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

Abstract

The invention provides an application of palovastatin in preparation of a medicine for treating acute brain injury after cerebral hemorrhage. It is found for the first time that parovastatin can reduce neuronal death and inflammation after cerebral hemorrhage, can improve neurological function impairment and encephaledema of SAH mice, and can reduce neurological injury and inflammation of SAH mice. The palovastatin has a great application prospect in preparation of medicines for treating acute brain injury after cerebral hemorrhage.

Description

Technical Field

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

[0002] Subarachnoid hemorrhage (SAH) is a serious emergency with high mortality and morbidity, accompanied by increased intracranial pressure, blood-brain barrier dysfunction, cortical dilatory depolarization, cerebral vasospasm, etc. Early brain injury (EBI) after SAH occurs through a series of important pathophysiological mechanisms, including inflammation, lipid peroxidation, apoptosis, ferroptosis, and tissue necrosis. Therefore, researchers have been committed to elucidating the underlying mechanisms of EBI and exploring 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 parovatin in the preparation of a drug for treating acute brain injury after cerebral hemorrhage. The present invention first discovered that parovatin can reduce neuronal death and inflammation after cerebral hemorrhage, improve neurological function damage and brain edema in SAH mice, and reduce nerve damage and inflammation in SAH mice. Parovatin has great application prospects in the preparation of drugs for treating acute brain injury after cerebral hemorrhage.

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

[0006] The 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 parovastatin is 1-10 uM.

[0011] Furthermore, the parovastatin 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 dihydroartemisinin DHA in the preparation of anti-liver cancer drugs. The present invention first discovered that parovatin can reduce neuronal death and inflammation after cerebral hemorrhage, improve neurological damage and brain edema in SAH mice, and reduce nerve damage and inflammation in SAH mice. Parovatin has great application prospects in the preparation of drugs for treating acute brain damage 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 briefly introduces 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 creative work.

[0015] Figure 1 Parovatin 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 parovatin; B is Dead / live fluorescence showing that parovatin (5uM) can significantly reduce the level of neuronal cell death after SAH; CD are Western blotting showing that parovatin can significantly reduce the level of neuronal inflammation after SAH.

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

[0017] Figure 3 Parovatin can improve nerve damage and inflammation in SAH mice; AB is Western blotting showing that parovatin can reduce neuroinflammation; CD is TUNEL fluorescence showing that parovatin can reduce the level of neuronal death at the base 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 implementations 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 implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0019] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes 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 by existing methods.

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

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

[0023] 1. Cell culture and establishment of in vitro SAH model

[0024] HT22 cells were treated with complete medium containing high glucose DMEM, supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin, and cultured in a 5% CO2 incubator at 37°C. Cells were passaged every 3-4 days until the confluence reached 80-90%. In the in vitro SAH experiment, HT22 cells were exposed to hemin (Hemin, HY-19424, MCE) to induce the cell model. In this study, hemin was dissolved in 0.1M NaOH. HT22 cells were treated with complete culture medium containing 150μM hemin for 24h 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). After 24 hours of Hemin treatment, CCK8 reagent was used to detect cell activity, Dead / live was used to detect cell death levels, and Western blot was used to extract HT22 cell proteins from each group to detect NLRP3, GSDMD-N and IL-1β levels. Statistical methods: CCK-8, Western blot and fluorescence staining of HT22 cells in the control group (Vehicle), experimental group (Hemin+DMSO) and treatment group (Hemin+Palo) were detected. Data are expressed as mean ± standard error, and one-way analysis of variance (ANOVA) and Tukey test were used for comparison among multiple groups. * indicates statistical difference when P < 0.05.

[0027] 2. Western blotting

[0028] Proteins of HT22 cells in each group were extracted and determined by BCA protein assay. Equal amounts of proteins 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 h and then incubated with appropriate primary antibodies overnight at 4°C. 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 h. After washing 3 times with TBST, immunolabeling was performed using an enhanced ECL kit, and the data were normalized to GAPDH levels.

