CN111228297B

CN111228297B - Application of fucoidan sulfate in promoting autophagy decomposition of ox-LDL (low-density lipoprotein) by foam cells

Info

Publication number: CN111228297B
Application number: CN202010071273.0A
Authority: CN
Inventors: 宋淑亮; 吉爱国
Original assignee: Shandong University
Current assignee: Shandong University
Priority date: 2020-01-21
Filing date: 2020-01-21
Publication date: 2021-03-19
Anticipated expiration: 2040-01-21
Also published as: WO2021147151A1; CN111228297A; KR20210096588A

Abstract

The invention discloses an application of fucosan sulfate in promoting autophagy of foam cells to decompose ox-LDL, which belongs to the technical field of pharmaceutical preparations.

Description

Application of fucoidan sulfate in promoting autophagy decomposition of ox-LDL (low-density lipoprotein) by foam cells

Technical Field

The invention belongs to the technical field of pharmaceutical preparations, and particularly relates to application of fucoidan sulfate in promoting autophagy of foam cells to decompose ox-LDL.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

With the aging and urbanization of society, the prevalence of unhealthy life style of residents and the increasing incidence of Cardiovascular and cerebrovascular diseases (CVD), it has become a worldwide problem. Atherosclerosis (AS) is the common basis for the onset of cardiovascular and cerebrovascular events such AS myocardial infarction and cerebral infarction, and the mechanism of AS is complex and the course of disease is long.

Among the pathological mechanisms of AS, abnormal lipid metabolism is an important component. Excess oxidized low density lipoprotein (ox-LDL) is the key to converting macrophages into foam cells. The specific process is that the monocyte enters endothelium through endothelium clearance, and is differentiated into macrophage under the action of inflammatory factor, etc., the scavenger receptor of the macrophage recognizes and phagocytoses ox-LDL in large quantity to form foam cell, and the foam cell is accumulated in large quantity to form lipid stripe and even atheromatous plaque. And it has been shown that in mouse and human atherosclerotic plaques, the autophagy-lysosomal system of foam cells is disrupted, so that excessively absorbed ox-LDL cannot be decomposed to accelerate the progression of AS.

Fucoidan is a sulfated polysaccharide containing high proportion of L-fucose and organic sulfate, and is mainly derived from brown algae. The current research shows that fucoidan has the functions of resisting inflammation, resisting tumor, resisting oxidation, reducing blood fat and inducing cancer cell autophagic apoptosis. However, the action of fucoidan on foam cells and the action mechanism thereof have not been reported.

Disclosure of Invention

In order to solve the problems in the prior art, the inventors have conducted long-term technical research and practical research and found that fucoidan sulfate can promote the removal of ox-LDL from foam cells. The new discovery can provide a new solution for preventing and treating atherosclerosis and a new medicine for treating cardiovascular and cerebrovascular diseases.

One of the objects of the present invention is to provide the use of fucoidan sulfate for promoting autophagy of foam cells to decompose ox-LDL.

The second object of the present invention is to provide a drug for promoting autophagy of foam cells to decompose ox-LDL.

It is a further object of the present invention to provide a pharmaceutical composition for promoting autophagy of foam cells to break down ox-LDL.

In order to achieve the purpose, the invention relates to the following technical scheme:

in a first aspect of the invention, there is provided the use of fucoidan sulfate for promoting autophagy of foam cells to break down ox-LDL.

Tests prove that the fucoidan sulfate has a better promoting effect on the removal of ox-LDL in foam cells, and the removal mechanism is determined to promote autophagy of cells, so that atherosclerosis can be effectively prevented and treated.

In a second aspect of the present invention, there is provided a drug for promoting autophagy of foam cells to decompose ox-LDL, wherein the active ingredient in the drug is fucoidan sulfate.

In some embodiments, the effective dose of the drug is 50 μ g/mL or more, and more preferably 800 μ g/mL.

