Application of psoralen in preparation of medicine for preventing and treating cerebral ischemia-reperfusion injury

Abstract

The invention discloses an application of psoralen in preparation of a medicine for preventing and treating cerebral ischemia-reperfusion injury. The invention firstly proposes and verifies that the psoralen can prevent and treat the cerebral ischemia reperfusion injury, including cerebral infarction, motor function injury, sensory function retardation or disappearance, equilibrium dysfunction and neurological function injury represented by reflex deficiency, and provides a new direction for mechanism research and treatment of the cerebral ischemia reperfusion injury.

Description

Technical Field

[0001] This invention relates to the field of natural product chemistry, and more particularly to the application of psoralen in the preparation of drugs for the prevention and treatment of cerebral ischemia-reperfusion injury. Background Technology

[0002] Ischemic stroke is one of the leading causes of death and long-term disability worldwide, posing a continuing challenge to public health. In clinical treatment, thrombolysis or anticoagulation are commonly used to restore blood supply to the brain, but this can potentially lead to cerebral ischemia-reperfusion injury. This phenomenon refers to a situation where, after a period of ischemia, although blood flow is restored to the brain, tissue damage is exacerbated, manifesting as nerve cell death, increased infarct area, and further deterioration of neurological function, severely impacting the patient's recovery process.

[0003] The mechanism of this injury is complex, with oxidative stress considered a core element. During reperfusion, the imbalance between the excessive production of reactive oxygen species (ROS) and the decreased antioxidant capacity of the body is disrupted, leading to redox disturbances, impaired mitochondrial function, enhanced lipid peroxidation, and activation of apoptosis and inflammation-related pathways. Under normal circumstances, ROS production and clearance are in dynamic equilibrium, but under reperfusion conditions, the explosive increase in ROS initiates a series of cellular cascade reactions, ultimately causing irreversible neuronal damage. Therefore, controlling ROS production in neural tissue has become an important research direction for the prevention and treatment of reperfusion injury. In addition, ferroptosis, a newly discovered mode of cell death, also plays a crucial role in reperfusion injury. It is characterized by the abnormal accumulation of intracellular iron ions, which catalyzes the production of highly reactive free radicals through the Fenton reaction, driving lipid peroxidation and leading to cell membrane structure destruction and cell death. Studies have shown that in experimental models of cerebral ischemia-reperfusion, nerve cells exhibit typical morphological changes of ferroptosis, such as mitochondrial shrinkage, increased membrane density, and reduced cristae structure. These changes collectively contribute to decreased cell activity and accelerate disease progression. Intervention targeting the ferroptosis pathway is therefore considered a potential treatment strategy.

[0004] Psoralen (PSR) is a natural furanocoumarin compound derived from the legume *Psoralea corylifolia*. Current research reports indicate that this compound possesses various pharmacological activities, including anticancer, anti-osteoporosis, antimicrobial, and antidepressant effects, and it has already found practical applications in the treatment of skin diseases and phototherapy. However, its role in the prevention and treatment of cerebral ischemia-reperfusion injury remains unclear. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to provide the application of psoralen in the preparation of drugs for the prevention and treatment of cerebral ischemia-reperfusion injury, especially drugs for the prevention of cerebral infarction caused by cerebral ischemia-reperfusion and the prevention and treatment of neurological function damage caused by cerebral ischemia-reperfusion.

[0006] Technical solution: The application of psoralen as described in this invention in the preparation of drugs for the prevention and treatment of cerebral ischemia-reperfusion injury.

[0007] Preferably, the CAS number of the psoralen is 66-97-7.

[0008] Preferably, the cerebral ischemia-reperfusion injury is caused by any one or more of ischemic stroke, cardiac arrest, and shock.

[0009] Preferably, the application is in the preparation of a drug for preventing cerebral infarction caused by cerebral ischemia-reperfusion; more preferably, the application is in the preparation of a drug for preventing and treating neurological function damage caused by cerebral ischemia-reperfusion; more preferably, the neurological function damage caused by cerebral ischemia-reperfusion is any one or more of motor function impairment, sensory impairment or loss, balance dysfunction, and reflex loss.

