CN114209680A - Application of astaxanthin in preparation of medicine for treating vascular dementia - Google Patents

Abstract

Compared with the prior art, the experimental research of the invention shows that the astaxanthin can reduce the neurotoxic protein A beta in the brain₄₂The expression and the inhibition of inflammatory reaction in the brain, thereby playing a role in protecting neurons and improving the learning and memory ability. Therefore, astaxanthin is used for treating vascular dementia and has a unique mechanism.

Description

Application of astaxanthin in preparation of medicine for treating vascular dementia

Technical Field

The invention belongs to the field of new application of astaxanthin, and particularly relates to application of astaxanthin in preparation of a medicine for treating vascular dementia.

Background

Vascular dementia (VaD) is a deterioration in brain cell function primarily caused by cerebrovascular disease, and is the second common type of dementia following senile dementia. Oxidative stress injury and neuroinflammatory response caused by low cerebral blood flow perfusion in patients with VaD are important factors causing cognitive dysfunction of patients. In recent years, the incidence of cerebrovascular diseases is increasing, and researchers at home and abroad pay more and more attention to the research of VaD. At present, the pathogenesis of VaD is not completely clear, and the research of effective VaD drug treatment strategies has become the focus of international attention today.

Astaxanthin (AST) is a natural carotenoid existing in marine organisms such as fish, shrimp, crab and algae, and has strong biological activities such as oxidation resistance and inflammation resistance. The unique chemical structure of AST makes it easy to penetrate blood brain barrier to treat central nervous system diseases.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the technical problems, the invention provides the application of astaxanthin in preparing the medicine for treating vascular dementia.

The technical scheme is as follows: in order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows:

application of astaxanthin in preparing medicine for treating vascular dementia is provided.

The chemical name of the astaxanthin is 3,3 ' -dihydroxy-4, 4 ' -diketone-beta, beta ' -carotene, and the chemical formula is C₄₀H₅₂O₄CAS registry number 472-61-7.

Further, the present invention also provides:

the astaxanthin is applied to the preparation of the medicine for protecting cerebral neuron damage caused by ischemia-reperfusion in vascular dementia.

Application of astaxanthin in preparation of A beta for reducing vascular dementia₄₂Application in protein expression drugs.

The astaxanthin is applied to the preparation of medicines for inhibiting intracerebral inflammatory reaction in vascular dementia.

A composition for treating vascular dementia, which comprises astaxanthin or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable auxiliary material.

Preferably, the dosage form of the composition is selected from one of tablets, powders, granules, capsules, oral liquid, sustained release preparations, injections, infusion solutions and sterile powder for injection.

Use of a composition comprising astaxanthin in the manufacture of a medicament for the treatment of vascular dementia.

Further preferred is:

the application of the composition containing astaxanthin in preparing medicines for protecting cerebral neuron damage caused by ischemia-reperfusion in vascular dementia is provided.

Application of composition containing astaxanthin in preparation of medicine for reducing A beta in vascular dementia₄₂Application in protein expression drugs.

The application of the composition containing astaxanthin in preparing medicines for inhibiting intracerebral inflammatory reaction in vascular dementia.

Has the advantages that: compared with the prior art, the experimental research shows that the astaxanthin can reduce the brain neurotoxicity protein Abeta₄₂The expression and the inhibition of inflammatory reaction in the brain, thereby playing a role in protecting neurons and improving the learning and memory ability. Therefore, astaxanthin is used for treating vascular dementia and has a unique mechanism.

Drawings

FIG. 1 shows the results of Neisseria staining of hippocampal tissues of mice in each group: 1a, sham group; 1B, model group; astaxanthin low dose group; astaxanthin high dose group; large scale 200 μm; the small scale indicates 50 μm, the neural cells containing nissl are blue, and n is 15.

FIG. 2 shows the hippocampal tissue Abeta of each group of mice₄₂Immunohistochemical staining results: 1a, sham group; 1B, model group; astaxanthin low dose group; astaxanthin high dose group; the large icon indicates 200 μm; the small scale in the black box indicates 50 μm, the brown region is the positive expression region of a β 42 protein, and n is 15.

