CN110585210B - Application of sparganium stoloniferum A in preparation of anti-ischemic cerebral apoplexy medicines - Google Patents

Abstract

The invention discloses application of scirpusin A in preparation of a medicine for resisting cerebral ischemic stroke. Specifically, the study of the invention finds that the sparganiin A has a remarkable inhibiting effect on calcium ion overload injury and hypoxia and glucose-deficient injury, can improve neurological deficit symptoms, and remarkably reduces the cerebral infarction area and cerebral index of rats with cerebral ischemia reperfusion injury; remarkably reduces the levels of TAX2, ET-1, TNF alpha, IL-beta, MDA and LDH activity in serum, has the functions of improving blood brain barrier and protecting neuron cells, is used for treating ischemic stroke, and has remarkable curative effect and small dosage. In addition, the sparganium stoloniferum element A is an effective component of the traditional Chinese medicine sparganium stoloniferum, has wide sources, is safe and low-toxicity, provides a new selection and powerful basis for the development of natural medicines for resisting cerebral ischemic stroke, and has wide development space and good application prospect.

Description

Application of sparganium stoloniferum A in preparation of anti-ischemic cerebral apoplexy medicines

Technical Field

The invention belongs to the technical field of medicines. More particularly, relates to application of scirpusin A in preparation of medicines for resisting cerebral ischemic stroke.

Background

Ischemic Stroke (Stroke) is a generic term for brain tissue necrosis due to stenosis or occlusion of blood supply arteries (carotid and vertebral) of the brain and insufficient blood supply to the brain. Cerebral ischemia includes four types, which are classified into Transient Ischemic Attack (TIA), reversible neurological dysfunction (RIND), progressive Stroke (SIE), and Complete Stroke (CS). TIA did not have cerebral infarction, while RIND, SIE and CS had different degrees of cerebral infarction. At present, the method for treating ischemic stroke mainly comprises the steps of taking chemical drugs or performing surgical treatment, the disease has poor prognosis, certain disability rate and mortality rate, and can be repeatedly attacked; in addition, chemical drugs have strong toxic and side effects on patients, and patients are easy to have dependence and drug resistance on the chemical drugs. Therefore, the development of a medicament with good curative effect, small toxic and side effect and obvious ischemic stroke resistance has important clinical application value.

Research shows that patent CN 108926566A discloses the application of scirpusin A in resisting blood stasis, and proves that scirpusin A has the effect of resisting blood stasis. Patent CN 103520160A discloses application of peptide compounds in rhizoma sparganii, and proves that the peptide compounds in rhizoma sparganii have anticoagulant and antithrombotic effects. However, blood stasis syndrome, coagulopathy and thrombosis are different from ischemic stroke in nature. The blood stasis syndrome refers to one syndrome type in the differentiation of traditional Chinese medicine, namely blood stasis refers to unsmooth blood circulation and blood stasis, and the blood stasis syndrome can be seen in various diseases; generally speaking, blood stasis refers to the condition of blood stasis when blood leaving the meridians cannot be timely dissipated and stagnates in a certain place, or the blood flow is obstructed and stagnates in the meridians or organs to form stagnation. Coagulopathy is due to the reduction or destruction of blood coagulation factors, and the main symptoms are generally bleeding, skin ecchymosis and the like. Thrombi are small pieces formed by the surface of blood flow within the vascular system at the sites of denudation or repair, and in the variable fluid-dependent type, they consist of insoluble fibrin, deposited platelets, accumulated white blood cells and entrapped red blood cells, with the main symptoms of dyspnea, atherosclerosis, varicose veins, traumatic thrombosis of the internal carotid artery, chest pain, etc. It can be seen that the pathogenesis and symptoms of ischemic stroke and blood stasis syndrome, coagulopathy and thrombosis have great essential differences.

At present, no research report about whether the scirpusin component in the rhizoma sparganii has the effect of resisting the ischemic stroke exists, and the research on whether the scirpusin component in the rhizoma sparganii has the effect of resisting the ischemic stroke can make up for the blank of the prior art and has important significance for guiding clinical medication and development of new drugs.

