CN117959281A

CN117959281A - Application of formononetin in preparing medicine for preventing and/or treating central nervous system inflammatory demyelinating diseases

Info

Publication number: CN117959281A
Application number: CN202410169122.7A
Authority: CN
Inventors: 王青; 刘健; 梁亚杰; 杨智超; 陈莹
Original assignee: Shanxi University of Chinese Mediciine
Current assignee: Shanxi University of Chinese Mediciine
Priority date: 2024-02-06
Filing date: 2024-02-06
Publication date: 2024-05-03

Abstract

The invention discloses application of formononetin in preparing a medicament for preventing and/or treating central nervous system inflammatory demyelinating diseases, and belongs to the technical field of biological medicines. The invention provides evidence that formononetin can significantly improve clinical symptoms of a bicyclohexanoyl dihydrazone (CPZ) demyelinating mouse, inhibit demyelination of the CPZ mouse, confirm that the action mechanism is related to abnormal activation of astrocytes and reduction of inflammatory reaction and oxidative stress in brain of the CPZ mouse, and show the capability of treating and preventing inflammatory demyelinating diseases of central nerves, wherein the mechanism is mainly formononetin for inhibiting inflammatory factors and oxidative stress. Provides a new application of the formononetin, widens the application range of the formononetin, and provides more possibilities for the application of the formononetin.

Description

Application of formononetin in preparing medicine for preventing and/or treating central nervous system inflammatory demyelinating diseases

Technical Field

The invention relates to the technical field of biological medicines, in particular to application of formononetin in preparing a medicine for preventing and/or treating inflammatory demyelinating diseases of the central nervous system.

Background

Central nervous inflammatory demyelinating diseases are a class of diseases characterized primarily by severe neuroinflammatory and extensive, primary demyelination. Among them, recurrent-remitting multiple sclerosis (multiple sclerosis, MS) and neuromyelitis optica (neuromyelitis optica spectrum disorders, NMOSD) are the most common demyelinating diseases, the course of which is usually represented by recurrent-remitting alternation, multiple attacks causing irreversible damage to neurological function, one of the main causes of non-traumatic disability in young and middle-aged people. Clinically, the immune injury which inhibits demyelination is a main treatment means, but only can relieve clinical symptoms, and does not prevent the progress of the disease.

Worsening neuroinflammation and oxidative stress are typical manifestations in the pathogenesis of MS, and are also one of the important contributors to increased production of pro-inflammatory cytokines and oxidative stress, leading to axonal injury, exacerbating demyelination, and are therefore effective therapeutic strategies for the treatment of demyelinating encephalopathy. Astrocytes (Astrocytes, AS) are the most abundant glial cells of the central nervous system (Central nervous system, CNS) that cover the entire CNS in a orderly and orderly manner in a continuous and substantially non-overlapping manner, to maintain homeostasis of the brain microenvironment by protecting and promoting remyelination, maintaining blood brain barrier integrity, regulating synaptic function and energy metabolism. In pathological conditions, a large number of reactive AS proliferate, exhibit different transcriptional states, respond to focal sites by changing their own functions and phenotypes, and have beneficial or detrimental effects. Such AS NMOSD, MS and animal models thereof-experimental allergic encephalomyelitis (Experimental autoimmune encephalomyelitis, EAE) and bicyclohexanoyl dihydrazone (cuprazone, CPZ) induced demyelination models, numerous reactive AS can be present which can damage neurons and myelin cells by releasing various inhibitory factors such AS keratan sulfate proteoglycans, oligodendrocyte myelin proteins and myelin-associated proteins, and inflammatory factors such AS tumor necrosis factor-alpha (Tumor necrosis factor-alpha, TNF-alpha), interleukin-1 alpha (Interleukin-1 alpha, IL-1 alpha) and complement 1q (Complement q, C1 q). Whereas targeting AS significantly alleviates demyelination, dalahmah et al found that Galectin-3 (gal-3) reduces myelin cell damage by inhibiting AS's inflammatory response. Thus, with the intensive research on the function of reactive As and its mechanism of action, it has become an effective target cell for protecting and promoting remyelination.