FIG. 3 shows Western blotting analysis of hippocampal tissue Abeta of each group of mice₄₂Protein expression: western blotting result; B. quantitative analysis (F: 53.312, P: 0.000), n: 15, aP compared to sham group<0.05; bP compared to model group<0.05; cP compared to astaxanthin high dose group<0.05。

FIG. 4 shows IL-4, IL-6 and Abeta of hippocampal tissues of each group of mice₄₂And (3) ELISA detection results: quantitative comparison of il-4 (F-65.613, P-0.000); quantitative comparison of il-6 (F-72.124, P-0.000); C.A.beta.₄₂Quantitative comparison results (F ═ 81.425, P ═ 0.000); aP compared to sham group<0.05; bP compared to model group<0.05; cP compared to astaxanthin high dose group<0.05；n＝10。

Detailed Description

The technical solutions of the present invention are further described in detail by the following specific examples, but it should be noted that the following examples are only used for describing the content of the present invention and should not be construed as limiting the scope of the present invention.

Example Experimental study of astaxanthin to improve learning and memory abilities of mice with vascular dementia

Materials and methods

1. Experimental Material

60 healthy male ICR cleaning-grade mice with the weight of 30-35 g are purchased from the center of Zhejiang experimental animals with the license number SCXK (Zhe) 2014-0001. AST (Shanghai Allantin reagent Co., Ltd., batch No. A114383, content not less than 98%), interleukin (interleukin, IL) -4, IL-6 cytokine determination kit (batch Nos. M150929-002a, M150826-004a, respectively, all purchased from Shenzhen Xin Bosheng Biotech Co., Ltd.), rabbit anti-mouse beta-amyloid peptide 42(amyloid beta-peptides42, Abeta. beta.) (Abeta.)₄₂) Antibody (batch number: ab10148, abcam), streptavidin-biotin-peroxidase complex technology (SABC), immunohistochemical staining kit, DA color development kit (wuhan bosch & d bioengineering ltd, lot numbers SV1022, 12J25C22, respectively).

2. Method of producing a composite material

2.1 establishment and experimental grouping of mouse VaD model: the mouse VaD model is established by clamping bilateral common carotid arteries, and the specific process is briefly described as follows: 10% chloral hydrate (300mg/kg) is injected into an abdominal cavity to anaesthetize a mouse, the mouse is fixed in a supine position, an incision is made in the middle of the neck, muscles and fascia are separated along the intramuscular lateral margin of the sternocleidomastoid to find two lateral carotid sheaths, the common carotid artery is clamped by a non-invasive artery clamp after the common carotid artery and the vagus nerve are stripped, so that the double-sided common carotid artery is blocked to cause global cerebral ischemia, the artery clamp is removed after 20min, and the vagus nerve close to the common carotid artery is prevented from being damaged in the operation. After a model is established for 24 hours, randomly dividing mice in an operation group into a sham operation group (sham), a model group (VaD), an AST low-dose group (15mg/kg, AST low-dose group) and an AST high-dose group (30mg/kg, AST high-dose group) and 15 mice in each group, and respectively carrying out intraperitoneal injection on astaxanthin vegetable oil solution with corresponding dose for 3 times per week for 6 weeks; the sham group only exposes bilateral carotid arteries separately, but does not clamp, and the rest of the procedure is the same as the surgical group. Sham and model mice were given equal amounts of vegetable oil.

2.2 training and positioning navigation and space exploration experiments: after 6 weeks, each group of experimental animals should be trained, positioned, navigated and subjected to space exploration experiments by using a water maze device. The Morris water maze mainly comprises a circular water pool and an automatic video recording and analyzing system, swimming tracks of mice can be recorded dynamically in real time, each group of mice are placed in the Morris water maze to search for a platform, the water maze is manually divided into 4 quadrants to record the time of the mice for searching for the platform within 2min, if the mice do not find the platform within 2min, the mice are guided to the platform by an experimenter and stay for 60s, the escape latency is recorded as 2min, and the mice are trained repeatedly for 5 d. Data are exported through experimental video analysis software, and the average escape latency is analyzed (the space acquisition capacity of the mouse is predicted, and the shorter the average escape latency is, the better the space acquisition capacity of the mouse is). And (3) removing the platform in the water maze after the 5-day water maze experiment is finished, allowing each group of mice to enter the water maze from the opposite side quadrant of the quadrant where the original platform is located, and measuring the times of crossing the original platform in 2min and the time of staying of the mice in each quadrant of the platform by using a Smart system so as to judge the memory storage and extraction reproduction capability of the mice.