Disclosure of Invention

The invention aims to solve the technical problem of making up the blank of the prior art, provides the application of the sparganium A in the preparation of the anti-ischemic stroke medicine and provides a powerful basis for the development of the anti-ischemic stroke natural medicine.

The invention aims to provide application of scirpusin A in preparation of a medicine for resisting cerebral ischemic stroke.

The invention also aims to provide a medicament for resisting the cerebral arterial thrombosis.

The above purpose of the invention is realized by the following technical scheme:

the invention proves that the sparganium A has a certain inhibiting effect on calcium ion overload injury and oxygen-deficient and sugar-deficient injury for the first time, can improve neurological deficit symptoms, and obviously reduces the cerebral infarction area and cerebral index of rats with cerebral ischemia reperfusion injury; and can obviously reduce the level of thromboxane A2(TAX2), endothelin 1(ET-1), tumor necrosis factor alpha (TNF alpha), interleukin beta (IL-beta), Malondialdehyde (MDA) and Lactate Dehydrogenase (LDH) in serum, can obviously reduce the permeability of blood brain barrier, has obvious effect of resisting ischemic stroke, can be used for preparing medicines for resisting ischemic stroke, and provides a powerful basis for the development of natural medicines for resisting ischemic stroke.

Therefore, the following applications should be within the scope of the present invention:

application of scirpusin A in preparing medicine for treating cerebral ischemic stroke is provided.

The application refers to the application of the scirpusin A in preparing the medicine capable of inhibiting the calcium ion overload damage or the anoxic and glucose-deficient damage of the cells of the cerebral ischemic stroke patient.

The application refers to the application of the scirpusin A in preparing the medicine capable of improving the nerve function defect symptom of the cerebral ischemic stroke patient or reducing the cerebral tissue infarction area.

The application refers to the application of scirpusin A in preparing a medicine capable of reducing the cerebral index of a patient with ischemic stroke.

The application refers to the application of the scirpusin A in preparing the medicines capable of reducing the level of thromboxane A2(TAX2), endothelin 1(ET-1), tumor necrosis factor alpha (TNF alpha), interleukin beta (IL-beta), Malondialdehyde (MDA) or the activity of Lactate Dehydrogenase (LDH) in the serum of a patient suffering from ischemic stroke.

The application refers to the application of scirpusin A in preparing medicines capable of reducing permeability of blood brain barrier.

The structural formula of the sparganium A (Cyc- (Tyr-Leu)) is shown as the formula (I):

in addition, the invention also provides a medicine for resisting cerebral arterial thrombosis, which comprises effective dose of sparganium stoloniferum A.

The medicine also comprises one or more pharmaceutically acceptable carriers, and is prepared into different dosage forms.

The invention has the following beneficial effects:

the invention provides application of scirpusin A in preparation of a medicine for resisting cerebral ischemic stroke. The invention proves that the scirpusin A has obvious inhibiting effect on calcium ion overload injury and oxygen-deficient and sugar-deficient injury for the first time, can improve neurological deficit symptoms and obviously reduce cerebral infarction area and cerebral index of rats with cerebral ischemia reperfusion injury; and can obviously reduce the levels of TAX2, ET-1, TNF alpha, IL-beta, MDA and LDH activity in serum, simultaneously has the functions of improving blood brain barrier and protecting neuron cells, is used for treating ischemic stroke, has obvious curative effect and small dosage.

In addition, the sparganium stoloniferum element A is an effective component of the traditional Chinese medicine sparganium stoloniferum, and has wide source, safety and low toxicity. Therefore, the sparganium A provides a new choice and powerful basis for developing natural medicines for resisting cerebral arterial thrombosis, and has wide development space and good application prospect.

Drawings

FIG. 1 is a graph showing the effect of KCl at various concentrations on the survival rate of PC12 cells.

FIG. 2 shows different concentrations of Na₂S₂O₄Results are presented as the effect on survival of PC12 cells.

FIG. 3 is a graph showing the results of infarct in brain tissue of rats in each group.

Detailed Description

The present invention is further illustrated by the following specific examples, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

Example 1 Effect of Sparganin A on inhibiting calcium ion overload injury and anoxia-glucose-deficiency injury of PC12 cells

First, experimental material

(1) Test cell

PC12 cell line (rat adrenal chromaffin tumor cells) was purchased from Shanghai cell resource center.

