Application of β,β-dimethylacryloyl akanin in the preparation of drugs for treating atherosclerosis

Abstract

The present invention belongs to the field of biomedicine technology, and in particular, relates to the use of β,β-dimethylacryloyl akanin in the preparation of a drug for treating atherosclerosis. The present invention first discovered and experimentally confirmed that β,β-dimethylacryloyl akanin can prevent and treat atherosclerosis; it has developed a new clinical indication for β,β-dimethylacryloyl akanin, which can inhibit β-gal activity and effectively intervene in the process of atherosclerosis; the compound β,β-dimethylacryloyl akanin provided by the present invention is applied to atherosclerosis, has the advantages of small side effects and good safety, and has good medicinal prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to the application of β,β-dimethylacryloyl acannin in the preparation of a drug for treating atherosclerosis. Background Art

[0002] With the aging of the population, aging-related diseases have become one of the major social problems. Accurate diagnosis and treatment of aging-related diseases is essential. Cellular senescence has become a potential important factor in aging-related diseases, manifested as irreversible inhibition of cell proliferation in response to various stresses. Atherosclerosis (AS) is an aging-related disease that has become a major cause of mortality and morbidity worldwide. Atherosclerotic plaques can lead to potentially catastrophic consequences, such as stroke and myocardial infarction. An important feature of the epidemiology of cardiovascular disease in China is the rapid and significant increase in the burden of atherosclerotic cardiovascular disease. In 2016, approximately 2.4 million people died of atherosclerotic cardiovascular disease, accounting for 61% of cardiovascular disease deaths and 25% of total deaths. AS has become a major public health issue, so it is necessary to explore new ways to treat AS and study new drugs for the treatment of AS.

[0003] Since ancient times, Chinese medicine has been used to treat a variety of diseases and improve human health. Medicinal plants have shown great potential in reducing β-galactosidase activity and treating diseases related to β-galactosidase disorders. Compared with traditional drugs, they have the advantages of multiple components, multiple targets, and multiple pathways. Therefore, screening suitable β-galactosidase inhibitors from natural drugs has important research significance. β,β-dimethylacryloyl akanin has anti-tumor, anti-inflammatory, and liver protection effects. In the prior art, there is no record of β,β-dimethylacryloyl akanin for the treatment of atherosclerosis. Summary of the invention

[0004] In view of the problems existing in the prior art, the present invention provides the use of β,β-dimethylacryloyl akanin in the preparation of a drug for treating atherosclerosis. The present invention evaluates the efficacy of β,β-dimethylacryloyl akanin in treating atherosclerosis and determines that β,β-dimethylacryloyl akanin has good efficacy and high safety in treating atherosclerosis.

[0005] The technical solution adopted by the present invention to achieve the above-mentioned purpose is:

[0006] The present invention provides an application of β,β-dimethylacryloyl akanin in preparing a medicine for treating atherosclerosis.

[0007] Preferably, β,β-dimethylacryloyl akanin is used in the preparation of a drug for improving the abnormal increase of β-gal in the process of atherosclerosis and inhibiting the concentration of β-gal.

[0008] Preferably, β,β-dimethylacryloyl akanin is used in the preparation of a drug for improving the abnormal increase of β-gal caused by cell senescence.

[0009] Preferably, β,β-dimethylacryloylakanin is used for preparing a drug for improving the abnormal increase of β-gal in the process of cell foaming.

[0010] The present invention also provides a medicine for preparing a medicine for treating atherosclerosis, wherein the active ingredient of the medicine is β,β-dimethylacryloyl acannin.

[0011] Preferably, the drug further comprises other medically acceptable carriers and excipients.

[0012] The drug containing β,β-dimethylacryloyl acannin provided by the present invention can be further prepared into any medically acceptable dosage form, including but not limited to tablets, capsules, granules, etc.

[0013] The present invention further provides a drug efficacy evaluation of β,β-dimethylacryloyl akanin for treating atherosclerosis, comprising the following steps:

[0014] (1) Establishment of RAW264.7 cell aging model and macrophage foaming model and evaluation of the pharmacological efficacy of β,β-dimethylacryloyl-acampine: A control group, a model group, a positive drug group, and a β,β-dimethylacryloyl-acampine group were established. Each group was injected with the fluorescent probe NFP-βgal for screening β-galactosidase inhibitors. The efficacy of β,β-dimethylacryloyl-acampine in the treatment of atherosclerosis was evaluated using laser confocal imaging.

