CN113952344A - Application of bufalin in preparation of medicine for treating atherosclerosis - Google Patents

Abstract

The invention discloses an application of bufalin in preparing a medicament for treating atherosclerosis. The invention proves that bufalin can treat atherosclerosis induced by high fat food through in vivo and in vitro experiments, and the bufalin possibly reduces the expression of ICAM-1 at the transcription level through an NF-kB passage, thereby reducing the adhesion of monocytes to HUVEC endothelial cells and trans-endothelial migration. The bufalin can treat atherosclerosis, has a protection effect on aorta, is safe and effective, and can provide a new target for treating atherosclerosis.

Description

Application of bufalin in preparation of medicine for treating atherosclerosis

Technical Field

The invention relates to the technical field of medicines, relates to a new medical application of bufalin, and particularly relates to an application of bufalin in preparation of a medicine for treating atherosclerosis.

Background

Bufalin (bufalinin) belongs to steroid compounds and is an effective active ingredient of Bufonis venenum, which is a traditional oriental Chinese medicine obtained by secretion and drying of skin gland and retroauricular gland of Bufo siccus. The molecular formula is C₂₄H₃₄O₄The chemical structural formula (abcam) is as follows:

bufalin is a potential inhibitor of SRC-3 by inhibiting Na⁺，K⁺ATPase increases intracellular Na⁺In a concentration of Na by adjusting⁺/Ca²⁺Pump activity leads to intracellular Ca²⁺Accumulation results in increased myocardial cell contractility and vasoconstriction. Bufalin has cardiotonic, antiinflammatory and antitumor effects.

(1) Cardiotonic effect: bufalin as Na⁺，K⁺ATPase inhibitors, which promote myocardial contractility by disrupting sodium pump function and increasing myocardial intracellular calcium ion concentration without altering heart rate, are clinically used for the treatment of heart failure, coronary heart disease, and other cardiogenic diseases.

(2) Anti-inflammatory action: venenum bufonis has long been used in china to treat throat inflammation and tonsillitis diseases. The venenum bufonis steroid compound can reduce the expression of inflammation related genes such as nitric oxide and prostaglandin E2 by inhibiting NF-kB signal channels, thereby reducing capillary permeability, reducing inflammatory exudation, and being beneficial to eliminating swelling caused by inflammation. Studies have shown that bufalin can reduce the expression of IL-1 beta, IL-6, TNF alpha, nitric oxide synthase and cyclooxygenase-2 by inhibiting NF-kB signal channel, and has the functions of anti-inflammation and analgesia.

(3) The anti-tumor effect is as follows: bufalin can induce cell necrosis by increasing receptor interaction protein RIP1/RIP3/PARP-1 pathway, thereby inhibiting the occurrence and development of breast cancer; bufalin can also inhibit auroraA and auroraB kinase activation by inhibiting PI3K-Akt pathway, inhibit cancer cell growth, and exert anti-tumor effect; the bufalin can also inhibit invasion and metastasis of hepatoma cells by inhibiting PI3K/AKT/mTOR pathway to target and regulate HIF-1 alpha expression. The bufalin has the potential of anticancer drugs, and provides a solid theoretical basis for the clinical application of the bufalin.

Atherosclerosis is the common pathological basis of many cardiovascular and cerebrovascular diseases such as myocardial infarction, cerebral apoplexy and the like. In recent years, with the improvement of living standard, the change of dietary structure and the accelerated development of the aging population of the world, the incidence and mortality of atherosclerosis and related diseases are increased year by year, which is a medical and social problem to be solved at present. Dysfunction of arterial endothelial cells is the first step in the formation of atherosclerotic lesions. At sites prone to atherosclerosis, decreased expression of eNOS and SOD results in impaired endothelial barrier leading to increased accumulation and retention of LDL and VLDL-containing apoB subcutaneously. Meanwhile, the activation of NF-kB channel at the part leads to the activation of endothelial cells, thereby increasing the expression of VCAM-1, ICAM-1, P selectin, TLR2 and cytokines (such as MCP-1 and IL-8); in addition, endothelial cell activation also increases reactive oxygen species levels, thereby converting oxidized LDL to oxLDL. Endothelial cell activation leads to a monocyte recruitment cascade including rolling, adhesion, activation, and translocating. Macrophages phagocytose oxLDL into foam cells, which in turn can secrete numerous growth factors and pro-inflammatory mediators, further damaging endothelium and exacerbating monocyte recruitment, promoting plaque growth and inflammatory responses.

