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

Abstract

The invention belongs to the field of biological medicine, and particularly relates to application of a compound in preparation of a medicine for preventing and treating aortic dissection, the compound is nobiletin, the chemical name of the compound is 3 ', 4', 5, 6, 7, 8-hexamethoxyflavone, and the CAS number is 478-01-3. The invention also correspondingly provides a pharmaceutical composition. The death rate of aortic dissection is high, treatment depends on surgical treatment, and the death rate is extremely high. At present, an effective medicine for preventing and treating the aortic dissection is lacked clinically, the nobiletin is screened and determined to be used as the medicine for preventing and treating the aortic dissection, the molecular mechanism of the nobiletin is clear that the nobiletin targets ferroptosis related protein SLC7A11 to improve ferroptosis of vascular smooth muscle cells, namely VSMC, and a new target spot is provided for treatment of the aortic dissection.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the application of a compound in the preparation of drugs for the prevention and treatment of aortic dissection. Background Technology

[0002] Aortic dissection (AD) is a serious and rapidly progressing disease with a high mortality rate, posing a significant threat to human health. In acute type A aortic dissection (ATAAD), the mortality rate increases by 1%–2% per hour within the first 48 hours, exceeds 70% within 7 days, and surpasses 90% within one year. As a "silent killer" with a mortality rate as high as 90%, AD's annual incidence is rising year by year, making it the third leading cause of vascular death in my country. Currently, AD treatment primarily relies on emergency surgery, which is characterized by significant trauma, high costs, high mortality, high difficulty, and numerous postoperative complications, imposing a huge economic burden on countries worldwide. Effective drug treatments for AD are currently lacking in clinical practice; therefore, finding effective prevention and treatment methods for AD is an urgent clinical challenge.

[0003] Patent application CN201510964906.X discloses the application of naringenin in the preparation of products for the prevention and / or treatment of aortic dissection. The drug provided by this invention for the prevention and / or treatment of aortic dissection is safe and low in toxicity, with strong pharmacological effects. Its raw materials are abundant, widely available, and inexpensive, and can be obtained from crude or monomeric naringin through hydrolysis, or extracted from various medicinal materials containing naringenin, or synthesized through other chemical methods. The treatment cost is low, the process is simple, and the yield is high; moreover, the efficacy is definite, and the effective dosage is low. This invention provides a new drug source for the prevention, diagnosis, detection, protection, treatment, and research of aortic dissection, and is easy to promote and apply, capable of generating significant social and economic benefits in a short period of time. The chemical name of naringenin is 4',5,7-trihydroxyflavanone, its CAS number is 480-41-1, and its molecular formula is C 15 H 12 O5 has the following molecular structure.

[0004] As can be seen from the effects described in the aforementioned patent applications, naringenin has some effect in the preparation of products for the prevention and / or treatment of aortic dissection, but its effect is weak and cannot be used as an effective drug for the prevention and treatment of aortic dissection in clinical practice. Therefore, there is a need in the art to develop a new compound for use in the preparation of drugs for the prevention and treatment of aortic dissection. Summary of the Invention

[0005] Nobiletin (NOB) is a chemically stable and safe O-methylated flavonoid compound mainly derived from the peel of citrus fruits (such as mandarins, oranges, and lemons). It possesses antioxidant, anti-inflammatory, mitochondrial function-improving, and metabolic homeostasis-maintaining functions. Studies have shown that nobiletin can inhibit ferroptosis, thereby alleviating cardiovascular diseases (CVDs) such as hypercholesterolemia, hypertension, and coronary heart disease. Ferroptosis is an iron-dependent lipid peroxidation (LPO)-driven programmed cell death, morphologically characterized by smaller mitochondria, reduced mitochondrial cristae, and increased mitochondrial membrane density. Ferroptosis is also a crucial pathological process in the development of aneurysm (AD). RvD1 can reduce ferroptosis and HMGB1 release through the macrophage FPR2 signaling pathway, thereby alleviating aortic inflammation and remodeling during the pathogenesis of abdominal aneurysm. Furthermore, molecules such as HMGB2 and PCSK9 may influence the development of AD by regulating vascular smooth muscle cell (VSMC) ferroptosis. Therefore, VSMC ferroptosis is an important molecular mechanism affecting the development of AD. Currently, there is no existing technology for preparing drugs for the prevention and treatment of aortic dissection using nobiletin (NOB), and it is even more unclear whether nobiletin can protect against the development of AD by improving VSMC ferroptosis.