Formononetin (formononetin, FMN) is also known as formononetin, english name: formononetin has chemical formula of C ₁₆H₁₂O₄, and is a bioactive isoflavone which is contained in common Chinese medicinal materials such as radix astragali, glycyrrhrizae radix, radix Puerariae, etc. The current pharmacological research finds that the traditional Chinese medicine composition has the functions of resisting oxidative stress, inflammation, apoptosis and the like. A great number of experiments prove that the FMN has certain treatment effects on the nervous system diseases such as Alzheimer disease, cerebral ischemia reperfusion, diabetic neuropathy, peripheral nervous system diseases, anxiety states and the like, but no report on the aspect of inflammatory demyelinating diseases of the central nervous system of formononetin has been found so far.

Disclosure of Invention

The invention aims to provide application of formononetin in preparing medicines for preventing and/or treating central nervous system inflammatory demyelinating diseases, so as to solve the problems in the prior art.

In order to achieve the above object, the present invention provides the following solutions:

The invention provides application of formononetin in preparing a medicament for preventing and/or treating central nervous inflammatory demyelinating diseases.

Preferably, the central nervous inflammatory demyelinating disease is multiple sclerosis, neuromyelitis optica or acute disseminated encephalomyelitis.

The invention also provides a pharmaceutical composition for treating central nervous inflammatory demyelinating diseases, comprising formononetin;

the central nervous inflammatory demyelinating disease is multiple sclerosis, neuromyelitis optica or acute disseminated encephalomyelitis.

The invention also provides a pharmaceutical formulation for treating central nervous inflammatory demyelinating diseases, comprising formononetin or the pharmaceutical composition and a pharmaceutically acceptable carrier.

Preferably, the pharmaceutically acceptable carrier is suitable for use in tablets, capsules, powders, pills, granules, oral liquids, injections or emulsions.

The invention also provides application of formononetin in preparing a medicament for inhibiting demyelination.

The invention also provides application of formononetin in preparing a medicament for inhibiting activation of astrocytes at myelin sheath sites.

The invention also provides application of formononetin in preparing a medicament for inhibiting inflammatory factors and protecting myelin sheath.

The invention also provides application of formononetin in preparing a medicament for inhibiting oxidative stress and protecting myelin sheath.

Based on the technical scheme, the invention has the following technical effects:

The invention provides evidence that formononetin can significantly improve clinical symptoms of CPZ demyelinating mice, inhibit demyelination of CPZ mice, confirm that the action mechanism is related to abnormal activation of astrocytes and reduce inflammatory reaction and oxidative stress in brains of CPZ mice, and shows the capability of treating and preventing central nerve inflammatory demyelinating diseases, wherein the mechanism is mainly that formononetin inhibits inflammatory factors and oxidative stress. Provides a new application of the formononetin, widens the application range of the formononetin, and provides more possibilities for the application of the formononetin.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 shows the effect of formononetin on CPZ mice moving in the plus maze closed arm and open field, wherein A is the effect of formononetin on the time percentage of CPZ mice moving in the plus maze closed arm and the number of times of entering the open arm in the experimental example of the application, and B is the effect of formononetin on the total distance of CPZ mice moving in the open field and the number of times of entering the central area in the experimental example of the application;

FIG. 2 shows the effect of formononetin on the brain corpus callosum region of CPZ mice, wherein A is the effect of formononetin on the demyelination of the brain corpus callosum region of CPZ mice detected by the black gold staining method in the experimental example of the application, and B is the effect of formononetin on the expression of MBP protein in the callus region of CPZ mice in the experimental example of the application;

FIG. 3 shows the effect of immunofluorescence staining on the expression of GFAP protein in the callus area of CPZ mice.

FIG. 4 shows the effect of formononetin on the expression of IL-1β, TNF- α, IL-6, IL-10, LPO and GSH in the brain of CPZ mice, wherein A is the effect of formononetin on the expression of IL-1β, TNF- α, IL-6 and IL-10 in the brain of CPZ mice in the experimental examples of the application, and B is the effect of formononetin on the expression of LPO and GSH in the brain of CPZ mice in the experimental examples of the application.

Detailed Description

Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless otherwise defined, 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 invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the application described herein without departing from the scope or spirit of the application. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present application. The specification and examples of the present application are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.

