CN115919835A

CN115919835A - Application of procyanidine in preparation of medicine for promoting central nervous system inflammatory demyelination regeneration

Info

Publication number: CN115919835A
Application number: CN202210786969.0A
Authority: CN
Inventors: 王青; 杨智超; 姜维佳; 元海军; 杨琬芳; 贾璐
Original assignee: Shanxi University of Chinese Mediciine
Current assignee: Shanxi University of Chinese Mediciine
Priority date: 2022-07-04
Filing date: 2022-07-04
Publication date: 2023-04-07

Abstract

The invention belongs to the technical field of medicines, and provides application of procyanidine in preparation of a medicine for promoting central nervous system inflammatory demyelination, wherein the procyanidine comprises procyanidine B1, procyanidine B2 and procyanidine B3 which are isomerides mutually, and the chemical molecular formula of the procyanidine B1, procyanidine B2 and procyanidine B3 is C ₃₀ H ₂₆ O ₁₂ . The procyanidins show good effectiveness on demyelination regeneration, and can effectively inhibit central nerve inflammatory factors. The application widens the application range of the procyanidine and provides more possibilities for the application of the procyanidine.

Description

Application of procyanidine in preparation of medicine for promoting central nervous system inflammatory demyelination regeneration

Technical Field

The application belongs to the technical field of medicines, relates to a new application of procyanidine, and particularly relates to an application of procyanidine in preparation of a medicine for promoting central nervous inflammatory demyelination regeneration.

Background

Inflammatory demyelinating CNS diseases are a group of diseases characterized primarily by an inflammatory response and extensive primary demyelination. Among them, multiple Sclerosis (MS) of relapsing-remitting type and neuromyelitis spectrum disease (NMOSD) are the most common demyelinating diseases, and the course of the disease usually shows that relapse and remitting are alternately performed, and multiple attacks cause irreversible damage to nerve function, and are one of the main causes of non-traumatic disability in middle-aged and young-aged people. Reducing inflammatory immune damage to myelin has long been an important target for therapy, but the ideal therapeutic effect has not been achieved.

Astrocytes (AS), the most abundant non-neuronal cell of the Central Nervous System (CNS), regularly and orderly cover the entire CNS in a continuous and essentially non-overlapping manner, and can maintain CNS homeostasis by participating in myelination, maintaining the blood-brain barrier, regulating synaptic function, and regulating energy metabolism. Once the center is damaged, AS responds rapidly, and a large number of reactive AS proliferate and exert beneficial or deleterious effects on peripheral neural and non-neural cells through significant transformation of their own functions and phenotypes. A large number of reactive AS's are present in demyelination models induced by NMOSD, MS and its animal models, experimental Allergic Encephalomyelitis (EAE) and dicyclohexanoneoxalyl dihydrazone (CPZ), and can damage neurons and myelin sheath cells by releasing various inhibitory factors such AS keratan sulfate, 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 (supplement 1q, C1q). Targeting AS, however, significantly ameliorates demyelination, AS found by Dalahmah et al, galectin-3 (gal-3 ) reduces oligodendrocyte damage by promoting proliferation and inflammatory responses of AS. Thus, with the intensive research on the function of reactive AS and its mechanism of action, it has gradually become an important class of potential target cells for protection and promotion of remyelination.

Procyanidins (procyanidins/procyanidins, PC) are widely present in plant tissues and organs such as grape seed, pine bark, lotus seed pot, apple, blueberry, lycium ruthenicum and rose hip, and are polyphenols obtained by condensing flavan-3-ols (catechin, epicatechin or gallic acid) in different numbers. Procyanidine is one of the most safe and effective antioxidants, has pharmacological effects of anti-inflammation, anti-malignant tumor, immunoregulation, anti-aging, neuroprotection and the like, but reports of procyanidine on central nervous inflammatory demyelinating diseases are not found so far.

Disclosure of Invention

Aiming at the problem of the development of the new application of the procyanidine, the application discovers that the procyanidine can promote the regeneration of mouse myelin by exploring the effect of the procyanidine on a chronic demyelination mouse induced by dicyclohexyl oxalyl dihydrazone (CPZ), and shows that the procyanidine has a wide application prospect in the treatment and prevention of demyelination diseases.

For the application of procyanidine, the application is mainly realized by the following technical scheme:

as one embodiment of the present application, there is provided the use of procyanidins in the preparation of a medicament for promoting central nervous system inflammatory demyelination.

The applicant performs behavioral detection on the demyelination model mouse through a mouse maze experiment, analyzes the behaviors of the mouse in a cross elevated maze experiment, an open field experiment and a T maze experiment, and results show that the procyanidine can obviously improve anxiety and working memory impairment of the demyelination model mouse.

The applicant performs staining analysis on brain tissues of demyelination model mice, performs solid Blue (LFB) staining on brain slices containing corpus callosum, evaluates demyelination form through color difference, and shows that LFB staining of a procyanidine treatment group becomes dark according to a judgment standard that the color of a demyelination area is lighter than that of normal tissues, so that procyanidine has a promoting effect on myelin restoration and regeneration of the demyelination area. By using FluoroMyelin ^TM Green myelin fluorescence probe stains the calluses of demyelinating model mice, and the result shows that the fluorescence intensity of the calluses of the procyanidine treatment group is recovered. Detected by immunofluorescence stainingMarkers of mature oligodendrocytes degrade myelin basic protein and myelin basic protein, and results show that procyanidins reverse the expression of degraded myelin basic protein and myelin basic protein in the calluses of demyelinating model mice. Staining analysis indicated that procyanidins could promote remyelination in demyelinating model mice.