[0006] This invention provides the application of a compound in the preparation of drugs for preventing and treating aortic dissection. The compound is noriheptacorlin, with the chemical name 3',4',5,6,7,8-hexamethoxyflavone and CAS number 478-01-3. Its molecular structure is as follows: .

[0007] The present invention also provides the use of pharmaceutically acceptable salts, esters, solvates, polymorphs or prodrugs of the compounds described above in the preparation of medicaments for the prevention and / or treatment of aortic dissection.

[0008] In one specific implementation, the molecular mechanism is that noriheptacorlins targets the ferroptosis-associated protein SLC7A11 to improve ferroptosis in vascular smooth muscle cells (VSMCs).

[0009] In one specific embodiment, the drug further comprises one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0010] In one specific embodiment, the dosage form of the drug is an oral preparation, an injection, a spray, a patch, or a sustained-release preparation.

[0011] The present invention also provides a pharmaceutical composition comprising an effective amount of the compound as described above or a pharmaceutically acceptable salt, ester, solvate, polymorph or prodrug thereof, and one or more pharmaceutically acceptable carriers, said pharmaceutical composition for the prevention and / or treatment of aortic dissection.

[0012] In one specific embodiment, the pharmaceutical composition is used in combination with at least one other drug for treating cardiovascular diseases.

[0013] In one specific embodiment, the other drugs for treating cardiovascular diseases are selected from at least one of antihypertensive drugs, beta-blockers, calcium channel blockers, angiotensin-converting enzyme inhibitors, angiotensin II receptor antagonists, or statins.

[0014] The present invention has at least the following beneficial effects: 1. The present invention screened and discovered the therapeutic effect of noriheptacortine on aortic dissection (AD). 2. The present invention clarified the effect of noriheptacortine in improving ferroptosis in vascular smooth muscle cells (VSMCs) by targeting the ferroptosis-related protein SLC7A11. Attached Figure Description

[0015] Figure 1 Gross images of the aorta of BAPN model mice in each group after 4 weeks of drinking water.

[0016] Figure 2 for Figure 1 Survival curves of mice in each group.

[0017] Figure 3 The incidence rates of AD (thoracic aortic dissection) and TAA (thoracic aortic aneurysm) in mice in the BAPN group and NOB+BAPN group were calculated.

[0018] Figure 4 To and Figures 1-2 Statistical analysis of aortic arch diameter in the same groups of mice.

[0019] Figure 5 To and Figures 1-2 The expression levels of MDA, SOD, and GSH / GSSG in the aorta of mice were different among the same groups; Figure A shows the difference in MDA (malondialdehyde) expression level in the aorta of mice among the groups, Figure B shows the difference in SOD (superoxide dismutase) expression level in the aorta of mice among the groups, and Figure C shows the difference in GSH / GSSG (reduced glutathione / oxidized glutathione) expression level in the aorta of mice among the groups.

[0020] Figure 6Figure 1 shows the effect of NOB on the expression of total iron ions and ferroptosis-related proteins in the aorta of a mouse AD model. Figure A shows the total iron ion content in the aorta of each group of mice, and Figure B shows the expression of SLC7A11 and GPX4 in the aorta of the mouse AD model detected by Western blot.

[0021] Figure 7 The diagram shows the docking of NOB with the ferroptosis-associated protein SLC7A11. The top diagram shows the binding of NOB to the two binding sites TYR244 and TYS198, while the bottom diagram is a 2D visualization of the binding site between NOB and SLC7A11.

[0022] Figure 8 This is an immunofluorescence image of primary aortic vascular smooth muscle cells. The green fluorescence image in the upper left corner is an SM22α staining image, the blue fluorescence image in the upper right corner is a DAPI staining image, and the Merge fluorescence image below is a combination of the two.

[0023] Figure 9 This is an immunofluorescence image of primary aortic fibroblasts. The red image in the upper left corner is a COL1A1 staining image, the blue image in the upper right corner is a DAPI staining image, and the Merge fluorescence image below is a combination of the two.

