Application of theaflavin in preparation of medicine for preventing and treating aortic dissection

Abstract

The invention discloses application of theaflavin in preparation of drugs for preventing and treating aortic dissection. According to the application of theaflavin provided by the invention, the theaflavin mediates macrophage polarization and inflammation imbalance by regulating and controlling an NLRP3 small body pathway, participates in attack of aortic dissection, and can effectively relieve occurrence and development of aortic dissection.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to the application of theaflavins in the field of prevention and treatment of aortic dissection. Background Art

[0002] This section merely provides background information related to the present disclosure and is not necessarily prior art.

[0003] Aortic dissection (AD) is a critical cardiovascular disease that seriously threatens the life and health of Chinese people. The onset population is mainly concentrated in the age group of 65-75 years old, and about 24 patients will be affected every year in 100,000 people. In addition to age, the main risk factors are hypertension, dyslipidemia, and genetic diseases that affect connective tissue, such as Marfan syndrome. The clinical manifestation of aortic dissection is a tear in the aorta's intima. Blood enters the junction of the aortic media and adventitia from the tear, causing separation of the middle layer of the aortic wall, accompanied by a very high risk of rupture. Therefore, its onset is often rapid and life-threatening. Exploring the influencing factors of the occurrence and development of aortic dissection, finding early marker changes and intervention sites at the tissue and molecular levels, and using this to quickly confirm the diagnosis and appropriate intervention treatment have always been hot topics and focuses of research in this field.

[0004] Black tea is one of the traditional teas originated in my country. It has always been loved by the public for its mellow taste and health benefits. Theaflavin is one of the main components of black tea. It is a general term for a class of compounds with benzophenones that are soluble in ethyl acetate and formed by the oxidation of polyphenols. Studies have shown that theaflavin can significantly reduce the mRNA level of inflammatory cytokines induced by lipopolysaccharide (LPS) by blocking the NF-κB signaling pathway. Theaflavin can also inhibit the inflammatory response caused by cerebral hemorrhage and sepsis caused by digestive system infection in vivo by inhibiting NF-κB and NLRP3 pathways. In addition, the NLRP3 body pathway and macrophage polarization, which are closely related to the inflammatory response, are considered to be one of the important factors affecting the progression of aortic dissection. However, it is not clear whether theaflavin can affect the progression of aortic dissection by regulating the NLRP3 body pathway and macrophage M1 polarization. In view of the beneficial effects of dietary theaflavins and their low toxicity, exploring the effects of theaflavins on the occurrence and development of aortic dissection through their regulatory effects on NLRP3 bodies and induction of macrophage M1 polarization has important implications for the prevention and treatment of aortic dissection. Summary of the invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide the use of theaflavins in the preparation of drugs for preventing and treating aortic dissection in view of the deficiencies in the prior art.

[0006] In order to solve the above technical problems, the present invention discloses the following technical solutions:

[0007] In a first aspect, the present invention discloses the use of theaflavins in the preparation of a drug for preventing and / or treating aortic dissection.

[0008] In a second aspect, the present invention discloses a drug for preventing and / or treating aortic dissection, wherein the drug comprises theaflavins.

[0009] The drug has any one or more of the following effects:

[0010] (a) Inhibit the occurrence of aortic dilatation and dissection;

[0011] (b) inhibiting aortic dilatation and dissection;

[0012] (c) Improvement of aortic intramural hematoma and false lumen;

[0013] (d) reduce the expression of inflammatory factors in aortic tissue;

[0014] (e) reducing the infiltration of inflammatory macrophages in aortic tissue;

[0015] (f) Reduce the M1 polarization of macrophages in the body;

[0016] (g) Reduced the protein expression level of NLRP3.

[0017] Wherein, the inflammatory factor is any one or more of IL-1β, IL-6 and TNF-α.

[0018] Wherein, the inflammatory macrophages are M1 macrophages.

[0019] In a third aspect, the present invention discloses the use of theaflavins in the preparation of drugs for inhibiting macrophage polarization.

