CN112245433A

CN112245433A - Application of ectoine substances in preparation of medicine for preventing and treating cerebral arterial thrombosis

Info

Publication number: CN112245433A
Application number: CN202011351358.0A
Authority: CN
Inventors: 钱林艺; 田昕; 董杰杰; 郭学平
Original assignee: Bloomage Biotech Co Ltd
Current assignee: Bloomage Biotech Co Ltd
Priority date: 2020-11-27
Filing date: 2020-11-27
Publication date: 2021-01-22
Anticipated expiration: 2040-11-27
Also published as: CN112245433B

Abstract

The invention discloses an application of ectoine substances in preparation of a medicine for preventing and treating cerebral arterial thrombosis, belonging to the technical field of medical application. The invention discovers for the first time that the ectoin substance can reduce the inflammatory reaction of microglia induced by LPS, has protective effect on both neurons and cerebral infarction of rats induced by ischemia-reperfusion, and can be used for developing new medicines for preventing or treating ischemic stroke.

Description

Application of ectoine substances in preparation of medicine for preventing and treating cerebral arterial thrombosis

Technical Field

The invention relates to a new application of ectoine substances, in particular to an application of ectoine substances in preparing a medicine for preventing and treating cerebral arterial thrombosis, belonging to the technical field of medical application.

Background

Stroke (stroke) is a sudden disorder of cerebral blood circulation, which results in oxygen and nutrient deficiency in brain tissue due to decreased supply of cerebral blood flow, and ultimately neuronal cell death and cerebral infarction. Ischemic stroke accounts for more than 85 percent of stroke, the proportion of ischemic stroke in China increases year by year, and the ischemic stroke has a trend of being younger, becomes the first cause of disability and death in China, and causes serious economic and social burden. In addition to high disability rate and high mortality rate, stroke has the characteristics of high morbidity, high recurrence rate and many complications, so that the prevention and treatment of ischemic stroke are particularly important. At present, the treatment method of stroke is limited, most of clinical medicines are urokinase, and the nerve function of most of stroke treated by the current medicines cannot be completely recovered. Therefore, the development of new therapeutic means for cerebral arterial thrombosis becomes a huge and urgent clinical demand.

Ectoin (Ectoin, chemical name: 2-methyl-1, 4, 5, 6, -tetrahydropyrimidine-4-carboxylic acid) is an amino acid derivative present in microorganisms, and belongs to cyclic amino acids. Since ectoin is derived from highly halophilic bacteria (Halomonas Elongata), ectoin is also called "halotolerant bacteria extract". Under the extreme conditions of high salt, high temperature and high ultraviolet radiation, the ectoin can prevent halophilic bacteria from being damaged. The chemical structural formula is as follows:

the existing research shows that the ectoin has good repairing and protecting effects on the skin, is one of bioengineering preparations adopted by high-grade cosmetics, and has the following biological functions: osmotic pressure gene inducer, anti-stress protection function, molecular chaperone function, radiation protection and moisture preservation function. Meanwhile, ectoin is effective for the treatment of inflammatory bowel disease, which is a disease characterized by intestinal dysbiosis.

At present, the application of ectoin in preventing and treating ischemic stroke is not found.

Disclosure of Invention

The invention discovers for the first time that the ectoin substance can reduce the inflammatory reaction of microglia induced by LPS and has protection effect on neurons and cerebral infarction of rats induced by ischemia-reperfusion. Experiments prove that the ectoine substances have excellent effect in the field of preventing and treating ischemic stroke and have research and development prospects.

Aiming at the research results, the invention provides the application of ectoin substances in preparing the medicine for preventing and treating cerebral arterial thrombosis. The new application of ectoin provides a new thought and direction for developing the medicine for preventing or treating cerebral arterial thrombosis.

Further, the ectoin substance comprises ectoin and derivatives thereof acceptable in the medical field. The derivatives of ectoin include hydroxy ectoin, ectoin sodium salt, ectoin potassium salt, etc.

Furthermore, the content of the ectoine substances in the medicine for preventing and treating the cerebral arterial thrombosis is effective treatment amount.

