Application of salvianolic acid A in preparation of medicine for preventing and treating advanced atherosclerosis vulnerable plaques

Abstract

The invention discloses application of salvianolic acid A in preparation of a medicine for preventing and treating vulnerable plaques of advanced atherosclerosis. The invention also discloses application of the salvianolic acid A combined with ferulic acid or the salt thereof in preparation of drugs for preventing and treating advanced atherosclerosis vulnerable plaques. Researches find that salvianolic acid A can promote apoptosis of senescent vascular smooth muscle cells and has a Senlytics effect; the salvianolic acid A stabilizes vulnerable atherosclerotic plaques by reducing the load of senescent vascular smooth muscle cells; the salvianolic acid A is in non-covalent binding with ASP54, ALA55, LYS58, ASP93, ASP102 and LYS112 of the HSP90AA1 to play a role in inhibition, and the ASP93 has the greatest contribution to binding free energy. The research of the invention is of great significance to the development and application of drugs with anti-aging effect for stabilizing advanced atherosclerotic vulnerable plaques.

Description

Technical Field

[0001] The invention relates to application of salvianolic acid A in preparing medicine for preventing and treating vulnerable plaques of late atherosclerosis, belonging to the technical field of medicines for treating atherosclerosis. Background Art

[0002] Atherosclerosis is the pathological basis of coronary heart disease, and its stability affects the risk of adverse cardiovascular and cerebrovascular events in the future. Aging is the main risk factor for atherosclerosis, and the increase in the load of aged vascular smooth muscle cells in atherosclerotic plaques drives the vulnerability of plaques. Anti-aging therapy brings new opportunities to innovate the prevention and treatment strategies of coronary heart disease, but currently there is still a lack of drugs with anti-aging effects to stabilize vulnerable plaques in advanced atherosclerosis.

[0003] Heat shock protein 90 (HSP90) inhibitors have been shown to induce apoptosis in senescent human fibroblasts, and these drugs that remove senescent cells are called Senolytics. However, the indications of HSP90 inhibitors currently in Phase II and Phase III clinical trials are all tumor diseases, and there have been no reports on HSP90 inhibitors with the drug effect of stabilizing vulnerable plaques in advanced atherosclerosis.

[0004] Salvianolic acid A (SAA) is a light yellow crystalline powder with a CAS number of 96574-01-5. Its chemical name is (R)-3-(3,4-dihydroxyphenyl)-2-(((E)-3-(2-((E)-3,4-dihydroxystyryl)-3,4-dihydroxy phenyl)acryloyl)oxy)propanoic acid and a molecular formula of C 26 H 22 O 10 , molecular weight is 494.4 g / mol, soluble in water, chemical structure is Figure 1 shown.

[0005] Salvianolic acid A is an active small molecule compound from the traditional Chinese medicine Salvia miltiorrhiza. The literature "Study on the intervention effect and anti-inflammatory mechanism of salvianolic acid A on ZDF rats with early atherosclerosis" (Zhejiang University of Chinese Medicine, 2015, Ma Quanxin) reported that 0.5-1 mg / kg of salvianolic acid A can reduce the area of ​​early atherosclerotic plaques in Zucker diabetic obese rats induced by high-fat diet combined with intraperitoneal injection of vitamin D3, which shows that salvianolic acid A can be used to prevent and treat diabetes-related early atherosclerosis. However, the Zucker diabetic obese rats induced by high-fat diet combined with intraperitoneal injection of vitamin D3 established diabetes-induced early calcified atherosclerosis, and its pathology showed the pathological characteristics of early atherosclerosis with endothelial damage as the main feature and calcification. The pathological characteristics of late atherosclerosis are completely different from those of early atherosclerosis. The vulnerable plaques of late atherosclerosis need to be continuously fed with high-fat diet to ApoE knockout mice (ApoE - / - ) for more than 12 weeks, pathologically, the pathological features are mainly senescence and phenotypic transformation of vascular smooth muscle cells and show vulnerable characteristics. - / - The mice lack APOE protein and develop normally, but show a significant increase in total plasma cholesterol levels and spontaneous atherosclerotic lesions. They are recognized and the most widely used atherosclerosis model mice, and their pathological characteristics are highly similar to those of humans. Therefore, it is not reasonable to infer from the records in this literature whether salvianolic acid A has a therapeutic effect on vulnerable plaques in advanced atherosclerosis.