[0015] (2) Establishment of zebrafish AS model and pharmacological evaluation of β,β-dimethylacryloyl-acampine: A control group, a probe-treated group, a model group, a positive drug group, and a β,β-dimethylacryloyl-acampine group were established. Except for the control group, each group was injected with the fluorescent probe NFP-βgal. The efficacy of β,β-dimethylacryloyl-acampine in treating atherosclerosis was evaluated using laser confocal imaging.

[0016] (3) In vivo pharmacodynamic validation based on an atherosclerosis mouse model: C57BL / 6J mice were used as the control group, APOE -- / --The mice were randomly divided into the model group, the positive drug group, the low, medium and high dose groups of β,β-dimethylacryloyl akanin. The mice in the blank group were fed with normal feed, and the mice in the other groups were fed with high-fat feed. After the experiment, the mice were killed and the aortic tissue was collected. After the probe treatment, the tissue was imaged by the small animal in vivo imaging system; the aortic root was stained with oil red, and the atherosclerotic plaques were observed by taking pictures.

[0017] Furthermore, the positive drug is dasatinib + quercetin.

[0018] Furthermore, in step (1), H2O2 was used to establish a RAW264.7 cell aging model, and ox-LDL was used to establish a macrophage foaming model.

[0019] Furthermore, in step (2), lipopolysaccharide (LPS) was used to induce zebrafish to establish an AS model.

[0020] Furthermore, in step (3), the mice in the control group and the model group were intragastrically administered with normal saline, the mice in the positive drug group were intragastrically administered with dasatinib + quercetin solution, and the mice in the β,β-dimethylacryloyl acanthate low, medium, and high dose groups were intragastrically administered with different doses of β,β-dimethylacryloyl acanthate solution.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) The present invention first discovered and experimentally confirmed that β,β-dimethylacryloyl akanin can prevent and treat atherosclerosis; it has opened up new clinical indications for β,β-dimethylacryloyl akanin, which can inhibit β-gal activity and effectively intervene in the process of atherosclerosis;

[0023] (2) The compound β,β-dimethylacryloylarachidine provided by the present invention is used for the treatment of atherosclerosis, has the advantages of small side effects and good safety, and has good medicinal prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The results of fluorescence imaging of endogenous β-gal in RAW264.7 cells induced by H2O2 using the probe NFP-βgal (scale bar 10 μm); a is the fluorescence imaging of endogenous β-gal in RAW264.7 cells under different treatments; b is the relative fluorescence intensity of different treatment groups in Figure A;

[0025] Figure 2The results of fluorescence imaging of endogenous β-gal in RAW264.7 cells induced by ox-LDL using the probe NFP-βgal (scale bar 10 μm); a is the fluorescence imaging of endogenous β-gal in RAW264.7 cells under different treatments; b is the relative fluorescence intensity of different treatment groups in Figure A;

[0026] Figure 3 The results of staining of senescence-related β-gal in the foaming process of RAW264.7 cells induced by ox-LDL using a commercial kit (scale bar 50 μm); A is the control group; B is the 10 μg / mL ox-LDL group; C is the 30 μg / mL ox-LDL group; D is the dasatinib + quercetin group; E is the β,β-dimethylacryloyl acanthate group;

[0027] Figure 4 The results of fluorescence imaging of endogenous β-gal in zebrafish induced by LPS using the probe NFP-βgal (scale bar 200 μm); a is the fluorescence imaging of endogenous β-gal in zebrafish under different treatments; b is the relative fluorescence intensity of different treatment groups in Figure A;

[0028] Figure 5 The results of fluorescence imaging determination of β-gal in mouse tissue using probe NFP-βgal; a is the fluorescence imaging of β-gal in mouse aorta tissue of control group, model group, positive drug group, β,β-dimethylacryloyl akanin low-dose group, β,β-dimethylacryloyl akanin medium-dose group, and β,β-dimethylacryloyl akanin high-dose group from left to right; b is the relative fluorescence intensity of different treatment groups in Figure A;

[0029] Figure 6 Oil Red O staining of mouse aortic arch tissue sections (bar 25 μm). DETAILED DESCRIPTION

[0030] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0031] The structural formula of the NFP-βgal probe (described in CN117886862A) used in the present invention is:

[0032]