The demyelination refers to central nervous system inflammatory demyelination related to virus infection, autoimmune reaction abnormality, heredity, environment and the like, and comprises multiple sclerosis, neuromyelitis optica and the like.

In this embodiment, the demyelination is a bicyclohexanoneoxalyl dihydrazone mediated chronic demyelination.

In this embodiment, the procyanidins improve the central nervous inflammatory microenvironment by modulating reactive astrocytosis, promoting the expression of cytokines beneficial for remyelination.

The applicant finds that procyanidins remarkably inhibit central inflammatory reaction by detecting the improvement effect of procyanidins on the central neuroinflammatory microenvironment of demyelinating model mice, and the mechanism of procyanidins is related to procyanidins regulation of reactive astrocyte proliferation.

In this embodiment, the procyanidins promote astrocyte secretion beneficial for remyelinating cytokines.

The cytokines beneficial for remyelination include at least one of Neurotrophic factor-3 (NT3), ciliary Neurotrophic factor (CNTF), gap junction protein 30 (Connexin 30, cx 30), and gap junction protein 47 (Connexin 47, cx 47).

The applicant detects cytokines beneficial to and harmful to differentiation and survival of Oligodendrocyte Precursor Cells (OPCs) by intervening inflammatory factor-activated astrocytes with procyanidins in vitro, and finds that procyanidins significantly promote expression of neurotrophin-3, ciliary neurotrophic factor, gap junction protein 30 and gap junction protein 47 at the gene level and the protein level. In addition, procyanidins were found to significantly increase CPZ-mediated smallness after further validation in vivoMouse callus GFAP ⁺ Expression of neurotrophic factor-3, ciliary neurotrophic factor, zonulin 30, and ciliary neurotrophic factor in astrocytes.

In the above embodiments of the present application, the procyanidin includes monomers and oligomers extracted from natural plants and artificially synthesized.

The natural plants include grape seed, black medlar, rose, cynomorium songaricum, acanthopanax, longan, cherokee rose, lotus seedpod, blueberry, seabuckthorn seed, blackcurrant, purple cabbage, xinjiang oleaster, cherry plum, european plum, mulberry, walnut, and the like.

Preferably, the procyanidin is catechin, epicatechin, or an oligomeric procyanidin formed by polymerization of catechin and epicatechin. The oligomeric procyanidin is specifically dimeric procyanidin.

More preferably, the dimeric procyanidin comprises procyanidin B1, procyanidin B2 and procyanidin B3 which are isomers of each other, and the chemical formula of procyanidin B1, procyanidin B2 and procyanidin B3 is C ₃₀ H ₂₆ O ₁₂ 。

Most preferably, the oligomeric procyanidin is procyanidin B2. The procyanidin B2 is isomer with strongest activity in dimer formed by polymerizing procyanidin monomers (catechin and epicatechin).

As another embodiment of the present application, a pharmaceutical preparation for treating a cns inflammatory demyelinating disease is provided, the pharmaceutical preparation comprising procyanidins.

The medicinal preparation is a preparation containing procyanidin alone or a preparation containing procyanidin composition.

The formulations may be prepared by methods conventional in the art, including tablets, capsules, granules, suspensions, emulsions, solutions, syrups or injections.

Preferably, the preparation contains 0.1-99.9 wt% of the procyanidin as an active ingredient.

The formulation further comprises a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier does not destroy the pharmaceutical activity of the procyanidins described herein.

The beneficial effect of this application does:

the application provides the regulation and control effect of procyanidine on reactive astrocyte hyperplasia, and shows the capacity of treating and preventing neuroinflammatory demyelinating diseases, and the mechanism of procyanidine is mainly that procyanidine promotes astrocytes to secrete cytokines which are beneficial to remyelination. Provides new application of the procyanidine, widens the application range of the procyanidine and provides more possibility for the application of the procyanidine.

Drawings

FIG. 1 is a comparison of the number of times procyanidine B2 enters the maze cross closed arm for CPZ mice in the experimental examples of the present application;

FIG. 2 is a comparison of the number of times procyanidin B2 entered the T maze bait arm for CPZ mice in the experimental examples of the present application;

FIG. 3 is a comparison of the total distance traveled by the procyanidine B2 in the CPZ mice in the open field and the distance traveled by the mice in the central region in the experimental examples of the present application;

FIG. 4 is a comparison of the blue-fixation staining image and average gray scale of procyanidine B2 on CPZ mouse corpus callosum in the experimental examples of the present application;

FIG. 5 is a comparison of fluorescence staining images and mean gray scale values of tissue procyanidine B2 on CPZ mouse callus FluoraMyelin in the examples of the present application;

FIG. 6 is a comparison of fluorescence staining images and mean gray values of procyanidine B2 on CPZ mouse callus dMBP and MBP in the experimental examples of the present application;

FIG. 7 is a comparison of the fluorescence staining image and the average gray value of procyanidin B2 on NG2 marker of CPZ mouse corpus callosum oligodendrocyte precursor cells in the experimental examples of the present application;

FIG. 8 is a comparison of the fluorescence staining image and mean gray value of procyanidin B2 versus GPR17, a marker for CPZ mouse callus oligodendrocyte precursor cells in the experimental examples of the present application;

FIG. 9 shows the effect of procyanidin B2 on neuroinflammatory factors in the brain of CPZ mice in the experimental examples of the present application;

FIG. 10 is the present inventionProcyanidine B2 in application experimental example to CPZ mouse multi-brain area GFAP ⁺ The effect of astrocyte number;

FIG. 11 shows the proanthocyanidin B2 pair GFAP in CPZ mouse brain in the experimental examples of the present application ⁺ Effect of Ki67 expression in astrocytes;