(2) Experimental reagent

(3) Laboratory apparatus

Second, the effect of sparganiin A on a PC12 cell calcium overload injury model

1. Experiment on influence of KCl at different concentrations on survival rate of PC12 cells

(1) Experimental methods

The method is established according to a model of PC12 cell calcium ion overload damage caused by KCl and is improved, and the specific operation steps are as follows:

taking out PC12 cells from the incubator, adding pancreatin for digestion, stopping digestion after cell shedding, centrifuging, adding serum-containing culture medium to prepare suspension, adding 100 μ L of the suspension into each well, inoculating into a 96-well plate, and adding 100 μ L of PBS around the wells to prevent edge effect. After the culture box is placed for 24 hours, KCl with different concentrations is prepared, wherein the KCl is 250mmol/L, 225mmol/L, 200mmol/L, 175mmol/L, 150mmol/L, 125mmol/L, 100mmol/L and 75mmol/L, each concentration corresponds to one group, a normal group is arranged, each group is provided with 5 multiple holes, each hole is added with 100 mu L of KCl solution, and the normal group is added with DMEM without serum. And sucking out liquid after 4h of damage, washing the liquid for 2-3 times by using PBS (phosphate buffer solution), adding culture media containing 10% serum into the liquid, and putting the liquid into an incubator to continue culturing for 24 h. And adding MTT, sucking out the liquid, adding dimethyl sulfoxide (DMSO) after 4h, placing in an incubator for 10min, measuring absorbance at 490nm, and calculating the survival rate.

(2) Results of the experiment

The results of the effect of different concentrations of KCl on the survival rate of PC12 cells are shown in fig. 1, and it can be seen that the concentration of KCl can affect the survival rate of PC12 cells, and the survival rate of PC12 cells decreases with the increase of the concentration of KCl; the survival rate of PC12 cells was 49.5% when the KCl concentration was 125mmol/L and the injury was 4 h.

2. Experiment for influence of sparganium A with different concentrations on survival rate of cells after calcium overload injury of PC12 cells

(1) Experimental methods

PC12 cells were prepared into a cell suspension, which was then plated in 96-well plates at 100. mu.L/well and cultured for 24 hours. After changing the solution, 5 groups of sparganiin A, positive group (20. mu. mmol/L edaravone), model group (serum-containing DMEM), and normal group (serum-containing DMEM) were prepared at different concentrations (0.4mmol/L, 0.33mmol/L, 0.267mmol/L, 0.2mmol/L, and 0.133mmol/L), and the resulting mixture was cultured for 24 hours in a total of 8 groups, each group being prepared with 5 duplicate wells. After changing the solution, 100. mu.L of serum-containing DMEM was added to the normal group, and 125mmol/L of KCl solution was added to the remaining groups. And (4) damaging for 4h, washing with PBS for 2-3 times, adding sparganin A with the same concentration as before into each group, adding edaravone into the positive group, adding DMEM containing serum into the model group and the normal group, and culturing for 24 h. MTT was added and the absorbance was measured at 490nm to calculate the survival rate.

(2) Results of the experiment

The survival rate of the cells after the PC12 cells are subjected to calcium overload injury by the sparganiin A with different concentrations is shown in Table 1, and it can be seen that the survival rate of the cells after the PC12 cells are subjected to calcium overload injury is remarkably improved along with the increase of the concentration of the sparganiin A and is higher than that of the model group; when the concentration of the scirpusin A is 0.4mmol/L, 0.33mmol/L and 0.267mmol/L, the difference is very significant compared with a model group (P is less than 0.01); when the concentration of the sparganium A is 0.2mmol/L, a significant difference (P is less than 0.05) exists compared with a model group; wherein, when the concentration of the sparganiin A is 0.4mmol/L, the survival rate of the cells after the PC12 cells are damaged by calcium overload is the highest, and is 68.2%.

TABLE 1 results of cell survival rate after calcium overload injury of PC12 cells by sparganiin A with different concentrations

Note: p < 0.05, as compared to model group; p < 0.01, as compared to model groups.

