Application of parovatine in the preparation of medicine for treating acute brain injury after cerebral hemorrhage

Abstract

The present invention provides the use of parovatine in the preparation of a medicament for treating acute brain injury following intracerebral hemorrhage. This invention, for the first time, discovers that parovatine can reduce neuronal death and inflammation following intracerebral hemorrhage, improve neurological impairment and cerebral edema in SAH mice, and reduce neurological damage and inflammation in SAH mice. Parovatine has great potential for use in the preparation of medicaments for treating acute brain injury following intracerebral hemorrhage.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to application of parovatine in preparing a medicine for treating acute brain injury after cerebral hemorrhage. Background Art

[0002] Subarachnoid hemorrhage (SAH) is a severe emergency with high mortality and morbidity, accompanied by increased intracranial pressure, blood-brain barrier dysfunction, cortical dilating depolarization, and cerebral vasospasm. Early brain injury (EBI) after SAH involves a series of important pathophysiological mechanisms, including inflammation, lipid peroxidation, apoptosis, ferroptosis, and tissue necrosis. Therefore, researchers have been working to elucidate the underlying mechanisms of EBI and explore potential therapeutic strategies.

[0003] Therefore, it is necessary to develop a drug for the treatment of acute brain injury after cerebral hemorrhage. Summary of the Invention

[0004] The present invention aims to provide the use of parovatine in the preparation of a medicament for treating acute brain injury following intracerebral hemorrhage. This invention, for the first time, discovers that parovatine can reduce neuronal death and inflammation following intracerebral hemorrhage, improve neurological impairment and cerebral edema in SAH mice, and reduce neurological damage and inflammation in SAH mice. Parovatine has great potential for use in the preparation of medicaments for treating acute brain injury following intracerebral hemorrhage.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] The present invention provides the use of parovatine in preparing a medicine for treating acute brain injury after cerebral hemorrhage.

[0007] Furthermore, the medicine also includes pharmaceutically acceptable excipients and carriers.

[0008] Furthermore, the auxiliary material includes at least one of a filler, a disintegrant, a binder, an excipient, a diluent, a lubricant, a sweetener or a colorant.

[0009] Furthermore, the dosage form of the drug includes at least one of granules, tablets, pills, capsules, and injections.

[0010] Furthermore, the added concentration of parovatin is 1-10 uM.

[0011] Furthermore, the parovatin reduces neuronal death and inflammation after cerebral hemorrhage, improves neurological function damage and brain edema in SAH mice, and reduces neurological damage and inflammation in SAH mice.

[0012] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0013] The present invention provides the use of parovatine in the preparation of a medicament for treating acute brain injury after cerebral hemorrhage. This invention is the first to discover that parovatine can reduce neuronal death and inflammation after cerebral hemorrhage, improve neurological impairment and cerebral edema in SAH mice, and reduce neurological damage and inflammation in SAH mice. Parovatine has great application prospects in the preparation of medicaments for treating acute brain injury after cerebral hemorrhage. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following is a brief introduction to the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0015] Figure 1 Parovatine can reduce neuronal death and inflammation after cerebral hemorrhage; A is CCK8 showing the changes in neuronal activity of Hemin-treated neurons with different concentrations of parovatine; B is Dead / live fluorescence showing that parovatine (5uM) can significantly reduce the level of neuronal cell death after SAH; CD are Western blotting showing that parovatine can significantly reduce the level of neuronal inflammation after SAH.

[0016] Figure 2 Parovatine can reduce neurological damage and brain edema in SAH mice; A is the Garacia score showing that different concentrations of parovatine can improve neurological damage in SAH mice; B is the brain water content test showing that different concentrations of parovatine can reduce the brain water content of the ipsilateral brain tissue of SAH mice; C is eosin blue staining showing that parovatine can reduce the permeability of brain tissue in SAH mice.

[0017] Figure 3 Parovatine can improve nerve damage and inflammation in SAH mice; AB are Western blotting results showing that parovatine can reduce neuroinflammation; CD are TUNEL fluorescence results showing that parovatine can reduce the level of neuronal death in the basal part of the ipsilateral brain tissue 24 hours after SAH. DETAILED DESCRIPTION

[0018] The present invention will be described in detail below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific embodiments and examples are for illustrating the present invention, rather than for limiting the present invention.

[0019] Throughout this specification, unless otherwise specified, the terms used herein should be understood as having the same meaning as commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the event of any conflict, the present specification shall take precedence.

[0020] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or obtained through existing methods.