The technical scheme of the invention is conventional in the field, and the reagents or raw materials are purchased from commercial sources or are disclosed.

The embodiment of the invention provides application of formononetin in preparing a medicament for preventing and/or treating central nervous inflammatory demyelinating diseases.

In some specific embodiments, the central inflammatory acute demyelinating disease is a bicyclohexanoyl dihydrazone-mediated central inflammatory acute demyelinating disease.

The demyelination refers to a central nervous inflammatory demyelinating disease related to autoimmune reaction abnormality, heredity, environment and the like, which is multiple sclerosis, neuromyelitis optica or acute disseminated encephalomyelitis.

The formononetin comprises monomers extracted from natural plants and synthesized. The natural plants comprise common plants or Chinese medicinal materials such as fig, coptis chinensis, phellodendron bark, red clover, astragalus root, liquorice, kudzuvine root and the like.

The embodiment of the invention provides a pharmaceutical composition for treating central nervous inflammatory demyelinating diseases, which comprises formononetin.

The embodiment of the invention provides a pharmaceutical preparation for treating central nervous inflammatory demyelinating diseases, which comprises formononetin or the pharmaceutical composition and a pharmaceutically acceptable carrier.

The preparation contains 0.1 to 99.9 weight percent of formononetin as an active ingredient; more preferably, the formononetin contains 30 to 99 percent of active ingredient by weight; most preferably, the formononetin as an active ingredient is contained in an amount of 90-99% by weight.

The formononetin and the pharmaceutical use thereof according to the application can comprise a "therapeutically effective amount" or a "prophylactically effective amount" of formononetin according to the application. "therapeutically effective amount" means an amount effective to achieve the desired therapeutic effect at the necessary dosage and time. The therapeutically effective amount of formononetin can vary depending on factors such as the condition, age, sex and weight of the individual, and the ability of the formononetin to elicit a desired response in the individual. A therapeutically effective amount also refers to an amount of the formononetin that has a beneficial therapeutic effect that exceeds any toxic or detrimental effect thereof. "prophylactically effective amount" means an amount effective to achieve the desired prophylactic effect at the necessary dosage and time. Because the prophylactic dose is administered to a subject prior to or early in the disease, the prophylactically effective amount is typically less than the therapeutically effective amount. It should be noted that the dosage value will vary depending on the type and severity of the condition to be alleviated, that is, the dosage or amount of formononetin as described herein when used in a patient, will generally be determined based on the age and weight of the patient or user and the physical condition or condition of the patient's symptoms. In addition, it will be appreciated that for any particular subject, the particular dosage regimen should be adjusted over time according to the individual needs and the professional judgment of the person administering or supervising the administration of the compositions, and that the dosage ranges set forth herein are exemplary only and are not limiting as to the scope or practice of the claimed compositions.

The pharmaceutically acceptable carrier is suitable for tablets, capsules, powder, pills, granules, oral liquid, injection or emulsion. The pharmaceutically acceptable carrier does not destroy the pharmaceutical activity of formononetin according to the application.

In some embodiments, the formononetin can be used alone or in combination with other components, which can be biologically active and therapeutically active chemicals that are not limited to the mechanism of treatment of formononetin, can be co-used with formononetin to treat central inflammatory demyelinating diseases, can have other functional effects, such as to alleviate side effects of formononetin during treatment, and can also serve as carriers, emulsifying, dispersing, etc. for example, only as an adjunct to the drug.

When the other component is used as a carrier, the other component is required to be a pharmaceutically acceptable carrier such as a physiologically acceptable solvent, a dispersion medium, a envelope, an antibacterial agent, an antifungal agent, an isotonic agent, a wetting agent, an emulsifying agent, a preservative, a buffer, or the like.