[0024] Figure 10 Immunofluorescence identification diagram for in situ detection of GPX4 and MMP9 protein expression levels. Red should represent GPX4 or MMP9 protein, and blue should represent the fluorescent dye DAPI.

[0025] Figure 11 This image shows immunofluorescence identification of changes in mitochondrial membrane potential, lipid peroxidation levels, and ferrous ion levels. In the first row, red fluorescence represents JC-1 aggregates, and green fluorescence represents JC-1 monomers. In the second row, red fluorescence represents unoxidized C11-BODIPY fluorescent probes, and green fluorescence represents oxidized C11-BODIPY fluorescent probes. The third row, red fluorescence represents the FerroOrange fluorescent probe.

[0026] Figure 12 This is an immunofluorescence pattern identifying changes in reactive oxygen species (ROS) levels. The green fluorescence represents the fluorescent probe DCFH-DA.

[0027] Figure 13 A schematic diagram of Western blotting of the expression of ferroptosis-related proteins SLC7A11 and GPX4 in different groups after NOB and H2O2 treatment.

[0028] Figure 14 Immunofluorescence images show the changes in apoptosis levels in different groups.

[0029] Figure 15 Immunofluorescence images showing changes in mitochondrial membrane potential in different groups.

[0030] Figure 16 Immunofluorescence images showing changes in lipid peroxidation levels in different groups.

[0031] Figure 17 Immunofluorescence images showing the intracellular ferrous ion concentrations in different cell groups.

[0032] Figure 18 Immunofluorescence images showing changes in total oxidative stress levels in different groups.

[0033] Figure 19 This is a schematic diagram of Western Blot analysis of the expression of ferroptosis-related proteins SLC7A11 and GPX4 in different groups after NOB and erastin / RSL3 treatment, where Figure A shows erastin and Figure B shows RSL3.

[0034] Figure 20 Immunofluorescence images showing changes in mitochondrial membrane potential in different groups.

[0035] Figure 21 Immunofluorescence images showing changes in lipid peroxidation levels in different groups.

[0036] Figure 22 Immunofluorescence images showing the intracellular ferrous ion concentrations in different cell groups.

[0037] Figure 23 Immunofluorescence images showing changes in total oxidative stress levels in different groups. Detailed Implementation

[0038] The CMap (Connectivity Map) database is an online pharmacogenomics database that systematically examined the effects of over 5,000 small molecule compounds on the gene expression profiles of various human cell lines using the L1000 platform (covering 978 landmark genes), thus establishing a drug-gene expression profile database. The database's logic is that if a drug can reverse the gene expression profiles of a specific disease, then that drug may have the potential to treat that disease; simultaneously, drugs with similar gene expression profiles may have potential therapeutic effects. Therefore, researchers can use the CMap database to screen drugs with specific gene expression profiles on a large scale, thereby rapidly discovering and screening potential therapeutic agents for diseases. Based on the reverse thinking of "from gene expression to drug discovery," the CMap database provides a completely new perspective on drug development and has become an economical and efficient drug screening tool. Doxycycline is a clinically used tetracycline antibiotic that can inhibit the development of mouse aortic aneurysms by improving VSMC apoptosis through the inhibition of matrix metalloproteinases (MMPs). The inventors used doxycycline as a seed drug and first searched for drugs similar to doxycycline in the CMap database. They identified noriheptacortine as a potential drug for treating AD, as shown in Table 1.

[0039] To determine whether NOB has a protective effect against AD, the inventors conducted experiments using a mouse BAPN (β-aminopropionitrile) model. They found that in the BAPN model, NOB could improve aortic lesions, reduce mortality, and decrease the incidence of TAA and AD in mice.

[0040] The inventors previously examined the expression of total iron content in the aorta of an AD mouse model and found that, compared with the blank control group, the iron content in the aorta of AD mice was significantly upregulated, and the expression of ferroptosis-related proteins (GPX4 and SLC7A11) was significantly downregulated. Compared with the BAPN model group, the iron content in the aorta of mice was significantly downregulated after NOB administration, and the expression of GPX4 and SLC7A11 was significantly upregulated, suggesting that NOB may improve the occurrence and development of AD by inhibiting aortic ferroptosis.