3. Experiment for influence of sparganiin A with different concentrations on LDH activity and MDA content of cell supernatant after PC12 cell calcium overload injury

(1) Experimental methods

PC12 cells were seeded in a 96-well plate and cultured for 24 hours. Changing the solution, setting 5 different concentrations (0.4mmol/L, 0.33mmol/L, 0.267mmol/L, 0.2mmol/L and 0.133mmol/L) of the sparganiin A group, the positive group (20 mu mmol/L edaravone), the model group and the normal group (DMEM containing serum is added in each case), and repeating 5 multiple wells, wherein each well is 100 mu L and culturing is carried out for 24 h. And (4) changing the liquid, adding DMEM containing serum into the normal group, adding KCl solution of 125mmol/L into the other groups, and damaging for 4 hours. And (3) changing the liquid, adding the scirpusin A with the same concentration as the scirpusin A in the scirpusin A group, adding edaravone in the positive group, adding DMEM containing serum in the model group and the normal group, and culturing for 24 h. Sucking out the liquid, centrifuging at 3000rpm/min for 10min, taking the supernatant, operating according to the instructions of Nanjing to build MDA and LDH kits of bioengineering research institute, measuring absorbance, calculating Malondialdehyde (MDA) content and Lactate Dehydrogenase (LDH) activity, and repeating for 2-3 times.

(2) Results of the experiment

When cells are subjected to ischemic injury, LDH and MDA are released. According to the content of MDA and the activity of LDH, the function of scirpusin A in resisting cerebral ischemia can be indirectly reflected. The results of the influence of sparganiin A with different concentrations on LDH activity and MDA content of cell supernatant after the PC12 cell calcium overload injury are shown in Table 2, and it can be seen that the LDH activity and the MDA content are gradually reduced with the increase of the sparganiin A concentration and are both lower than those of the model group; when the concentration of the scirpusin A is 0.4mmol/L, 0.33mmol/L, 0.267mmol/L and 0.2mmol/L, compared with a model group, the difference is very significant (P is less than 0.01); when the concentration of the sparganiin A is 0.133mmol/L, a significant difference (P is less than 0.05) exists compared with a model group; wherein, when the concentration of the sparganin A is 0.4mmol/L, the LDH activity and the MDA content of the cell supernatant after the PC12 cell calcium overload injury are the lowest.

TABLE 2 results of the effect of sparganiin A at different concentrations on LDH activity and MDA content of cell supernatant after calcium overload injury of PC12 cells

Note: p < 0.05, as compared to model group; p < 0.01, as compared to model groups.

Action of scirpusin A on PC12 cell oxygen sugar deprivation damage model

1. Different concentrations of Na₂S₂O₄Effect on survival of PC12 cells

(1) Experimental methods

PC12 cells were made into suspension, 100 μ L was added to each well, seeded in 96-well plates, and PBS was added around one circle. After being placed in an incubator for 24 hours, Na with different concentrations is prepared₂S₂O₄11.25mmol/L, 10mmol/L, 8.75mmol/L, 7.5mmol/L, 6.25mmol/L, 5mmol/L, 3.75mmol/L, 2.5mmol/L, 1.25mmol/L, each concentration corresponds to one group, and a normal group, 5 multiple wells are arranged, each well is 100 mu L, the normal group is 100 mu L DMEM, after 4h, liquid is sucked out, each group is added with culture medium containing 10% serum, and the culture is continued in an incubator. After 24h, the absorbance at 490nm was measured, and the cell viability was measured on the basis of the absorbance.

(2) Results of the experiment

Different concentrations of Na₂S₂O₄Effect on the survival of PC12 cellsAs shown in FIG. 2, it can be seen that Na is present₂S₂O₄Can affect the survival rate of PC12 cells, along with Na₂S₂O₄The survival rate of the PC12 gradually decreases with the increase of the concentration; na (Na)₂S₂O₄The survival rate of PC12 cells was 48.7% at a concentration of 5mmol/L and 4h after injury.