[0029] 3. The paraffin sections of brain tissue were placed in xylene I and xylene II, anhydrous ethanol and gradient alcohol for dewaxing, then washed with deionized water 3 times, 5 minutes each time, soaked in 0.01M citrate buffer and placed in a microwave for heat repair for 30 minutes, and then washed with PBS 3 times, 5 minutes each time. The membrane was permeabilized with 0.4% permeabilization solution at room temperature for 30 minutes, then washed 3 times with PBS, and blocked with 5% BSA for 1 hour. HT22 cells were fixed with formaldehyde for 10 minutes, then incubated with 0.25% Triton-X 100 for 30 minutes, and then blocked with 5% BSA for 1 hour. The primary antibody dilution was added dropwise and incubated overnight at 4°C in the dark. The primary antibodies were mouse anti-NeuN (66836-1-lg, Proteintech), rabbit anti-RARγ (A23233, abclonal) and rabbit anti-ACSL4 (A20414, abclonal). Then washed with PBS 3 times, 5 minutes each time. Incubate brain tissue or cells with corresponding fluorescent secondary antibodies at room temperature for 1.5 hours, then wash with PBS three times, 5 minutes each time. Pipette an appropriate amount of staining solution containing DAPI, mounting medium and anti-fluorescence quenching, and seal the slides with coverslips, 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 parovastatin, the neuronal activity increased significantly. Subsequent treatment of cells with 5uM parovastatin significantly reduced neuronal cell death ( Figure 2AB). 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 2 CD). These results suggest that RAR activation can reduce neuronal damage in vitro.

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

[0033] 1. Establishment of SAH mouse model

[0034] Eight-week-old mice were selected to establish the SAH mouse model by intravascular puncture using thread plugs: male C57BL6 / J mice were provided by the Animal Center of Wuhan University and maintained under room temperature of 22°C, relative humidity of 50-60%, and a light-dark cycle of 12:12h. Food and water were provided free of charge. All experimental procedures were approved by the Institutional Animal Care and Utilization Committee of Wuhan University. The mouse SAH model was established by intravascular puncture.

[0035] 2. Experimental Methods

[0036] The experiment was divided into 4 groups: Sham (control group), SAH (experimental group), SAH+Palo (treatment group) and SAH+DMSO (treatment group). The Garcia neurological function score was used, with a maximum total score of 18 points, divided into 6 parts, each with a score of 0-3 points: spontaneous activity, symmetry of forelimb movement, outward extension of forepaws, body proprioception and reaction to touch of tentacles. Brain tissue was collected 24 hours after SAH and divided into left and right cerebral hemispheres and brainstem. The water content of brain tissue was detected by dry-wet method. Encephalon blue staining was used to detect brain tissue barrier damage.

[0037] 3. Experimental Results

[0038] Compared with the control group, the neurological function of 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 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 3 AC). The results showed that parovatin can improve the neurological damage and reduce the degree of brain edema in SAH mice.

[0039] Example 3: Parovatin can reduce nerve damage and inflammation in SAH mice

[0040] 1. Establishment of SAH mouse model

[0041] The establishment of SAH mouse model was the same as in Example 2.

[0042] 2. Experimental Methods

[0043] Sham (control group), SAH (experimental group), SAH+Palo (treatment group 1) and SAH+DMSO (treatment group 2). Brain tissues on the same side were collected 24 hours after SAH, and the brain tissue proteins of each group were extracted for Western blot detection of NLRP3, GSDMD-N and IL-1β levels. TUNEL fluorescence staining was used to detect the level of neuronal damage in brain tissues of each group. Statistical methods: Neurological function scores, brain edema, Western blot and fluorescence staining of mice in the control group, experimental group (SAH), treatment group (SAH+Palo) and treatment group (SAH+DMSO). Data are expressed as mean ± standard error. One-way analysis of variance (ANOVA) and Tukey test were used for comparison among multiple groups. * indicates statistical difference when 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, parovastatin 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, parovastatin significantly reduced the level of neuronal damage in the ipsilateral cerebral cortex of mice ( Figure 3 CD). These results indicate that parovastatin can reduce the level of neural 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 series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0047] Although preferred embodiments of the present invention have been described, additional changes and modifications may be made to these embodiments by those skilled in the art once the basic inventive concepts are known. 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 present 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 present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Use of parovatin in the preparation of a drug 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, an excipient, a diluent, 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 parovastatin is 1-10 uM.

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

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

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

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