From a specific classification, the pharmaceutically acceptable carrier refers to a conventional pharmaceutical carrier in the pharmaceutical field, and includes one or more excipients such as starch or water; disintegrants such as microcrystalline cellulose and the like; a lubricant such as one or more of talc, calcium stearate, magnesium stearate, polyethylene glycol, or the like; a binder such as one or more of pregelatinized starch, dextrin, cellulose derivative, alginate, gelatin, or polyvinylpyrrolidone, etc.; osmotic pressure regulators such as one or more of glucose, sucrose, sorbitol, mannitol, etc.; fillers such as one or more of starch or lactose, etc.; diluents such as water and the like; absorption promoters such as quaternary ammonium compounds and the like; a lubricant such as one or more of glycerin or magnesium stearate, etc.; disintegrants such as one or more of agar, calcium carbonate or sodium bicarbonate, etc.; surfactants such as cetyl alcohol and the like; an adsorption support such as one or more of kaolin or saponite, etc.; in addition, other adjuvants such as one or more of flavoring agent or sweetener can be added into the composition.

The formononetin pharmaceutical preparation can be formulated into various dosage forms by conventional methods known in the art, such as by mixing the active ingredient with one or more carriers and then formulating the desired dosage form. For example, formononetin can be further prepared into granules, capsules, tablets, pills and other dosage forms by adding proper auxiliary materials, wherein the auxiliary materials can be one or more selected from starch, dextrin, lactose, silicon dioxide, calcium hydrophosphate, cyclodextrin, microcrystalline cellulose, sodium carboxymethyl cellulose, magnesium stearate, talcum powder and the like.

The dosage form includes one or more of tablets, capsules, granules, suspensions, emulsions, solutions, syrups or injections, etc., and one or more administration routes of oral administration or injection (including one or more of intravenous injection, intravenous drip, intramuscular injection or subcutaneous injection, etc.) are adopted.

The embodiment of the invention provides application of formononetin in preparing a medicament for inhibiting demyelination.

In some embodiments, the demyelination is chronically demyelinating mediated by cyclohexanone dihydrazone.

The formononetin improves the inflammatory microenvironment of the central nervous system by inhibiting inflammatory factors and oxidative stress, and inhibits demyelination. The inventors have found that formononetin significantly inhibits the inflammatory response of the central nervous system by examining the improvement of the central nervous inflammatory microenvironment in demyelinating model mice, the mechanism of which may be related to the inhibition of reactive astrocyte proliferation by formononetin.

The inhibited proinflammatory factors express IL-1 beta, TNF-alpha and IL-6, and the promoted proinflammatory factors express IL-10; the reduced oxidative stress product is LPO and the increased antioxidant substance is GSH.

The embodiment of the invention provides application of formononetin in preparing a medicament for inhibiting activation of astrocytes at a myelin sheath part.

The embodiment of the invention provides application of formononetin in preparing a medicament for inhibiting inflammatory factors and protecting myelin sheath.

The embodiment of the invention provides application of formononetin in preparing a medicament for inhibiting oxidative stress and protecting myelin sheath.

It should be noted that, the technical scheme of the invention is formed based on network pharmacology:

CPZ is a copper ion chelator, whose induced demyelination model is a classical animal research model simulating MS demyelination and regeneration, with obvious MS lesions processes and characteristics, and is widely used to study classical models of central demyelination and remyelination, which have neurotoxicity that can lead to the occurrence of pathological changes of MS, such as oligodendrocyte apoptosis, demyelination, inflammatory response, oxidative stress, etc., by disrupting copper homeostasis in the central nervous system. In addition, in the CPZ mouse model, cognition and behavior abnormality, such as memory decline, mania, anxiety and the like, may occur in the later stage, and the application detects the influence of FMN on CPZ mouse behaviours, and discovers that the medicament can obviously improve the cognition function, mania, anxiety and the like. Myelination is a typical manifestation of MS onset, MBP is a marker protein that constitutes myelin, and the corpus callosum in the brain of mice is the main site of demyelination in CPZ model mice. According to the application, two staining methods of TrueGold and immunofluorescence staining are used for respectively detecting myelination loss of the callus region of the acute CPZ demyelinating mice, compared with a normal group, the CPZ+NS group callus region shows remarkable myelination loss, and the loss of MBP expression is accompanied, so that the death of myelin cells is shown, and FMN can obviously improve the pathological conditions.

Therefore, the inventor proposes the following technical scheme: application of formononetin in preparing medicine for treating central nervous inflammatory demyelinating diseases is provided.