FIG. 12 is a graph of the effect of procyanidin B2 on beneficial and detrimental remyelination cytokines secreted by primary astrocytes in vitro in the experimental examples of the present application;

FIG. 13 shows the effect of procyanidin B2 on primary astrocytes NT3, CX47, CX30 and CNTF expression in vitro in the experimental examples of the present application;

FIG. 14 shows the effect of procyanidin B2 on the expression of NT3, CX47, CX30 and CNTF in CPZ mouse brain in the experimental examples of this application;

FIG. 15 shows the proanthocyanidin B2 pair GFAP in CPZ mouse brain in the experimental examples of the present application ⁺ The effect of NT3 expression in astrocytes;

FIG. 16 shows the proanthocyanidin B2 pair GFAP in CPZ mouse brain in the experimental examples of the present application ⁺ The effect of CNTF expression in astrocytes;

FIG. 17 shows the proanthocyanidin B2 pair GFAP in CPZ mouse brain in the experimental examples of the present application ⁺ The effect of CX30 expression in astrocytes;

FIG. 18 shows the proanthocyanidin B2 pair GFAP in CPZ mouse brain in the experimental examples of the present application ⁺ Effect of CX47 expression in astrocytes.

Detailed Description

The technical solutions of the present application will be described clearly and completely with reference to specific embodiments of the present application, and it should be apparent that the described embodiments are only a part of the embodiments of the present application, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.

The technical solution of the present application is formed based on the unexpected finding that:

astrocytes (astrocytes) are the most widely distributed cell type in the brain of mammals, and are the largest mass of glial cells. It has many protrusions, which extend to fill in the cell body of nerve cells and between the protrusions, and plays a role in supporting and separating nerve cells, and participates in the formation of blood brain barrier. Astrocytes respond to some neuroactive substances by producing and secreting certain neurotransmitters and expressing certain neurotransmitter receptors.

Demyelinating diseases are nervous system diseases in which loss of the nerve myelin is the main or primary lesion and axons, soma and glia are relatively weakly damaged, and may occur in the central or peripheral nervous system. The pathological change is that the myelin sheath of nerve fiber is lost, and nerve cells are relatively intact, so that the transmission of nerve impulse is affected. Acute demyelinated nerve myelin can be regenerated, and more rapidly and completely, with little effect on functional recovery. Chronic demyelinating neuropathies do not recover fully function because of the marked proliferation of regenerating schwann cells that undergo repeated demyelination and myelination, the nerve becomes thicker, and axonal loss occurs.

Astrocytes also play a key role in the pathogenesis of demyelination, and under the action of sustained adverse stimuli, astrocytes rapidly activate, proliferate and hypertrophy in a process known as "reactive astrocytosis", highly expressing Glial Fibrillary Acidic Protein (GFAP), further worsening the central nervous inflammatory microenvironment through changes in their phenotype and function, and being a pathological hallmark of central nervous inflammatory demyelinating diseases. However, the applicant has found the contradictory phenomenon that procyanidins have a promoting effect on astrocytes and a treating and preventing effect on demyelinating diseases.

To explain this phenomenon, the applicant has conducted an intensive analysis on the therapeutic and preventive effects of procyanidins in demyelinating diseases. The effectiveness of procyanidine on a dicyclohexyl oxalyl dihydrazone mouse model is proved by a myelin staining experiment through establishing a dicyclohexyl oxalyl dihydrazone (CPZ) -induced chronic demyelinating mouse model, and the mechanism reason that the procyanidine promotes astrocyte proliferation is used for treating/preventing the demyelinating disease is proved by an immunofluorescence staining method, an RT-PCR method, an enzyme-linked immunosorbent assay (Elisa) and Western blot, and the procyanidine can promote astrocytes to secrete cytokines beneficial to myelin regeneration.

Therefore, the applicant proposes a technical solution: application of procyanidin in preparing medicine for treating central nervous system inflammatory demyelinating diseases is provided.

In the present application, the procyanidin may be used alone or in combination with other components, such as bioactive and therapeutic chemicals, which are not limited to the mechanism of procyanidin therapy, and may be used together with procyanidin to treat central neurite demyelinating diseases, or have other functional effects, such as alleviating side effects of procyanidin during therapy, and may be used as a carrier, emulsion, dispersion, etc. only as an adjuvant for drugs.

When the other component serves as a carrier, it is required that the other component is a pharmaceutically acceptable carrier such as a physiologically acceptable solvent, dispersion medium, integument, antibacterial agent, antifungal agent, isotonic agent, wetting agent, emulsifier, preservative, buffer, and the like.

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

Pharmaceutical preparations of procyanidins can be prepared in various dosage forms by conventional methods of manufacture well known in the art, for example by mixing the active ingredient with one or more carriers and then formulating into the desired dosage form. For example, the procyanidin described herein can be further formulated into granules, capsules, tablets, pills, etc. by adding suitable excipients, which can be one or more selected from starch, dextrin, lactose, silicon dioxide, calcium hydrogen phosphate, cyclodextrin, microcrystalline cellulose, sodium carboxymethylcellulose, magnesium stearate, talc, etc.

The dosage form comprises one or more of tablets, capsules, granules, suspensions, emulsions, solutions, syrups or injections, and the like, and adopts one or more administration routes of oral administration or injection (comprising one or more of intravenous injection, intravenous drip, intramuscular injection or subcutaneous injection, and the like).

The preparation containing the procyanidin preferably contains 0.1-99.9 wt% of the procyanidin as an active ingredient, more preferably 30-99 wt% of the procyanidin as an active ingredient, and most preferably 90-99 wt% of the procyanidin as an active ingredient.