[0010] Preferably, the drug contains psoralen or its pharmaceutically acceptable salts, solvates, or hydrates as active ingredients; more preferably, the drug also contains pharmaceutically acceptable excipients; more preferably, the pharmaceutically acceptable excipients include any one or more of excipients, diluents, lubricants, flow aids, wetting agents, emulsifiers, pH buffers, solubilizers, cosolvents, or solvents.

[0011] Preferably, the dosage form of the drug includes tablets, capsules, granules, powders, chewable tablets, effervescent tablets, sustained-release tablets, microcapsules, injections, infusions, suspensions, patches, suppositories, transdermal patches, microemulsions, liposomes, and nanoparticles.

[0012] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The present invention is the first to propose and verify that psoralen can prevent and treat cerebral ischemia-reperfusion injury, including cerebral infarction and motor function damage, sensory dullness or loss, balance dysfunction, and neurological function damage represented by reflex loss, providing a new direction for the study of the mechanism and treatment of cerebral ischemia-reperfusion injury. Attached Figure Description

[0013] Figure 1 Representative images of cerebral infarction in mice after different treatments following cerebral ischemia-reperfusion injury;

[0014] Figure 2 Statistical graph showing the infarct area of ​​cerebral ischemia-reperfusion mice after different treatments;

[0015] Figure 3 Statistical graph of the results of modified neurological function severity scores after different treatments in mice with cerebral ischemia-reperfusion injury;

[0016] Figure 4 Statistical graph of grip strength test results in mice after different treatments following cerebral ischemia-reperfusion injury;

[0017] Figure 5 Representative H&E staining images of brain tissue from mice subjected to different treatments after cerebral ischemia-reperfusion injury;

[0018] Figure 6 Statistical graph of H&E staining scores in mice after different treatments following cerebral ischemia-reperfusion injury;

[0019] Figure 7 Statistical graph of ELISA results for ferroptosis-related indicators in brain tissue of mice after different treatments following cerebral ischemia-reperfusion.

[0020] Figure 8 Representative images of ROS fluorescence detection in brain tissue of mice after different treatments following cerebral ischemia-reperfusion injury;

[0021] Figure 9 Statistical graph of quantitative results of ROS fluorescence detection in brain tissue of mice after different treatments in cerebral ischemia-reperfusion mice;

[0022] Figure 10 Figure showing the results of detecting the protein expression levels of GPX4 and SLC7A11 in the brain tissue of mice after different treatments following cerebral ischemia-reperfusion injury;

[0023] Figure 11 Statistical graph showing the quantitative results of protein levels of GPX4 and SLC7A11 in brain tissue of mice after different treatments following cerebral ischemia-reperfusion injury;

[0024] Figure 12 A statistical graph showing the results of detecting the mRNA expression levels of GPX4 and SLC7A11 in the brain tissue of mice after different treatments following cerebral ischemia-reperfusion injury;

[0025] Figure 13 A statistical graph showing the cell viability test results of different treatments in the oxygen-glucose deprivation / reperfusion cell model;

[0026] Figure 14 Representative images for detecting cell proliferation levels after different treatments in an oxygen-glucose deprivation / reperfusion cell model;

[0027] Figure 15 A statistical graph showing the results of cell proliferation level detection after different treatments in an oxygen-glucose deprivation / reperfusion cell model;

[0028] Figure 16 Representative images of cell morphology after different treatments in the oxygen-glucose deprivation / reperfusion cell model;

[0029] Figure 17Figure 1 shows the results of detecting the protein expression levels of GPX4 and SLC7A11 after different treatments in an oxygen-glucose deprivation / reperfusion cell model.

[0030] Figure 18 Statistical graph showing the quantitative results of GPX4 and SLC7A11 protein levels after different treatments in an oxygen-glucose deprivation / reperfusion cell model;

[0031] Figure 19 A statistical graph showing the results of detecting the mRNA expression levels of GPX4 and SLC7A11 after different treatments in an oxygen-glucose deprivation / reperfusion cell model.

[0032] Figure 20 Figure 1: Flow cytometry results of ROS levels in an oxygen-glucose deprivation / reperfusion cell model after different treatments.