The main reagent in the embodiment of the invention:

Formononetin (F828304) was purchased from Shanghai Milin Biochemical technologies Co., ltd, CPZ (370-81-0) was purchased from Sigma Co;

Myelin basic protein (Myelin basic protein, MBP, abcam, ab 40390);

glial fibrillary acidic protein (glial fibrillary acidic protein, GFAP, oasis Biofarm, OB-PGP 055);

Alexa Fluor 488-labeled goat anti-mouse IgG (H+L) and Alexa Fluor 594-labeled goat anti-rabbit IgG (H+L) were purchased from Invitrogen under the designations A-11001 and A-11012, respectively;

TNF- α, IL-1β, IL-6, IL-10 kits were purchased from Invitrogen under the designations 88-7324-88, 88-7013A-88, 88-7064-22, 88-7105-22, respectively;

Lipid peroxide test boxes (LPO) and glutathione test boxes (GSH) are purchased from Nanjing, and the product numbers are A106-1 and A061-1 respectively;

BCA protein quantification kit was purchased from Monad/monad, cat No. PQ10101S;

TrueGold myelin staining kit was purchased from European Siemens Biotechnology Inc., cat# BK-AC001.

The main instrument in the embodiment of the invention comprises:

frozen microtomes (Leica, germany, model: CM 1950);

elevated plus maze, open field experimental analysis system (ravode life technologies limited);

electronic analytical balance (model: PL303, metrehler Toli, switzerland);

A high-speed cryocentrifuge (Eppendorf, germany, model number 5840);

microplate reader (Burteng apparatus, model: SYNERGY/H1);

a fluorescence microscope (model number: BX51 of Olympus corporation, japan).

Example 1

1. Test method

① Experimental mice were grouped and dosed:

C57BL/6 male mice (8-10 weeks) were purchased from beijing velarix and randomly split into three groups (10 per group) according to body weight, and two parallel controls were set up for each group:

normal group (Normal): normal feed is fed for 6 weeks, and physiological saline is injected intraperitoneally from week 5;

CPZ treatment group (cpz+ns): the feed containing 0.2% of cyclohexanone oxalyl dihydrazone was fed continuously for 6 weeks for molding, and physiological saline was injected intraperitoneally from week 5 once a day, 200. Mu.L each time. Immunofluorescent staining of TrueGold and MBP demonstrated significant loss of myelin sites in the model group compared to the normal group (fig. 2).

Formononetin treatment group (cpz+fmn): modeling of feed containing 0.2% cyclohexanone oxalyldihydrazone was fed continuously for 6 weeks, starting with week 5, with intraperitoneal injection of FMN (30 mg/kg/d), once daily, 200 μl each.

② Behavioural testing

Behavioural experiments were performed after the end of the above dosing procedure:

(1) Elevated plus maze test (elevated plus maze, EPM) mice have explored new environment behaviors due to curious psychology, but they are naturally terrible, and mice with normal cognitive ability and stable emotion have evading behaviors to the high place. EPM is formed by crisscross two arms with equal length, wherein one arm, namely a closed arm, is enclosed by a baffle; the other is that the periphery of the open arm is not provided with a baffle; the center cross non-baffled region is the central region and the EPM is about 50cm from the ground. The mice were placed in the central area during the experiment, and the trajectory data of the movement of the mice was recorded with a camera in a quiet environment for 5min until all mice were detected. Between experiments, the maze was rinsed with 70% alcohol to eliminate odor effects. The mice were analyzed for the percentage of time they were active in the closed arm and the number of times they entered the open arm after the end of the experiment.

(2) Open field experiment (open FIELD TEST, OFT): the open field experiment is a method for evaluating the autonomous behavior, exploring behavior and tension of experimental animals in a new environment, and detecting the movement track of the experimental animals. Before the test, the mice are placed in a square opaque open field box at the bottom, after the mice are adapted to the environment for 1min, the test is started for 5min, and the movement track data of the mice are recorded. To eliminate the smell of mice, each mouse was removed after the experiment was completed and sprayed with 70% alcohol on the box used for the experiment to avoid affecting the next mouse behavioral movement trace. The total distance travelled and the number of times the mice entered the central zone were analysed after the end of the experiment.