Procyanidins of the present application and pharmaceutical uses thereof may comprise a "therapeutically effective amount" or a "prophylactically effective amount" of procyanidins of the present application. A "therapeutically effective amount" is an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic effect. A therapeutically effective amount of procyanidins may vary according to factors such as the condition, age, sex and weight of the individual and the ability of the procyanidin to elicit a desired response in the individual. A therapeutically effective amount also refers to an amount of the procyanidin that has a beneficial therapeutic effect over any toxic or detrimental effects thereof. A "prophylactically effective amount" is an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic effect. Because prophylactic doses are used in subjects before or at an early stage of the disease, the prophylactically effective amount is generally less than the therapeutically effective amount. It should be noted that the dosage value will vary depending on the type and severity of the disease to be alleviated, i.e., when administered to a patient, the procyanidin dosage or amount described herein will generally be determined based on the age and weight of the patient or user, as well as the physical condition or condition of the patient's symptoms. In addition, it is to be understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need 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 do not limit the scope or practice of the claimed compositions.

The action mechanism of procyanidin is verified by combining with a specific experimental example.

1. Experimental Material

(1) Grouping and dosing of experimental mice:

c57BL/6 male mice were purchased from beijing vindolizhihua (8-10 weeks) and randomized into four groups by weight (12 per group) with three parallel controls per group:

normal group (Nor): feeding with common feed for 12 weeks;

CPZ treatment group (CPZ + NS): the feed containing 0.2% of bicyclohexanoneoxalyl dihydrazone was fed for 10 consecutive weeks for modeling, and the normal feed was fed from the 11 th week and the normal saline was intraperitoneally injected.

Procyanidin B2 treated group (Nor + PC-B2): ordinary feed is fed for 10 weeks, and PC-B2 (50 mg/kg/d) is injected from 11 weeks;

procyanidin B2 treatment group (CPZ + PC-B2): the feed containing 0.2% bicyclohexanoneoxalyldihydrazone was fed for 10 consecutive weeks, modeled, and ordinary feed was fed from week 11 onward, and was intraperitoneally injected with PC-B2 (50 mg/kg/d).

(2) Tissue preparation

Mice in a normal group, a CPZ treatment group, a procyanidine B2 treatment group and a procyanidine B2 treatment group are randomly selected at the end of 12 weeks for killing, normal saline is perfused into the heart of the mice, then 4% paraformaldehyde is perfused, brains are taken, fixed by 4% paraformaldehyde for 6 hours, and then dehydrated for 24 hours at 4 ℃ by using 30% sucrose solution. The dehydrated brain tissue was embedded in OTC, stored in a-80 ℃ freezer, cut into 10 μm slices using a cryostat during staining, and stored at 4 ℃ for histopathological staining.

Mice of a normal group, a CPZ treatment group, a procyanidine B2 treatment group and a procyanidine B2 treatment group are randomly selected after 12 weeks to be sacrificed, normal saline is perfused only, brains are taken out and homogenized in RIPA lysis buffer containing protease inhibitor (100 x), after being lysed for 1h on ice, supernatants are collected by centrifugation at 13000g for 20min at 4 ℃, and the supernatants are stored in a refrigerator at-80 ℃ after being subpackaged for enzyme-linked immunosorbent assay (ELISA) and Western blot experiments.

(3) Primary astrocyte culture

Anesthetizing newborn C57BL/6 mice 2-7d with ice at low temperature for 2-10min, sterilizing the head and neck with 75% ethanol, taking out the brain, placing in ice-bath high-sugar DMEM (Gibco, 11995040), dissecting meninges under a stereomicroscope, and shearing the brain tissue into 1mm with sterilized ophthalmic scissors ³ Of the chip (a). The tissue fragments were placed in 0.25% trypsin EDTA solution (Thermo, R001100), incubated with gentle shaking for 10min, and then the trypsin reaction was stopped by adding complete medium. The complete medium was high-glucose DMEM containing 10% fetal bovine serum. The digested tissue was centrifuged at 300g for 5min, the supernatant carefully removed and resuspended in high glucose DMEM, and the tissue separated into single cell suspensions through a 40 μm Nylon membrane. The obtained single cell suspension was inoculated in a T75 culture flask to obtain primary mixed glial cells. The flasks were previously coated with 100. Mu.g/ml polylysine (molecular weight 15-30 ten thousand) and then left overnight at 4 ℃, washed three times with PBS and dried before use. The fresh complete culture solution is replaced after the primary mixed glial cells are cultured for 2 days, and the culture solution is replaced every 3 days, and usually 7-11 days of mixed glial cells are spread on the bottom of the culture flask. The confluent flask was placed on a shaker for 24h shaking at 180rpm to remove Microglia (Microglia, MG). Fresh astrocyte medium (ScienCell, 1801) was then added and shaken at 240rpm for 6h to remove Oligodendrocyte Precursor Cells (OPCs). The remaining cells were differentially attached for 30min prior to the experiment and further purified to obtain Astrocytes (AS).

(4) Establishment of astrocyte activation model and pharmaceutical intervention

Normal group (Nor): preparing the obtained primary Astrocytes (AS);

astrocyte activation Model group (Model): incubating primary astrocytes with 3ng/ml IL-1 alpha (PeproTech, 315-01A), 30ng/ml TNF-alpha (PeproTech, 215-11A) and 400ng/ml C1q (MyBioSource, MBS 143105) for 24h to establish inflammatory factor activating AS;

procyanidin B2 intervention group (PC-B2): PC-B2 at 50. Mu.g/ml was added to the astrocyte activation model and incubated for 24h to interfere with astrocyte activation.

2. Statistical method

The data of each group are described as mean ± standard deviation and statistically analyzed using GraphPad Prism 8.0 software (GraphPad software, la Jolla, CA). After one-element analysis of variance (ANOVA), tukey's post-hoc test was used for the inter-group comparisons, and single comparisons were evaluated by unpaired T-test. p <0.05 indicates that the difference is statistically significant.

