Application of (20S)-ginsenoside Rh1 in prevention and improvement of cerebral arterial thrombosis

Abstract

The invention discloses application of (20S)-ginsenoside Rh1 in preparation of drugs for preventing and / or improving cerebral arterial thrombosis, and relates to the technical field of medical application of natural drugs. The invention verifies that (20S)-ginsenoside Rh1 up-regulates the expression of an anti-apoptosis gene by promoting STAT3 phosphorylation and translocation to a cell nucleus, so that the (20S)-ginsenoside Rh1 plays a role in protecting neuronal injury induced by sugar oxygen deprivation. In MCAO model mice, the (20S)-ginsenoside Rh1 obviously relieves the symptom of ischemic cerebral apoplexy and has positive influence on neurodegenerative events related to cerebral infarction, and the anti-apoptosis and neuroprotection effects are most prominent. Mechanism research shows that (20S)-ginsenoside Rh1 plays a therapeutic role in PC12 cell and MCAO model mice by activating a JAK2 / STAT3 signal channel.

Description

Technical Field

[0001] The present invention relates to the technical field of medical applications of natural medicines, and particularly to the application of (20S)-ginsenoside Rh1 in the prevention and improvement of ischemic stroke. Background Art

[0002] Stroke (i.e., stroke) is the second leading cause of death globally, second only to ischemic heart disease. According to the pathological type, stroke can be divided into ischemic stroke (accounting for 60%-80% of new cases) and hemorrhagic stroke, of which the former is caused by acute occlusion of cerebral blood vessels, resulting in ischemia and hypoxia of brain tissue. At present, the core strategy of clinical treatment is to quickly open the occluded blood vessels (such as intravenous thrombolysis, endovascular thrombectomy), but cerebral ischemia-reperfusion injury (CIRI) may be triggered after blood flow recovery, aggravating neurological damage.

[0003] The pathological mechanism of CIRI is complex and involves multiple pathways such as oxidative stress, inflammatory response, calcium overload and apoptosis. Among them, apoptosis is one of the main forms of nerve cell death. Signal Transducer and Activator of Transcription 3 (STAT3), as a key regulatory protein, participates in the regulation of cell proliferation, differentiation and apoptosis and plays an important role in neuroprotection. The specific mechanism is as follows: after Janus Kinase 2 (JAK2) is activated, it catalyzes the tyrosine phosphorylation of STAT3 (p-STAT3), promotes STAT3 to form a homodimer and translocates to the nucleus, and then initiates the transcriptional expression of anti-apoptotic genes (such as Bcl-2, Survivin), ultimately inhibiting nerve cell apoptosis and reducing CIRI damage.

[0004] Ginseng is a natural herb mainly distributed in Asian countries, and its dried roots and rhizomes are widely used in both the medical and health care fields. (20S)-ginsenoside Rh1 is a tetracyclic triterpenoid dammarane-type saponin extracted from ginseng, and has various biological activities such as immunomodulation, neuroprotection, anti-tumor and anti-skin aging. However, the specific role of (20S)-ginsenoside Rh1 in the treatment of ischemic stroke has not been reported yet. Therefore, the present invention proposes the application of (20S)-ginsenoside Rh1 in the prevention and improvement of ischemic stroke to solve the problems existing in the prior art. Summary of the Invention

[0005] In view of the above problems, the object of the present invention is to propose the application of (20S)-ginsenoside Rh1 in the prevention and improvement of ischemic stroke. By using a cell injury model induced by glucose and oxygen deprivation (OGD / R) and a mouse model of middle cerebral artery occlusion (MCAO model), the mechanism of (20S)-ginsenoside Rh1 in neuroprotection is studied, providing new ideas and methods for the prevention and improvement of ischemic stroke.

[0006] To achieve the object of the present invention, the present invention is realized through the following technical solutions:

[0007] The application of (20S)-ginsenoside Rh1 in the preparation of a drug for preventing and / or improving ischemic stroke.