Currently, the treatment for myocardial hypertrophy mainly comprises: 1) lipid-lowering drug therapy: statins (Statins) and non-statin lipid lowering drugs (PCSK9 inhibitors); 2) non-lipid lowering therapy: antiplatelet agents; 3) anti-inflammatory drugs: IL-1. beta. antibody Cannabumab (canakinumab); 4) lifestyle interventions, and the like. The above treatment methods have limited effectiveness, and new therapeutic target drugs are urgently needed to be found.

Disclosure of Invention

The invention aims to provide application of bufalin in preparing a medicament for treating atherosclerosis.

In order to achieve the purpose, the technical scheme of the invention is as follows:

in one aspect of the invention, the application of bufalin or pharmaceutically acceptable salts, solvates, stereoisomers and tautomers thereof in preparation of drugs for treating atherosclerosis is provided.

Preferably, the concentration of bufalin is 1.0 mg/kg.

Preferably, atherosclerosis refers to high fat food-induced atherosclerosis.

The pharmaceutically acceptable salts include the compounds of the present invention reacted with inorganic or organic acids to form conventional pharmaceutically acceptable salts. For example, conventional pharmaceutically acceptable salts can be prepared by reacting a compound of the present invention with inorganic acids including hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, sulfamic acid, phosphoric acid and the like, or organic acids including citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, naphthalenesulfonic acid, ethanesulfonic acid, naphthalenedisulfonic acid, maleic acid, malic acid, malonic acid, fumaric acid, succinic acid, propionic acid, oxalic acid, trifluoroacetic acid, stearic acid, pamoic acid, hydroxymaleic acid, phenylacetic acid, benzoic acid, salicylic acid, glutamic acid, ascorbic acid, p-aminobenzenesulfonic acid, 2-acetoxybenzoic acid, isethionic acid and the like; or a sodium, potassium, calcium, aluminum or ammonium salt of a compound of the invention with an inorganic base; or the methylamine, ethylamine or ethanolamine salt of a compound of the invention with an organic base.

"pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances which are suitable for human use and must be of sufficient purity and sufficiently low toxicity. By "compatible" is meant herein that the components of the composition can be admixed with the compounds of formula I of the present invention, pharmaceutically acceptable salts thereof or solvates thereof and mixtures thereof without significantly diminishing the efficacy of the active ingredient. Examples of pharmaceutically acceptable carrier moieties are cellulose and its derivatives (e.g., sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (e.g., stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g., soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (e.g., propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (e.g., sodium lauryl sulfate), colorants, flavors, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

The drug can be introduced into body such as muscle, intradermal, subcutaneous, intravenous, mucosal tissue by injection, spray, nasal drop, eye drop, penetration, absorption, physical or chemical mediated method; or mixed with other materials or encapsulated and introduced into body.

If necessary, one or more pharmaceutically acceptable carriers can be added into the medicine. The carrier comprises diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorption carriers, lubricants and the like which are conventional in the pharmaceutical field.

Bufalin or pharmaceutically acceptable salts, esters, solvates, stereoisomers, tautomers and prodrugs thereof are used as active ingredients, and are used alone or in combination or are prepared into various dosage forms with other medicines, auxiliary materials and the like, wherein the dosage forms include various forms such as tablets, powder, pills, injections, capsules, films, suppositories, ointments, granules and the like. The medicaments in various dosage forms can be prepared according to the conventional method in the pharmaceutical field.

To achieve the above object, the present invention is verified by the following experiments:

1. animal experiments: the invention takes ApoE-/-mice with C57/BL6 background as research objects, establishes an atherosclerosis model by feeding high fat food, and evaluates the treatment effect of bufalin on atherosclerosis by establishing a prevention model and a treatment model of bufalin. The application dosage of bufalin is 1mg/kg, and is administered by intraperitoneal injection once a day and 6 times a week for 13 weeks. At the end of the experiment, mice were sacrificed, aorta was taken for oil red O staining, blood sampling was performed to detect blood lipid and inflammatory factor, and the results showed that bufalin has a certain protective effect on atherosclerosis in the prevention model (figure 1) and treatment model of bufalin. Bufalin may partially inhibit ICAM-1 expression through NF- κ B pathway, thereby reducing atherosclerosis. Then, the inventor detects the blood fat and inflammatory factor level in the serum, and the result shows that the bufalin does not influence the blood fat level in the prevention model, and the bufalin raises the level of total cholesterol and high-density lipoprotein in the serum in the treatment model; bufalin significantly reduces the level of IL-6 in serum in the prevention model, and substantially reduces the levels of IL-6, TNF α, and IL-1 β in serum in the treatment model.