2.3 Niey staining: after the behavioral testing of all experimental animals was completed, the rats were sacrificed by intraperitoneal injection of 10% excess chloral hydrate. Taking out whole brain, cutting coronal section by section, cutting off brain tissue before visual cross and after cerebellum, embedding and continuously slicing the left middle brain tissue, numbering the brain tissue slices of each group, selecting slices with the same serial number for Nie dyeing, dewaxing xylene, dehydrating with gradient ethanol, adding distilled water, dyeing neuron Nie body according to standard steps shown in Nie dyeing kit specification, dehydrating with anhydrous ethanol, removing xylene, sealing with neutral gum, and observing under an optical microscope.

2.4 Aβ₄₂Immunohistochemical staining: when immunohistochemical staining was performed, 5 μm sections of the same sequence number were selected and the sections were guaranteed to have a classical hippocampal structure (contiguous CA1, CA3 and DG 3 regions). Taking the hippocampal tissue slices, and carrying out Abeta according to the specification of the SABC immunohistochemical staining kit₄₂Immunohistochemical staining, the specific steps are briefly described as follows: tissue sections are processed by absolute ethyl alcohol until the gradient concentration ethanol is obtainedAdding distilled water for hydration, and incubating with 3% hydrogen peroxide at room temperature for 30min to inactivate endogenous peroxidase; washing with PBS for 20 min. Adding blocking solution (5% sheep serum + 1% BSA + 0.05% Tween-20) to block the slide for 30min at room temperature; removing excess blocking solution, adding rabbit anti-mouse Abeta₄₂Polyclonal antibody (1:200), incubating at room temperature for 1h, washing with PBS for 10min, adding biotinylated goat anti-rabbit IgG (1:200), incubating at room temperature for 30min, and washing with PBS for 10 min. Adding HRP-labeled ovalbumin, and incubating for 30 min. And (3) after the PBS is washed, DAB is added for color development, ethanol is gradually dehydrated, and the neutral resin is sealed. Blank control was performed with PBS instead of primary antibody. Serial histological sections with similar cross-sectional levels were taken from each mouse for observation.

2.5 Western blotting analysis of Abeta₄₂Expressing: 15 mice were taken from each group, brain hippocampal tissue was isolated, total protein was extracted from RIPA lysate, and the total protein was measured with BCA kit. Taking 70 mu g protein sample, 15 mu l system, adding rabbit anti-mouse Abeta after electrophoresis, membrane transfer and sealing₄₂Incubating the antibody (1:500) at 4 ℃ overnight, adding goat anti-rabbit secondary antibody (1:3000) for hybridization reaction for 1h, incubating at room temperature for 2h, washing with membrane washing buffer (TBST) for 3 times, each time for 10min, and developing. Western blotting imaging was analyzed by quantitative One 4.6.2 software, and the relative expression of each histone and internal reference GAPDH was calculated.

2.6 Hippocampus brain tissues IL-4, IL-6 and Abeta₄₂Content determination: respectively taking the brain hippocampus tissues of each group of mice, weighing, adding pure water according to a ratio of 1: 9(m/V), ultrasonically homogenizing the tissues at low temperature, centrifuging at 4 ℃ and 10000r/min for 10min, and taking the supernatant for later use. Determination of IL-4, IL-6 and Abeta in Hippocampus tissues by ELISA kit₄₂Protein content, the specific operation steps are strictly carried out according to the kit instructions.

3. Statistical analysis

Processing was performed with the SPSS 17.0 statistical software package. Data of the experiment are mean. + -. standard deviation

The mean comparison between two groups adopts t test, the comparison between multiple sample means adopts One-Way ANOVA test, the water maze data adopts repeated design variance analysis, and P is less than 0.05, and the difference is statisticThe meaning of learning.

Second, result in

1 Water maze test results

As shown in table 1, on days 4 and 5, the average escape latency of mice in the VaD group was significantly higher than that of the sham group, and the difference was statistically significant (P was less than 0.05), while the platform crossing times and residence time of mice in the VaD group were significantly lower than those of the sham group (P was less than 0.05); compared with the VaD group, mice in the different concentration AST intervention group had significantly decreased escape latency on both day 4 and day 5 (P both <0.05), while platform crossing times and residence time were significantly increased (P < 0.05); in addition, the avoidance latency was significantly decreased on both day 4 and day 5 of the AST high dose group (P both <0.05), and the number of platform crossings and residence time were significantly increased (P both <0.05) compared to the AST low dose group.