[0008] The application of (20S)-ginsenoside Rh1 in the preparation of a drug for relieving the motor disorder and the decline of learning and memory ability in ischemic stroke.

[0009] The application of (20S)-ginsenoside Rh1 in the preparation of a drug for enhancing the activity of nerve cells in cerebral ischemic stroke.

[0010] The application of (20S)-ginsenoside Rh1 in the preparation of a drug for inhibiting the expression of proteins related to nerve cell apoptosis in ischemic stroke.

[0011] The application of (20S)-ginsenoside Rh1 in the preparation of a drug for targeting the JAK2-STAT3 axis signaling pathway to relieve nerve cell damage.

[0012] The application of (20S)-ginsenoside Rh1 in the preparation of a drug for reducing the infarct area in ischemic stroke.

[0013] A drug for preventing / improving ischemic stroke, wherein the active ingredient of the drug comprises (20S)-ginsenoside Rh1.

[0014] The present invention also provides the application of (20S)-ginsenoside Rh1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating ischemic stroke.

[0015] A further improvement lies in that: the medicament comprises an effective amount of (20S)-ginsenoside Rh1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[0016] A further improvement lies in that: the pharmaceutically acceptable carrier comprises a filler, a diluent, a binder, a disintegrant, an emulsifier and a non-toxic and side-effect-free drug carrier.

[0017] A further improvement lies in that: the dosage form of the medicament comprises tablets, granules, oral liquid preparations, drops, injection preparations and capsule preparations.

[0018] A further improvement lies in that: the drug is prepared in the form of a single-dose drug.

[0019] A further improvement lies in that: the single-dose drug contains 1 - 1000 mg of (20S)-ginsenoside Rh1 or a pharmaceutically acceptable salt thereof.

[0020] The beneficial effects of the present invention are as follows: The present invention verifies that (20S)-ginsenoside Rh1 plays a protective role against glucose and oxygen deprivation-induced neuronal damage by promoting STAT3 phosphorylation and translocation to the nucleus, and upregulating the expression of anti-apoptotic genes. In MCAO model mice, (20S)-ginsenoside Rh1 significantly alleviates ischemic stroke symptoms and has a positive impact on neurodegenerative events related to cerebral infarction, among which the anti-apoptotic and neuroprotective effects are the most prominent. Mechanistic studies have shown that (20S)-ginsenoside Rh1 exerts a therapeutic effect in PC12 cells and MCAO model mice by activating the JAK2 / STAT3 signaling pathway. These findings reveal the potential of (20S)-ginsenoside Rh1 as a new drug for the treatment of ischemic stroke and provide a new strategy for clinical treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0022] Figure 1 It is a schematic diagram showing the effects of different hypoxia times and drug concentrations on the viability of PC12 cells in Example 1 of the present invention;

[0023] Figure 2 It is a schematic diagram showing the effects of ginsenoside Rh1 on oxidative stress and mitochondrial function of PC12 cells in Example 2 of the present invention;

[0024] Figure 3 It is a schematic diagram showing the analysis of histological staining results of different brain regions after MCAO by ginsenoside Rh1 in Example 3 of the present invention;

[0025] Figure 4 It is a schematic diagram showing that ginsenoside Rh1 alleviates brain damage caused by MCAO in mice by inhibiting apoptosis in Example 4 of the present invention;

[0026] Figure 5 It is a schematic diagram showing the exploration of potential targets and mechanisms of ginsenoside Rh1 in the treatment of CIRI based on network pharmacology in Example 5 of the present invention;

[0027] Figure 6 It is a schematic diagram of the negative regulation of the Jak2 / STAT3 signaling pathway by ginsenoside Rh1 in Example 6 of the present invention;

[0028] Figure 7 It is a schematic diagram of ginsenoside Rh1 promoting the nuclear translocation of STAT3 through KPNA3 in Example 7 of the present invention;

[0029] Figure 8 It is the chemical structural formula of ginsenoside Rh1 of the present invention. Detailed implementation manners

[0030] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present invention.