Further, the ischemic stroke is cerebral ischemia reperfusion injury.

Furthermore, the medicine for preventing and treating the ischemic stroke refers to various types and various dosage forms of medicines for preventing and treating the ischemic stroke, and the ectoine substances serving as the active ingredients of the medicine can be used singly or together with other active medicine ingredients. The dosage form of the medicine can be various feasible dosage forms, such as tablets, capsules, granules, oral liquid, dripping pills, water injection, freeze-dried powder injection, sterile powder injection and the like.

Further, the capsule is a sustained-release capsule or a controlled-release capsule.

In another aspect, the present invention provides a pharmaceutical composition for preventing and treating ischemic stroke, wherein the active ingredient of the pharmaceutical composition comprises ectoin.

Further, the dosage form of the medicament is in accordance with the previous definition.

Further, the amount of ectoin in the unit dose of the drug is a therapeutically effective amount. The unit dose drug refers to the unit dose or the smallest unit used of a pharmaceutical package. Such as single tablet, single capsule, single-package granule or dripping pill, single bottle of oral liquid, single needle, etc.

Further, the medicine also comprises medically usable auxiliary materials. The auxiliary materials are necessary auxiliary materials for preparing various different dosage forms, and comprise one or more of a filling agent, an adhesive, a lubricating agent, a disintegrating agent, a cosolvent, a surfactant and the like.

The invention discovers for the first time that the ectoin substance can reduce the inflammatory reaction of microglia induced by LPS, has protective effect on both neurons and cerebral infarction of rats induced by ischemia-reperfusion, and can be used for developing a new medicament for preventing or treating ischemic stroke.

Detailed Description

In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments. It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

The test materials used in the examples of the present invention are all conventional in the art and commercially available. The experimental procedures, for which no detailed conditions are indicated, were carried out according to the usual experimental procedures or according to the instructions recommended by the supplier.

Example 1: effect of ectoin on LPS-induced microglial inflammatory response

The effect of the ectoine on the inflammatory response of LPS-induced microglia was examined with reference to the method of the effect of the ethanol extract of Hippocampus kelloggi on the induction of BV2 microglia inflammatory response by lipopolysaccharide (proceedings of Guangdong university of oceans, 2019, 39 (1): 90-96). The method comprises the following specific steps:

1. the test method comprises the following steps:

1.1 cell culture:

the microglia BV2 was inoculated into DMEM high-glucose medium (containing 1% by volume double antibody) containing 10% by volume fetal bovine serum and incubated at 37 ℃ with 5% CO₂And culturing in an incubator with saturated humidity, carrying out subculture every 2-3d, and inoculating the BV2 cells in a 24-well plate when the BV2 cells are attached to the wall and grow well, and carrying out subsequent experiments.

1.2Griess method for detecting NO content released by each group of cells:

taking BV2 cells in logarithmic growth phase at 1X 10⁵The density of each well was inoculated into 24-well plates (5 wells per group), and a control group (medium without addition of ectoin and LPS) and a model group (medium containing only ectoin and LPS) were providedThe culture medium with 100ng/ml LPS), the drug I group (the culture medium with 100ng/ml LPS after 24h pretreatment by 5ng/ml ectoin), and the drug II group (the culture medium with 100ng/ml LPS after 24h pretreatment by 5ng/ml hydroxyectoin), and the supernatant in each well is collected after 24h addition of LPS, and the content of NO in each group is detected by using a Griess kit according to the specification.

1.3ELISA assay for TNF- α, IL-1 β and IL-6 levels released by each group of cells:

taking BV2 cells in logarithmic growth phase at 1X 10⁵Each well was inoculated at a density of 5 wells in a 24-well plate (each group was provided with 5 duplicate wells), and a control group (medium without addition of ectoin and LPS), a model group (medium containing only 100ng/ml LPS), a drug I group (medium +100ng/ml LPS after 24h pretreatment with 5ng/ml ectoin), and a drug II group (medium +100ng/ml LPS after 24h pretreatment with 5ng/ml hydroxyectoin) were set, and supernatants from the wells were collected 24h after addition of LPS, and the contents of TNF-. alpha., IL-1. beta., and IL-6 in each group were measured using an ELISA kit according to the instructions thereof.