[0006] Ferulic acid (FA) is a colorless to pale yellow crystalline powder with a CAS number of 1135-24-6. Its chemical name is 3-(4-hydroxy-3-methoxyphenyl)-2-propenoic acid and its molecular formula is C 10 H 10 O4, molecular weight is 194.18 g / mol, its sodium salt sodium ferulate is soluble in water, chemical structure is as follows Figure 2 shown. Summary of the invention

[0007] In view of the above-mentioned prior art, the present invention provides a new use of salvianolic acid A - the use of salvianolic acid A as a heat shock protein 90α inhibitor in the preparation of a drug for preventing and treating vulnerable plaques in advanced atherosclerosis, and the use of salvianolic acid A in combination with ferulic acid in the preparation of a drug for preventing and treating vulnerable plaques in advanced atherosclerosis.

[0008] The present invention is achieved through the following technical solutions:

[0009] Application of salvianolic acid A in the preparation of medicines for preventing and treating vulnerable plaques of advanced atherosclerosis.

[0010] Furthermore, in specific applications, the drug is used alone, the dosage is 10 mg / kg, and the administration method is intraperitoneal injection.

[0011] Furthermore, in specific applications, the drug is used alone, and the dosage for in vitro stress-induced aged vascular smooth muscle cells is 100 μM.

[0012] Application of salvianolic acid A combined with ferulic acid or its salt in the preparation of a drug for preventing and treating vulnerable plaques in late atherosclerosis.

[0013] Furthermore, the ferulate salt is sodium ferulate.

[0014] Furthermore, in specific applications, the combination drug is used with a dosage of 10 mg / kg, and the administration method is intraperitoneal injection.

[0015] Furthermore, in specific applications, the combination of drugs is used, and the dosage of 100 μM salvianolic acid A combined with 50 μM ferulic acid to the in vitro stress-induced aged vascular smooth muscle cells.

[0016] A pharmaceutical composition for treating vulnerable plaques of advanced atherosclerosis, the active ingredients of which are salvianolic acid A and ferulic acid or its salt.

[0017] Furthermore, the ferulate salt is sodium ferulate.

[0018] Furthermore, the effective concentration of salvianolic acid A is 100 μM, and the effective concentration of ferulic acid is 50 μM.

[0019] The present invention has found through research that salvianolic acid A has the following uses and effects:

[0020] (1) Salvianolic acid A is an inhibitor of HSP90α and can promote the protein degradation of HSP90α in vascular smooth muscle cells.

[0021] (2) The Kd of salvianolic acid A binding to human HSP90α is 24.5 μM; HSP90α protein is encoded by HSP90AA1 (HGNC: 5253, NCBI Gene: 3320, Ensembl: ENSG00000080824).

[0022] (3) Salvianolic acid A promotes apoptosis of aged vascular smooth muscle cells and has a senolytic effect.

[0023] (4) Tannic acid A stabilizes vulnerable atherosclerotic plaques by reducing the load on aging vascular smooth muscle cells.

[0024] (5) Salvianolic acid A non-covalently binds to ASP54, ALA55, LYS58, ASP93, ASP102, and LYS112 of HSP90AA1, exerting an inhibitory effect, among which ASP93 contributes the most to the binding free energy.

[0025] (6) Salvianolic acid A combined with ferulic acid synergistically reduced the load on aged vascular smooth muscle cells and stabilized vulnerable atherosclerotic plaques, and the effect was better than that of single administration of salvianolic acid A or ferulic acid.

[0026] The present invention has found through experimental research that salvianolic acid A has a stabilizing effect on vulnerable plaques of advanced atherosclerosis, and the effect is significant. It is also found that salvianolic acid A combined with ferulic acid has a better technical effect. The research of the present invention is of great significance to the development and application of drugs with anti-aging effects for stabilizing vulnerable plaques of advanced atherosclerosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 : The chemical structural formula of salvianolic acid A.