[0031] Single-cell RNA sequencing is a high-throughput technology that can effectively analyze the cellular and molecular changes in CIRI. By analyzing single-cell data, it is found that neuronal apoptosis plays a key role in IRI. As a core link in immune regulation, neuronal apoptosis has great therapeutic potential. Neurological function assessment tests (such as mNSS score, grip strength test, and rotarod test) are important indicators for evaluating animal models of central nervous system diseases. It can effectively reflect the neurological status and lesion degree of the model. Immunohistochemistry technology realizes the qualitative, localization, and quantitative analysis of antigens through antigen-antibody reactions and is widely used in biomedical research. Western blotting analyzes protein expression through gel electrophoresis and antibody staining and is a core method in molecular biology research. Flow cytometry uses fluorescence labeling and laser detection to achieve multi-parameter analysis of single cells and is widely used in cell biology research. Immunofluorescence technology uses fluorescently labeled antibodies as probes to enable high-resolution localization and qualitative analysis of specific antigens in tissues or cells. Co-immunoprecipitation (Co-IP) is a protein interaction research technology based on the specific binding of antigen and antibody and is widely used in the analysis of signaling pathways and protein complexes.

[0032] Example 1

[0033] This example verifies the inhibitory effect of (20S)-ginsenoside Rh1 on the reduction of viability of PC12 cells (rat adrenal pheochromocytoma cell line) induced by oxygen-glucose deprivation (OGD), and the operation is as follows:

[0034] 1.1 Cell culture: PC12 cells were inoculated into DMEM containing 10% fetal bovine serum (FBS) and 100 μg / mL streptomycin, and cultured in a humid environment at 37°C (CO2 concentration was 5%). When the cell density reached about 80% under microscopic observation, 0.25% trypsin was used for digestion and centrifugation for subculture. At the same time, cells were inoculated into the well plates required for the experiment. The seeding density in the 96-well plate was about 4.5×10 4 cells / mL, the seeding density in the 12-well plate was about 2.5×10 5 cells / mL, and the inoculation amount in the 6-well plate was about 4×10 5 cells / mL.

[0035] 1.2 Glucose and oxygen deprivation treatment: PC12 cells were inoculated into a 4.2 mm 2 culture dish and cultured with DMEM containing 10% fetal bovine serum for 24 h. Subsequently, they were washed 3 times with PBS, changed to a sugar-free medium, and incubated in a triple-gas incubator for 2 h, 4 h, 6 h, 8 h, and 24 h respectively. The conditions of the triple-gas incubator were set as 1% O2, 5% CO2, and 94% N2. After hypoxia treatment, the sugar-free medium was discarded and changed to DMEM containing 10% FBS. The cells in the normal control group were not subjected to glucose and oxygen deprivation treatment, and only the medium was uniformly changed at the end of the hypoxia treatment to maintain synchronization. Finally, all cells were placed in a conventional incubator and continued to be cultured for 20 - 24 h.

[0036] 1.3 MTT assay to detect cell viability: PC12 cells were inoculated into 96-well plates and cultured with complete medium for 20 - 24 h. They were pretreated with 1 μM, 5 μM, and 10 μM of (20S)-ginsenoside Rh1 for 2 h respectively, and then subjected to glucose and oxygen deprivation treatment for 2 h, 4 h, 6 h, 12 h, and then reoxygenated and cultured for 24 h. After the treatment, 0.5 mg / mL of MTT (prepared with DMEM) was added to each well and incubated at 37°C for 3 h. After discarding the medium, 100 μL of DMSO was added to dissolve the formazan crystals, and the absorbance value at a wavelength of 570 nm was measured using an enzyme-linked immunosorbent assay detector. The effects of different hypoxia times and drug concentrations on the viability of PC12 cells are as Figure 1 shown ( Figure 1 A in Figure 1 : After reoxygenation for 24 h after different hypoxia time treatments of PC12 cells, the cell viability was detected by MTT assay, Figure 8 B in

[0037] Example 2

[0038] This example verifies the protective effect of (20S)-ginsenoside Rh1 on the neurotoxicity of PC12 cells induced by glucose and oxygen deprivation, and the operation is carried out according to the following steps:

[0039] The cell culture method is the same as 1.1 in Example 1. Inoculate PC12 cells into the well plates required for the experiment and culture them with complete medium for 20 - 24 h. Pretreat with 1 μM and 5 μM of (20S)-ginsenoside Rh1 for 2 h respectively, then perform glucose and oxygen deprivation treatment for 6 h and reoxygenation culture for 24 h.