③ Sample material and sample preparation

Randomly extracting 5 mice from each group after the behavioural experiment is finished, anaesthetizing the mice with pentobarbital sodium (50 mg/kg), carrying out heart infusion with normal saline until the liver turns white, then carrying out heart infusion with 4% paraformaldehyde, taking brain tissue, fixing the paraformaldehyde, then placing the brain tissue in 10%, 20% and 30% sucrose solution for gradient dehydration, then embedding the brain tissue in OTC, quickly freezing the brain tissue with liquid nitrogen, and placing the brain tissue in a refrigerator at-80 ℃ for later use; the brain tissue sections were cut into 10 μm sections for immunofluorescence staining using a frozen microtome.

The remaining 5 mice of each group were heart perfused with physiological saline alone, brain tissue was weighed, placed in sterile EP tubes, lysates were added by weight, and ground to a homogenized state. Cracking on ice for 30min, and shaking and mixing once every 5min. Then transferred to a centrifuge at 4℃for 15min at 12000 rpm. Transferring the supernatant to a new sterile EP tube by using a pipette, measuring the protein concentration, and sub-packaging to obtain the brain tissue protein solution. In order to reduce protein degradation, split charging was performed and placed in a-80 ℃ refrigerator for subsequent ELISA detection.

④ TrueGold dyeing

Freezing the prepared brain tissue slice, and then baking at 37 ℃ for 30min; diluting the required dosage of the dye and the dye finishing agent by double distilled water; adding a dye, placing in a wet box, dying at 45 ℃ for 30min in a dark place, rinsing the slice with PBS for 2 times each for 1min, adding a dying agent, incubating at 45 ℃ for 2-3min, rinsing with PBS for 3 times each for 1min, taking the gray matter and white matter which can be distinguished obviously as standards, sealing the slice after the water is absorbed by water absorption paper, and observing and collecting pictures under a microscope.

⑤ Immunofluorescent staining to detect MBP and GFAP expression

Washing the prepared brain tissue frozen section with PBS for 3 times for 5min each time; the primary antibody was added to PBS containing 0.3% Triton X-100 and 1% BSA, suspended and then dropped onto the sections, and incubated overnight at 4℃in a wet box. Washing with PBS for 3 times for 5min, adding secondary antibody of corresponding species, incubating at room temperature for 1h, washing with PBS for 3 times again, sealing with DAPI-containing anti-fluorescence quenching sealing liquid, observing the result under a fluorescence microscope after 10min, and analyzing fluorescence density with Image J fiji 2.14.0.

⑥ ELISA method and trace enzyme labeling method for detecting content of brain tissue protein liquid TNF-alpha, IL-1 beta, IL-6, IL-10, LPO and GSH

And (3) taking each group of the brain tissue protein liquid, operating according to the specification steps of ELISA and a micro-enzyme labeling method kit, reading Optical Density (OD) values in an enzyme labeling instrument, and detecting the contents of TNF-alpha, IL-1 beta, IL-6, IL-10, LPO and GSH in the mouse brain tissue protein liquid.

According to the embodiment of the invention, GRAPHPAD PRISM 8.0.2 software is adopted for statistical analysis, metering data conforming to normal distribution is expressed by x+/-s, single-factor analysis of variance (ANOVA) is adopted for comparison among multiple groups, and t-test is adopted for comparison between every two groups. p <0.05 is statistically significant for differences.

2. Results

2.1 Behavioural detection

In the cross elevated maze Experiment (EPM), the mice are sensitive to the change of the external environment, like to live in the dark environment, like to live at night, and are afraid of high naturally, so the invention counts the time percentage of the mice moving in the closed arm and the times of entering the open arm. As shown in fig. 1a, EPM results showed that the percentage of time that cpz+ns group mice were active in the closed arm was reduced (p < 0.0001) and the number of times that they entered the open arm was increased (p < 0.001) compared to the normal group. The percentage of time that the cpz+fmn group mice were active in the closed arm was increased (p < 0.0001) and the number of open arms entered was decreased (p < 0.001) compared to the cpz+ns group.