Experimental example 1 behavioural assay

The cross elevated maze consists of two metal arms which are mutually vertical and crossed, wherein one metal arm is a closed arm, the other metal arm is an open arm, all metal arm platforms are 50 cm higher than the ground, and the anxiety degree of a mouse is detected by detecting the times and the distance of the mouse entering the closed arm. The T-shaped maze is composed of two selection arms and a starting arm above the center of a square, the food is placed on the left selection arm to be used as a bait arm, and the T-shaped maze measures the working memory of a mouse by detecting the times of the mouse entering the bait arm and the distance of the mouse walking in the bait arm. In the open field experiment, the mice have the property of avoiding the open field on one hand and the curiosity of exploring the center of the open field on the other hand, the conflict between the two causes anxiety, and the anxiety degree of the mice is evaluated by detecting the moving distance and the entering frequency of the mice in the central area. The established normal group (Nor), CPZ treatment group (CPZ + NS), procyanidine B2 treatment group (Nor + PC-B2) and procyanidine B2 treatment group (CPZ + PC-B2) mice are respectively placed in the plus maze, T maze and open field for autonomous activity for 10min, three parallel controls are set for each group of mice, and the results are shown in figures 1-3 and table 1.

TABLE 1 mouse behavioural assays

^* p<0.05， ^** p<0.01 and ^* p<0.05vs CPZ+NS

as can be seen from the data in table 1, in the elevated plus maze, the number of times the procyanidin B2-treated mice entered the closed arm and the moving distance of the closed arm were significantly increased compared to the CPZ-treated mice (fig. 1), indicating that PC-B2 could significantly improve the anxiety level of the CPZ-mediated mice. In the T maze trial, the CPZ-treated mice entered the bait arm less frequently and at a shorter distance than the blank group, indicating impaired working memory in the CPZ-treated mice, while the procyanidin B2-treated mice entered the bait arm more frequently (fig. 2), indicating that PC-B2 can significantly improve CPZ-mediated impaired working memory in the mice. In open field experiments, total movement distance in open field and movement distance in central area of procyanidin B2 treated mice were significantly shortened compared to CPZ treated mice (fig. 3), indicating that PC-B2 can significantly improve the anxiety level of CPZ-mediated mice.

Experimental example 2 Proanthocyanidins B2 significantly promotes remyelination

Selecting mouse brain slices of the prepared normal group, CPZ treatment group, procyanidine B2 treatment group and procyanidine B2 treatment group, carrying out pathological staining on the brain slices, setting three parallel controls in each group, observing the staining condition of each slice of each group, and evaluating the influence of procyanidine B2 on demyelination form.

1) Fast blue dyeing

Each group of brain sections containing the corpus callosum was stained with Fast Blue (LFB), which assesses demyelination morphology by color differences, with demyelination areas lighter than normal tissue. The procedure for the staining procedure with fast blue staining reagent (GENMED, GMS 80019.2) was performed according to the kit instructions. The staining images were each photographed by a Leica upright fluorescent microscope, and the obtained staining images were quantitatively analyzed by Image Pro Plus software for three section images of each group of mice.

As shown in fig. 4 and table 2, LFB staining of the normal group mice was uniformly distributed and dark, calluses of the CPZ-treated group mice were light, LFB staining of the procyanidin B2-treated group was dark, and the average gray levels were 214.6, 124.8, and 180, respectively, which were significant compared to the CPZ-treated group.

2) Chemical dyeing

By using FluoroMyelin ^TM Green (Invitrogen, F34651) fluorescent myelin staining stains groups of brain sections containing callus. The staining images were respectively photographed by a leica upright fluorescence microscope, and the obtained staining images were quantitatively analyzed by Image Pro Plus software for three section images of each group of mice.

As shown in FIG. 5 and Table 2, the mean gray level of fluorescence intensity was 21.93 for the normal group and 12.41 for the callus of the mice in the CPZ-treated group, which was significantly lower than that of the normal group, whereas the mean gray level of fluorescence intensity of the callus of the procyanidin B2-treated group treated with PC-B2 reached 17.1, indicating that the fluorescence intensity of the callus of the mice in the CPZ-treated group was restored by the treatment with PC-B2.

3) Immunofluorescence staining

The brain sections of each group of mice were placed in PBS buffer for 10min, the embedding agents were washed off, placed in 1% BSA and blocked at room temperature for 60min, and washed three times with PBS for 10min each. anti-Degraded Myelin basic protein antibody (Degraded Myelin basic protein, dMBP, EMD Millipore Co rp, AB 5864), anti-Myelin basic protein antibody (MBP, abcam, AB 40390), anti-neuroglia/glial antigen-2 antibody (Neural/glial antigen-2, NG, respectively ₂ EMD Millip ore Corp, clone 13238), anti-G protein-coupled receptor 17 antibody (G protein-coupled receptor 17, GPR17, bioworld, BS5741) were incubated overnight at 4 ℃ and after three washes in PBS, incubated with species-related FITC or Cy 5-labeled fluorescent secondary antibodies for 2 hours at room temperature. Nuclei are stained with staining reagents including 4', 6-diamino-2-phenylindole (DAPI). The staining images were each picked up by a leica upright fluorescence microscope, and the obtained staining images were quantitatively analyzed by Image Pro Plus software for three sections of each group of mice.