2. In vitro cytology experiments: primary endothelial cell HUVEC is taken as a research object, firstly bufalin is used for treating for 24 hours, then TNF alpha or IL-1 beta is used for treating HUVEC cells, samples are collected after 6 hours, and the expression conditions of SRC-3, ICAM-1, p-p65 and p65 are detected. In addition, THP-1 cells were used to examine the effect of bufalin on monocyte adhesion and transendothelial migration ability. The results show that bufalin can reduce ICAM-1 expression at the transcriptional level through NF-kB pathway, thereby reducing monocyte adhesion to HUVEC endothelial cells and transendothelial migration.

Bufalin may down-regulate ICAM-1 expression via the NF- κ B pathway, thereby reducing monocyte adhesion to endothelial cells and transendothelial migration.

The invention discloses the following technical effects:

(1) bufalin can effectively treat atherosclerosis induced by high-fat food, and a specific mechanism is related to NF-kB signal channels.

(2) Bufalin may reduce ICAM-1 expression through the NF- κ B pathway, thereby reducing monocyte adhesion to HUVEC endothelial cells and transendothelial migration.

(3) The bufalin has the function of treating atherosclerosis, is safe and effective, expands the application range of the bufalin, and provides a new target for treating atherosclerosis.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. For reasons of space, they will not be described in detail.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments will be briefly described below, and it is obvious that the following drawings are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.

FIG. 1: chanduling prevention model mouse aorta gross oil red O staining condition and aorta plaque statistical result chart

A: a representative graph of aortoceros gross oil red O staining of a bufalin prevention model mouse;

b: statistical results of aortic plaque in bufalin prophylaxis model mice (x, P < 0.001).

FIG. 2: chanduling treatment model mouse aorta gross oil red O staining condition and aorta plaque statistical result chart

A: representative graph of aortosis gross oil red O staining of bufalin treatment model mouse;

b: statistical results of aortic plaques in bufalin-treated model mice (x, P < 0.01).

FIG. 3: graph of expression of SRC-3, ICAM-1, P-P65 and P65 in the aortas of bufalin prophylactic model (., P < 0.05).

FIG. 4: map of SRC-3, ICAM-1, P-P65 and P65 in the aorta of bufalin treatment model (, P < 0.05).

FIG. 5: graph of protein expression of SRC-3, ICAM-1, p-p65 and p65 in HUVEC cells treated with bufalin for 24 hours and further treated with TNF α or IL-1 β.

FIG. 6: bufalin was treated for 24 hours, calcein-labeled THP1 monocytes were added, and the results were further treated with TNF α or IL-1 β.

A: a fluorescence microscopy picture;

b: number of THP1 adhering to HUVECs compared to total HUVEC number (. prime, P < 0.001).

FIG. 7: HUVEC cells were plated in the upper chamber of a transwell, treated with bufalin for 24 hours, added to calcein-labeled THP1 monocytes, and then treated with TNF α or IL-1 β.

A: a fluorescence microscopy picture;

b: number of THP1 cells in the lower chamber of the transwell (, P < 0.05).

FIG. 8: mRNA expression profiles of ICAM-1 from HUVEC cells treated with bufalin for 24 hours and then TNF α or IL-1 β (x, P < 0.01;. P < 0.001).

A: TNF alpha treatment group

B: IL-1 beta treated group

FIG. 9: HUVEC cells transfected ICAM-1 promoter, then use bufalin treatment for 24 hours, and then TNF alpha or IL-1 beta treatment for 6 hours of the luciferase activity map (. about.P, P < 0.01;. about.P, P < 0.001).

A: TNF alpha treatment group

B: IL-1 beta treated group

FIG. 10: SRC-3 and P65 recruitment patterns (. about.P.0.01;. about.P.0.001).

FIG. 11: and after 24 hours of bufalin treatment, the SRC-3 and ICAM-1 expression profiles of ICAM-1 overexpression by ICAM-1 expression plasmids were transfected.

FIG. 12: after 24 hours of bufalin treatment, ICAM-1 expression plasmid was transfected to overexpress ICAM-1, calcein-labeled THP1 monocytes were added, and then TNF α or IL-1 β was used to treat the results.