[0033] Figure 21 Statistical graph of flow cytometry results of ROS level quantification in oxygen-glucose deprivation / reperfusion cell model after different treatments;

[0034] Figure 22 Representative images of ROS fluorescence detection after different treatments in an oxygen-glucose deprivation / reperfusion cell model;

[0035] Figure 23 Statistical graph of ROS fluorescence detection quantitative results after different treatments in oxygen-glucose deprivation / reperfusion cell models;

[0036] Figure 24 A statistical graph showing the ELISA results of ferroptosis-related indicators after different treatments in an oxygen-glucose deprivation / reperfusion cell model. Detailed Implementation

[0037] The technical solution of the present invention will be further described below.

[0038] Example 1: Construction of an animal model of cerebral ischemia-reperfusion and evaluation of treatment effects

[0039] A mouse model of middle cerebral artery occlusion / reperfusion (MCAO / R) was established using the suture occlusion method.

[0040] Male C57BL / 6 mice, aged 8-10 weeks and weighing 25-28 g, were purchased from the Animal Center of Nantong University. The mice were housed in a specific pathogen-free (SPF) animal barrier environment, and all experiments were approved by the Animal Ethics Committee of Nantong University. The mice were randomly divided into four groups: a normal control group (n=6); an MCAO / R group (saline) (n=6); an MCAO / R + edaravone treatment group (EDA15) (n=6); and an MCAO / R + psoralen treatment group (PSR 20) (n=10).

[0041] The MCAO / R model was established as follows: Mice were anesthetized by intraperitoneal injection of 10% chloral hydrate (300 mg / kg), and their body temperature was maintained at 36.5-37.5℃ using a constant-temperature heating plate. A midline cervical incision was made, and the right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were separated. The proximal end of the CCA was ligated, and the bifurcation of the CCA and the proximal end of the ECA were blocked with an arterial clamp. A small oblique incision was made below the CCA slipknot, and a waxed nylon suture plug (0.26 mm in diameter) was inserted, advancing to 18-22 mm (stopping upon encountering resistance). The suture plug was fixed, the proximal end of the ECA was ligated, and the wound was sutured. After 90 minutes of ischemia, the suture plug was pulled out to restore blood flow, completing reperfusion. In the sham surgery group, only a cervical incision and vascular exposure were performed, without inserting a suture plug.

[0042] Immediately after reperfusion, the mice were administered medication. The EDA 15 group received an intraperitoneal injection of 15 mg / kg edaravone (purchased from MedChemExpress LLC., catalog number HY-B0099, CAS number 89-25-8), and the PSR 20 group received an intraperitoneal injection of 20 mg / kg psoralen (purchased from MedChemExpress LLC., catalog number HY-N0053, CAS number 66-97-7). The sham-operated group and the model group received the same volume of physiological saline. The medication was administered once daily for 10 consecutive days. After the administration period, the mice were euthanized, and brain tissue and blood samples were collected for subsequent testing.

[0043] 1. Infarct area and neurological function testing

[0044] Detection of cerebral infarction area: Mouse brain tissue was taken, frozen at -20℃ for 30 min, and then cut into 2 mm thick sections in the coronal plane. The sections were placed in 2% 2,3,5-triphenyltetrazolium chloride (TTC) solution (MCE, HY-D0714) and incubated at 37℃ in the dark for 30 min. Images were acquired using an optical microscope, and the infarct area was analyzed using ImageJ software. Normal brain tissue was shown in red, and infarct foci were shown in white. The percentage of cerebral infarction area to total brain volume was calculated using the following formula: Percentage of cerebral infarction area (%) = White area / (White area + Red area) × 100.

[0045] The results are as follows Figure 1 , 2 As shown, compared with the sham-operated group mice, the model group mice showed a significant increase in the area of ​​cerebral infarction (pale white due to lack of ischemic enzyme activity) in TTC staining, indicating that the brain damage in the model group mice was more severe. After treatment with 20 mg / kg PSR, the area of ​​cerebral infarction was significantly reduced, similar to the positive drug EDA treatment group, preliminarily showing that PSR has an effect in alleviating MCAO / R disease.

[0046] Neurological function testing: Testing was conducted within 24 hours after the last administration. The modified neurological severity score (mNSS) was used to assess neurological function, with a score range of 0-18, where a higher score indicates more severe neurological deficits. Grasp strength testing was also performed, using a mouse grip strength meter to measure the duration of grip strength maintained by the forelimbs of mice, and the grip strength time was recorded and the differences between groups were statistically analyzed.