As shown in fig. 6 and table 2, when the marker MBP of the mature oligodendrocyte was detected by the immunofluorescent staining method, the fluorescence intensities of the normal group and the procyanidin B2-treated group were both significantly higher than those of the CPZ-treated group, while the fluorescence intensity of the procyanidin B2-treated group reached 19.27; for marker dMBP, the fluorescence intensity of marker MBP was higher in both the normal group and the procyanidin B2-treated group than in the CPZ-treated group, whereas the fluorescence intensity of procyanidin B2-treated group decreased to 6.969. Thus, treatment with PC-B2 reversed the expression of dMBP and MBP in the calluses of the CPZ-treated group.

As shown in FIGS. 7 to 8 and Table 2, the marker NG for oligodendrocyte precursor cells was detected ₂ And GPR17, found NG ₂ And GPR17 reduced expression in CPZ-treated mice, procyanidin B2-treated group after PC-B2 treatment, NG ₂ And significantly increased levels of GPR 17.

Table 2 effect of procyanidin B2 on remyelination

^* p<0.05， ^** p<0.01 and ^* p<0.05vs CPZ+NS

the staining experiment shows that the procyanidine B2 can obviously promote the regeneration of demyelination of a CPZ mouse model.

Experimental example 3 Proanthocyanidins B2 promotes astrocytosis

1) Effect of procyanidin B2 on inflammatory factors in vivo

The slow repair of myelin in a mouse chronic model mediated by CPZ is closely related to central nerve inflammation, and therefore, the applicant examined the effect of PC-B2 on CPZ-mediated central nerve inflammation in mice. And detecting related cytokines by adopting an Elisa method through the mouse brain tissue supernatant of the prepared normal group, the CPZ treated group, the procyanidine B2 treated group and the procyanidine B2 treated group. Changes in tumor necrosis factor-alpha (TNF-alpha, DY 410), interleukin-6 (IL-6, DY406), interleukin-1 beta (IL-1 beta, DY 401), and interleukin-10 (IL-10, DY417) were detected using a sandwich ELISA kit from R & D, three parallel controls per group.

TABLE 3 procyanidin B2 inhibits inflammatory responses of the central nervous system

^* p<0.05， ^** p<0.01 and ^* p<0.05vs CPZ+NS

as can be seen from fig. 9 and table 3, the inflammatory factors in the brain of the CPZ-treated group were higher than those of the normal group and the procyanidin B2-treated group, and the inflammatory factors of the mice of the procyanidin B2-treated group were inhibited to some extent by the treatment of PC-B2, especially TNF- α and IL-6, indicating that the inflammatory response of the central nerve mediated by CPZ was significantly inhibited by PC-B2.

2) In vivo procyanidin B2 vs. GFAP ⁺ Effect of astrocyte count

In order to investigate the mechanism of PC-B2 for inhibiting central nervous inflammatory response, the conditions of astrocytes in mouse brains were detected, and PC-B2 was found to promote CPZ-mediated proliferation of astrocytes in mouse multiple brain regions.

Brain sections of mice in the normal group, the CPZ-treated group, the procyanidin B2-treated group, and the procyanidin B2-treated group were taken and placed in PBS buffer for 10min, embedded reagent was washed off, placed in 1% bsa, blocked at room temperature for 60min, and washed three times with PBS for 10min each. After incubation with anti-Glial Fibrillary Acidic Protein (GFAP) antibody and anti-Ki 67 antibody, respectively, overnight at 4 ℃ and three washes with PBS, they were incubated with species-related FITC or Cy 5-labeled fluorescent secondary antibodies for 2 hours at room temperature. Nuclei are stained with staining reagents including 4', 6-diamino-2-phenylindole (DAPI). And the grey values of the sections were quantitatively analyzed by Image Pro Plus software.

TABLE 4GFAP ⁺ Conditions of astrocytosis

^* p<0.05， ^** p<0.01 and ^* p<0.05vs CPZ+NS

as can be seen from FIG. 10 and Table 4, the mouse models of the normal group and PC-B2-treated group had intracerebral polyencephalic GFAP regions ⁺ Astrocytes were not very different in number, although the PC-B2-treated group was GFAP ⁺ There was some increase in astrocyte number, but no significance, indicating that PC-B2 was present against GFAP in normal mice ⁺ Astrocyte proliferation had no significant effect; mouse model intracerebral multiple brain area GFAP mediated by CPZ ⁺ The number of astrocytes is obviously increased compared with that of the normal group, which indicates that CPZ can promote GFAP of normal mice ⁺ Proliferation of astrocytes, CPZ mouse model treated with PC-B2, discovery of GFAP ⁺ The number of astrocytes is further obviously increased, which shows that PC-B2 can obviously increase GFAP of intracerebral multiple brain areas of CPZ mouse model ⁺ Astrocyte number. Meanwhile, as shown in FIG. 11 and Table 4, mouse models GFAP of the normal group and PC-B2-treated group were found ⁺ The expression of Ki67 in astrocytes is not greatly different, the expression level of the CPZ group is obviously increased compared with that of a normal group, and the expression level of Ki67 in a PC-B2 treatment group is further increased compared with that of the CPZ group, which shows that PC-B2 can obviously improve the CPZ-mediated GFAP of intracerebral areas of mice ⁺ Expression of Ki67 in astrocytes. Thus, PC-B2 increased the intracerebral polyencephalic GFAP of the CPZ mouse model ⁺ Mechanism of astrocyte number and promotion of GFAP by PC-B2 ⁺ Ki67 expression in astrocytes was correlated.