[0041] The inventors performed molecular docking analysis on NOB with ferroptosis-associated proteins GPX4, SLC7A11, HOMX-1, and NRF2, finding that NOB can stably bind to SLC7A11 with a docking score of -6.1 kcal / mol. The docking score is a scoring function provided by the Schrödinger software, used to evaluate the binding ability between the ligand and the target protein during molecular docking.

[0042] The inventors constructed a H2O2-induced HAVSMC ferroptosis cell model using primary human aortic vascular smooth muscle cells (HAVSMCs); and using NOB intervention, they found that H2O2 can induce downregulation of SLC7A11 and GPX4 expression, while NOB administration can upregulate the expression of SLC7A11 and GPX4.

[0043] Erastin is a small molecule ferroptosis agonist that can inhibit Xc - The system inhibits cystine uptake, thereby reducing GPX activity, increasing ROS, and ultimately leading to ferroptosis. RSL3 is a VADC-independent ferroptosis agonist that induces LPO by binding to and inactivating GPX4, ultimately leading to ferroptosis. The inventors induced ferroptosis in HAVSMCs using Erastin / RSL3 separately and found that significant ferroptosis was observed in HAVSMCs after Erastin / RSL3 administration. NOB administration significantly alleviated Erastin / RSL3-induced ferroptosis in HAVSMCs.

[0044] Example 1

[0045] CMap database screening identified NOB as a candidate drug for treating Alzheimer's disease (AD). The inventors, using the CMap database built on the L1000 platform and doxycycline as a seed drug, searched for potential AD treatments. Among the top 30 drugs in terms of score, chuanxiongpietin, a traditional Chinese medicine monomer compound, was found. It can be extracted from natural products, has few side effects, a large extraction yield, and high safety. Table 1 shows the top 30 drugs in the CMap database selected using doxycycline as a seed drug. All 30 drugs in Table 1 are classified as CP, i.e., cell line perturbation.

[0046] Table 1

[0047] Example 2

[0048] This study investigated the protective effect of NOB on an animal model of atrial hyperplasia (AD). A mouse BAPN model was established, and NOB or saline was administered by gavage to explore the protective effect of NOB on AD in vivo. The results showed that in the aorta of the mouse BAPN model, NOB improved aortic lesions, reduced mortality, decreased the incidence of thoracic aortic aneurysms and aortic dissections, and downregulated aortic oxidative stress levels. (See details below.) Figures 1-5 . Figures 1-5 This indicates that NOB has a protective effect against BAPN-induced AD in a mouse model. The experiment consisted of four groups: a Control group, a NOB group, a BAPN group, and a NOB+BAPN group (n=30). Figure 1Gross images of the aorta of BAPN model mice in each group after 4 weeks of drinking water. Figure 2 for Figure 1 Survival curves of mice in each group. Saline is the control group, and BAPN is the BAPN+Saline group. Figure 2 The horizontal axis represents time (number of days), and the vertical axis represents the survival rate (%). Figure 3 The incidence rates of AD (thoracic aortic dissection) and TAA (thoracic aortic aneurysm) in mice in the BAPN group and NOB+BAPN group were calculated. Figure 3 The vertical axis represents the percentage (%), with yellow Normal indicating normal, red Rupture indicating rupture (AD), and blue TAA indicating thoracic aortic aneurysm. Figure 4 To and Figures 1-2 Statistical analysis of aortic arch diameter in mice from the same groups. The vertical axis in the figure represents the aortic arch diameter of the mice, in mm. Figure 5 To and Figures 1-2 The expression levels of MDA, SOD, and GSH / GSSG in the aorta of mice were observed across the same groups. Figure A shows the difference in MDA (malondialdehyde) expression levels in the aorta of mice across the groups, with the vertical axis representing mmol / mg protein. Figure B shows the difference in SOD (superoxide dismutase) expression levels in the aorta of mice across the groups, with the vertical axis representing SOD activity, in U / mg protein. Figure C shows the difference in GSH / GSSG (reduced glutathione / oxidized glutathione) expression levels in the aorta of mice across the groups, with the vertical axis representing the GSH / GSSG ratio, in %. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0049] Example 3

[0050] This embodiment demonstrates that NOB can inhibit aortic ferroptosis in a mouse AD model. The inventors examined the total iron ion content in the aorta of a mouse BAPN model and found that the BAPN model induces an upregulation of total iron ion content in the aorta, and NOB administration significantly inhibited this upregulation. Figure 6 A). To further clarify the expression of ferroptosis-related proteins, the inventors used Western blot to detect the expression of SLC7A11 and GPX4 in the aorta of a mouse AD model. They found that the expression of SLC7A11 and GPX4 in the aorta of BAPN model mice was significantly downregulated, and NOB administration improved the expression of SLC7A11 and GPX4. In the figure, CTRL is the control, and Tubulin is a microtubule protein used as an internal control. Figure 6 B).