[0047] mNSS assessment results are as follows Figure 3 As shown, compared with the sham-operated group mice, the MCAO / R model group mice had significantly higher scores and severely impaired neurological function. After PSR treatment, the scores were significantly reduced and were comparable to those of the EDA drug treatment group.

[0048] Mouse grip strength test results as follows Figure 4 As shown, the grip strength maintenance time of mice in the MCAO / R model group was significantly shorter than that in the sham-operated group, reflecting a decline in limb motor function. However, after PSR treatment, it was significantly improved and comparable to the grip strength maintenance time in the EDA drug treatment group, indicating that the muscle dysfunction caused by cerebral ischemia injury was significantly alleviated.

[0049] 2. Brain tissue pathology and ferroptosis marker detection

[0050] 2.1 Pathological examination of brain tissue: Brain tissue was fixed overnight with 4% paraformaldehyde, dehydrated in a gradient manner, embedded in paraffin, and cut into 3μm sections; after dewaxing and hydration, hematoxylin-eosin (H&E) (MCE, HY-K0315) staining was performed, and the pathological changes of brain tissue were observed by optical microscopy to assess cell damage and interstitial condition.

[0051] The results of H&E staining of brain tissue are as follows Figure 5 , 6 As shown, the brain tissue cells of the sham-operated group mice had normal morphology and intact structure, while the model group showed obvious cell necrosis and structural disorder. However, after PSR treatment, the degree of brain tissue cell damage was reduced, and the cell morphology and structure were closer to normal. The H&E staining results of brain tissue in each group were scored according to Table 1 below. The statistical results also confirmed that PSR can improve the pathological damage of brain tissue caused by cerebral ischemia-reperfusion.

[0052] Table 1 Scoring rules for H&E staining results in brain tissue

[0053]

[0054] 2.2 ELISA Detection of Ferrocyte Indicators: Brain tissue homogenate was centrifuged at 12000 rpm for 30 min at 4℃, and the supernatant was collected. The supernatant was then analyzed using the Beyotime Lipid Oxidation (MDA) Detection Kit (Catalog No. S0131S), the Sangon Biotech 4-Hydroxynonenoic Acid (4-HNE) ELISA Kit (Catalog No. D751041), and the Elite Ferrous Ion Colorimetric Assay Kit (Catalog No. E-BC-K773-M) to detect malondialdehyde (MDA), 4-hydroxynonenoic acid (4-HNE), and ferrous ions (Fe2+) in the supernatant. 2+ ) level.

[0055] The results are as follows Figure 7 As shown, compared with the sham-operated group, the MCAO / R model group mice had higher levels of MDA and Fe in their brain tissue. 2+ The levels of 4-HNE were significantly increased, while the level of GSH was significantly decreased. These results indicate that there is a high level of ferroptosis in MCAO / R model mice. After PSR treatment, MDA and Fe were significantly downregulated. 2+ The levels of 4-HNE were significantly increased, while the level of GSH was also significantly increased. These changes in ferroptosis markers were consistent with those in the EDA drug treatment group.

[0056] 2.3 ROS Immunofluorescence Detection: After dewaxing and hydration of 3 μm sections of brain tissue, 10 μM of DCFH-DA fluorescent probe (purchased from MedChemExpress LLC., catalog number HY-D0940) was added and incubated at 37℃ for 30 min; after washing with PBS, nuclear staining was performed using DAPI, and images were observed and acquired using a fluorescence microscope. The ROS fluorescence intensity was analyzed using ImageJ software.

[0057] The results are as follows Figure 8-9 As shown, the ROS fluorescence signal intensity in the brain tissue of MCAO / R model mice was significantly higher than that in the sham-operated group. After PSR treatment, the fluorescence signal intensity was significantly reduced, which was similar to the results of EDA drug treatment, and could reduce the generation of ROS after cerebral ischemia-reperfusion.