[0033] Example 1 Cell experiment

[0034] (1) Establishment of RAW264.7 cell senescence model and pharmacodynamic evaluation of β,β-dimethylacryloylarachidine: After digestion, centrifugation, and resuspension, the cells were counted and the cell density was adjusted to 8×10 based on the results obtained by the counter. 4 / mL, 1mL of cell suspension was inoculated in each well of the cell culture dish and incubated overnight in a 37℃, 5% CO2 incubator. The cells were observed to be in good growth condition the next day. The cell culture medium was replaced with a drug-containing culture medium solution with different drug concentrations. The experimental groups were divided into control group (NFP-βgal probe), H2O2 modeling group (NFP-βgal probe + H2O2), positive drug group (NFP-βgal probe + H2O2 + 10μM dasatinib + 50μM quercetin), and active monomer group (NFP-βgal probe + H2O2 + 100μM β, β-dimethylacryloyl acannin), and laser confocal imaging was performed on the above four groups of cells.

[0035] (2) Establishment of macrophage foaming model and pharmacodynamic evaluation of β,β-dimethylacryloyl-arcanin: The cell culture medium was replaced with a drug-containing culture medium solution with different drug concentrations. The experimental groups were divided into control group (NFP-βgal probe), ox-LDL modeling group (NFP-βgal probe + ox-LDL), positive drug group (NFP-βgal probe + ox-LDL + 10μM dasatinib + 50μM quercetin), and active monomer group (NFP-βgal probe + ox-LDL + 100μM β,β-dimethylacryloyl-arcanin). Laser confocal imaging was performed on the above four groups of cells.

[0036] (3) Senescence-associated β-gal staining: The activity changes of β-gal during the foaming process of RAW264.7 cells were determined using a commercial senescence-associated β-gal staining kit (Biyuntian, Shanghai, China). RAW264.7 cells were seeded in 6-well plates, and macrophage foaming was induced using different concentrations of ox-LDL, followed by drug intervention using 100 μM β,β-dimethylacryloyl acanthate. RAW264.7 cells were fixed and incubated with β-gal working solution in a sterile incubator at 37°C CO2 overnight. Random images of each well were captured using an optical microscope.

[0037] (4) Analysis of test results:

[0038] Establishment of RAW264.7 cell senescence model and pharmacodynamic evaluation of β,β-dimethylacryloyl-arcanin: Hydrogen peroxide (H2O2) was used to establish a cell senescence model. The experimental results are shown in Figure 1After RAW264.7 cells were stimulated with H2O2, the red fluorescence intensity increased significantly, indicating an increase in endogenous β-gal, which also confirmed the view that excessive β-gal is produced during cell aging. In addition, the inhibitory effects of dasatinib combined with quercetin and β,β-dimethylacryloyl akanin on β-gal were evaluated. Compared with the model group, the fluorescence signal emitted by RAW264.7 was significantly reduced, among which the fluorescence intensity of the positive drug group decreased by about 2.3 times, while that of the β,β-dimethylacryloyl akanin group decreased by about 2.1 times. The above data show that β,β-dimethylacryloyl akanin can improve the abnormal increase of β-gal caused by cell aging.

[0039] Establishment of macrophage foaming model and pharmacodynamic evaluation of β,β-dimethylacryloyl-arcanin: Oxidized low-density lipoprotein (ox-LDL) was used to establish a macrophage foaming model. The experimental results are shown in Figure 2 After RAW264.7 cells were stimulated by ox-LDL, the red fluorescence intensity increased significantly, indicating an increase in endogenous β-gal, which also confirmed the view that excessive β-gal is produced during the macrophage foaming process. In addition, the inhibitory effects of dasatinib combined with quercetin and β,β-dimethylacryloyl akanin on β-gal were evaluated respectively. Compared with the model group, the fluorescence signal emitted by RAW264.7 was significantly reduced. The above data show that β,β-dimethylacryloyl akanin can improve the abnormal increase of β-gal during the cell foaming process.

[0040] Senescence-associated β-gal staining: To further verify the reliability of the fluorescence imaging results, a commercial kit was used to study the senescence-associated β-gal activity in macrophage foaming. Figure 3 After RAW264.7 cells were stimulated with different concentrations of ox-LDL, the staining depth increased significantly, indicating an increase in endogenous β-gal, which was consistent with the fluorescence imaging results. In addition, the inhibitory effect of dasatinib combined with quercetin and β,β-dimethylacryloyl akanin on β-gal was further evaluated. Compared with the model group, the staining depth of the treatment group was significantly reduced. The above data further indicate that β,β-dimethylacryloyl akanin can improve the abnormal increase of β-gal caused by the cell foaming process.