2. And (3) test results:

2.1 Effect of ectoine on LPS-induced NO release in BV2 cells:

the results of the examination of the effect of ectoin on LPS-induced NO release in BV2 cells are shown in table 1.

As can be seen from table 1, BV2 cells released NO significantly increased 24h after LPS stimulation (model group) compared to the control group; after the ectoin substances are adopted for pretreatment, the release of NO by BV2 cells induced by LPS can be obviously reduced.

2.2 Effect of ectoin on LPS-induced concentrations of TNF- α, IL-1 β and IL-6, pro-inflammatory factors in BV2 cells:

the results of the observation of the effect of ectoin on the concentration of LPS-induced pro-inflammatory factors TNF-alpha, IL-1 beta and IL-6 in BV2 cells are shown in Table 2.

As can be seen from Table 2, compared with the control group, the concentrations of the pro-inflammatory factors TNF-alpha, IL-1 beta and IL-6 of BV2 cells after 24h of LPS stimulation (model group) are all increased remarkably; after the ectoine substances are adopted for pretreatment, the release of BV2 cells TNF-alpha, IL-1 beta and IL-6 induced by LPS can be obviously reduced.

Example 2: protective effect of ectoin on neurons:

the protective effect of ectoin on neurons was examined with reference to the method of patent CN109908154A, which is as follows:

1. the test method comprises the following steps:

1.1 establishment of rat Primary cortical neuron cell oxygen sugar deprivation/reoxygenation (OGD/R) model:

adding sodium dithionite (Na) into low-sugar DMEM medium₂S₂O₄) Dissolving the dry powder sufficiently to obtain a final concentration of 20mM, and adding NaHCO₃Adjusting the pH value to 7.2 to obtain the anoxic liquid. The primary cortical neuron cells are cultured in a 24-well plate cell culture plate for 7-10 days, and cells with good growth state are selected for experiment. The experiment comprises a blank control group, a model control group and an ectoine administration group.

The blank control group is continuously cultured by using the culture medium, the culture medium is removed after 24 hours, and equal volume of maintenance culture medium (Neurobasal Media 49ml + B27-Supplement 1ml) is added; continuously culturing the model control group with the culture medium, removing the culture medium after 24h, and adding an equal volume of anoxic liquid; the culture medium in the 24-well plate was gently aspirated before administration, washed with PBS buffer 2 times, cultured with a medium containing 5ng/ml ectoin, and 24 hours later, the medium containing the drug was removed and replaced with an anoxic solution. After incubation in the incubator for 2h, the supernatant was completely removed, washed twice with PBS buffer and then maintenance medium was added. Then put at 37 ℃ and 5% CO₂After the incubator is continuously cultured for 2h, the modeling is completed, and the neuron cells can be used for experiments.

1.2 Effect of ectoin on survival rate of neurons after hypoxia and reoxygenation injury:

after molding, the cell culture plate is placed under an inverted microscope to observe the growth of cortical neuron cells and the cell damage condition. After removing the medium from the 24-well plate, 50. mu.L of MTT solution (5mg/ml) was added to each well after adding 450. mu.L of the maintenance medium, and the plate was incubated at 37 ℃ for 4 hours. After discarding the MTT-containing medium, 200. mu.L of DMSO solution was added to each well, and the mixture was shaken on a shaker for 10 min. 150 μ L of each well was removed and transferred to a 96-well plate, and the OD value of each well was measured at 490nm of a microplate reader to reflect the survival of the cells.

2. And (3) test results:

the protective effect of ectoin on neurons is shown in table 3.

As can be seen from table 3, the survival rate of the neuron cells in the model control group was significantly decreased after the hypoxia/reoxygenation injury of the neuron cells, compared to the blank control group; the survival rate of the neuron cells after the model is made can be obviously improved by adopting the ectoin for treatment.