[0058] 2.4 Molecular biological detection of glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11 protein (SLC7A11):

[0059] Western blot analysis: Total protein was extracted from brain tissue using the Neo-Syneema Columnar Animal Tissue and Cell Total Protein Extraction Kit (Catalog No. WB3050), followed by SDS-PAGE electrophoresis. After transfer and blocking, the tissue was incubated overnight at 4°C with GPX4 (Proteintech, Catalog No. 30388-1-AP) and SLC7A11 (Proteintech, Catalog No. 26864-1-AP) primary antibodies. After rinsing, the tissue was incubated at room temperature for 2 h with secondary antibody (Proteintech, Catalog No. SA00001-2). The tissue was then developed using the Neo-Syneema High-Sensitivity ECL Chemiluminescence Kit (Catalog No. P10100), and the grayscale values ​​of the developed images were analyzed using ImageJ software.

[0060] The results are as follows Figure 10 , 11 As shown, compared with the sham-operated group, the protein expression levels of GPX4 and SLC7A11 in the brain tissue of MCAO / R model mice were significantly reduced. However, with PSR treatment, the expression levels of these two anti-ferroptosis proteins were significantly upregulated, and the trend was similar to that of the EDA drug treatment group.

[0061] RT-PCR assay: Total RNA was extracted using the Novizan FastPure Complex Tissue / Cell Total RNA Isolation Kit (catalog number RC113-01) and reverse transcribed into cDNA using the Novizan HiScript III All-in-one RTSuperMix Perfect for qPCR Kit (catalog number R333-01). The primers used were as follows: GPX4 upstream primer: 5'-tctcagccaaggacatcgac-3'; GPX4 downstream primer: 5'-ggccaggattcgtaaaccac-3'; SLC7A11 upstream primer: 5'-cccagatatgcatcgtcctt-3'; SLC7A11 downstream primer: 5'-cgtctgaaccacttgggttt-3'; GAPDH upstream primer: 5'-gtgaaggtcggtgtgaacg-3'; GAPDH downstream primer: 5'-tgccgtgagtggagtcatac -3', RT-PCR was performed using the Novizan ChamQ Universal SYBR qPCR MasterMix kit (catalog number Q711-02). The reaction conditions were: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 10 s; 60℃ annealing and extension for 30 s, for 35 cycles. Fluorescence values ​​were collected during the extension phase of each cycle. GADPH was used as an internal control, and Ct values ​​were calculated. The relative expression levels of GPX4 and SLC7A11 were calculated using the ΔΔCt method.

[0062] The results are as follows Figure 12 As shown, compared with the sham-operated group, the mRNA levels of GPX4 and SLC7A11 in the brain tissue of the model mice were significantly reduced. After PSR treatment, the mRNA levels of both were significantly restored, which is consistent with the results of the EDA drug treatment group.

[0063] Example 2: Validation of the effect of PSR on the HT22 cell oxygen deprivation / reperfusion (OGD / R) model

[0064] Construction of the OGD / R model: Mouse hippocampal neurons HT22 cells (Procell, CL-0697) were routinely cultured in DMEM medium (Procell, PM150210) containing 10% fetal bovine serum (Procell, 164220) at 37℃ and 5% CO2. Cells were seeded into culture plates, and when confluence reached 70%, the medium was replaced with glucose-free DMEM. The plates were then placed in a tri-gas incubator (1% O2, 5% CO2, 94% N2) and cultured at 37℃ for 3 hours under hypoxic and glucose-deficient conditions. The culture was then replaced with complete DMEM medium and continued for another 24 hours to complete the OGD / R model construction.

[0065] An OGD / R model group and an OGD / R+PSR (10 μM) treatment group were set up. The drug treatment was added at the beginning of reperfusion (i.e., when changing to DMEM complete medium), and cells were collected after 24 h of culture for subsequent detection.

[0066] 1. Cell viability detection

[0067] Cells from each group were seeded in 96-well plates. When the cell density reached 70±10%, the corresponding concentration of drug was added to each group for treatment. Each drug treatment was performed in triplicate. The treatment lasted for 24 h. After treatment, 100 μL of DMEM basal medium containing 10% CCK8 reagent (purchased from Novizan, catalog number A311-01) was added to each well. The absorbance value at 450 nm was measured by microplate reader. The cell viability of the other groups was calculated with the cell viability of the normal group as 100%.

[0068] Test results as follows Figure 13 As shown, compared with the normal group, the cell activity of the OGD / R model group was significantly reduced, while the cell activity of the PSR treatment group was significantly increased compared with the OGD / R model group, proving that PSR can restore the activity of HT22 cells after OGD / R damage.