[0051] Figure 6Figure 1 shows the effect of NOB on the expression of total iron ions and ferroptosis-related proteins in the aorta of a mouse AD model. Figure A is a schematic diagram of the total iron ion content in the aorta of each group of mice, with the vertical axis representing the total iron ion content in the aorta, in μg / mg protein. Figure A shows that NOB inhibits the upregulation of total iron ion content in the aorta of the mouse BAPN model. Figure B is a Western blot diagram of the expression of SLC7A11 and GPX4 in the aorta of the mouse AD model. Figure B shows that NOB improves the expression of SLC7A11 and GPX4 in the aorta of the mouse BAPN model. ****P < 0.0001.

[0052] Example 4

[0053] In this embodiment, molecular docking revealed that NOB can bind to SLC7A11 and enhance its stability, thereby improving VSMC ferroptosis. This invention used molecular docking technology to predict whether NOB could bind to ferroptosis-related proteins such as SLC7A11. The results showed that NOB can bind to SLC7A11 and enhance its thermal stability, with a docking score (binding energy) of -6.1 kcal / mol. Figure 7 ). Figure 7 The diagram shows the docking of NOB with the ferroptosis-associated protein SLC7A11. The top diagram is a schematic diagram of NOB binding to two binding sites, TYR244 and TYS198, and the bottom diagram is a 2D visualization of the binding site of NOB to SLC7A11. Figure 7 Molecular docking suggests that NOB can bind to SLC7A11 and enhance its thermal stability. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0054] Example 5

[0055] This embodiment found that H2O2 can induce oxidative stress and ferroptosis in HAVSMCs. This invention has established extraction techniques for primary aortic vascular smooth muscle cells and fibroblasts, and performed staining identification. Figure 8 and Figure 9 To determine whether H2O2 can induce ferroptosis in HAVSMCs, we used IF to detect the molecular expression levels of GPX4 and MMP9 in HAVSMCs after H2O2 treatment. The results showed that H2O2 significantly upregulated the expression level of MMP9 protein and downregulated the expression level of GPX4 protein in HAVSMCs; NOB inhibited the changes in the expression levels of MMP9, MMP2, and GPX4 proteins induced by H2O2. Figure 10The inventors then examined changes in mitochondrial membrane potential and lipid peroxidation levels after H2O2 treatment, finding that H2O2 could induce a decrease in mitochondrial membrane potential and lipid peroxidation in HAVSMCs; FerroOrange (a fluorescent probe specifically designed for detecting ferrous ions) was used to detect intracellular ferrous ions (Fe). 2+ The expression level was analyzed, and it was found that H2O2 can significantly upregulate intracellular Fe in HAVSMCs. 2+ level( Figure 11 Finally, we examined the reactive oxygen species (ROS) levels in HAVSMCs after H2O2 treatment and found that H2O2 could induce a significant increase in ROS levels in HAVSMCs. Figure 12 ).

