Application of ferulic acid in preparation of medicine for treating subarachnoid hemorrhage

Abstract

The invention discloses application of ferulic acid in preparation of a medicine for treating subarachnoid hemorrhage. The method comprises the following steps: carrying out mouse experiments, constructing a subarachnoid hemorrhage mouse model, carrying out ferulic acid drug intervention, comparing with a control group, respectively carrying out nervous system scoring, SAH scoring and brain water content scoring, carrying out HE staining on mouse brain tissues, extracting mouse brain tissue RNA, and carrying out IL6, IL-1beta, TNF and IL-10 related index detection. A control group and a drug administration group are found to have significant differences. Mouse experiments find that ferulic acid can well improve a series of sequelae after subarachnoid hemorrhage, such as activity decline and hemiplegia, a reliable product is provided for subarachnoid hemorrhage, and the effect of improving SAH sequelae is achieved.

Description

Technical Field

[0001] The present invention relates to the use of ferulic acid in the preparation of a medicament for treating subarachnoid hemorrhage, and belongs to the field of pharmaceutical technology. Background Art

[0002] Subarachnoid hemorrhage (SAH) caused by rupture of intracranial aneurysms is a critical emergency in neurosurgery, characterized by high incidence, high disability rate, high fatality rate, and high recurrence rate. Incidence: Aneurysmal subarachnoid hemorrhage (aSAH) accounts for about 85% of all spontaneous subarachnoid hemorrhages. Worldwide, the overall annual incidence of aSAH is approximately 9.1 / 100,000, while in Beijing, the annual incidence of aSAH is 2 / 100,000, slightly lower than the world's overall level. Mortality rate: The average mortality rate of aSAH ranges between 27% and 44%. The results of a hospital-based prospective multi-center study showed that the cumulative mortality rates of Chinese aSAH patients at 28 days, 3 months, 6 months, and 12 months after onset were 16.9%, 21.2%, 23.6%, and 24.6% respectively.

[0003] Intracranial aneurysms are the main cause of subarachnoid hemorrhage, accounting for about 51% of all cases. Other etiologies include cerebral AVM (arteriovenous malformation), hypertension arteriosclerosis, etc. The inducing factors for rupture of intracranial aneurysms are complex and diverse, including emotional agitation, strenuous exercise, forced defecation, coughing, etc. Among them, emotional agitation is one of the more common inducing factors. The prognosis of aSAH is affected by many factors, including the inducing factor of onset, aneurysm size, Hunt-Hess grading, cerebral vasospasm, etc. The recurrence rate after rupture of intracranial aneurysms is relatively high, and is related to multiple factors such as the inducing factor of onset, vascular factors, and disease course.

[0004] The research on subarachnoid hemorrhage (aSAH) after intracranial aneurysms is of extremely important significance, not only because its high incidence, high disability rate, and high fatality rate pose a serious threat to human health, but also because this disease involves complex pathophysiological processes, posing high requirements for medical technology and treatment strategies.

[0005] At present, the clinical treatment of this disease mainly includes craniotomy and endovascular intervention. However, surgical treatment has great risks, and even if the surgery is successful, complications may remain, such as intracranial infection, hydrocephalus, cerebral vasospasm, nerve dysfunction, limb dysfunction, deep vein thrombosis in the lower extremities, etc. Although many current techniques are used to treat subarachnoid hemorrhage, the treatment effects of some sequelae are not satisfactory. In the past, many traditional Chinese medicine compounds have been verified to be able to treat subarachnoid hemorrhage and related sequelae, but the compounds have disadvantages such as complex decocting methods, unclear active ingredients, and expensive medicinal materials. We aim to find a medicament that can effectively improve the prognosis of subarachnoid hemorrhage. Summary of the Invention

[0006] In order to solve the current problems of high risk and sequelae in surgical treatment, through the research and screening of ferulic acid and related targets of subarachnoid hemorrhage, the present invention has found that ferulic acid can well improve subarachnoid hemorrhage in mice, providing a reliable product and achieving the effect of improving SAH.

[0007] The present invention is achieved through the following technical solutions:

[0008] The first object of the present invention is to provide the use of ferulic acid in the preparation of a drug for the treatment of subarachnoid hemorrhage.