[0028] Figure 2 : The chemical structural formula of ferulic acid.

[0029] Figure 3 : Results of multiple tissue immunofluorescence staining, where DAPI stands for 4',6-diamidino-2-phenylindole, used to mark cell nuclei; αSMA stands for α-smooth muscle actin, used to mark vascular smooth muscle cells; p16 stands for cyclin-dependent kinase inhibitor 2A protein, used to mark senescent cells; Merge indicates their superposition display.

[0030] Figure 4 : HE staining results, MASSON staining results and Oil Red O staining results.

[0031] Figure 5 :Western Blot detection results.

[0032] Figure 6 : Measurement results of surface plasmon resonance technology.

[0033] Figure 7 :Interaction mode of salvianolic acid A-HSP90α.

[0034] Figure 8 : Energy decomposition results of amino acid residues.

[0035] Fig. 9 : Dose-response curve of salvianolic acid A.

[0036] Fig.10 : Dose-response curve of ferulic acid.

[0037] Fig.11 : Results of in situ staining of apoptotic cells. DETAILED DESCRIPTION

[0038] The present invention will be further described below in conjunction with the embodiments. However, the scope of the present invention is not limited to the following embodiments. It will be appreciated by those skilled in the art that various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention.

[0039] The instruments, reagents, and materials involved in the following examples, unless otherwise specified, are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through regular commercial channels. The experimental methods and detection methods involved in the following examples, unless otherwise specified, are all conventional experimental methods and detection methods already available in the prior art.

[0040] Experiment 1: Salvianolic acid A stabilizes ApoE - / - Advanced vulnerable atherosclerotic plaques in mice and the effect of reducing the burden of senescent vascular smooth muscle cells in plaques

[0041] (1) Model establishment and drug intervention

[0042] Select 8-week-old male ApoE - / - 72 mice were used, and 12 8-week-old male C57BL / 6J mice of the same genetic background were used as a normal control group. A high-fat diet (containing 0.15% cholesterol and 21% fat) was fed for 20 weeks to establish a late-stage atherosclerosis model. All experimental animals were caged, with 5 animals per cage, and were given free access to food and water, and were raised in an environment with 12 hours of light and dark. - / - The mice were randomly divided into 6 groups, 12 mice in each group, and 12 C57BL / 6J mice were used as the normal control group. - / - After the mice were fed a high-fat diet for 8 weeks, they were given corresponding drug intervention for 12 weeks starting from the 9th week. The specific intervention measures are as follows:

[0043] Normal control group (Chow): Normal C57BL / 6J mice were intraperitoneally injected with an equal volume of normal saline daily;

[0044] Disease model group (HFD): ApoE - / - Mice were intraperitoneally injected with an equal volume of normal saline daily;

[0045] Salvianolic acid A group (HFD+SAA): ApoE - / - Mice were given salvianolic acid A 10 mg / kg by intraperitoneal injection daily;

[0046] Ferulic acid group (HFD+FA): ApoE - / - Mice were given 10 mg / kg sodium ferulate intraperitoneally daily for intervention;

[0047] Salvianolic acid A-ferulic acid combined group (HFD+SAA+FA): ApoE - / - Mice were given salvianolic acid A 10 mg / kg and sodium ferulate 10 mg / kg by intraperitoneal injection daily;

[0048] Salvia miltiorrhiza-Chuanxiong decoction group (HFD+Salvia miltiorrhiza+Chuanxiong): ApoE - / - The mice were given daily oral administration of Danshen and Chuanxiong decoction (Danshen crude drug 1.95 g / kg + Chuanxiong crude drug 1.95 g / kg);

[0049] Positive control group (HFD+Rosuvastatin): ApoE - / - Mice were given rosuvastatin calcium 10 mg / kg by oral gavage daily.