[0041] Example 2 Establishment of zebrafish AS model and pharmacodynamic evaluation of β,β-dimethylacryloylarachidonic acid

[0042] (1) Select 3dpf wild-type zebrafish and transfer them to culture bottles and incubate them in a 27°C constant temperature incubator. Observe the growth status every day. Replace the E3 medium with drug-containing medium solutions with different drug concentrations. The experiment is divided into the following groups:

[0043] (A) Control group: E3 medium was added to the culture flask and cultured at 27°C. Before imaging, the zebrafish were transferred to a confocal dish and fluorescence imaging analysis was performed under the same conditions as cell imaging.

[0044] (B) Probe treatment group: E3 medium containing 10 mM fluorescent probe was added to the culture flask and cultured at 27°C for 30 min. Before imaging, the zebrafish were transferred to a confocal dish, washed three times with PBS, and then subjected to fluorescence imaging analysis;

[0045] (C) AS model group: 3 dpf zebrafish were cultured with E3 medium containing 10 mg / mL LPS for 10 days, and the medium containing 10 mM fluorescent probe was added to the LPS modeling flask and cultured for another 30 min. Before imaging, the zebrafish were transferred to a confocal dish, washed three times with PBS, and then subjected to fluorescence imaging analysis;

[0046] (D) Positive drug group: 3 dpf zebrafish were cultured with E3 medium containing 10 mg / mL LPS for 10 days, and then treated with medium containing 10 mM dasatinib and 50 mM quercetin for 2 hours. The medium containing 10 mM fluorescent probe was added to the culture dish and cultured for another 30 minutes. Before imaging, the zebrafish were transferred to a confocal dish, washed three times with PBS, and then subjected to fluorescence imaging analysis;

[0047] (E) Active monomer group: 3 dpf zebrafish were cultured with E3 medium containing 10 mg / mL LPS for 10 days, and then treated with medium containing 100 mM β,β-dimethylacryloyl akanin for 2 hours. Medium containing 10 mM fluorescent probe was added to the culture dish and cultured for another 30 minutes. Before imaging, the zebrafish were transferred to a confocal dish, washed three times with PBS, and then subjected to fluorescence imaging analysis.

[0048] Laser confocal imaging was performed on the above five groups of zebrafish respectively.

[0049] (2) Analysis of experimental results: The AS model was established in zebrafish induced by lipopolysaccharide (LPS). Figure 4 After zebrafish were stimulated with LPS, the red fluorescence intensity increased significantly, indicating an increase in endogenous β-gal, which also confirmed the view that excessive β-gal would be produced during the AS process. In addition, the inhibitory effects of dasatinib combined with quercetin and β,β-dimethylacryloyl akanin on β-gal were evaluated respectively. Compared with the model group, the fluorescence signal was significantly reduced after drug intervention. The above data show that β,β-dimethylacryloyl akanin can improve the abnormal increase of β-gal during the AS process.

[0050] Example 3 In vivo pharmacodynamics validation based on atherosclerosis mouse model

[0051] (1) Experimental animals and grouping: C57BL / 6L and APOE- / - mice (eight weeks old) were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. All animals were housed in cages, with six in each cage. Before the start of the study, the mice were acclimated to the environment for seven days, with free access to food and water, and exposed to a 12-h day-night cycle at a relative humidity of 60±5% and a room temperature of 23±1°C. After one week of animal adaptive feeding, C57BL / 6J mice were selected as the control group, and APOE- / - mice were randomly divided into the model group, positive drug group, β,β-dimethylacryloyl acanthate low (5 mg / kg / day), medium (10 mg / kg / day), and high dose (15 mg / kg / day) groups, with six in each group. It was divided into two stages. In the first stage, modeling was carried out. After one week of animal adaptive feeding, the mice in the blank group were fed with normal feed, and the mice in the other groups were fed with high-fat feed for 12 weeks. In the second stage of drug administration, mice in the control group and model group were gavaged with 0.1 mL of normal saline, mice in the positive drug group were gavaged with 0.1 mL of dasatinib + quercetin solution, and mice in the low-, medium-, and high-dose groups of β,β-dimethylacryloyl acanthate were gavaged with different doses of 0.1 mL of β,β-dimethylacryloyl acanthate solution. The experimental period was 20 weeks in total.