A: a fluorescence microscopy picture;

b: the number of THPs 1 adhering to HUVECs was compared to the total HUVEC number (. about.p < 0.05;. about.p, 0.01;. about.p < 0.001).

FIG. 13: HUVEC cells were plated in the upper chamber of a transwell, treated with bufalin for 24 hours, transfected with ICAM-1 expression plasmid to overexpress ICAM-1, added to calcein-labeled THP1 monocytes, and treated with TNF α or IL-1 β.

A: fluorescence microscope

B: the number of THP1 cells in the lower chamber of the transwell was plotted (. about.P < 0.01;. about.P < 0.001).

FIG. 14: a possible action mechanism diagram of bufalin in treating atherosclerosis is shown.

Detailed Description

The inventor of the application researches extensively and deeply, finds that bufalin can improve atherosclerosis, down-regulates ICAM-1 mRNA expression, reduces monocyte adhesion and transendothelial migration capability, and can be used for treating atherosclerosis and related diseases. On the basis of this, the present invention has been completed.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures for which specific conditions are not indicated in the following examples are generally carried out according to conventional conditions (e.g.as described in Sambrook et al, molecular cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989)) or according to the conditions as recommended by the manufacturer. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

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. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.

Example 1 in vivo atherosclerotic pharmacological Activity assay

1. Animal experiments: ApoE with C57/BL6 background in the invention^-/^-The mice are taken as study objects, an atherosclerosis model is established by feeding high fat food, and the treatment effect of bufalin on atherosclerosis is evaluated by establishing a prevention model and a treatment model of bufalin.

Prevention model: ApoE^-/^-Mice were fed high fat diet for 13 weeks while 1mg/kg bufalin was intraperitoneally injected once a day, 6 times a week for 13 weeks continuously. At the end of the experiment, mice were sacrificed, aortas were taken for oil red O staining, blood sampling for blood lipid and inflammatory factors. Control and bufalin treated ApoE^-/^-Representative images of aortic gross oil red O staining and aortic plaque statistics for mice are shown in fig. 1. The expression of SRC-3, ICAM-1, p-p65 and p65 in the aorta in the bufalin prophylactic model is shown in FIG. 3.

The treatment model is as follows: ApoE^-/^-After the mice were fed high-fat diet for 10 weeks, 1mg/kg bufalin was further intraperitoneally injected once a day, 6 times a week, for 13 weeks. At the end of the experiment, mice were sacrificed, aortas were taken for oil red O staining, blood sampling for blood lipid and inflammatory factors. Control and bufalin treatmentApoE of (a)^-/^-Representative images of aortic gross oil red O staining and aortic plaque statistics for mice are shown in fig. 2. The expression of SRC-3, ICAM-1, p-p65 and p65 in the aorta in the bufalin therapy model is shown in FIG. 4.

Research results show that the bufalin plays a certain role in protecting atherosclerosis in a prevention model (figure 1) and a treatment model (figure 2) of the bufalin. Bufalin may partially inhibit ICAM-1 expression through the NF- κ B pathway (fig. 3 and 4), thereby reducing atherosclerosis.

2. Subsequently, the inventors examined the blood lipid and inflammatory factor levels in serum, and the results showed that bufalin did not affect the blood lipid levels in the prevention model, while bufalin increased the levels of total cholesterol and high density lipoprotein in serum in the treatment model (table 1); bufalin significantly reduced levels of IL-6 in serum in the prophylactic model, and IL-6, TNF α, and IL-1 β in serum in the therapeutic model (table 2).

TABLE 1 control and bufalin treated ApoE^-/^-Blood glucose and blood lipid levels in the serum of mice

TABLE 2 control and bufalin treated ApoE^-/^-Expression level of inflammatory factor in mouse serum

Example 2 in vitro cytology experiments

Primary endothelial cell HUVEC is taken as a research object, firstly bufalin is used for treating for 24 hours, then TNF alpha or IL-1 beta is used for treating HUVEC cells, samples are collected after 6 hours, and the expression conditions of SRC-3, ICAM-1, p-p65 and p65 are detected. In addition, THP-1 cells were used to examine the effect of bufalin on monocyte adhesion and transendothelial migration ability.

HUVEC cells were treated with bufalin for 24 hours, then TNF α or IL-1 β, and harvested after 6 hours to examine the protein expression of SRC-3, ICAM-1, p-p65 and p 65. The results are shown in FIG. 5.