[0020] In a fourth aspect, the present invention discloses a drug for inhibiting macrophage polarization, wherein the drug comprises theaflavins.

[0021] In a fifth aspect, the present invention discloses a pharmaceutical preparation comprising i) theaflavin; and ii) a pharmaceutically acceptable excipient.

[0022] In a sixth aspect, the present invention discloses the use of the above-mentioned pharmaceutical preparation in preparing a drug for preventing and / or treating aortic dissection, and / or in preparing a drug for inhibiting macrophage polarization.

[0023] The therapeutic effective rate of the medicine or preparation of the present invention is 1-2.2 mg theaflavins / kg, such as 1.2-2 mg theaflavins / kg, 1.4-1.8 mg theaflavins / kg.

[0024] The present invention provides the application of theaflavins to mediate macrophage polarization and inflammatory imbalance by regulating the NLRP3 body pathway, and participate in the pathogenesis of aortic dissection. The method comprises administering theaflavins to mice orally to reduce aortic dissection formed by BAPN modeling at the tissue level; and adding theaflavins to the culture supernatant to inhibit the activation of the NLRP3 body pathway and macrophage M1 polarization induced by LPS and adenosine triphosphate (ATP).

[0025] Beneficial effects:

[0026] By gavaging mice with theaflavins and treating macrophages with theaflavins, the NLRP3 body pathway and macrophage M1 polarization were inhibited, which effectively alleviated the occurrence and development of aortic dissection. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0028] Figure 1 The survival curves of mice treated with 0.5% BAPN in drinking water and 0.5% BAPN in drinking water and theaflavins by oral gavage.

[0029] Figure 2 The differences in the incidence of various aortic events were observed between the administration of 0.5% BAPN in drinking water and the combined administration of 0.5% BAPN in drinking water and theaflavins by oral gavage.

[0030] Figure 3 This is a representative gross image of the mouse aorta: the mice were administered with normal drinking water, 0.5% BAPN drinking water, and 0.5% BAPN drinking water and theaflavins by oral gavage. After the modeling was completed, the mice were euthanized and the aortic tissues were dissected and observed and photographed under a microscope.

[0031] Figure 4 The representative statistics of the aortic diameter of mice: normal drinking water, 0.5% BAPN drinking water, and 0.5% BAPN drinking water and theaflavins were administered by intragastric administration. Cardiac ultrasound was performed at week 0, week 2, and week 4 to observe the formation of aortic aneurysms / dissections, and the maximum inner diameter of the aorta was measured at week 4. ***P<0.001, ****P<0.0001.

[0032] Figure 5 Representative images of EVG staining and Masson staining of mouse aorta: aortic tissues of mice administered with normal drinking water, 0.5% BAPN drinking water, and 0.5% BAPN drinking water and theaflavin by oral gavage were dissected and embedded in paraffin, sliced, and EVG staining, Masson staining and scanning were performed.

[0033] Figure 6 The effect of theaflavins on the mRNA expression levels of various inflammatory factors in the macrophage M1 polarization model: After 500nM PMA stimulation for 16h to induce THP1 cells to differentiate into macrophages, the cells were treated with PBS, LPS (500ng / mL) + ATP (5mM), and LPS (500ng / mL) + theaflavins + ATP (5mM) for 4h, and the mRNA expression levels of macrophage inflammatory factors were detected by qPCR. *P<0.05, **P<0.01.

[0034] Figure 7 The effect of theaflavins on the expression level of NLRP3 protein in the macrophage M1 polarization model: After 500nM PMA stimulation for 16h to induce THP1 cells to differentiate into macrophages, the cells were treated with PBS, LPS (500ng / mL) + ATP (5mM), and LPS (500ng / mL) + theaflavins + ATP (5mM) for 4h, and the protein was extracted by RIPA method, and the expression level of NLRP3 protein was detected by Western Blot and quantified. *P<0.05.