[0040] 2.1 ROS staining: Transfer the cells to a 1.5 mL EP tube, centrifuge at 12,000 rpm for 15 min at 4 °C, and discard the supernatant. Add 10 μM DCFH-DA reagent (diluted 1:1000 with DMEM medium), incubate in the dark at 37 °C for 60 min. Wash twice with PBS, and measure the fluorescence intensity using an Infinite M200 PRO multimode microplate. The excitation wavelength is 488 nm and the emission wavelength is 525 nm.

[0041] 2.2 JC-1 staining method: Discard the medium, add 50 μL of JC-1 staining solution (diluted 1:250 with DMEM medium), mix well, and incubate at 37 °C for 1 h. After incubation, wash twice with PBS. Then observe under a fluorescence microscope.

[0042] 2.3 Western blotting experiment: Discard the medium, wash three times with PBS, add RIPA buffer containing protease and phosphatase inhibitors, collect in a 1.5 mL EP tube, shake and lyse on ice for 30 min, centrifuge at 12,000 rpm for 15 min at 4 °C, and take the supernatant. Determine the protein concentration by the BCA method, heat in a metal bath at 95 °C for 10 min. After the samples are electrophoresed on SDS-PAGE at 150 V for 50 min, electrotransfer at 300 mA for 90 min to transfer the proteins on the gel to the PVDF membrane. Block with 3% BSA solution for 1 - 2 h, incubate with the primary antibody (1:1000) overnight at 4 °C. Wash with TBST for 10 min × 3 times, incubate with the secondary antibody (1:2000) at room temperature for 2 h. Wash with TBST for 10 min × 3 times. Detect the protein bands using a chemiluminescence or multicolor fluorescence imaging system (Bio-Rad Gel DocXR). Quantitatively process the protein bands using Image J and Prism 10 software. Detect and quantitatively analyze the expression levels of Bcl2, Bax, and Cleaved-Caspase3 proteins. The effects of ginsenoside Rh1 on oxidative stress and mitochondrial function in PC12 cells are as Figure 2 shown ( Figure 2A in: After OGD / R treatment, different concentrations of ginsenoside Rh1 were administered, and the intracellular ROS level was detected by fluorescence staining; Figure 2 B in: JC-1 staining was used to evaluate the change of mitochondrial membrane potential; Figure 2 C in: Western blot was used to detect the effects of different concentrations of ginsenoside Rh1 on the expression of Bcl-2, Bax, and cleaved caspase-3 after OGD / R treatment, with β-actin as the internal reference; Figure 2 D in: Figure 2 Quantitative analysis of the Western blot results in C above).

[0043] 2.4 Flow cytometry experiment: The cells were digested with trypsin and collected in a 1.5 mL EP tube, centrifuged at 1000 g for 15 min, the supernatant was discarded, resuspended with PBS, and centrifuged again at 1000 g for 5 min. After discarding the supernatant, 200 μL of Binding Buffer was added to resuspend to an appropriate concentration. Subsequently, 5 μL of Annexin V-FITC and 10 μL of propidium iodide (PI) were added to each group, incubated at room temperature in the dark for 30 min, and finally detected using a flow cytometer.