[0050] (2) Test items

[0051] After the drug intervention, mice in each group were taken to prepare frozen sections of aortic root tissue for H&E (Hematoxylin-Eosin) staining, Masson staining, Oil Red O staining, and multiple tissue immunofluorescence staining.

[0052] H&E staining: Frozen sections were rewarmed at room temperature for 30 minutes, fixed with tissue fixative for 15 minutes, and rinsed with running water. The sections were treated with HD constant staining pretreatment solution for 1 minute. The sections were dyed with hematoxylin staining solution for 3-5 minutes, washed with tap water, differentiated with differentiation solution, washed with tap water, blued with blue reversion solution, and rinsed with running water. The sections were dehydrated in 95% alcohol for 1 minute, and stained in eosin staining solution for 15 seconds. The sections were sequentially placed in anhydrous ethanol I for 2 minutes-anhydrous ethanol II for 2 minutes-anhydrous ethanol III for 2 minutes-n-butanol I for 2 minutes-n-butanol II for 2 minutes-xylene I for 2 minutes-xylene II for 2 minutes to make them transparent, and sealed with neutral gum.

[0053] Masson staining: Frozen sections were rewarmed at room temperature for 30 minutes, fixed with tissue fixative for 15 minutes, and rinsed with running water. The sections were immersed in 2.5% potassium dichromate mordant overnight and rinsed with running water. The sections were immersed in Weigert iron hematoxylin stain for 1 minute and washed with tap water. The sections were briefly differentiated in 1% hydrochloric acid alcohol differentiation solution for a few seconds and washed with tap water. The sections were immersed in Ponceau acid fuchsin for 6 minutes and rinsed with tap water. The sections were immersed in 1% phosphomolybdic acid solution for 1 minute, without washing with water, and directly stained in 2.5% aniline blue solution for 30 seconds after draining. The sections were rinsed and differentiated with 1% acetic acid, dehydrated in two cylinders of anhydrous ethanol, placed in a third cylinder of anhydrous ethanol for 5 minutes, and transparentized with xylene for 5 minutes. The sections were sealed with neutral gum and observed and photographed under a microscope.

[0054] Oil Red O staining: Rewarm at room temperature for 30 minutes. Immerse the slices in Oil Red O working solution for 10 minutes in the dark. Take out the slices, leave them for 3 seconds, then immerse them in two cylinders of 60% isopropanol for 5 seconds. Then, immerse the slices in two cylinders of pure water for 10 seconds each time. Use hematoxylin staining solution to stain the cell nuclei. Add glycerol gelatin sealing agent to seal the slices, and observe and take pictures under the microscope.

[0055] Multiple tissue immunofluorescence staining: Rewarm at room temperature for 30 minutes, fix with paraformaldehyde for 10 minutes. Block with 3% H2O2 for 10 minutes, add TBST to wash for 5 minutes × 2 times, and wash off H2O2. Add 40% Goat Serum to block at room temperature for 30 minutes. Add pre-diluted primary antibody, incubate at room temperature for 1 hour, add TBST to wash for 5 minutes × 3 times, and wash off unbound primary antibody. Add fluorescent secondary antibody to incubate at room temperature for 20 minutes, add TBST to wash for 5 minutes × 3 times. Incubate TSA dye at room temperature for 10 minutes, add TBST to wash for 5 minutes × 3 times. Add rapid stripping buffer, wash at room temperature for 20 minutes, add TBST to wash for 5 minutes × 3 times. Add 40% Goat Serum to block for 30 minutes. Add pre-diluted second primary antibody, incubate at room temperature for 1 hour, add TBST to wash for 5 minutes × 3 times. Incubate the second secondary antibody at room temperature for 20 minutes, add TBST to wash for 5 minutes × 3 times. The second TSA dye was incubated at room temperature for 10 min, and TBST was added to wash for 5 min × 3 times. DAPI (1:500) was incubated at room temperature for 10 min, and TBST was added to wash for 5 min × 2 times. The slides were sealed with neutral resin, observed and photographed under a fluorescence microscope.