2. Effect of sparganiin A at different concentrations on cell survival rate of PC12 cells after oxygen deprivation injury experiment (1) experiment method

PC12 cells were seeded in a 96-well plate and cultured for 24 h. After changing the solution, 5 different concentrations (0.4mmol/L, 0.33mmol/L, 0.267mmol/L, 0.2mmol/L and 0.133mmol/L) of sparganiin group A, positive group (20. mu. mmol/L edaravone), model group (adding serum-containing DMEM), normal group (adding serum-containing DMEM), and 8 groups in total were set, and 5 duplicate wells were set and cultured for 24 hours. Changing the solution, adding 5mmol/L Na₂S₂O₄Normal group was supplemented with serum-free DMEM and damaged for 4 h. And (3) changing the solution, adding sparganiin A with the same concentration as before into each group, adding edaravone into the positive group, adding DMEM containing serum into the normal group and the model group, and culturing for 24 h. MTT was added and the absorbance was measured at 490nm to calculate the survival rate.

(2) Results of the experiment

The results of the influence of sparganium A in different concentrations on the survival rate of cells after oxygen deprivation injury of PC12 cells are shown in Table 3, and it can be seen that the survival rate of PC12 cells is gradually improved with the increase of the concentration of sparganium A, and the survival rate is higher than that of the model group; when the concentration of the scirpusin A is 0.4mmol/L and 0.33mmol/L, compared with a model group, the difference is very significant (P is less than 0.01); when the concentration of the scirpusin A is 0.267mmol/L and 0.2mmol/L, the significant difference (P is less than 0.05) exists compared with the model group; wherein, when the concentration of the sparganiin A is 0.4mmol/L, the cell survival rate is highest after the PC12 cells are damaged by oxygen deprivation, and is 71.8%.

TABLE 3 Effect of different concentrations of Sparganin A on cell viability after oxygen deprivation injury of PC12 cells

Note: p < 0.05, as compared to model group; p < 0.01, as compared to model groups.

3. Experiment for influence of sparganium A with different concentrations on LDH activity and MDA content of cell supernatant after oxygen sugar deprivation injury of PC12 cells

(1) Experimental methods

PC12 cells were seeded in a 96-well plate and cultured for 24 h. Changing the solution, setting 5 sparganiin A groups with different concentrations (0.4mmol/L, 0.33mmol/L, 0.267mmol/L, 0.2mmol/L and 0.133mmol/L), positive groups (20 mu mmol/L edaravone), model groups (adding a culture medium containing serum) and normal groups (adding a culture medium containing serum), repeating for 5 times of multiple wells, and culturing for 24 h. Changing the solution, adding 5mmol/L Na₂S₂O₄The normal group was incubated for 4 hours with serum-containing medium. And (3) changing the solution, adding the scirpusin A with the same concentration as the previous solution, adding edaravone into the positive group, adding DMEM containing serum into the normal group and the model group, and culturing for 24 h. Sucking out the liquid, centrifuging at 3000rpm/min for 10min, taking the supernatant, operating according to the instructions of Nanjing to build MDA and LDH kits of bioengineering research institute, measuring absorbance, calculating LDH activity and MDA content, and repeating for 2-3 times.

(2) Results of the experiment

The results of the influence of sparganium A with different concentrations on the LDH activity and the MDA content of the cell supernatant after the PC12 cells are deprived of oxygen sugar and damaged are shown in Table 4, and it can be seen that the LDH activity and the MDA content are gradually reduced with the increase of the sparganium A concentration and are lower than those of the model group; in terms of LDH activity, when the concentration of the scirpusin A is 0.4mmol/L, 0.33mmol/L and 0.267mmol/L, compared with the model group, there is a very significant difference (P < 0.01); when the concentration of the scirpusin A is 0.2mmol/L and 0.133mmol/L, a significant difference exists compared with a model group (P < 0.05). In terms of MDA content, when the concentration of the scirpusin A is 0.4mmol/L, 0.33mmol/L, 0.267mmol/L and 0.2mmol/L, compared with a model group, the difference is very significant (P is less than 0.01); when the concentration of the scirpusin A is 0.133mmol/L, a significant difference exists compared with a model group (P is less than 0.05). Wherein, when the concentration of the scirpusin A is 0.4mmol/L, the LDH activity and MDA content of cell supernatant after the PC12 cells are damaged by oxygen sugar deprivation are the lowest.

TABLE 4 Effect of different concentrations of Sparganin A on LDH activity and MDA content of cell supernatant after oxygen deprivation injury of PC12 cells

Note: p < 0.05, as compared to model group; p < 0.01, as compared to model groups.