[0044] Example 3

[0045] This example verified the improvement effect of (20S)-ginsenoside Rh1 on the cerebral infarction area and motor dysfunction in mice with ischemic stroke, and the operation was carried out according to the following steps:

[0046] Forty C57 / B6L mice were randomly divided into 4 groups, with 10 mice in each group. The weight of each mouse was weighed before administration, and the administration dose was calculated according to the weight. Group A was the blank control group (administered with an equal amount of normal saline); Group B was the model group (administered with an equal amount of normal saline); Group C was the model group + (20S)-ginsenoside Rh1 1 mg / kg; Group D was the model group + (20S)-ginsenoside Rh1 5 mg / kg. In this example, a mouse model of cerebral ischemia-reperfusion was constructed by the middle cerebral artery occlusion method (MCAO). After occluding the middle cerebral artery of the mouse for 1 h, the thread embolism was pulled out, the wound was sutured, and then the mouse was placed back in the cage and raised in a 25 °C room temperature environment, with sufficient drinking water and soft food provided.

[0047] 3.1 Behavioral tests: On the 1st, 4th, 7th, and 14th days after surgery, relevant behavioral data of C57 / B6L mice in the control group, model group, and drug administration groups (1 mg / kg, 5 mg / kg) were recorded. For the grip strength test, mice were placed in the test environment 15 - 20 minutes before the experiment to adapt. When starting the test, the mice were placed on the grip strength meter, and their tails were gently pulled to make their forelimbs / hindlimbs grasp the force measuring rod, and the maximum grip strength value (ending when the mouse fell off) was recorded. Each group of mice was measured 3 times repeatedly, and the average value was taken as the final result. Rotarod test: Mice were placed in the test environment 2 days before the experiment to adapt. When starting the test, the mice were placed with their backs facing the rotation direction of the rod, and it was ensured that all four limbs were in contact with the surface of the rotarod. The rotarod speed was gradually increased at an acceleration of 20 rpm / min, and the staying time of the mice on the rotarod and the rotarod speed were recorded. Each group was tested 3 times repeatedly, and the average value was taken as the final result. mNSS score, the judgment criteria include the following aspects:

[0048] (1) Motor function detection: Observe and record the spontaneous activity state of the mice, and judge whether there are abnormal manifestations such as limb paralysis, difficulty walking, stereotyped circling, body tilt, or movement asymmetry.

[0049] (2) Sensory function detection, gently touch the four limbs of the mice with a cotton swab and a thin needle, and observe and record their responses to tactile stimuli.

[0050] (3) Reflex function detection, place the mice in the supine position, and record the time required for them to return to the normal posture (normal is 1 - 2 s); lift the mouse's tail to make it suspended in the air, and observe whether the forelimbs show a flexed posture.

[0051] Each aspect accounts for 6 points. The total score range of 1 - 6 points indicates mild injury, 7 - 12 points indicates moderate injury, and 13 - 18 points indicates severe injury.

[0052] 3.2 TTC staining: 24 hours after perfusion, mice were euthanized with 0.25 mg / ml chloral hydrate and decapitated to take the brain. The removed brain tissue was cut into 4 complete slices with a thickness of about 2 mm along the coronal plane, soaked in 1% TTC solution (prepared with PBS), and incubated at 37 °C in the dark for 20 minutes. Finally, the brain slices were fixed in 4% paraformaldehyde.

[0053] 3.3, HE staining (hematoxylin-eosin staining method): After perfusion of each group of mice with 4% paraformaldehyde, the mice were sacrificed, the brains were removed and immersed in 4% paraformaldehyde for fixation for 24 h, and washed with PBS for 5 min × 3 times. They were successively immersed in 70%, 80%, 90%, 95% and 100% ethanol for gradient dehydration for 15 min each. They were permeabilized in xylene I and xylene II for 10 min each, infiltrated with soft wax and hard wax for 1 h each, and sectioned coronally at 5 μm. The cut paraffin sections were immersed in xylene for dewaxing for 10 min × 2 times. They were rehydrated with gradient ethanol (100%, 95%, 85%, 75%) for 3 min each, and immersed in distilled water for 2 min. They were stained with hematoxylin solution for 40 min, differentiated for 30 s, and rinsed with tap water 2 times. They were stained with 0.5% eosin solution for 30 s, dehydrated with gradient ethanol for 3 s each, and then washed with fresh 100% ethanol for 1 min. They were cleared in xylene for 1 min × 2 times, and observed under a fluorescence microscope after mounting.