[0056] Western Blot detection of aorta HSP90α protein level: prepare 10% PAGE gel, inject prestained protein marker and protein sample into the loading well, set the voltage to 80V for electrophoresis, set the voltage to 120V when the protein runs to the bottom of the upper gel, continue electrophoresis, and stop electrophoresis when the protein runs to the bottom of the lower gel. Use wet transfer method, install the transfer device in sequence, bury the transfer tank in ice, constant current 400mA, and set the time for transfer according to the molecular weight. Put the membrane into 1× protein-free fast blocking solution and block it at room temperature for 15min. Immerse the membrane in pre-diluted HSP90α primary antibody and incubate it in a refrigerator at 4℃ overnight. After washing with 1×TBST buffer for 5min×5 times, immerse the membrane in pre-diluted secondary antibody (1:5000) and incubate it at room temperature for 1h, and wash it with 1×TBST buffer for 5min×5 times. Add developer to the membrane for color exposure, capture the image and measure the gray value.

[0057] (3) Results and conclusions

[0058] The results of multiple tissue immunofluorescence staining were as follows Figure 3As shown in the figure, it can be seen that the salvianolic acid A group, ferulic acid group, salvianolic acid A-ferulic acid combination group and Salvia miltiorrhiza and Chuanxiong decoction group can effectively reduce the load of senescent vascular smooth muscle cells in plaques (p16 + αSMA + area), stable advanced atherosclerotic vulnerable plaques.

[0059] HE staining results Figure 4 As shown, it can be seen that the necrotic core area of ​​atherosclerotic plaques in the salvianolic acid A group, ferulic acid group, salvianolic acid A-ferulic acid combination group and Salvia miltiorrhiza and Chuanxiong decoction group was significantly smaller than that in the disease model group, and the effect of the salvianolic acid A-ferulic acid combination was significantly better than that of salvianolic acid A alone, and was also significantly better than that of ferulic acid alone.

[0060] The results of MASSON staining are as follows Figure 4 As shown, the thickness of the atherosclerotic plaque fibrous cap in the salvianolic acid A group, ferulic acid group, salvianolic acid A-ferulic acid combination group and Salvia miltiorrhiza and Chuanxiong decoction group was significantly greater than that in the disease model group. Moreover, the effect of the salvianolic acid A-ferulic acid combination was significantly better than that of salvianolic acid A alone and that of ferulic acid alone.

[0061] Oil red O staining results Figure 4 As shown, the total area of ​​atherosclerotic plaques in the salvianolic acid A group, ferulic acid group, salvianolic acid A-ferulic acid combination group and Salvia miltiorrhiza and Chuanxiong decoction group were significantly smaller than that in the disease model group. Moreover, the effect of the salvianolic acid A-ferulic acid combination was significantly better than that of salvianolic acid A alone, and was also significantly better than that of ferulic acid alone.

[0062] Western Blot test results Figure 5 As shown, the aortic HSP90α in the salvianolic acid A group, ferulic acid group, salvianolic acid A-ferulic acid combination group and Salvia miltiorrhiza and Chuanxiong decoction group were significantly lower than that in the disease model group.

[0063] Experiment 2: Salvianolic acid A promotes apoptosis of aged vascular smooth muscle cells by inhibiting HSP90α through senolytic effects

[0064] (I) Salvianolic acid A directly acts on HSP90α

[0065] (1) Detection

[0066] Surface plasmon resonance technology: Biacore T200 was used to evaluate the binding affinity of salvianolic acid A to human HSP90α protein. Human recombinant HSP90α protein was diluted in sodium acetate buffer and flowed into the sensor chip CM5 at a rate of 10 μL / min for 7 minutes. Uncoupled protein was rinsed with 1M ethanolamine-hydrochloride (pH 8.0). Diluted salvianolic acid A was injected into the fixed protein surface in sequence, and the binding sensor map was recorded.

[0067] Cycloheximide (CHX) chase assay: To evaluate the stability of HSP90α protein, cycloheximide chase assay was performed. Cells were treated with 50 μM CHX at 0, 2, 4, 6, 8, 10, and 12 hours. Cell lysates were collected and subjected to Western blot analysis using β-actin as a loading control.