Example 2 drug effect experiment of Sparganin A on cerebral ischemia reperfusion injury of rats

First, experimental material

(1) Laboratory animal

SPF SD rat (male, weight 250 ~ 300g), experimental rat by Guangdong province medical experiment animal center provide, license number: SCXK (Yue) 2013-. Is raised by experimental animal center of Guangdong university of pharmacy.

(2) Laboratory apparatus

(3) Experimental reagent

Second, drug effect experiment method and experiment result of scirpusin A on cerebral ischemia reperfusion injury of rat

1. Grouping and administration of experimental animals

The male SD rats are randomly divided into two batches, one batch is used for researching the protection effect of the scirpusin A on brain tissues in the cerebral arterial thrombosis, and the other batch is used for researching the protection effect of the scirpusin A on the blood brain barrier of the cerebral arterial thrombosis.

Each batch of male SD rats was randomly divided into high, medium and low dose groups of scirpusin a, sham operated group, positive group (nimodipine), model group, total 6 groups. Dissolving the scirpusin A and the nimodipine in 0.5 percent of CMC-Na (weighing 0.5g of CMC-Na to be dissolved in water, and fixing the volume to 100mL), respectively administering 0.0606g/kg, 0.0303g/kg and 0.01515g/kg to the high, medium and low dose components of the scirpusin A, administering 20mg/kg to the positive group, and administering 0.5 percent of CMC-Na to the pseudo-operation group and the model group. The MCAO model is constructed by operation after 4 days of continuous gavage administration (1 mL of administration is given per 100g of body weight) and 2 hours of administration on the fourth day (12 hours before model building are performed and water is not forbidden), and the stomach is gavaged for 1 time after 8 hours after model building.

2. Establishment of rat focal cerebral ischemia reperfusion Model (MCAO)

After 2h of the last intragastric administration, a Middle Cerebral Artery Occlusion (MCAO) reperfusion model of rats is constructed by adopting a wire-embolus method, and a sham operation group does not plug wires and the other operations are the same. Injecting 10% chloral hydrate into abdominal cavity of a rat for anesthesia (3mL/kg), after complete anesthesia, supinely fixing the rat on an operating table, clipping hair, smearing iodophor, performing median longitudinal incision (25 cm) in the neck, separating the right common carotid artery and the external carotid artery of the rat, ligating the proximal ends of the two arteries, clamping the internal carotid artery by a micro artery clamp, cutting a small opening at the far-end (about 5cm away from the bifurcation of the common carotid artery) of CCA ligation by an ophthalmic scissors, inserting a embolus, loosening an ICA artery clamp, rapidly adjusting the strength and direction of the embolus, and inserting the embolus from the CCA into the ICA until the initial area of the anterior cerebral artery, slightly applying force to insert the embolus after meeting resistance, and then stopping. After the wound is sutured, 1mL of physiological saline is injected into the abdominal cavity of the rat, the embolism line is pulled out to the marked black spot after 2 hours of ischemia, and then the perfusion is carried out for 24 hours. (refer to Longa improvement method) after the rat is anesthetized and revived, the nerve injury symptom scoring is carried out by using the Longa method, rat brain tissues are taken, TTC staining is carried out on partial brain tissues, partial brain tissues are preserved (used for Western blot), and rat TAX in serum is measured by using a kit₂ET-1, TNF alpha, IL-beta and MDA content,Activity of LDH.

3. Neurological functional score and determination of infarct size

(1) Experimental methods

Neurological impairment was scored on 4 points: no obvious symptoms of neurological deficit: 0 minute; failure to fully extend the left forelimb: 1 minute; and (3) rotating and walking to the left side: 2 min; when walking, the utility model is inclined leftwards: 3 min; inability to walk, occurrence of disturbance of consciousness: and 4, dividing. And (5) observing the condition after the rat revives, grading and recording, wherein the molding is successful after more than 1 point. Dead rats and rats with unsuccessful molding were removed.