[0021] The present application will be described in detail below with reference to examples and experimental data.

[0022] Example 1: Parovatine can reduce neuronal death and inflammation after cerebral hemorrhage

[0023] 1. Cell Culture and Establishment of In Vitro SAH Model

[0024] HT22 cells were maintained in complete medium containing high-glucose DMEM supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin and cultured at 37°C in a 5% CO2 incubator. Cells were passaged every 3-4 days until they reached 80-90% confluency. For in vitro SAH experiments, HT22 cells were exposed to hemin (HY-19424, MCE) to induce a cell model. In this study, hemin was dissolved in 0.1M NaOH. HT22 cells were treated with complete medium containing 150µM hemin for 24 hours to simulate the SAH model in vitro.

[0025] 2. Experimental Methods

[0026] 1. The experiment was divided into 3 groups: Vehicle (control group), Hemin + DMSO (experimental group) and Hemin + Palo (parovatin) (treatment group) were used. After 24 hours of hemin treatment, cell viability was assessed using CCK-8 assay, and cell death was assessed using Dead / Live assay. Proteins from HT22 cells in each group were extracted and Western blot analyzed for NLRP3, GSDMD-N, and IL-1β levels. Statistical Methods: HT22 cells in the control (Vehicle), experimental (Hemin + DMSO), and treatment (Hemin + Palo) groups were analyzed using CCK-8 assay, Western blot, and fluorescent staining. Data are expressed as mean ± standard error. One-way analysis of variance (ANOVA) and Tukey's test were used for comparisons among multiple groups. * indicates statistically significant P < 0.05.

[0027] 2. Western blotting

[0028] Protein was extracted from HT22 cells in each group and measured using the BCA protein assay. Equal amounts of protein were separated by electrophoresis on 8-12% SDS-PAGE gels. Proteins were transferred to PVDF membranes by electrophoresis. The membranes were sealed with 5% skim milk for 1 hour and then incubated with appropriate primary antibodies overnight at 4°C. The PVDF membranes were incubated with various primary antibodies, including rabbit anti-ACSL4 (A20414, abclonal), rabbit anti-NOX4 (A3656, abclonal), rabbit anti-NLRP3 (A24294, abclonal), rabbit anti-GSDMD-N (PA5-116745, Invitrogen), rabbit anti-IL-1β (A16288, abclonal), rabbit anti-GPX4 (A11243, abclonal), and rabbit anti-GAPDH (AC001, abclonal), at 4°C overnight. After washing 3 times with TBST, the PVDF membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature for 1.5 hours. After washing 3 times with TBST, immunolabeling was performed using an enhanced ECL kit, and the data were normalized to GAPDH levels.

[0029] 3. Brain tissue paraffin sections were dewaxed in xylene I and II, then in anhydrous ethanol and a graded alcohol solution. Sections were then washed three times with deionized water for 5 minutes each. Sections were then immersed in 0.01 M citrate buffer and heat-fixed in a microwave for 30 minutes. Sections were then washed three times with PBS for 5 minutes each. The membranes were permeabilized with 0.4% permeabilization buffer for 30 minutes at room temperature, washed three times with PBS, and blocked with 5% BSA for 1 hour. HT22 cells were fixed with formaldehyde for 10 minutes, incubated with 0.25% Triton-X 100 for 30 minutes, and then blocked with 5% BSA for 1 hour. Primary antibody dilutions were added and incubated overnight at 4°C in the dark. Primary antibodies used were mouse anti-NeuN (66836-1-1g, Proteintech), rabbit anti-RARγ (A23233, abclonal), and rabbit anti-ACSL4 (A20414, abclonal). Sections were then washed three times with PBS for 5 minutes each. Incubate brain tissue or cells with the corresponding fluorescent secondary antibody at room temperature for 1.5 hours, then wash three times with PBS for 5 minutes each. Aspirate an appropriate amount of staining solution containing DAPI, mounting medium, and anti-fluorescence quencher, then cover the slides with a coverslip and store in the dark.

[0030] 3. Experimental Results

[0031] Compared with the control group, the cell activity in the experimental group was significantly reduced. After treatment with different concentrations of parovatine, the neuronal activity increased significantly. Subsequent treatment with 5uM parovatine significantly reduced neuronal cell death ( Figure 1AB). Compared with the control group, the expression of NLRP3, GSDMD-N and IL-1β in neurons of the experimental group was significantly increased. Compared with the experimental group, the expression of NLRP3, GSDMD-N and IL-1β in neurons of the treatment group was significantly decreased ( Figure 1 CD). These results indicate that RAR activation can reduce neuronal damage in vitro.