[0068] (2) Results: Salvianolic acid A significantly promoted the protein degradation of HSP90α. The results of surface plasmon resonance technology were as follows: Figure 6 As shown, the Kd of salvianolic acid A binding to human HSP90α is 24.5 μM.

[0069] (II) Key amino acids for the binding of salvianolic acid A to HSP90α

[0070] (1) Detection

[0071] Molecular docking: Molecular docking was performed simultaneously using Autodock Vina (version 1.2.0), LeDock (version 1.0), and UCSF DOCK (version 6.10). The reference structure was HSP90α bound to 17-DMAG (PDB ID: 1OSF), in which water molecules and 17-DMAG were removed. The position of 17-DMAG in 17-DMAG-bound HSP90α was set as the center point. The size of the grid box was set to To ensure insertion into the active site pocket of HSP90α.

[0072] Molecular dynamics simulation: Molecular dynamics simulation was performed using Gromacs (version 2019.3) software. The simulation conditions were carried out at a static temperature of 300K and normal pressure (1Bar), the force field used was Amber99sb-Idn [AMBER99SB-ILDN protein, nucleic AMBER94 (Lindorff-Larsen et al., Proteins 78, 1950-58, 2010)], the solvent was water molecules (Tip4p water model) and the total charge of the simulation system was neutralized by adding an appropriate number of sodium ions. The molecular dynamics simulation system first used the steepest descent method to minimize the energy, and then performed 500,000 steps of isothermal isochoric ensemble (NVT) equilibrium and isothermal isobaric ensemble (NPT) equilibrium, respectively, with a coupling constant of 0.1ps and a duration of 500ps. Finally, a free molecular dynamics simulation with a total duration of 50ns was run.

[0073] (2) Results: The interaction pattern of salvianolic acid A and HSP90α is as follows Figure 7 As shown, salvianolic acid A non-covalently binds to ASP54, ALA55, LYS58, ASP93, ASP102, and LYS112 of HSP90AA1 to exert an inhibitory effect. The results of the amino acid residue energy decomposition are shown in Figure 8 As shown, it can be seen that ASP93 contributes the most to the binding free energy.

[0074] (III) Dose-effect analysis of the synergistic effect of salvianolic acid A and ferulic acid on selective elimination of stress-induced aged vascular smooth muscle cells

[0075] (1) Detection

[0076] Human aortic vascular smooth muscle cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C and 5% CO2. 200 μM palmitate-10% BSA was given to induce stress-induced senescence of human aortic smooth muscle cells, and PBS-10% BSA was used as a control. The specific intervention measures are as follows:

[0077] Non-senescent VSMC+salvianolic acid A intervention group: after administration of PBS-10% BSA, gradient concentrations of SAA (10, 20, 40, 80, 160, 320, 640 μM) were added for intervention for 24 h;

[0078] Senescent VSMC+salvianolic acid A intervention group: after administration of palmitate-10% BSA, gradient concentrations of SAA (10, 20, 40, 80, 160, 320, 640 μM) were added for intervention for 24 h;

[0079] Non-senescent VSMC+salvianolic acid A combined with ferulic acid intervention group: after administration of PBS-10% BSA, 100 μM salvianolic acid A and gradient concentrations of FA (10, 20, 40, 80, 160 μM) were added for 24 h;

[0080] Aged VSMC+salvianolic acid A-ferulic acid intervention group: after administration of palmitate-10% BSA, 100 μM salvianolic acid A and gradient concentrations of FA (10, 20, 40, 80, 160 μM) were added for 24 h;

[0081] (2) Results: The dose-response curve of salvianolic acid A is shown in Fig. 9 As shown in the figure, it can be seen that salvianolic acid A has an effect on the EC 50 The survival rate of normal vascular smooth muscle cells was significantly reduced when the concentration of salvianolic acid A reached 160 μM. Based on this, 100 μM was selected as the dose for subsequent SAA intervention.