Further, the medicine also comprises one or more pharmaceutically or dietetically acceptable auxiliary materials. The adjuvants can be solid or liquid. Solid form preparations include powders, tablets, dispersible granules, capsules, pills and suppositories. Powders and tablets may contain from about 0.1% to about 99.9% of the active ingredient. Suitable solid excipients may be magnesium carbonate, magnesium stearate, talc, sugar or lactose. Tablets, powders, pills and capsules are solid dosage forms suitable for oral administration. Liquid forms include solutions, suspensions and emulsions, or oral solutions with added sweeteners and contrast agents. In addition, it can be made into small water injection for injection, lyophilized powder for injection, infusion solution or infusion solution.

In a third aspect of the invention, a pharmaceutical composition is provided comprising the above-described agent in combination with at least one other agent for promoting autophagy of foam cells to break down ox-LDL.

Preferably, the pharmaceutical composition is a solid oral preparation, a liquid oral preparation or an injection.

Further preferably, the pharmaceutical composition is a tablet, a dispersible tablet, an enteric-coated tablet, a chewable tablet, an orally disintegrating tablet, a capsule, a sugar-coated agent, a granule, a dry powder, an oral solution, a small water injection for injection, a freeze-dried powder injection for injection, a large infusion solution or a small infusion solution.

The invention has the beneficial effects that:

the fucoidan sulfate can promote autophagy of cells to decompose excessive ox-LDL in foam cells, and provides a new direction for restoring the foam cells into normal macrophages, thereby achieving the effect of preventing and treating arteriosclerosis. The invention provides a new idea for the application of the fucosan sulfate.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1 is a graph showing the effect of ox-LDL on macrophages in example A: effect of ox-LDL at 0-120 μ g/mL on macrophage RAW264.7 survival; b: (ii) cholesterol ester/total cholesterol content in macrophages after ox-LDL treatment of 0-120. mu.g/mL. P < 0.05vs Group 0 μ g/mL; p < 0.01vs Group 0 μ g/mL;

FIG. 2 is a graph of oil red O staining after ox-LDL treatment in the examples;

FIG. 3 is the pharmacological activity test chart of fucoidan sulfate on foam cells in the examples, A-G: dyeing oil red O after fucosan sulfate treatment; h: the effect of fucoidan on macrophage survival; i: the effect of fucoidan on cholesterol ester content in foam cells, p < 0.05vs Model; p < 0.01vs Model; p < 0.001vs Model;

FIG. 4 is a graph showing the effect of fucoidan on the level of mRNA of a foam autophagy marker protein in examples; a: LC3B mRNA expression level; b: p62 mRNA expression level; c: TFEB mRNA expression level; d: lamp1 mRNA expression level. P < 0.05vs Model; p < 0.01vs Model; p < 0.001vs Model;

FIG. 5 is a graph showing the effect of fucoidan on the amount of expression of autophagy marker proteins in foam cells in examples, (A) Western blot results of autophagy marker proteins, and (B) ImageJ data processing results. P < 0.05vs Model; p < 0.01vs Model; p < 0.001vs Model; #, p < 0.05vs Control; p < 0.01vs Control; p < 0.001vs Control;

FIG. 6 is a graph showing the effect of fucoidan on foam cell lipid metabolism after 3-MA treatment in example;

FIG. 7 is a graph of the effect of fucoidan sulfate on lipid metabolism after treatment with Bafilomycin A1 in example;

figure 8 is the cholesterol ester content after autophagy inhibitor treatment in the examples, a: cholesterol ester content after 1.5h of 5mM 3-MA treatment; b: cholesterol ester levels changed after 2h treatment with 100nM Bafilomycin A1. P < 0.05; p < 0.01; p < 0.001.

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

Examples

The macrophage RAW264.7 was used to construct a foam cell model:

the density is 5 multiplied by 10⁴The cells were inoculated with RAW264.7 at a concentration of 80. mu.g/mL in a 96-well plate/24-well plate, incubated with ox-LDL at a concentration of 0, 20, 40, 80, 100, 120. mu.g/mL for 24 hours after attachment, stained with lipid droplets and assayed for cholesterol ester content using oil red O and cholesterol kits, respectively, and the appropriate molding concentration of the foam cells was determined to be 80. mu.g/mL from FIGS. 1 and 2.