[0032] Example 2: Parovatine can improve neurological impairment and brain edema in SAH mice

[0033] 1. Establishment of SAH Mouse Model

[0034] A SAH mouse model was established in 8-week-old mice using intravascular puncture using a suture embolization technique. Male C57BL6 / J mice were provided by the Wuhan University Animal Center and housed at 22°C, 50–60% relative humidity, and a 12:12 h light-dark cycle. Food and water were provided free of charge. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Wuhan University. The SAH mouse model was established using intravascular puncture.

[0035] 2. Experimental Methods

[0036] The experiment was divided into four groups: sham (control), SAH (experimental), SAH + Palo (treatment), and SAH + DMSO (treatment). The Garcia neurological function score was used, with a maximum score of 18, divided into six components, each scored 0-3: spontaneous activity, forelimb movement symmetry, forepaw extension, proprioception, and response to vibrissae. Brain tissue was collected 24 hours after SAH and divided into the left and right cerebral hemispheres and brainstem. Brain tissue water content was determined using the dry-wet method. Echinops blue staining was used to assess brain barrier damage.

[0037] 3. Experimental Results

[0038] Compared with the control group, the neurological function of the mice in the experimental group was significantly reduced, the water content of the ipsilateral brain was significantly increased, and the blood-brain barrier was significantly damaged. Compared with the experimental group, the neurological function of the mice in the treatment group was improved to a certain extent, and the water content of the ipsilateral brain and the damage of the blood-brain barrier were reduced (see Figure 2 AC). The results showed that parovatin could improve neurological impairment and reduce brain edema in SAH mice.

[0039] Example 3: Parovatine reduces nerve damage and inflammation in SAH mice

[0040] 1. Establishment of SAH Mouse Model

[0041] The SAH mouse model was established in the same manner as in Example 2.

[0042] 2. Experimental Methods

[0043] Mice were divided into the sham (control) group, the SAH (experimental) group, the SAH + Palo group (treatment group 1), and the SAH + DMSO group (treatment group 2). Brain tissue was collected from the ipsilateral side 24 hours after SAH, and protein was extracted from brain tissues of each group for Western blot analysis of NLRP3, GSDMD-N, and IL-1β levels. TUNEL staining was also performed to assess neuronal damage in the brain tissues of each group. Statistical methods: Neurological function scores, brain edema, Western blot analysis, and fluorescence staining were performed in the control, experimental (SAH), treatment (SAH + Palo) groups, and treatment (SAH + DMSO) groups. Data are expressed as mean ± standard error. One-way analysis of variance (ANOVA) and Tukey's test were used for comparisons among multiple groups. * indicates statistical significance at P < 0.05.

[0044] 3. Experimental Results

[0045] Compared with the control group, the expression of NLRP3, GSDMD-N and IL-1β in the ipsilateral cerebral cortex of the experimental group was significantly increased. Compared with the experimental group, parovatin could significantly reduce the expression levels of NLRP3, GSDMD-N and IL-1β in the ipsilateral cerebral cortex of mice ( Figure 3 AB). In addition, parovatin can significantly reduce the level of neuronal damage in the ipsilateral cerebral cortex of mice ( Figure 3 CD). These results indicate that parovatin can reduce nerve damage and inflammation in SAH mice.

[0046] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements, but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0047] Although preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they are aware of the basic inventive concepts. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the invention. Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the invention. Thus, the present invention is intended to include such changes and modifications as fall within the scope of the claims and their equivalents.

Claims

1. Use of parovatine in the preparation of a medicament for treating acute brain injury after cerebral hemorrhage.

2. The use according to claim 1, characterized in that The medicine also includes pharmaceutically acceptable excipients and carriers.

3. The use according to claim 1, characterized in that The auxiliary materials include at least one of a filler, a disintegrant, a binder, a lubricant, a sweetener or a colorant.

4. The use according to claim 1, characterized in that The dosage form of the drug includes at least one of granules, tablets, pills, capsules and injections.

5. The use according to claim 1, characterized in that The added concentration of parovatin is 1-10 uM.

6. The use according to claim 1, characterized in that The parovatine reduces neuronal death and inflammation after cerebral hemorrhage.

7. The use according to claim 1, characterized in that Parovatine improves neurological impairment and brain edema in SAH mice.

8. The use according to claim 1, characterized in that Parovatine reduces nerve damage and inflammation in SAH mice.

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