[0009] In one embodiment of the present invention, the subarachnoid hemorrhage is subarachnoid hemorrhage after intracranial aneurysm.

[0010] In one embodiment of the present invention, ferulic acid can treat subarachnoid hemorrhage by reducing the expression of inflammatory indexes IL-6, Il-1β, Tnf-a and increasing the expression of Il-10.

[0011] In one embodiment of the present invention, ferulic acid is administered after subarachnoid hemorrhage.

[0012] In one embodiment of the present invention, the administration dose of ferulic acid is 50-150 mg / kg / d.

[0013] In one embodiment of the present invention, the dosage form of ferulic acid is a liquid dosage form or a solid dosage form.

[0014] In one embodiment of the present invention, the liquid dosage form is an oral solution.

[0015] In one embodiment of the present invention, the solid dosage form is tablets, capsules or granules.

[0016] In one embodiment of the present invention, the drug further comprises a pharmaceutically acceptable excipient.

[0017] In one embodiment of the present invention, the excipient includes one or more of a diluent, a filler, a binder, a wetting agent, an absorption promoter, a surfactant, a lubricant, and a stabilizer.

[0018] The beneficial effects of the present invention:

[0019] In this invention, a mouse model of subarachnoid hemorrhage was constructed through mouse experiments, and ferulic acid drug intervention was carried out. Compared with the control group, neurological scores, SAH scores, brain water content scores were respectively performed, and HE staining was performed on the mouse brain tissue. Mouse brain tissue RNA was extracted, and related indicators of IL6, IL-1β, TNF, and IL-10 were detected, and significant differences were found between the control group and the drug administration group. Through mouse experiments, we found that ferulic acid can well improve a series of sequelae after subarachnoid hemorrhage, such as decreased activity ability, hemiplegia, etc., providing a reliable product for subarachnoid hemorrhage and achieving the effect of improving SAH sequelae. Brief Description of the Drawings

[0020] 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.

[0021] Figure 1 Neurological scores of mice in different groups at each time point;

[0022] Figure 2 SAH scores of mice in different groups at each time point;

[0023] Figure 3 Brain water content of mice in different groups at each time point;

[0024] Figure 4 Brain tissue of mice in different groups at each time point;

[0025] Figure 5 HE staining diagrams of paraffin sections of brain tissue of mice in different groups at each time point;

[0026] Figure 6 Il-6 index data in brain tissue RNA of mice in different groups at each time point;

[0027] Figure 7 IL-1β index data in brain tissue RNA of mice in different groups at each time point;

[0028] Figure 8 Tnf index data in brain tissue RNA of mice in different groups at each time point;

[0029] Figure 9 Il-10 index data in brain tissue RNA of mice in different groups at each time point. Detailed Embodiments

[0030] The present invention will be further described below in conjunction with specific examples. These examples are only used to illustrate the present invention and not to limit the scope of the present invention. In addition, after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

[0031] Source of raw materials

[0032] Mice: Purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd., breed: C57BL / 6JNIFDC, male, age: 8 weeks, body weight: 20 - 22 g;

[0033] Ferulic acid was purchased from Shanghai Tongtian Biotech Co., Ltd.

[0034] The technical solution of the present invention will be described in detail below in conjunction with specific examples. In the following examples, unless otherwise specified, the reagents, materials and equipment used can be obtained from commercial sources, or prepared by conventional methods, or commonly used in this industry.

[0035] Example 1:

[0036] We constructed a mouse subarachnoid hemorrhage model by the method of injecting blood into the prechiasmatic cistern. The mice were divided into a Sham group: a small hole was drilled in the right front of the anterior fontanelle of the mouse, and no blood was injected into the subarachnoid space. After the sham operation, the mice were given 150 μl of gastric perfusion water daily. Model group mice: A small hole was drilled in the right front of the anterior fontanelle of the mouse, and 50 μl of the mouse's own blood was injected into the subarachnoid space. After the operation, the mice were given 150 μl of gastric perfusion water daily. Drug group mice: A small hole was drilled in the right front of the anterior fontanelle of the mouse, and 50 μl of the mouse's own blood was injected into the subarachnoid space. After the operation, the mice were given 150 μl of ferulic acid solution daily (prepared according to the dosing amount of 100 mg / kg / d).