In open field experiments (OFT), mice are naturally afraid of open field, on the other hand have curiosity of exploring fresh things, and the two psychological conflicts can cause mania and anxiety, which affect behaviours. The present invention thus records the total distance traveled by the mice and the number of times the mice entered the central area. As shown in fig. 1B, the OFT results showed that the total distance traveled by the cpz+ns group of mice in the open field was increased (p < 0.0001) and the number of times of entry into the central region was also increased (p < 0.0001) compared to the normal group. The total distance traveled by the cpz+fmn mice in the open field was reduced (p < 0.001) and the number of entries into the central region was also reduced (p < 0.01) compared to the cpz+ns group. The results are shown in FIG. 1 and Table 1.

The behavioral detection result shows that the formononetin can obviously improve the cognitive function, mania, anxiety and other emotions of the demyelinating model mouse.

TABLE 1 influence of formononetin on CPZ mouse behaviours

Note that: ^*p<0.05,^**p<0.01,^*** p <0.001 and ^**** p <0.0001vs CPZ+NS.

2.2 Formononetin significantly inhibits demyelination

Selecting prepared normal group, CPZ treatment group and formononetin treatment group of mouse brain slices, performing pathological staining on the brain slices, setting three parallel controls in each group, observing the staining condition of each slice in each group, and evaluating the influence of formononetin on demyelination.

Myelin protection in acute CPZ demyelinated mice was tested by two staining methods, trueGold and immunofluorescence staining, respectively. In black gold staining (fig. 2 a), the cpz+ns group mice had large area demyelination of the brain corpus callosum region (p < 0.0001) compared to the normal group; compared with the CPZ+NS group, the brain callus area demyelination area of the CPZ+FMN group is obviously reduced (p < 0.0001). In fluorescent staining (B in fig. 2), the MBP staining fluorescence intensity of the cpz+ns group callus was significantly reduced (p < 0.001) compared to the normal group; compared to the cpz+ns group, the cpz+fmn group had increased fluorescence intensity of the callus MBP staining (p < 0.05), as shown in table 2.

The brain sections containing the callus are used for black gold (TrueGold) staining, the demyelination area is estimated through the color difference, the result shows that the dark-colored area of the black gold staining of the formononetin treatment group is larger, and the inhibition effect of the formononetin on the demyelination is shown according to the judgment standard that the color of the demyelination area is lighter than that of normal tissues.

Immunofluorescence is adopted to dye myelin basic protein (Myelin basic protein, MBP) in the callus area of a demyelinating model mouse, the result shows that the fluorescence intensity of the callus of a formononetin treatment group is higher than that of a model group, and the result shows that the formononetin reverses the expression of MBP in the callus of the demyelinating model mouse.

Demyelination refers to central neuritic demyelination associated with viral infections, abnormal autoimmune responses, inheritance, environment, etc., and includes multiple sclerosis, neuromyelitis optica, etc. Staining analysis indicated that formononetin could inhibit demyelination in demyelinating model mice.

TABLE 2 Effect of formononetin on CPZ mice on demyelination and MBP expression in the corpus callosum region

Note that: ^*p<0.05,^**p<0.01,^*** p <0.001 and ^**** p <0.0001vs CPZ+NS.

2.3 Immunofluorescence staining method for detecting influence of formononetin on expression of GFAP in brain of CPZ mice

Since abnormally activated Astrocytes (AST) can impair the morphology and function of myelin cells by secreting inflammatory factors and oxidative stress, it is an important way for them to participate in demyelinating lesions. It is necessary to detect the state of an AST. In immunofluorescent staining, the number of callus GFAP ⁺ AST was significantly increased (p < 0.05) in the cpz+ns group of mice compared to the normal group; after drug intervention, the GFAP ⁺ AST numbers were significantly reduced (p < 0.05) in the cpz+fmn group of mice, see fig. 3, table 3, indicating that FMN may reduce inflammatory response and oxidative stress by inhibiting abnormal activation of the focal zone GFAP ⁺ AST.

TABLE 3 Effect of formononetin on GFAP expression in CPZ mice

Note that: ^*p<0.05,^**p<0.01,^*** p <0.001 and ^**** p <0.0001vs CPZ+NS.