[0054] 3.4, Nissl staining: The taken brain tissue was fixed, washed, paraffin-embedded, sectioned and dewaxed and hydrated routinely. Then the sections were stained with Nissl staining solution for 15 min, and then placed in 70% ethanol for differentiation for several seconds. Finally, routine dehydration, permeabilization and mounting were carried out, and observation was carried out under a fluorescence microscope.

[0055] 3.5, TUNEL staining: After the taken brain tissue was fixed, washed, paraffin-embedded, sectioned and dewaxed and hydrated routinely. The sections were immersed in PBS containing 0.3% Triton X-100 for permeabilization at room temperature for 5 min, and washed with PBS for 5 min × 3 times. After dropping the blocking solution, the TUNEL reaction solution was added, and incubated at 37 °C in the dark for 90 min, and rinsed with PBS for 5 min × 3 times. The labeled reaction termination solution was dropped, and reacted at 37 °C in the dark for 30 min, and washed with PBS. The Streptavidin-HRP working solution was added, and reacted at room temperature in the dark for 30 min, and washed with PBS. Then the DAB chromogenic working solution was dropped, the color development was observed under the microscope, and the reaction was terminated with distilled water. After rinsing with tap water, hematoxylin solution was dropped for counterstaining for 3 min, and rinsed thoroughly with tap water again. Observation was carried out under a fluorescence microscope after mounting. The histological staining results analysis of ginsenoside Rh1 on different brain regions after MCAO was as Figure 3 shown ( Figure 3 A in Figure 3 : After the establishment of the MCAO model, the brain tissue sections of different brain regions were stained with H&E; Figure 3 B in

[0056] Example 4

[0057] This example verifies the inhibitory effect of (20S)-ginsenoside Rh1 on neuronal injury and apoptosis in mice with ischemic stroke, and the operation is carried out according to the following steps:

[0058] Forty C57 / B6L mice were randomly divided into 4 groups, with 10 mice in each group. Before administration, the weight of each mouse was weighed, and the administration dose was calculated according to the weight. Group A was the blank control group (administered an equal amount of normal saline); Group B was the model group (administered an equal amount of normal saline); Group C was the model group + (20S)-ginsenoside Rh1 1 mg / kg; Group D was the model group + (20S)-ginsenoside Rh1 5 mg / kg. In this example, the middle cerebral artery occlusion method (MCAO) was used to construct a mouse cerebral ischemia-reperfusion model. After occluding the middle cerebral artery of the mouse for 1 h, the suture was removed, the wound was sutured, and then the mouse was placed back in the cage and raised in a room temperature environment of 25 °C, and sufficient drinking water and soft food were provided.

[0059] 4.1 Immunofluorescence detection: The mice in each group were sacrificed after perfusion with 4% paraformaldehyde, and the brain was taken and fixed for 24 h, and washed 3 times with PBS. The brain tissue was dehydrated successively with 10%, 20%, and 30% sucrose gradients. After embedding with OCT, frozen sections were made with a thickness of 20 μm. The sections were washed with PBS for 3 min × 3 times. A 0.1% Triton X-100 solution (prepared with PBS) was added dropwise for permeabilization for 1 h, washed with PBS for 10 min × 3 times, and then 10% BSA (prepared with PBS) was added dropwise for blocking for 1 h. The primary antibody (1:500) was added dropwise and incubated overnight at 4 °C, washed with PBS for 10 min × 3 times, the secondary antibody (1:800) was added dropwise, incubated in the dark at room temperature for 60 min, and washed with PBS for 10 min × 3 times. Finally, DAPI nuclear staining was added dropwise and the slices were sealed, and observed under a fluorescence microscope.