EXAMPLE 4 Proanthocyanidins B2 promoting astrocyte secretion of cytokines beneficial for remyelination

1) Proanthocyanidins B2 promoting gene expression beneficial for remyelination in primary astrocytes in vitro

In order to explore the mechanism of action of PC-B2 on astrocytes to promote remyelination, in vitro PC-B2 was used to intervene in astrocytes activated by IL-1 α, TNF- α and C1q after 24h incubation, protooncogenes (C-Fos), neuregulin (Neuregin-1), ciliary neurotrophic factor (CNTF), insulin-like growth factor (IGF-1), neurotrophic factor-3 (NT 3), connexin 30 (Cx 30) and gap connexin 47 (Cx 47) which are beneficial to differentiation and survival of oligodendrocytes, fibronectin (Fibronectin), chemokine 10 (CXCL 10), platelet-derived growth factors (PDGF and basic fibroblast growth factor 2), and the like were detected by RT-PCR.

Three parallel control groups were set for each of the established normal group (Nor), astrocyte activation Model group (Model), and procyanidin B2 dry pre-group (PC-B2). After the cells were washed twice with PBS for each group of astrocytes, they were collected and then used with RNAioso Plus ⁺ (Takara, 9108) Total RNA was extracted from the cells. Extracting total RNA with PrimeScript ^TM 1st Strand cDNA Synthesis Kit (Takara, 6110A) was reverse transcribed into first Strand cDNA. Use of TB

Premix Ex Taq ^TM Quantitative PCR amplification was performed by ROX plus (Takara, RR42 LR) and CFX96 Optics Module fluorescent quantitative PCR instrument (Bio-Rad) using 2 ^-ΔΔCt The mRNA expression level was calculated. PCR primers were synthesized as shown in Table 5 by Shanghai BioTNT.

TABLE 5 primer sequences for RT-PCR

TABLE 6 Effect of procyanidin B2 on Gene expression of astrocytes beneficial or detrimental to oligodendrocyte precursor cells

^* p<0.05， ^** p<0.01 and ^* p<0.05vs Model

as can be seen from fig. 12 and table 6, the expression levels of neurotrophic factor-3 (NT 3), gap junction protein 47 (CX 47), gap junction protein 30 (CX 30), and ciliary neurotrophic factor (CNTF), which are beneficial to differentiation and survival of oligodendrocyte precursor cells, were significantly increased for astrocytes activated with IL-1 α, TNF- α, and C1q, as compared to the astrocyte activation model group, after the PC-B2 stem cells had been treated. However, protooncogenes, neuromodulators, insulin-like growth factors and fibronectin, chemokine 10, platelet-derived growth factors (PDGF and basic fibroblast growth factor FGF 2) which are beneficial to the differentiation and survival of OPCs have no significant difference with the astrocyte activation model, and the fibronectin, the chemokine 10 and the platelet-derived growth factors (PDGF and basic fibroblast growth factor FGF 2) which are harmful to the differentiation and survival of OPCs. It is demonstrated that PC-B2 can promote the expression of genes beneficial for remyelination in primary astrocytes.

2) Proanthocyanidins B2 promotes in vitro cytokine expression beneficial for remyelination in primary astrocytes

The conditions of astrocytes expressing neurotrophic factor-3 (NT 3), gap junction protein 47 (CX 47), gap junction protein 30 (CX 30) and ciliary neurotrophic factor (CNTF) were further detected at the protein level in vitro using enzyme-linked immunosorbent assay (ELISA) and Western blot assay.

And replacing the established normal group (Nor), astrocyte activation Model group (Model) and procyanidine B2 dry pre-group (PC-B2) with fresh culture solution, continuously culturing for 24h, collecting supernatant, subpackaging and storing in a refrigerator at-80 ℃ for enzyme-linked immunosorbent assay (ELISA). The cytokine was detected using the ELISA kit of Vancouv, in pg/ml. The cells of each group are respectively homogenized in RIPA lysis buffer containing protease inhibitor (100 x), after being lysed for 1h on ice, the supernatant is collected by centrifugation at 13000g for 20min at 4 ℃, and the supernatant is stored in a refrigerator at minus 80 ℃ after being subpackaged for Western blot experiment. Protein concentration was determined using BCA protein assay kit, equal amounts of protein (30. Mu.g) were separated by SDS-PAGE and half-dried on PVDF membrane, blocked with 5% skim milk powder, and incubated with anti-NT 3 antibody (Abcam, ab 263864), anti-CNTF antibody (Abcam, ab 270992), anti-CX 30 (Abcam, ab 200866), anti-CX 47 (Invitrogen, PA 5-39184) and anti-Tubulin (Bioworld, AP 0064) antibodies, respectively, overnight at 4 deg.C, after three washes with TBST, and incubated with horseradish peroxidase (HRP) -labeled secondary antibody for 1h at room temperature. After washing, protein bands were visualized using the Enhanced Chemiluminescence (ECL) system (GE Healthcare Life Sciences). Finally, the quantification software Image J was used to measure the optical density of the internal reference, tubulin, with three parallel controls per group.

Table 7 procyanidin B2 cytokine expression beneficial for remyelination in primary astrocytes in vitro

^* p<0.05， ^** p<0.01 and ^* p<0.05vs Model

as can be seen from fig. 13 and table 7, the expression levels of neurotrophic factor-3, gap junction protein 47, gap junction protein 30 and ciliary neurotrophic factor, which are beneficial to remyelination, in astrocytes activated by IL-1 α, TNF- α and C1q were significantly lower than those of primary astrocytes in the normal group, while the expression levels of neurotrophic factor-3 (NT 3), gap junction protein 47 (CX 47), gap junction protein 30 (CX 30) and ciliary neurotrophic factor (CNTF), which are beneficial to remyelination, were significantly increased after the intervention of PC-B2, and the procyanidin B2 stem group was significantly different from the astrocyte activation model group.