After 24h of perfusion, weighing, carrying out intraperitoneal injection of 10% chloral hydrate, carrying out abdominal aorta blood sampling, opening the skull of a rat to take out the brain (removing the cerebellum and the olfactory bulb part), cleaning the brain tissue by PBS, then sucking the water of the brain tissue by using filter paper, weighing the wet weight of the brain tissue by using an electronic balance, calculating the brain index of the rat, then putting the brain tissue into a refrigerator with the temperature of-20 ℃ for freezing for 10min, taking out and placing the brain tissue on a brain model, slicing, and cutting the brain into 5 slices along the crossing part of the optic nerve and the 2mm part of the optic nerve. The sections were stained in 0.5% TTC (protected from light), forward stained and counter stained for 5min each, then taken out and placed in 4% paraformaldehyde fixing solution, photographed 24h later, the infarct area of brain tissue was measured with ImageJ, and the percentage of infarct area was calculated and the data was recorded.

(2) Results of the experiment

The infarction results of the brain tissues of the rats in each group are shown in fig. 3, and it can be seen that no infarction focus appears in the sham operation group, while the infarction focus appears in the model group, and the infarction conditions of the high, medium and low dose groups of scirpusin A are obviously reduced compared with the model group. Infarct size is an important indicator of response to brain tissue damage. The results of neurological function score and infarct size of the brain tissues of the rats in each group are shown in table 5, and it can be seen that the infarct size and neurological score are gradually increased with the increase of the concentration of sparganin A, but are lower than those of the model group; compared with the model group, the infarct size and the neurological score of the middle and low dose groups (respectively administered with 0.0303g/kg and 0.01515g/kg) of sparganiin A are very significantly different (P is less than 0.01); compared with the model group, the infarct size and the neurological score of the high-dose group (0.0606 g/kg of administration) of the sparganium A are significantly different (P is less than 0.05); the bursin A is shown to have the function of inhibiting the cerebral ischemia injury of rats.

TABLE 5 neurological function scores and infarct size results in the brain tissue of various groups of rats

Note: p < 0.05, as compared to model group; p < 0.01, as compared to model groups.

4. Determination of MDA content and LDH activity in serum of rats of each group

(1) Experimental methods

After the abdominal aorta of the rat was bled, the blood was allowed to stand for a while and centrifuged (3000rpm/min, 4 ℃, 10 min). After centrifugation, the supernatant was collected and placed in a-80 ℃ refrigerator to measure the MDA content and the LDH activity (according to the kit instruction of Nanjing institute of bioengineering).

(2) Results of the experiment

The activity of LDH and the content of MDA can indirectly respond to the apoptosis and necrosis of neuron cells in brain tissues to a certain extent. The results of the MDA content and the LDH activity in the serum of each group of rats are shown in Table 6, and it can be seen that the MDA content and the LDH activity are gradually improved with the increase of the concentration of the sparganiin A, but are lower than those of the model group; compared with the model group, the LDH activity and MDA content of the high, medium and low dose groups (respectively administered with 0.0606g/kg, 0.0303g/kg and 0.01515g/kg) of the scirpusin A are very different significantly (P is less than 0.01).

The brain index can reflect the damage condition of brain tissue to a certain extent, and the brain tissue is damaged to cause edema. The results of the brain indexes of the rats in each group are shown in table 7, and it can be seen that the brain indexes of the rats are improved with the increase of the concentration of the scirpusin A, but are all lower than those of the rats in the model group; compared with the model group, the brain indexes of the high, medium and low dose groups (respectively administered with 0.0606g/kg, 0.0303g/kg and 0.01515g/kg) of the sparganin A are very different significantly (P is less than 0.01).

TABLE 6 results of MDA content and LDH activity in serum of rats of each group

Note: p < 0.01, as compared to model groups.

TABLE 7 results of brain index of each group of rats

Note: p < 0.01, as compared to model groups.

5. Determination of content of TAX2, ET-1, TNF alpha and IL-beta in serum of rats of each group

(1) Experimental methods

Serum was removed from a freezer at-80 ℃ and the contents of thromboxane A2(TAX2), endothelin 1(ET-1), tumor necrosis factor alpha (TNF α), interleukin beta (IL- β) were determined (according to the kit instructions of Nanjing institute of bioengineering).