[0082] The combined intervention was performed by adding gradient doses of ferulic acid to 100 μM salvianolic acid A. The dose-response curve of ferulic acid is shown in Fig.10 As shown, see, EC of ferulic acid 50 The optimal ratio of SAA-FA for selective clearance of senescent vascular smooth muscle cells in vitro was 100 μM salvianolic acid A combined with 50 μM ferulic acid.

[0083] (IV) Salvianolic acid A promotes apoptosis of stress-induced aged vascular smooth muscle cells

[0084] (1) Detection

[0085] Human aortic vascular smooth muscle cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C and 5% CO2. 200 μM palmitate-10% BSA was given to induce stress-induced senescence of human aortic smooth muscle cells, and PBS-10% BSA was used as a control. The specific intervention measures are as follows:

[0086] Blank control group: PBS-10% BSA was added, and an equal volume of PBS was added for 24 h;

[0087] Model group: after administration of palmitate-10% BSA, an equal volume of DMSO was added for intervention for 24 h;

[0088] Salvianolic acid A group: after administration of palmitate-10% BSA, 100 μM salvianolic acid A was added for intervention for 24 h;

[0089] Salvianolic acid A-ferulic acid combined group: After administration of palmitate-10% BSA, 100 μM salvianolic acid A and 50 μM ferulic acid were added for intervention for 24 h.

[0090] In situ staining of apoptotic cells: The mitochondrial membrane potential-dependent red fluorescent probe MitoTracker RedCMXRo is used to label living cells that maintain mitochondrial membrane potential, the cell apoptosis green fluorescent probe Annexin V-FITC is used to label cells that have undergone apoptosis or necrosis, and the blue fluorescent Hoechst 33342 is used to label the cell nucleus.

[0091] (2) Results: The results of in situ staining of apoptotic cells are as follows Fig.11 As shown, it can be seen that 100 μM salvianolic acid A intervention for 24 hours significantly induced the apoptosis of stress-induced aged vascular smooth muscle cells, while 100 μM salvianolic acid A combined with 50 μM ferulic acid synergistically enhanced the apoptotic effect on stress-induced aged vascular smooth muscle cells.

[0092] The above examples are provided to those skilled in the art to fully disclose and describe how to make and use the claimed embodiments, and are not intended to limit the scope of the disclosure herein. Modifications that are obvious to those skilled in the art will fall within the scope of the appended claims.

Claims

1. Application of salvianolic acid A in the preparation of drugs for preventing and treating vulnerable plaques in advanced atherosclerosis.

2. The use according to claim 1, characterized in that: When used specifically, the drug is used alone at a dosage of 10 mg / kg by intraperitoneal injection.

3. The use according to claim 1, characterized in that: In specific application, the drug is used alone, and the dosage for in vitro stress-induced aged vascular smooth muscle cells is 100 μM.

4. Use of salvianolic acid A in combination with ferulic acid or its salts in the preparation of a drug for preventing and treating vulnerable plaques in advanced atherosclerosis.

5. The use according to claim 4, characterized in that: The ferulate salt is sodium ferulate.

6. The use according to claim 4 or 5, characterized in that: In specific applications, the combination drug is used with a dosage of 10 mg / kg and the administration method is intraperitoneal injection.

7. The use according to claim 4 or 5, characterized in that: In specific application, the drug is used in combination, and the dosage of the drug to the in vitro stress-induced aged vascular smooth muscle cells is 100 μM salvianolic acid A combined with 50 μM ferulic acid.

8. A pharmaceutical composition for treating vulnerable plaques in advanced atherosclerosis, characterized in that: The active ingredients are salvianolic acid A and ferulic acid or its salt.

9. The pharmaceutical composition for treating vulnerable plaques in advanced atherosclerosis according to claim 8, characterized in that: The ferulate salt is sodium ferulate.

10. The pharmaceutical composition for treating vulnerable plaques in advanced atherosclerosis according to claim 8, characterized in that: Furthermore, the effective concentration of salvianolic acid A is 100 μM, and the effective concentration of ferulic acid is 50 μM.

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