[0060] 4.2 Western blot experiment: After removing the olfactory bulbs from the brain tissue, it was divided and weighed (μg), and 12 times the volume of RIPA buffer containing protease and phosphatase inhibitors (μL) was added according to the weight, placed in an EP tube containing grinding beads, and ground for 30 min. Centrifuged at 15000 rpm for 15 min at 4 °C, and the supernatant was collected. Western blotting was performed. The protein expression levels of Bcl2, Bax, and Cleaved-Caspase3 were detected and analyzed. Ginsenoside Rh1 alleviated the brain injury caused by MCAO to mice by inhibiting apoptosis as Figure 4 shown ( Figure 4 A in Figure 4 : Evaluation of the effect of different concentrations of ginsenoside Rh1 on the survival of neurons in the brain tissue after MCAO model treatment by NeuN immunofluorescence staining; Figure 4C in: Detection of apoptotic cells in brain tissue by TUNEL staining).

[0061] Example 5

[0062] This example verifies the potential action targets and regulatory pathways of (20S)-ginsenoside Rh1 on cerebral ischemia-reperfusion injury, and operates according to the following steps:

[0063] Use an online tool to obtain the potential target genes of (20S)-ginsenoside Rh1, and collect CIRI disease target genes from the CIRI-related database. The common target genes of (20S)-ginsenoside Rh1 and CIRI are obtained through intersection analysis, and an interaction network is constructed and pathway analysis is carried out by combining network tools to further screen key pathways and targets, and explore the potential targets and mechanisms of ginsenoside Rh1 in the treatment of CIRI based on network pharmacology as Figure 5 shown ( Figure 5 A in: The Venn diagram shows the common action targets between ginsenoside Rh1 and CIRI; Figure 5 B in: The compound-target interaction network of ginsenoside Rh1; Figure Five C and D in: Two main functional clusters extracted based on the protein-protein interaction network (PPI), representing the main functional gene groups in the potential targets; Figure 5 E and F in: KEGG pathway enrichment analysis is performed on the two main functional clusters of C and D respectively).

[0064] Example 6

[0065] This example verifies that the JAK2 / STAT3 pathway inhibitor reverses the neuroprotective effect of (20S)-ginsenoside Rh1 on oxygen-glucose deprivation (OGD)-induced neurons, and operates according to the following steps:

[0066] The cell culture method is the same as 1.1 in Example 1. PC12 cells are seeded in 12-well plates and cultured with complete medium for 20-24 h. Pretreat with 1 μM and 5 μM of (20S)-ginsenoside Rh1 for 2 h respectively, then perform oxygen-glucose deprivation for 6 h and reoxygenation culture for 24 h. Perform western blotting assay. Detect and analyze the protein expression levels of JAK2, P-JAK2, STAT3 and P-STAT3, and take β-actin as the reference, and analyze P-JAK2 and P-STAT3 with JAK2 and STAT3 as the controls respectively;

[0067] PC12 cells were pretreated with 1 μM and 5 μM of (20S)-ginsenoside Rh1 for 2 h, followed by glucose and oxygen deprivation for 6 h and reoxygenation for 24 h. Western blot assay was performed. The protein expression levels of Bcl-2, Bax, and cleaved-caspase-3 were analyzed. Ginsenoside Rh1 negatively regulated the Jak2 / STAT3 signaling pathway as Figure 6 shown ( Figure 6 A in Figure 6 : Effects of different concentrations of ginsenoside Rh1 on the expression of p-JAK2, JAK2, p-STAT3, and STAT3 after OGD / R treatment detected by Western blot, with β-actin as the internal reference; Figure 6 B in Figure 6 : Figure 6 Quantitative analysis of the Western blot results in A in Figure 6 ).

[0068] Example 7

[0069] This example verified the effect of the binding of (20S)-ginsenoside Rh1 to STAT3 on the nuclear translocation of neurons induced by glucose and oxygen deprivation, and the operation was as follows:

[0070] The cell culture method was the same as 1.1 in Example 1. PC12 cells were seeded in the well plates required for the experiment and cultured with complete medium for 20 - 24 h. They were pretreated with 1 μM and 5 μM of (20S)-ginsenoside Rh1 for 2 h, followed by glucose and oxygen deprivation for 6 h and reoxygenation for 24 h or 20 h.