[0035] Figure 8 for Figure 7 Quantization diagram of . DETAILED DESCRIPTION

[0036] The present invention can be better understood according to the following examples. However, it is easy for those skilled in the art to understand that the contents described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0037] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0038] Example

[0039] 1. Modeling and grouping

[0040] In order to clarify the role of theaflavin in aortic dissection, we fed 4-week-old wild-type male C57BL / 6J mice with normal drinking water (Sham group), drinking water containing 0.5% BAPN (BAPN group), drinking water containing 0.5% BAPN and theaflavin (BAPN+Theaflavin group, theaflavin dose was 10 mg / kg / d) by gavage. Blood pressure and cardiac ultrasound were measured before the model was established, two weeks later, and four weeks later. The model was terminated after 4 weeks, and the aortic vascular tissues of the mice were removed after euthanasia.

[0041] 2. Survival rate, incidence of various aortic events, aortic tissue

[0042] After 4 weeks of modeling, there was no death or aortic dilatation in the normal drinking water group, while the mortality rate of aortic rupture in the BAPN group reached 40%, and the surviving mice generally developed aortic dissection, aneurysm or severe dilatation. The mice fed with BAPN water and given theaflavins by gavage had less rupture death, dissection aneurysm formation and severe dilatation ( Figure 1 , Figure 2 We found that no mice in the normal drinking water group had aortic dissection and significant dilatation. BAPN drinking water administration significantly induced aortic dilatation and dissection in mice, and theaflavins significantly reduced the incidence of these events ( Figure 3 , Figure 4 ).

[0043] 3. EVG and Masson staining of aorta

[0044] The aortic tissues of mice in different groups were paraffin-embedded, sectioned, and stained with EVG and Masson.

[0045] (1) Immediately after dissection, obtain mouse aortic tissue and immerse it in 4% PFA for 4-24 hours. After fixation, dehydrate the tissue so that it can be embedded in water-insoluble paraffin. Soak the tissue in increasing concentrations of a dehydrating agent such as ethanol to gradually dehydrate it. Then, incubate in xylene to replace the dehydrating agent and make the tissue transparent. Heat the paraffin to 60°C to embed the tissue, then let it harden overnight and slice the tissue using a microtome.

[0046] (2) EVG staining: Dewax the sections and stain them with the prepared Verhoeff's stain solution for 1-3 minutes at room temperature until the color turns dark black. After rinsing with ddH2O, differentiate with 2% ferric chloride solution for 10-20 seconds. Observe the elastic fibers under a microscope and they will appear black, while the background will appear gray. After a brief rinsing with ddH2O, counterstain with Van Gieson's solution for 10-15 seconds and wash with anhydrous ethanol. Dehydrate with anhydrous ethanol, make transparent with xylene, and seal with neutral gum.

[0047] (3) Masson staining: paraffin sections were dewaxed to water, rinsed with ddH2O, and then stained with Regaud hematoxylin or Weigert iron hematoxylin for 5–10 min; differentiated with 1% hydrochloric acid alcohol for 5–15 s, and washed thoroughly with water; added 0.1–1% lithium carbonate for 5 min to increase the degree of blueing, and washed with water; used Masson acidic Ponceau red solution for 5–10 min; washed with 2% glacial acetic acid aqueous solution for 1 min; differentiated with 1% phosphomolybdic acid aqueous solution for 3–5 min, and washed with 2% glacial acetic acid aqueous solution for 1 min; stained directly with aniline blue aqueous solution or 1% light green aqueous solution for 1–2 min without washing; washed with 0.2% glacial acetic acid aqueous solution for 1 min; dehydrated with 95% ethanol for 2–3 s, dehydrated with anhydrous ethanol 3 times for 5 s each, transparentized with xylene 3 times for 1 min each, and sealed with neutral gum.

[0048] We also found that the number of elastic fiber breaks in the aorta middle layer of mice drinking BAPN water was significantly higher than that of the normal drinking water group, and many individuals had obvious aortic intramural hematoma and false lumen; however, oral administration of theaflavins could significantly improve this phenomenon ( Figure 5 ).