[0052] (2) Fluorescent probe NFP-βgal was used for fluorescence imaging of β-gal in mouse tissues: After C57BL / 6J mice were killed, aortic tissues were quickly collected and washed three times with PBS buffer solution. Each group of tissues was incubated with PBS buffer solution containing NFP-βgal probe (50 μM) for 30 minutes, and then washed three times with PBS buffer solution to remove excess NFP-βgal. Finally, the tissues were imaged using the IVIS Lumina LT small animal in vivo imaging system. Excitation wavelength λ ex =520nm, wavelength range λ em =600-700nm.

[0053] (3) Oil red staining of aortic root:

[0054] Fixation: Take out the previously made slices of the aortic root from the low-temperature refrigerator at -20℃, place them at room temperature for 15 minutes, and then immerse them in 4% paraformaldehyde for 15 minutes to fully fix them. Rinse them with running water for half an hour to wash away the excess paraformaldehyde. Staining: After fixing the slides, soak them in 60% isopropanol twice, 1 minute each time. Stain them with oil red dye for 15 minutes, differentiate them with 60% isopropanol for 30 seconds, rinse them with running water, and observe them under the microscope. The lipid droplets in the plaques are red.

[0055] Differentiation: Shake off the water, add hematoxylin to stain for 5 minutes, rinse with running water, differentiate with 1% hydrochloric acid alcohol for 3 seconds, and immerse in bluing solution to return to blue.

[0056] Sealing: After natural air drying, seal the slide with glycerol glue. After the slide is dry, take photos and observe immediately to prevent fading.

[0057] (4) Statistical analysis method: SPSS 25.0 software was used to analyze the macro-indicator data. The data were expressed as mean ± standard deviation. Normal distribution test and variance homogeneity test were performed. When p < 0.05, the difference was considered statistically significant, and when p < 0.01, it was considered to be very significantly different.

[0058] (5) Analysis of test results:

[0059] NFP-βgal probe is used for fluorescence imaging of β-gal in AS mouse tissue: In order to evaluate the inhibitory effect of β,β-dimethylacryloyl akanin on β-gal in aortic tissue of AS mice induced by high-fat diet, the aortic tissue of each group of mice was collected and cultured with NFP-βgal probe (50μM), and finally fluorescence imaging was performed. The experimental results are shown in Figure 5 , the model group had obvious red fluorescence, indicating a high β-gal content. After treatment with β,β-dimethylacryloyl akanin, the fluorescence intensity signal was significantly reduced. And with the increase in the concentration of β,β-dimethylacryloyl akanin, the inhibitory effect was more obvious. After fluorescence data analysis, the fluorescence intensity of the high-concentration β,β-dimethylacryloyl akanin treatment was significantly reduced compared with the model group (P<0.01). This shows that β,β-dimethylacryloyl akanin has good potential in inhibiting β-gal concentration and delaying the progression of AS.

[0060] Oil red O staining of aortic root: test results are shown in Figure 6 We used Oil Red O staining to observe the aortic arch, where atherosclerotic plaques are prone to form, to intuitively understand the progression of atherosclerosis in each group of mice. Compared with the control group mice, which had almost no atherosclerotic plaques, the model group mice had significantly more atherosclerotic plaques in the aortic sinus. After treatment with β,β-dimethylacryloyl acanthine, the aortic sinus fat tissue and plaques of the mice were significantly reduced.

[0061] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the protection scope of the present invention.

Claims

1. Application of β,β-dimethylacryloylakanin in the preparation of drugs for the treatment of atherosclerosis.

2. The use according to claim 1, characterized in that: Application of β,β-dimethylacryloyl akanin in the preparation of drugs for improving the abnormal increase of β-gal in the process of atherosclerosis and inhibiting the concentration of β-gal.

3. The use according to claim 1, characterized in that: Application of β,β-dimethylacryloyl akanin in the preparation of a drug for improving the abnormal increase of β-gal caused by cell senescence.

4. The use according to claim 1, characterized in that: Application of β,β-dimethylacryloyl akanin in the preparation of a drug for improving the abnormal increase of β-gal in the process of cell foaming.

Images