TABLE 1 mean escape latency and space exploration experimental results for each group of mice

1. The residence time of the mouse in the quadrant where the platform is located in the space exploration experiment accounts for the percentage of the total experiment time; aP <0.05 compared to sham group; bP <0.05 compared to model group; cP <0.05 compared to astaxanthin high dose group

2. Results of brain histopathology

The results of Niger staining of brain tissue of each group of mice are shown in FIG. 1. Compared with the sham group, after 6 weeks, neurons in CA1, CA3 and DG regions of hippocampal tissues of the brain of mice in the VaD group are obviously solidified and contracted and deeply stained, the neurons are obviously lost, gaps around the cells are enlarged and become vacuole-like lesions, and the cells are not arranged in a hierarchical manner. After the dry prognosis of AST treatment with different concentrations, the number of mouse brain tissue Neisseria staining positive neurons is obviously increased, the neuron shrinkage degree is reduced, the intercellular spaces are not vacuolated, particularly, the neurons in the CA1, CA3 and DG areas of mouse brain tissue hippocampus in an AST high-dose intervention group are clearly visible, and the neurons and the interstitial structures thereof are complete and approximate to the level of a sham group.

Immunohistochemical results for a β 42 protein in various regions of hippocampal brain tissue of each group of mice are shown in fig. 2. Abeta (beta)₄₂The protein expression region was dark brown. A beta of each region of hippocampus of VaD group mice₄₂The positive expression of the protein is obviously higher than that of the sham group; after the prognosis of different AST concentrations, Abeta of each region of mouse hippocampal tissues₄₂The protein expression is obviously reduced compared with the VaD group, and the effect is obviously dose-dependent.

Western blotting analysis of Hippocampus tissues Abeta of various groups of mice₄₂Protein expression

Hippocampus tissue Abeta of each group of mice₄₂The Western blotting detection results of protein expression are shown in FIG. 3, and the statistical results show that compared with the sham group, the hippocampal Abeta of mice in the VaD group₄₂Protein expression is significantly increased (P)<0.05); after different forms of AST treatment intervention, Abeta of mouse hippocampal region₄₂The protein is obviously reduced compared with the VaD group (P is equal to<0.05), and the mice hippocampal tissue Abeta with AST high dose group prognosis₄₂Protein expression is obviously reduced compared with AST low dose group and model group (P is equal to<0.05)。

4. Hippocampus cerebri tissue IL-4, IL-6 and Abeta₄₂Results of content measurement

Hippocampus tissues IL-4, IL-6 and Abeta of each group of mice₄₂The results of the content measurement are shown in FIG. 4. Mouse hippocampal IL-4 expression was significantly reduced in the VaD group compared to the sham group (P)<0.05), IL-6 and Abeta₄₂Significantly increased expression (P)<0.05); compared with the VaD group, the IL-4 content of the hippocampal tissue of the mice subjected to different-concentration AST treatment prognosis is remarkably increased (P)<0.05), and IL-6 and Abeta₄₂The content is remarkably reduced (P)<0.05), and the results are dose-dependent, AST high dose groups mice hippocampal IL-4, IL-6 and Abeta₄₂The content was close to the sham group level.

The Morris water maze experiment utilizes the astronomy that a mouse does not like swimming and tends to escape water to search for a platform hidden in the water, and the mouse uses different surrounding markers to distinguish the position of the platform after continuous training, so that stable spatial cognitive memory is formed and is widely used for evaluating the learning and memory functions of the mouse. The main reason for the obvious difference of the average escape latency of the platforms of each group on the 4 th day and the 5 th day of the water maze is that as the training days increase, the sham group with normal learning and memory ability gradually forms stable memory for the platform, so that the time for searching the platform (average escape latency) is obviously shorter than that of the mice in the VaD group with impaired learning and memory ability and without stable memory for the platform (P < 0.05). The results of the space exploration experiments also further confirm that mice in the VaD group have obvious decline in learning and memory abilities compared with those in the sham group. In addition, consistent with previous literature reports, pathological results show that neuronal cells in hippocampal CA1, CA3 and DG regions are shrunk and the number of neurons positive for Niger staining is obviously reduced after 6 weeks in mice in VaD group, which indicates that ischemia reperfusion can cause the mice to be damaged in learning and memory functions. After AST treatment with different concentrations is used for prognosis, water maze and space exploration experiments show that the learning memory ability and the space memory ability of the mouse are obviously improved compared with those of a VaD group, the effect is dose-dependent, meanwhile, Niger dyeing shows that the quantity of hippocampal tissue neurons and Niger corpuscles of different dose intervention groups of AST is obviously higher than that of the VaD group, and the neurons are arranged in order, which shows that the AST can protect cerebral neuron damage caused by ischemia reperfusion, thereby improving the learning and cognitive dysfunction of VaD animals.