[0069] 2. Cell proliferation detection

[0070] Cells from each group were seeded into 96-well plates. When the cell density reached 70±10%, the corresponding concentration of drug was added to each group for treatment. Each drug treatment was performed in triplicate. After 24 h of treatment, 50 μM EdU reagent (purchased from Novizan, catalog number A413-01) was added and incubated at 37℃ for 2 h. After fixation with 4% paraformaldehyde solution at room temperature for 10 min, permeabilization was performed at room temperature using Beyotime immunostaining permeabilization solution (catalog number P0096) for 15 min. Then, Click reaction solution (purchased from Novizan, catalog number A413-01) was added and incubated in the dark for 30 min. The slides were then mounted using Beyotime anti-fluorescence quenching mounting solution and observed under a fluorescence microscope. The percentage of EdU-positive cells was counted.

[0071] The results are as follows Figure 14 , 15 As shown, compared with the normal group, the EdU fluorescence of cells in the OGD / R model group was significantly reduced, indicating that the proliferation rate of cells in the OGD / R model group was significantly reduced. However, in the PSR treatment group, we found that the EdU fluorescence intensity was restored, and its positive rate was significantly higher than that of the OGD / R model group, indicating that PSR can promote the proliferation activity of HT22 cells after OGD / R injury.

[0072] 3. Cell morphology observation

[0073] Cells from each group were seeded in 6 cm culture dishes. When the cell density reached 70±10%, the corresponding concentration of drug was added to each group for treatment. Each drug treatment was performed in triplicate. The treatment lasted for 24 h. Cell morphology of each group was observed under an optical microscope, cell adhesion was recorded, and photos were taken for comparison and analysis.

[0074] The results are as follows Figure 16 As shown, the normal group of HT22 cells had normal morphology and were tightly adhered to the wall, while the OGD / R model group cells were significantly shrunken and loosely adhered to the wall. With PSR treatment, the cell morphology became closer to that of the normal group, and the cell activity was improved. This further illustrates that PSR can alleviate the damage to the morphology of HT22 cells in the OGD / R model.

[0075] 4. Detection of reactive oxygen species (ROS) and ferroptosis indicators

[0076] 4.1 Molecular biological detection

[0077] Cells from each group were seeded in 6-well plates. When the cell density reached 70±10%, the corresponding concentration of drug was added to each group for treatment. Each drug treatment was performed in 3 replicates. After 24 h of treatment, the cells were collected.

[0078] Western blot analysis: Total protein was extracted using NewSyne Western blot and IP cell lysis buffer (catalog number P70100), and the protein expression levels of GPX4 and SLC7A11 were determined according to the method described in Example 1.

[0079] The results are as follows Figure 17 , 18 As shown, the expression levels of GPX4 and SLC7A11 proteins in HT22 cells in the OGD / R model were significantly lower than those in the normal group, while the expression of these two proteins was significantly restored in the PSR treatment group, indicating that PSR can promote the expression of antiferroptosis protein in HT22 cells in the OGD / R model.

[0080] RT-PCR detection: After extracting total RNA using the Novizan FastPure Complex Tissue / Cell Total RNA Isolation Kit (catalog number RC113-01), the relative expression levels of GPX4 and SLC7A11 mRNA were determined according to the method described in Example 1.

[0081] The results are as follows Figure 19 As shown, compared with the normal group, the mRNA expression of GPX4 and SLC7A11 in HT22 cells in the OGD / R model was significantly reduced, and this reduction could be significantly restored by PSR. This experiment shows that PSR can regulate the expression of GPX4 and SLC7A11 in OGD / R cells at the transcriptional level.

[0082] 4.2 Flow Cytometry Detection of ROS: Cells from each group were seeded in 6-well plates. When the cell density reached 70±10%, the corresponding concentration of drug was added to each group for treatment. Each drug treatment was performed in triplicate. After 24 h of treatment, cells were collected, and 5 μM of DCFH-DA fluorescent probe was added. The cells were incubated at 37℃ for 30 min. The level of intracellular ROS in each group was characterized by detecting the fluorescence intensity of the FITC channel using flow cytometry.

