.

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