[0049] 4. RNA extraction and RT-qPCR experiment of mouse aorta tissue

[0050] In order to clarify whether theaflavins affect aortic dissection by regulating the NLRP3 body pathway and macrophage M1 polarization, we verified it at the mRNA level and protein level. After the aorta was removed after the animal modeling, we extracted the aortic tissue RNA and performed RT-qPCR experiments.

[0051] 4.1 RNA extraction from mouse aorta tissue

[0052] (1) Fresh mouse aorta tissue was quickly frozen with liquid nitrogen and quickly transferred to a mortar pre-cooled with liquid nitrogen. The aorta tissue was ground with a pestle while continuously adding liquid nitrogen until it was ground into powder.

[0053] (2) The ground aortic tissue was transferred to a centrifuge tube, and 500 μl of FreeZol Reagent (Vazyme) was added for every 50 mg of tissue. The tissue was vortexed to fully lyse the tissue and then allowed to stand at room temperature for 5 min.

[0054] (3) Add Dilution Buffer (Vazyme) to the above lysate, with 100 μl of Dilution Buffer for every 500 μl of FreeZol Reagent. Close the centrifuge tube cap tightly, vortex until the solution is fully mixed, and let it stand at room temperature for 5 min.

[0055] (4) Centrifuge at 11,200 rpm (12,000 × g) at room temperature for 15 min. Carefully pipette the supernatant into a new centrifuge tube, add an equal volume of isopropanol, mix thoroughly by inverting, and let stand at room temperature for 10 min.

[0056] (5) Centrifuge at 11,200 rpm (12,000 × g) for 10 min at room temperature and carefully discard the supernatant.

[0057] (6) Add 1 ml of 75% ethanol (prepared with RNase-free ddH2O). Tap the bottom of the tube gently to suspend the precipitate, invert it several times, centrifuge at 9,100 rpm (8,000 × g) at room temperature for 3 min, and discard the supernatant.

[0058] (7) Repeat step (6), discard the supernatant and place at room temperature to dry, add 100 μl RNase-free ddH2O to dissolve the precipitate, and vortex at room temperature to fully dissolve the RNA precipitate.

[0059] 4.2 RT-qPCR

[0060] (1) Genomic DNA removal

[0061]

[0062] (2) Reverse transcription reaction

[0063]

[0064] (3)SYBR Green I chimeric fluorescence qPCR reaction

[0065]

[0066] Note: Primer Premier 5 software was used to assist in the design of primers. All primers were synthesized by GenScript Biotech Co., Ltd.

[0067] We found that compared with the control group, the mRNA expression levels of M1 macrophage proinflammatory factors such as IL-1β, IL-6, and TNF-α in the aorta tissue of mice in the BAPN group were significantly increased, while this trend was significantly inhibited in the theaflavins gavage group ( Figure 6 ).

[0068] 5. Cell Protein Lysis and Western-Blot

[0069] To further verify that theaflavins can inhibit the NLRP3 body pathway, we used PMA to induce THP-1 cells to differentiate into macrophages, and used LPS+ATP to stimulate macrophages to transform into M1 phenotype, and then gave theaflavins intervention. The groups were PBS, LPS (500ng / mL) + ATP (5mM), LPS (500ng / mL) + theaflavins + ATP (5mM). Then the Western-Blot experiment was performed as follows:

[0070] (1) Use clean instruments to dissect mouse aortic tissue, place the tissue in a centrifuge tube and immerse it in liquid nitrogen for quick freezing, then place it on ice and immediately homogenize it. Add 300 μL of lysis buffer to every 5 mg of tissue, homogenize it with an electric homogenizer, and shake it continuously at 4°C for 2 hours.

[0071] (2) Centrifuge at 11,200 rpm (12,000 × g) at room temperature for 15 min. Gently remove the centrifuge tube from the centrifuge and place it on ice. Aspirate the supernatant and transfer it to a new tube pre-cooled on ice. Discard the precipitate.