[0083] The results are as follows Figure 20 , 21 As shown, the ROS level of HT22 cells in the OGD / R model was significantly higher than that in the normal group, while the ROS level in the PSR treatment group was significantly lower than that in the OGD / R model group, proving that PSR can effectively reduce the generation of intracellular ROS in the OGD / R model.

[0084] 4.3 ROS fluorescence observation: Cells from each group were seeded in 6-well plates. When the cell density reached 70±10%, the corresponding concentration of drug was added to each group for treatment. Each drug treatment was performed in triplicate. After treatment for 24 h, the cells were fixed with 4% paraformaldehyde at room temperature for 10 min, and then 10 μM of DCFH-DA fluorescent probe was added and incubated at 37℃ for 30 min. After washing with PBS, nuclear staining was performed using DAPI. The cells were observed and images were acquired using a fluorescence microscope. The ROS fluorescence intensity was analyzed using ImageJ software.

[0085] The results are as follows Figure 22 , 23 As shown, the ROS fluorescence intensity of HT22 cells in the OGD / R model was significantly higher than that in the normal group. After PSR treatment, the ROS fluorescence intensity was significantly reduced, which is consistent with the trend obtained by flow cytometry. This strongly confirms that PSR can reduce the ROS level of HT22 cells in the OGD / R model, thereby achieving the purpose of treating the disease by inhibiting ferroptosis.

[0086] 4.3 ELISA Detection of Ferrocyte Indicators: Cells from each group were seeded into 6-well plates. When the cell density reached 70±10%, the corresponding concentration of drug was added to each group for treatment. Each drug treatment was performed in triplicate. After 24 h of treatment, cells were collected, and total protein was extracted using Western blotting with cytotoxicity assay (Catalog No. P70100). The levels of MDA, 4-HNE, and Fe in total protein were detected using the Beyotime Lipid Oxidation (MDA) Detection Kit (Catalog No. S0131S), the Sangon Biotech 4-Hydroxynonenoic Acid (4-HNE) ELISA Kit (Catalog No. D751041), and the Ellansé Ferrous Ion Colorimetric Assay Kit (Catalog No. E-BC-K773-M). 2+ The level.

[0087] The results are as follows Figure 24 As shown, compared with the normal group, the levels of MDA, 4-HNE, and Fe²⁺ in HT22 cells of the OGD / R model group were significantly increased, while the level of GSH was decreased. After PSR treatment, the above indicators were restored to the levels of the normal group. These results further indicate that PSR can significantly inhibit ferroptosis, thereby alleviating the activity of OGD / R cells and ultimately achieving the goal of treating cerebral ischemia-reperfusion injury.

Claims

1. The application of psoralen in the preparation of drugs for the prevention and treatment of cerebral ischemia-reperfusion injury.

2. The application according to claim 1, characterized in that, The CAS number of the psoralen is 66-97-7.

3. The application according to claim 1, characterized in that, The cerebral ischemia-reperfusion injury refers to cerebral ischemia-reperfusion injury caused by any one or more of ischemic stroke, cardiac arrest, and shock.

4. The application according to claim 1, characterized in that, The application is in the preparation of drugs for preventing cerebral infarction caused by cerebral ischemia-reperfusion.

5. The application according to claim 1, characterized in that, The application is in the preparation of drugs for preventing and treating neurological damage caused by cerebral ischemia-reperfusion.

6. The application according to claim 5, characterized in that, The neurological function impairment caused by cerebral ischemia-reperfusion includes any one or more of the following: motor function impairment, sensory impairment or loss, balance dysfunction, and reflex loss.

7. The application according to any one of claims 1-6, characterized in that, The drug contains psoralen or its pharmaceutically acceptable salts, solvates, or hydrates as active ingredients.

8. The application according to claim 7, characterized in that, The drug also contains pharmaceutically acceptable excipients.

9. The application according to claim 8, characterized in that, The pharmaceutically acceptable excipients include any one or more of the following: excipients, diluents, lubricants, glidants, wetting agents, emulsifiers, pH buffers, solubilizers, cosolvents, or solvents.

10. The application according to claim 1, characterized in that, The dosage forms of the drugs include tablets, capsules, granules, powders, chewable tablets, effervescent tablets, sustained-release tablets, microcapsules, injections, infusions, suspensions, patches, suppositories, transdermal patches, microemulsions, liposomes, and nanoparticles.

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