[0037] At 24 h, 48 h, 72 h, and 8 d after subarachnoid hemorrhage, the nervous system scores of each group of mice were measured, and the SAH scores of the mouse brain tissues were taken at each time point, and the brain water content was detected.

[0038] (1) Nervous system score: The highest total score is 18 points, and each item is scored from 0 to 3 points. The higher the score, the lower the injury.

[0039] Test content: 1. Spontaneous activity (highest score 3 points) 2. Symmetry of forelimb movement (highest score 3 points) 3. Extension of forepaws outward (highest score 3 points) 4. Rock climbing (highest score 3 points) 5. Somatosensation of the body (highest score 3 points) 6. Response to whisker touch (highest score 3 points).

[0040] Subjects: Any kind of mice, without prior training, but the mice should be adapted to the test environment and the experimenter. Steel wire: Used for climbing test. Cotton swab: Used for physical proprioception and response to tactile test. Ethanol: Used for cleaning between tests. Test area: A clean table with enough space to place the animals and two cages. During the test, there are no other personnel in the room. Lighting: Well-lit.

[0041] Before the test: The mice were placed in the test room for at least 1 hour before the test to minimize the influence of stress on behavior during the test.

[0042] 1. Spontaneous activity (observing the activity in the test cage for 5 minutes) Normal (3 points): Approaches the four walls of the cage. Slightly affected (2 points): Hesitates but still explores the walls. Moderately affected (1 point): Moves very little but eventually approaches the walls. Severely affected (0 points): Does not move.

[0043] 2. Symmetry of forepaw movement (When the animal is lifted off the ground by the base of the tail, the extension and movement of the forepaws can be observed) Normal (3 points): The forepaws are symmetrical. Slight (2 points): The limbs on the damaged side do not extend as well as the normal side. Moderate (1 point): The limbs on the damaged side do not extend but are close to the body. Severe (0 points): The limbs on the damaged side are completely immobile.

[0044] 3. Forepaw extension (When the animal is lifted by the base of the tail and allowed to walk on the table, the forepaw extension and forepaw walking are observed. When the animal is dragged backward by the base of the tail, the use of the forepaws can also be observed). Normal (3 points): The forepaws are outstretched and the animal can walk in a straight line with the forepaws. Slight (2 points): The forepaws are outstretched and the animal walks with the forepaws but towards the damaged side. Moderate (1 point): The damaged forepaw does not extend well and the forepaw walking is severely biased towards the damaged side. Severe (0 points): The damaged forepaw does not extend outwards or move during walking.

[0045] 4. Climbing (When the animal is lifted by the tail and placed on the wire mesh on one side of the cage, the climbing is observed) Normal (3 points): Grasps the steel wire and easily climbs onto the home cage. Moderate (2 points): Climbs to the cage, but the damaged paw does not find the wire during climbing. Severe (1 point): The paw cannot grasp the steel wire and cannot climb to the cage.

[0046] 5. Physical proprioception (When the animal is separated while crawling on the floor, the reaction is observed). Normal (3 points): Startles after checking both sides or prodding both sides. Moderate (2 points): Checks the damaged side after checking the damaged side. Severe (1 point): No reaction after being damaged.

[0047] 6. Response to whisker touch (The response can be observed when the whiskers are brushed separately on both sides with a long and thin stick. The stick should approach from behind to ensure that the animal cannot see the stick during the test. In the first few brushes, its behavior should be scored because too many brushes will make the animal accustomed to the touched whisker) Normal (3 points): After touching the whisker with the stick, the head is brushed on one side, or the head retreats from the stick. Moderate (2 points): After brushing the whisker on the affected side, the head moves away from the stick more slowly compared to the unaffected side. Severe (1 point): No response when the brush touches the whisker on the damaged side.

[0048] The results are as Figure 1 shown. The neurological scores of the mice in the ferulic acid administration intervention group (Drug group) at each time point were significantly higher than those in the Model group and gradually approached those in the Sham group.