After 24 hours of treatment with bufalin, calcein-labeled THP1 monocytes were added and treated with TNF α or IL-1 β for 6 hours, and photographed by a fluorescence microscope to calculate the ratio of the number of THP1 adhering to HUVEC to the number of total HUVEC. The results are shown in FIG. 6.

HUVEC cells were plated in the upper chamber of the transwell, treated with bufalin for 24 hours, added with calcein-labeled THP1 monocytes, treated with TNF α or IL-1 β for 6 hours, photographed by a fluorescence microscope, and the number of THP1 cells in the lower chamber was counted, and the results are shown in FIG. 7.

HUVEC cells were treated with bufalin for 24 hours and then TNF α or IL-1 β, and harvested after 3 hours and 6 hours to detect ICAM-1 mRNA expression, as shown in FIG. 8.

The luciferase activity of HUVEC cells transfected with ICAM-1 promoter, treated with bufalin for 24 hours and then treated with TNF α or IL-1 β for 6 hours was assayed, and the results are shown in FIG. 9.

HUVEC were treated with bufalin for 24 hours, then TNF α or IL-1 β for 6 hours, and cells were harvested for ChIP experiments to detect recruitment of SRC-3 and p65 to the ICAM-1 promoter, as shown in FIG. 10.

HUVEC were treated with bufalin for 24 hours, then transfected with ICAM-1 expression plasmid to overexpress ICAM-1, and the protein was collected and tested for expression of SRC-3 and ICAM-1, with the results shown in FIG. 11.

After HUVEC were treated with bufalin for 24 hours, ICAM-1 was overexpressed by transfection of ICAM-1 expression plasmid, calcein-labeled THP1 monocytes were added, and then treated with TNF α or IL-1 β for 6 hours, photographed by a fluorescence microscope, and the ratio of the number of THP1 adhering to HUVEC to the number of total HUVECs (., P < 0.05;. P, P < 0.01;. P, P < 0.001) was calculated, and the results are shown in FIG. 12.

HUVEC cells were plated in the upper chamber of the transwell, treated with bufalin for 24 hours, transfected with ICAM-1 expression plasmid to overexpress ICAM-1, added to calcein-labeled THP1 monocytes, treated with TNF α or IL-1 β for 6 hours, photographed by fluorescence microscopy, and the number of THP1 cells in the lower chamber (. about.P., P < 0.01;, P < 0.001) was counted, as shown in FIG. 13.

The results show that bufalin can reduce ICAM-1 expression at the transcriptional level through NF-kB pathway, thereby reducing monocyte adhesion to HUVEC endothelial cells and transendothelial migration (see FIGS. 5-13), and the possible action mechanism is shown in FIG. 14.

Although specific embodiments of the invention have been described above, it will be understood by those skilled in the art that the specific embodiments described are illustrative only and are not limiting upon the scope of the invention, and that equivalent modifications and variations can be made by those skilled in the art without departing from the spirit of the invention, which is to be limited only by the appended claims.

Claims (8)

1. The application of bufalin or a pharmaceutically acceptable salt thereof is characterized in that the bufalin or the pharmaceutically acceptable salt thereof is used for preparing a medicine for treating atherosclerosis.

2. Use according to claim 1, characterized in that: the concentration of the bufalin is 1.0 mg/kg.

3. The use of claim 1, wherein said atherosclerosis is high fat food-induced atherosclerosis.

4. The use of claim 1, wherein the medicament further comprises a pharmaceutically acceptable carrier.

5. The use of claim 2, wherein the pharmaceutically acceptable carrier is selected from the group consisting of: diluent, excipient, filler, adhesive, wetting agent, disintegrating agent, absorption enhancer, surfactant, adsorption carrier and lubricant.

6. The use of claim 1, wherein the bufalin or a pharmaceutically acceptable salt thereof, or the medicament is formulated as a tablet, powder, pill, injection, capsule, film, suppository, paste, or granule.

7. The use of claim 1, wherein the medicament is introduced into the body by injection, spray, nasal drip, eye drip, osmotic, absorption, physical or chemical mediated methods.

8. The use of claim 1, wherein the treatment is:

(1) downregulating ICAM-1 expression;

(2) reduce monocyte adhesion to endothelial cells and transendothelial migration;

(3) reducing IL-6 levels in serum;

(4) reducing levels of TNF α in serum; or

(5) Reduce IL-1 beta level in serum.

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