The present invention also initially investigates the molecular mechanism of AST treatment of VaD. Long-term hypoxic hypoperfusion of VaD results in high expression of a β. A beta is obtained by the proteolytic cleavage of beta Amyloid Precursor Protein (APP), and under pathological conditions, abnormal hydrolysis of APP leads to the increasing of A beta, and after aggregation, A beta oligomer is formed or high beta folding is aggregated to form insoluble A beta fiber or plaque, so that neurotoxicity is generated. A beta is mainly A beta₄₀And Abeta₄₂Two subtypes. And Abeta₄₀In contrast, Abeta₄₂Has the characteristics of low solubility, higher neurotoxicity and the like, and plays a key role in plaque polymerization. It has been shown that A.beta.₄₂Deposition can activate microglia to release proinflammatory cytokines such as IL-6 and the like, so that astrocytes are activated, proliferated and released to inflammatory proteins, and A beta is promoted₄₂Conversion to neurotoxic insolublePrecipitating to further activate microglia to release inflammatory factors so as to form a chronic inflammatory response. IL-4 is a major anti-inflammatory factor secreted by glial cells and is capable of providing a danger signal to neural stem cells, the level of which indirectly reflects the level of inflammation in the brain. Immunohistochemical staining and Western blotting analysis of groups of mice from 6 weeks later, and IL-4, IL-6 and Abeta₄₂ELISA detection results show that the expression of hippocampal Abeta protein and the content of inflammatory factor IL-6 of mice in the VaD group are obviously higher than those in the sham group, and the content of anti-inflammatory factor IL-4 is lower than that in the sham group, which indicates that animals in the VaD group have obvious inflammatory response, and the results are consistent with the above documents. After different dose of AST treatment, the mouse hippocampal region Abeta₄₂The protein expression and IL-6 content are obviously reduced compared with that of the VaD group, the IL-4 content is obviously increased, and the effect is dose-dependent, which indicates that the AST can reduce the A beta in the brain of the VaD mouse₄₂The expression of protein and inflammation related factor IL-6 can raise the content of anti-inflammatory factor IL-4, reduce inflammation reaction and play the role of protecting neuron.

In conclusion, the research of the invention proves that astaxanthin can reduce A beta₄₂The expression of the protein and the inhibition of inflammatory reaction in brain play a role in protecting neurons and improve the learning and cognitive functions of a mouse model with vascular dementia.

Claims (10)

1. Application of astaxanthin in preparing medicine for treating vascular dementia is provided.

2. The use according to claim 1, wherein the astaxanthin is used in the preparation of a medicament for protecting cerebral neuronal damage caused by ischemia-reperfusion in vascular dementia.

3. Use according to claim 1, wherein astaxanthin is used in the preparation of a composition for reducing A β in vascular dementia₄₂Application in protein expression drugs.

4. The use according to claim 1, wherein the use of astaxanthin in the manufacture of a medicament for inhibiting an inflammatory response in the brain in vascular dementia.

5. A composition for treating vascular dementia, which comprises astaxanthin or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable auxiliary material.

6. The composition according to claim 5, wherein the composition is in a dosage form selected from one of tablets, powders, granules, capsules, oral liquids, sustained release preparations, injections, infusions, and sterile powders for injections.

7. Use of a composition comprising astaxanthin in the manufacture of a medicament for the treatment of vascular dementia.

8. The use according to claim 7, wherein the composition comprises astaxanthin for the preparation of a medicament for the protection of cerebral neuronal damage caused by ischemia-reperfusion in vascular dementia.

9. Use according to claim 7, wherein the composition comprising astaxanthin is used in the preparation of a composition for reducing A β in vascular dementia₄₂Application in protein expression drugs.

10. Use according to claim 7, wherein the composition comprises astaxanthin for the preparation of a medicament for the inhibition of inflammatory reactions in the brain in vascular dementia.

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