Example 3: protective effect of ectoin on cerebral infarction of ischemia-reperfusion-induced rat

The protective effect of ectoin on the cerebral infarction of rats induced by ischemia-reperfusion is examined by referring to the method of patent CN109908154A, which is as follows:

1. the test method comprises the following steps:

1.1, constructing a cerebral infarction model:

and (3) blocking the middle cerebral artery of the SD rat by using a wire plug to carry out ischemia reperfusion on the SD rat so as to prepare a cerebral infarction model.

1.2 test grouping and handling:

SD rats successfully modeled were randomly divided into 3 groups: model group, positive drug group and ectoine group; wherein, the positive drug group is injected with much gold in the tail vein at a dose of 15mg/kg, the ectoine group is injected with ectoine in the tail vein at a dose of 15mg/kg, and the model group is administered with the same amount of physiological saline for 3 days continuously.

1.3 investigation indexes:

(1) neurological scoring

Animals were graded for behavioral deficits according to the method of Bederson, with the following criteria:

0 minute: no neurological symptoms were observed;

1 minute: when the tail is lifted and suspended, the operation of the animal shows that the contralateral forelimb shows that the wrist and elbow are bent, the shoulder is rotated inwards, the elbow is expanded outwards and is tightly attached to the chest wall;

and 2, dividing: the animal is placed on a smooth plane, and when the side shoulder of the operation is pushed to move towards the opposite side, the resistance is reduced;

and 3, dividing: when the animal walks freely, the animal rotates towards the opposite side of the operation or rotates around;

4, dividing; flaccid paralysis, no spontaneous movement of limbs.

(2) Measurement of cerebral infarction volume

The rats were sacrificed by decapitation, the whole brain was removed and weighed. Making four coronary cutting knives at the positions 2mm before and after the visual intersection, cutting into five slices, quickly placing the brain slices into 5ml of phosphoric acid buffer solution containing 1% TTC, incubating in a dark place for 30min, turning over every 7-8 min in the incubating process, taking out the brain slices after incubating for 30min, taking a picture by using a digital camera, separating a pale area (infarct area) and a non-pale area (normal area) by using ophthalmic forceps, and calculating the infarct percentage as follows:

percent (%) infarct is pale area weight/(pale area weight + non-pale area weight) × 100%.

2. And (3) test results:

the results of the examination of the protective effect of ectoin on cerebral infarction of rats induced by ischemia-reperfusion are shown in table 4.

As can be seen from table 4, compared with the model group, the ectoine group had a significant tendency to improve neurological deficits, and could significantly reduce the percentage of cerebral infarction in the cerebral infarction model animals; and compared with positive drugs, the protective effect of the rat cerebral infarction induced by the ischemic reperfusion of the ectoin is greatly improved.

The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. The application of ectoine substances in preparing medicines for preventing and treating cerebral arterial thrombosis is provided.

2. Use according to claim 1, characterized in that: the ectoin substance comprises ectoin and derivatives thereof which are acceptable in the field of medicine.

3. Use according to claim 1, characterized in that: the derivatives of ectoin comprise hydroxyl ectoin, ectoin sodium salt and ectoin potassium salt.

4. Use according to any one of claims 1 to 3, characterized in that: the content of the ectoine in the medicine is effective amount for treatment.

5. Use according to any one of claims 1 to 3, characterized in that: the cerebral ischemic stroke is cerebral ischemia reperfusion injury.

6. Use according to any one of claims 1 to 3, characterized in that: the medicament for preventing and treating the cerebral arterial thrombosis is in the dosage form of tablets, capsules, granules, oral liquid, dropping pills, water injection, freeze-dried powder injection or sterile powder injection.

7. Use according to claim 6, characterized in that: the capsule is a slow release capsule or a controlled release capsule.

8. A pharmaceutical composition for preventing and treating cerebral ischemic stroke is characterized in that: the active ingredient comprises ectoin.

9. The medicament of claim 8, wherein: the content of the ectoin in the unit dose medicament is therapeutically effective amount.

10. The medicament of claim 8, wherein: the medicine also comprises medically usable auxiliary materials; preferably, the auxiliary materials comprise a filler, a binder, a lubricant, a disintegrant, a cosolvent or a surfactant.