2.4 Inhibition of inflammatory response and oxidative stress in CPZ mice by FMN

In order to detect the influence of FMN on the inflammatory response of CPZ mice, the invention uses ELISA method to detect the inflammatory related factors such as IL-1 beta, TNF-alpha, IL-6, IL-10 and the like on the brain tissue protein liquid. As shown in fig. 4a, the secretion levels of the pro-inflammatory factors IL-1β, TNF- α and IL-1β were significantly increased in the cpz+ns group compared to the normal group (p <0.05, p <0.0001 and p <0.0001, respectively). Secretion levels of TNF- α, IL-6, IL-1β were down-regulated after administration of CPZ+FMN groups compared to CPZ+NS groups (p <0.01, p <0.0001, and p <0.0001, respectively). Furthermore, the secretion level of the anti-inflammatory factor IL-10 was significantly reduced in the CPZ+NS group compared to the normal group (p < 0.01). The CPZ+FMN group up-regulated IL-10 secretion levels (p < 0.05) after administration compared to the CPZ+NS group.

The results show that FMN can effectively inhibit secretion of inflammatory factors of CPZ mice. In order to explore the influence of FMN on CPZ mouse oxidative stress, the invention uses ELISA method to detect the expression condition of oxidative stress related proteins such as LPO, GSH and the like on brain tissue protein liquid. As shown in fig. 4B, LPO protein expression was significantly increased in brains of mice in the cpz+ns group compared to the normal group (p < 0.001), and expression of LPO protein was reduced after administration of the cpz+fmn group compared to the cpz+ns group (p < 0.05); the expression of GSH protein in the brains of mice in the cpz+ns group was significantly reduced (p < 0.001) compared to the normal group, and the expression of GSH protein was increased (p < 0.01) after the administration of cpz+fmn compared to the cpz+ns group.

TABLE 4 formononetin inhibits inflammatory response and oxidative stress in the brain of CPZ mice

Note that: ^*p<0.05,^**p<0.01,^*** p <0.001 and ^**** p <0.0001vs CPZ+NS.

In summary, the embodiment of the invention discovers that formononetin can increase the time percentage of CPZ mice moving in a closed arm, reduce the total moving distance of CPZ mice in open field, reduce the demyelination of brain corpus callosum areas of CPZ model mice, enhance the fluorescent intensity of callus MBP staining, reduce GFAP expression, down regulate TNF-alpha, IL-6, IL-1 beta and LPO expression, up regulate IL-10 and GSH expression, and shows that formononetin can alleviate the multiple sclerosis of CPZ mice, obviously inhibit the demyelination, and effectively alleviate the verification reaction and oxidative stress of mice. The formononetin has wide application prospect in the treatment and prevention of demyelinating diseases.

It is to be understood that the above examples of the present invention are provided for clarity of illustration only and are not limiting of the embodiments of the present invention. Other variations or modifications of the above description will be apparent to persons of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. Any modification, equivalent replacement, improvement, etc. which come within the spirit and principles of the invention are desired to be protected by the following claims.

Claims

1. Use of formononetin in the preparation of a medicament for the prevention and/or treatment of inflammatory demyelinating diseases of the central nervous system.

2. The use according to claim 1, wherein the central neuritic demyelinating disease is multiple sclerosis, neuromyelitis optica or acute disseminated encephalomyelitis.

3. A pharmaceutical composition for treating central nervous inflammatory demyelinating diseases, comprising formononetin;

4. A pharmaceutical formulation for the treatment of central nervous inflammatory demyelinating diseases comprising formononetin or the pharmaceutical composition of claim 3 and a pharmaceutically acceptable carrier.

5. The pharmaceutical formulation of claim 4, wherein the pharmaceutically acceptable carrier is suitable for use in a tablet, capsule, powder, pill, granule, oral liquid, injection, or emulsion.

6. Use of formononetin in the preparation of a medicament for inhibiting demyelination.

7. Use of formononetin in the preparation of a medicament for inhibiting activation of astrocytes in myelin sheath region.

8. Application of formononetin in preparing medicine for inhibiting inflammatory factor and protecting myelin sheath is provided.

9. Application of formononetin in preparing medicine for inhibiting oxidative stress and protecting myelin sheath is provided.