(2) Results of the experiment

The brain tissue damage often produces inflammation, the content of TNF alpha and IL-beta is measured, and whether the scirpusin A can reduce inflammatory factors and whether related coagulation factors can be reduced or not can be observed. The results of the contents of TAX2, ET-1, TNF alpha and IL-beta in the serum of rats in each group are shown in Table 8, and it can be seen that the contents of TAX2, ET-1, TNF alpha and IL-beta in the serum of rats are all increased with the increase of the concentration of sparganiin A, but are all lower than those of the model group; compared with the model group, the serum contents of TAX2, ET-1, TNF alpha and IL-beta in the middle and low dose groups (respectively administered with 0.0303g/kg and 0.01515g/kg) of the sparganiin A are very different (P is less than 0.01).

TABLE 8 results of the serum levels of TAX2, ET-1, TNF α and IL- β in rats of each group

Note: p < 0.05, as compared to model group; p < 0.01, as compared to model groups.

6. Investigation of blood brain barrier permeability in groups of rats

(1) Experimental methods

After constructing MCAO model, the male SD rat revives and then carries out neurological scoring, the line bolt is pulled out after cerebral ischemia is 2h, the rat which is not successful in modeling and dies is removed, after 22 h of reperfusion, 2% Evans Blue (EB) is injected into tail vein of the rat, the Evans Blue is diluted to 2% by normal saline, eyes and skin of the rat turn Blue after a plurality of seconds, the rat is killed after 2h, and brain tissue is taken out. After the brain tissue of the rat is taken out, the brain tissue is washed in PBS for 3 times, the brain tissue is put into a centrifuge tube and is cut into pieces, 2mL of 50% trichloroacetic acid is added, the rat brain tissue is placed in a water bath at the temperature of 60 ℃ for 24 hours, and then the rat brain tissue is centrifuged at the temperature of 4 ℃ and at the rpm/min of 3000 for 10 minutes, and the supernatant is taken out.

2% EB as 1: 10000. 1: 5000. 1: 1000. 1: 500 dilution, absorbance at 632nm and standard curve generation. And measuring the absorbance of the supernatant of each group at 632nm, and calculating the content of evans blue of each group according to the standard curve.

(2) Results of the experiment

When cerebral ischemia can destroy the blood brain barrier, and the permeability of the blood brain barrier can be changed, so that the destruction degree and the permeability of the blood brain barrier can be evaluated according to the content of Evans blue. The results of the neurological scores and Evans blue content of the rats in each group are shown in Table 9, and it can be seen that the neurological scores and Evans blue content of the rats are improved with the increase of the concentration of the sparganin A, but are lower than those of the model group; the neurological score and Evans blue content of the sparganiin A medium and low dose groups (0.0303 g/kg and 0.01515g/kg respectively) were significantly different from those of the model group (P < 0.01).

The evans blue content of the sparganium A low-dose group is 8.98 mu g/mL, and the significant difference exists compared with that of the model group.

TABLE 9 neurological scoring and Evans blue content results for various groups of rats

Note: p < 0.05, as compared to model group; p < 0.01, as compared to model groups.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims (6)

1. The application of a compound with the following structural formula in preparing a medicine for resisting cerebral ischemic stroke is disclosed in the formula (I):

2. the use according to claim 1, wherein the use is for the preparation of a medicament for inhibiting calcium overload damage or hypoxic-glucose-deprivation damage in cells of a patient with ischemic stroke, comprising administering to the patient a compound according to claim 1.

3. The use according to claim 1, wherein the use is for the preparation of a medicament for improving the symptoms of neurological deficit or reducing the infarct size in brain tissue of a patient suffering from ischemic stroke, comprising administering to the patient a compound according to claim 1.

4. The use according to claim 1, wherein the use is for the manufacture of a medicament for reducing the cerebral index in patients with ischemic stroke, comprising a compound according to claim 1.

5. The use of claim 1, wherein the use is for the manufacture of a medicament for reducing the level of TAX2, ET-1, tnfa, IL- β, MDA or LDH activity in the serum of a patient with ischemic stroke, comprising administering to the patient a compound of claim 1.

6. The use according to claim 1, wherein the use is for the manufacture of a medicament for decreasing the permeability of the blood-brain barrier, comprising a compound according to claim 1.

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