3) Proanthocyanidins B2 promotes the expression of cytokines beneficial for remyelination in the center of mice

Enzyme-linked immunosorbent assay (ELISA) and Westernblot assay were used to further detect the expression of neurotrophic factor-3 (NT 3), gap junction protein 47 (CX 47), gap junction protein 30 (CX 30) and ciliary neurotrophic factor (CNTF) in the mouse center at the protein level.

Mice of the established normal group, CPZ treatment group, procyanidin B2 treatment group and procyanidin B2 treatment group are sacrificed, physiological saline is perfused only, the brain is taken out and homogenized in RIPA lysis buffer containing protease inhibitor (100 x), after being lysed for 1h on ice, the supernatant is collected by centrifugation at 13000g for 20min at 4 ℃ and is used for enzyme-linked immunosorbent assay (ELISA) and Westernblot experiment. The cytokine was detected using the ELISA kit of Van. Kogyo, in pg/ml. Protein concentration was measured by BCA protein assay kit, and equal amounts of protein (30. Mu.g) were separated by SDS-PAGE and semidried on PVDF membrane, blocked with 5% skim milk powder, and then treated with anti-NT 3 antibody (Abcam, ab 263864) and anti-CNTF antibody (Abcam, ab 270992), respectively; anti-CX 30 (Abcam, ab 200866), anti-CX 47 (Invitrogen, PA 5-39184) and anti-Tubulin (Bioworld, AP 0064) antibodies were incubated overnight at 4 ℃ and after three TBST washes, incubated with horseradish peroxidase (HRP) -labeled secondary antibody for 1h at room temperature. After washing, protein bands were visualized using the Enhanced Chemiluminescence (ECL) system (GE Healthcare Life Sciences). Finally, the quantification software Image J was used to measure the optical density of the internal reference, tubulin, each set of three parallel controls.

Table 8 procyanidin B2 promotes expression of cytokines beneficial for remyelination in vivo

As can be seen from fig. 14 and table 8, the expression levels of neurotrophic factor-3, gap junction protein 47, gap junction protein 30 and ciliary neurotrophic factor, which are beneficial to remyelination, in the center of the mice in the CPZ-treated group were significantly lower than those in the center of the mice in the normal group, while the expression levels of neurotrophic factor-3, gap junction protein 47, gap junction protein 30 and ciliary neurotrophic factor, which are beneficial to remyelination, were significantly increased after the treatment with PC-B2, and the procyanidin B2-treated group was significantly different from the CPZ-treated group.

4) Proanthocyanidin B2 in vivo promotes astrocyte to secrete cell factor beneficial to remyelination

The applicant simultaneously verifies the effect of procyanidin B2 on astrocyte cytokines in vivo. Brain sections of mice in the normal group, the CPZ-treated group, the procyanidin B2-treated group and the procyanidin B2-treated group were taken, tissue sections of the mice in each group were placed in PBS buffer for 10min, the embedding agent was washed off, the mice were placed in 1% BSA for blocking at room temperature for 60min, and the mice were washed three times with PBS for 10min each, and three parallel controls were set for each group. After incubation with anti-Glial Fibrillary Acidic Protein (GFAP) antibody, anti-neurotrophic factor-3 (NT 3) antibody, anti-ciliary neurotrophic factor (CNTF) antibody, gap junction protein 47 (CX 47) antibody and gap junction protein 30 (CX 30) antibody, respectively, overnight at 4 ℃, three washes with PBS, incubation with species-related FITC or Cy 5-labeled fluorescent secondary antibodies for 2 hours at room temperature. Nuclei were stained with a stain including 4', 6-diamino-2-phenylindole (DAPI). The stained images were taken by a come card upright fluorescence microscope and three sections of each group of mice were quantitatively analyzed by Image Pro Plus software.

TABLE 9 procyanidin B2 promotes astrocyte neurotrophic factor secretion in vivo

^* p<0.05， ^** p<0.01 and ^* p<0.05vs CPZ+NS

as can be seen from fig. 15 to 18 and table 9, the expression levels of NT3, CNTF, CX30 and CX47 in mouse GFAP + astrocytes were significantly reduced in the CPZ-treated group compared to the normal group and the procyanidin B2-treated group, while the expression levels of NT3, CNTF, CX30 and CX47 in mouse GFAP + astrocytes were significantly increased in the PC-B2-treated group compared to the CPZ-treated group.

Although embodiments of the present application have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the application, the scope of which is defined in the appended claims and their equivalents.

Claims

1. Application of procyanidine in preparing medicine for promoting CNS inflammatory demyelination regeneration is provided.

2. Use according to claim 1, characterized in that the demyelination is a bicyclohexanoneoxalyl dihydrazone mediated chronic demyelination.

3. The use of claim 1, wherein the procyanidins improve the cns inflammatory microenvironment by modulating reactive astrocytosis, promoting the expression of cytokines beneficial for remyelination.

4. The use of claim 1, wherein the procyanidins promote astrocyte secretion of beneficial remyelinating cytokines.

5. The use of claim 4, wherein the cytokines conducive to remyelination comprise at least one of neurotrophin-3, ciliary neurotrophic factor, gap junction protein 30, or gap junction protein 47.

6. The use as claimed in claim 1, wherein the procyanidin is catechin, epicatechin, or dimeric procyanidin formed by polymerization of catechin and epicatechin.

7. The use as claimed in claim 6, wherein the dimeric procyanidin comprises procyanidin B1, procyanidin B2 and procyanidin B3 as isomers of each other, and the chemical formula of procyanidin B1, procyanidin B2 and procyanidin B3 is C ₃₀ H ₂₆ O ₁₂ 。

8. Use according to claim 7, wherein the procyanidin is procyanidin B2.

9. A pharmaceutical product for treating cns inflammatory demyelinating diseases, said pharmaceutical product comprising procyanidins.