[0071] 7.1 CETSA experiment: The medium was discarded, and the cells were washed 3 times with PBS (containing protease inhibitors). Subsequently, the cells were resuspended with PBS and equally divided into PCR tubes, with 3 replicates set for each group. They were heated at 37 °C, 45 °C, 55 °C, and 65 °C for 3 min respectively. NP40 buffer was added for resuspension, and they were frozen and thawed 3 times in liquid nitrogen, centrifuged at 20,000 g for 20 min at 4 °C, and the supernatant was taken for Western blotting assay for analysis and comparison.

[0072] 7.2 DARTS experiment: The medium was discarded, and after routine washing, lysis, and centrifugation, the protein concentration was measured and samples were prepared. The samples were divided into two groups: the experimental group was added with (20S)-ginsenoside Rh1 solution, and the control group was added with an equal volume of solvent, and they were incubated at 37 °C for 30 min. An equal amount of protease (1:1000) was added to both groups and incubated at 37 °C for 30 min. Subsequently, Western blotting experiment was performed to detect and compare the expression levels of STAT3 and P-STAT3 proteins in the two groups.

[0073] 7.3 Co-Immunoprecipitation (Co-IP) Assay: The treated brain tissues (using the same method as in 4.2 of Example 4) and the proteins of PC12 cells (using the same method as in 2.3 of Example 2) were added to RIPA buffer supplemented with protease and phosphatase inhibitors, collected in 1.5 mL EP tubes, and lysed with shaking on ice for 30 min. After centrifugation, half of the supernatant was reserved as the input group, and the remaining supernatant was incubated with the antibody overnight at 4°C. The next day, protein A / G magnetic beads were added and incubated at room temperature for 2 h. The beads were washed three times with lysis buffer. Western blotting was performed to detect and analyze the expression of STAT3 protein. Ginsenoside Rh1 promoted the nuclear translocation of STAT3 through KPNA3 as Figure 7 shown in Figure 7 A: Immunofluorescence staining was used to observe the nuclear translocation of STAT3 in brain tissues after treatment with ginsenoside Rh1 in the MCAO model; Figure 7 B: Western blot was used to detect the expression levels of p-JAK2, JAK2, p-STAT3, and STAT3 in the cytoplasm and nucleus of PC12 cells after OGD / R treatment, with β-actin as the internal reference; Figure 7 C: STAT3 was used as the Co-IP antibody, and immunoblotting (IB) was performed to detect KPNA3; Figure 7 D: KPNA3 was used as the Co-IP antibody, and IB was performed to detect STAT3).

[0074] Example 8

[0075] This example verified the effect of (20S)-ginsenoside Rh1 on the binding of JAK2 and STAT3, and the operations were as follows:

[0076] The Co-IP assay was performed using the same method as in 7.3 of Example 7. The expression levels of JAK2, P-JAK2, STAT3, and P-STAT3 proteins were detected and analyzed.

[0077] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Use of (20S)-ginsenoside Rh1 in the preparation of a medicament for preventing and / or improving ischemic stroke.

2. Use of (20S)-ginsenoside Rh1 in the preparation of a medicament for relieving motor disorders and decline in learning and memory ability in ischemic stroke.

3. Use of (20S)-ginsenoside Rh1 in the preparation of a medicament for enhancing the activity of nerve cells in cerebral ischemic stroke.

4. Use of (20S)-ginsenoside Rh1 in the preparation of a medicament for inhibiting the expression of proteins related to nerve cell apoptosis in ischemic stroke.

5. Use of (20S)-ginsenoside Rh1 in the preparation of a medicament for targeting the JAK2-STAT3 axis signaling pathway to relieve nerve cell damage.

6. Use of (20S)-ginsenoside Rh1 in the preparation of a medicament for reducing the infarct area in ischemic stroke.

7. A drug for preventing / ameliorating ischemic stroke, characterized in that: The active ingredient of the medicament comprises (20S)-ginsenoside Rh1.

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