[0072] (3) Take a small amount of lysate for protein quantitative analysis, add 1 / 4 volume of 5× loading buffer to the remaining lysate and boil it at 100°C for 5 minutes for reduction and denaturation.

[0073] (4) Gel running: Load equal amounts of protein and molecular weight markers into the wells of the SDS-PAGE gel. The total protein loading amount of the aorta tissue homogenate is 20-30 μg, and run the gel at 100 V for 1-2 hours.

[0074] (5) Transfer: Activate PVDF with methanol for 1 min and rinse with transfer buffer before preparing the transfer sandwich. The transfer time and voltage are optimized according to the molecular weight of the target protein.

[0075] (6) Blocking: After the transfer, remove the PVDF membrane and immerse the membrane in TBST containing 5% skimmed milk powder (TBS is added with Tween-20 at a concentration of 0.05%) for blocking for 1 h.

[0076] (7) Primary antibody incubation: After blocking, incubate the PVDF membrane with primary antibody at an appropriate dilution in blocking buffer and shake at 4°C overnight.

[0077] (8) Secondary antibody binding: Wash the membrane with TBST for 10 min × 3 times. Incubate the membrane with the recommended dilution of the coupled secondary antibody in blocking buffer at room temperature for 1 hour. Wash the membrane again with TBST for 10 min × 3 times.

[0078] (9) ECL color development: Mix ECL color development solution A and B and evenly add them onto the membrane surface. Expose the membrane in a dark place and observe the results.

[0079] The results showed that the protein expression level of NLRP3 in macrophages was significantly increased after LPS+ATP stimulation, and this increase trend could be significantly inhibited by theaflavins ( Figure 7 , Figure 8 ).

[0080] The above experimental results can prove that theaflavins can effectively inhibit the NLRP3 body pathway and macrophage M1 polarization.

[0081] The above experimental results can fully prove that the activation of the NLRP3 body pathway and the M1 polarization of macrophages are involved in the development of aortic dissection, and theaflavins can effectively slow down and inhibit this process. By increasing the intake of theaflavins, the occurrence of aortic dissection can be effectively prevented. Therefore, we believe that theaflavins can be used as a new important drug for the prevention of aortic dissection in clinical practice, and have potential clinical application value in the prevention and treatment of aortic dissection.

[0082] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. The use of theaflavins in the preparation of drugs for preventing and / or treating aortic dissection.

2. A drug for preventing and / or treating aortic dissection, characterized in that: The drug includes theaflavins.

3. The drug according to claim 2, characterized in that The drug has any one or more of the following effects: (a) Inhibit the occurrence of aortic dilatation and dissection; (b) inhibiting aortic dilatation and dissection; (c) Improvement of aortic intramural hematoma and false lumen; (d) reduce the expression of inflammatory factors in aortic tissue; (e) reducing the infiltration of inflammatory macrophages in aortic tissue; (f) Reduce the M1 polarization of macrophages in the body; (g) Reduced the protein expression level of NLRP3.

4. The drug according to claim 3, characterized in that The inflammatory factor is any one or more of IL-1β, IL-6 and TNF-α.

5. The drug according to claim 3, characterized in that The inflammatory macrophages are M1 macrophages.

6. Application of theaflavins in the preparation of drugs for inhibiting macrophage polarization.

7. A drug for inhibiting macrophage polarization, characterized in that: The drug includes theaflavins.

8. A pharmaceutical preparation, characterized in that The invention comprises i) theaflavins; ii) pharmaceutically acceptable auxiliary materials.

9. Use of the pharmaceutical preparation according to claim 8 in the preparation of a drug for preventing and / or treating aortic dissection and / or inhibiting macrophage polarization.

10. The use according to claim 1, 6 or 9, characterized in that: The therapeutically effective amount of the drug or preparation is 1-2.2 mg / kg.

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