[0049] (2) SAH score: The basal ganglia can be divided into 6 parts, and each area is scored according to the degree of subarachnoid hemorrhage: 0, no subarachnoid hemorrhage; 1, a small amount of subarachnoid hemorrhage; 2, moderate hemorrhage with obvious arteries; 3, blood clots covering all arteries within the segment. The score is calculated as the sum of the scores of these six areas, and the grade ranges from 0 to 18. Mild: 0 - 7 points, Moderate: 8 - 12 points, Severe: 13 - 18 points.

[0050] The results are as Figure 2 shown. The SAH score of the Drug group was significantly lower than that of the Model group and was close to that of the Sham group.

[0051] (3) Brain water content: Immediately after the animal is sacrificed, the brain is taken. After weighing the wet weight of the brain tissue, it is immediately placed in a constant temperature electric oven. The oven temperature is generally set from 60 - 110 °C for 24.0 - 72.0 h. If the brain mass change does not exceed 0.2 mg after 72.0 h, it is considered to have reached a constant mass, and then the dry weight is weighed. The formula for calculating brain water content is (wet weight - dry weight) / wet weight × 100. Whether to separate and weigh the left and right brains separately depends on the experimental requirements.

[0052] The results are as Figure 3 shown. The brain water content of the Drug group was significantly lower than that of the mice in the Model group and was close to that of the Sham group.

[0053] The brain tissues of the mice at each time point are as Figure 4 shown. It can be clearly found that at the 24 h time point, there was the most blood in the subarachnoid space of the mice in the Model group. At each time point, the amount of blood in the subarachnoid space of the mice in the Drug (ferulic acid treatment group) group was close to that of the Sham group and was significantly less than that of the Model group.

[0054] Furthermore, the brain tissues of the mice at each time point were separately sectioned into paraffin sections and stained with HE. The results are asFigure 5 As shown, from the HE staining results, it can be seen that the number of dead cells in the HE sections of the mice in the Drug group was significantly less than that in the Model group and was close to that in the Sham group.

[0055] RNA was extracted from the mouse brain tissue at each point, and PCR detection of inflammation-related indicators such as Il-6, IL-1β, Tnf, and Il-10 was carried out. The results are as Figures 6 to 9 shown. From the PCR results, it can be seen that after applying ferulic acid in mice with subarachnoid hemorrhage, the expression of related inflammatory indicators IL-6, Il-1β, and Tnf-a can be significantly reduced, and the expression of Il-10 can be increased to anti-inflammation.

[0056] Inflammatory response in the nervous system is a major factor in brain injury after subarachnoid hemorrhage. Ferulic acid can effectively reduce the expression of pro-inflammatory factors and increase the expression of anti-inflammatory factors to alleviate local inflammation, thereby reducing brain tissue damage and improving nerve function.

[0057] The embodiments provided above are not intended to limit the scope covered by the present invention, nor are the described steps intended to limit their execution order. Obvious improvements made by those skilled in the art to the present invention in combination with the existing well-known common sense also fall within the protection scope defined by the claims of the present invention.

Claims

1. Use of ferulic acid in the preparation of a medicament for treating subarachnoid hemorrhage.

2. The application according to claim 1, wherein The subarachnoid hemorrhage is subarachnoid hemorrhage after intracranial aneurysm.

3. The application according to claim 1, wherein The ferulic acid can treat subarachnoid hemorrhage by reducing the expression of inflammatory indexes IL-6, Il-1β, Tnf-a and increasing the expression of Il-10.

4. The application according to claim 1, characterized in that The ferulic acid is administered after subarachnoid hemorrhage.

5. The application according to claim 4, characterized in that The dosage of the ferulic acid is 50-150 mg / kg / d.

6. The application according to claim 1, characterized in that, The dosage form of the ferulic acid is a liquid dosage form or a solid dosage form.

7. The application according to claim 6, wherein The liquid dosage form is an oral solution.

8. The application according to claim 6, wherein The solid dosage form is tablets, capsules or granules.

9. The application according to claim 1, wherein The medicament further comprises a pharmaceutically acceptable excipient.

10. The application according to claim 9, wherein The excipient includes one or more of a diluent, a filler, a binder, a wetting agent, an absorption enhancer, a surfactant, a lubricant, a stabilizer.

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