Pharmacological activity of fucoidan sulfate (commercially available sigma) on foam cells test:

the density is 5 multiplied by 10⁴The RAW264.7 of each/mL is inoculated on a 24-well plate, the experiment is divided into a blank control group and an experiment group, after 24 hours of adherence, the blank control group is given with a culture medium, the experiment group is incubated with 80 mug/mL ox-LDL for 24 hours, then 100, 200, 400, 800 mug/mL fucosan sulfate and 342.3 mug/mL trehalose are respectively given, after 24 hours of treatment, the MTT experiment detects the cell survival rate, and oil red O and cholesterol kits are respectively used for carrying out lipid drop staining and cholesterol ester content determination, and as can be seen from figure 3, the fucosan sulfate can eliminate the ox-LDL in foam cells.

Fucoidan sulfate can promote autophagy of foam cells.

The density is 5 multiplied by 10⁴Each/mL of RAW264.7 was inoculated in a 6-well plate, the experiment was divided into a blank control group and an experimental group, the blank control group was administered with a culture medium overnight, the experimental group was administered with 80. mu.g/mL of ox-LDL, after 24 hours of treatment, the experimental group was administered with 200, 400, 800. mu.g/mL of fucoidan sulfate and 342.3. mu.g/mL of trehalose, and after 24 hours of treatment, proteins and mRNAs were extracted from cells using a cell lysate and an RNA extraction kit, respectively.

Using RevertAid^TMThe First Strand cDNAsynthesis Kit reverse transcribes the isolated mRNA into complementary cDNA and determines the effect of fucoidan sulfate on foam autophagy from the gene level using qRT-PCR for expression level determination of the autophagy markers p62, LC3B, Lamp1 and TFEB.The extracted protein is subjected to autophagy markers LC3B, p62, TFEB, Atg4B, Atg5, Atg16 and other protein expression level measurement by using Western-Blot, and the influence of fucoidan sulfate on the autophagy of foam cells is determined from the protein level. As can be seen from fig. 4 and 5, fucoidan sulfate can promote autophagy of foam cells.

The density is 5 multiplied by 10⁴Each/mL of RAW264.7 was inoculated in a 96-well plate/24-well plate, a blank control group was given with the culture medium 24h after adherence, an experimental group was given 80 μ g/mL of ox-LDL, pretreatment was performed for 1.5h and 2h with an autophagy inhibitor 5mM 3-MA (3-Methyladenine) and 100nM Bafilomycin A1 respectively 24h after treatment, a blank control group and a model control group were given with the culture medium, a drug treatment group was given 200, 400, 800 μ g/mL of fucoidan sulfate and 342.3 μ g/mL of trehalose respectively 24h, and lipid drop staining and cholesterol ester content determination were performed respectively using oil red O and cholesterol kits. As shown in fig. 6, 7 and 8, the autophagy inhibitor reversed the effect of fucoidan sulfate on the clearance of foam cell ox-LDL.

Conclusion and significance: experiments successfully construct a foam cell model by using ox-LDL, and the fucoidan sulfate can decompose excessive ox-LDL in foam cells through oil red O staining and the content measurement of cholesterol ester. In addition, by determining the mRNA and protein expression levels of autophagy markers in the foam cells after fucoidan sulfate treatment, the ox-LDL elimination effect of fucoidan sulfate was achieved by promoting autophagy in the cells and was further verified by autophagy inhibitors. The results provide a new direction for the foam cells to recover to normal macrophages and provide a new idea for the application of the fucoidan.

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

Claims

1. Use of fucoidan sulfate to promote autophagy of foam cells in an in vitro environment to degrade ox-LDL, said use not encompassing diagnosis and treatment of disease.

2. Use according to claim 1, characterized in that: the effective dose of the fucoidan sulfate is 50 mug/mL or more.

3. Use according to claim 2, characterized in that: the effective dose of the fucoidan sulfate is 800 mug/mL.