[0056] Figure 8 This is an immunofluorescence identification image of primary aortic vascular smooth muscle cells. The green fluorescent image in the upper left corner is an SM22α staining image. SM22α is smooth muscle 22α, an actin-binding protein highly expressed in vascular smooth muscle cells. The blue fluorescent image in the upper right corner is a DAPI staining image. DAPI is a fluorescent dye that can strongly bind to DNA (200×). The Merge fluorescent image below shows the binding of the two. Figure 9 This is an immunofluorescence image of primary aortic fibroblasts. The red image in the upper left corner is a COL1A1 staining image. COL1A1 is the collagen type I α1 chain gene, which is a key gene encoding type I collagen. The blue image in the upper right corner is a DAPI staining image. The Merge fluorescence image below is a combination of the two, 200×. Figure 10 Immunofluorescence identification diagram for in situ immunofluorescence detection of ferroptosis protein GPX4 and MMP9 protein (matrix metalloproteinase 9) expression levels. Red should represent GPX4 or MMP9 protein, and blue should represent the fluorescent dye DAPI. Figure 11 This image shows immunofluorescence identification of changes in mitochondrial membrane potential, lipid peroxidation levels, and ferrous ion levels. In the first row, red fluorescence represents JC-1 aggregates, and green fluorescence represents JC-1 monomers. In the second row, red fluorescence represents non-oxidized C11-BODIPY, and green fluorescence represents oxidized C11-BODIPY. The third row of red fluorescence represents FerroOrange, a fluorescent probe specifically designed for detecting ferrous ions. Figure 12 This is an immunofluorescence pattern identifying changes in reactive oxygen species (ROS) levels. The green fluorescent component DCFH-DA, or 2',7'-dichlorofluorescein diacetate, is a fluorescent probe for detecting intracellular ROS. Figures 8-12 The scale bars in all of them are 50 μm.

[0057] Example 6

[0058] This embodiment confirms that NOB can inhibit H2O2-induced ferroptosis in HAVSMCs. Based on the H2O2-induced HAVSMC ferroptosis cell model, we further used NOB for pretreatment. To determine whether NOB has a protective effect against H2O2-induced HAVSMC ferroptosis, we first used Western blotting (WB) to detect the expression of ferroptosis proteins SLC7A11 and GPX4. The results showed that H2O2 significantly downregulated the expression levels of SLC7A11 and GPX4 proteins in HAVSMCs; NOB could improve the changes in SLC7A11 and GPX4 protein expression levels induced by H2O2. Figure 13 Then, the inventors examined the apoptosis levels of HAVSMC cells after NOB and H2O2 treatment, and found that NOB could significantly improve H2O2-induced apoptosis in HAVSMCs. Figure 14 Further analysis of mitochondrial membrane potential and lipid peroxidation levels revealed that NOB could improve H2O2-induced decrease in HAVSMC mitochondrial membrane potential and lipid peroxidation. Figure 15 and Figure 16 ); intracellular ferrous ions (Fe) 2+ Level detection revealed that NOB can improve H2O2-induced intracellular Fe in HAVSMCs. 2+ Horizontal change ( Figure 17 Finally, the levels of reactive oxygen species (ROS) in HAVSMCs were measured after NOB and H2O2 treatment. It was found that NOB could alleviate the H2O2-induced increase in ROS levels in HAVSMCs. Figure 18 ). Figures 13-18 NOB inhibits H2O2-induced oxidative stress and ferroptosis in HAVSMCs. Figure 13 This is a schematic diagram of Western blot analysis of the expression of ferroptosis-related proteins SLC7A11 and GPX4 in different groups after NOB and H2O2 treatment. Figure 14 The images show immunofluorescence identification of changes in apoptosis levels in different groups. The green fluorescent Annexin V in the image is a reagent for detecting cell apoptosis, and the red fluorescent PI in the image is also a reagent for detecting cell apoptosis. Figure 15 Immunofluorescence identification images of mitochondrial membrane potential changes in different groups; red fluorescence represents JC-1 aggregates, and green fluorescence represents JC-1 monomers. Figure 16Immunofluorescence images show the changes in lipid peroxidation levels in different groups; red fluorescence indicates non-oxidized C11-BODIPY, i.e., unoxidized C11-BODIPY fluorescent probes, and green fluorescence indicates oxidized C11-BODIPY, i.e., oxidized C11-BODIPY fluorescent probes. Figure 17 Immunofluorescence identification images of intracellular ferrous ion concentrations in different groups of cells, where red fluorescence represents FerroOrange, a fluorescent probe specifically designed for detecting ferrous ions. Figure 18 Immunofluorescence identification images of changes in total oxidative stress levels in different groups, where the green fluorescent DCFH-DA is 2',7'-dichlorofluorescein diacetate, a fluorescent probe for detecting intracellular reactive oxygen species. Figures 14-18 The scale bars in all of them are 50 μm.

[0059] Example 7

[0060] This embodiment demonstrates that NOB inhibits ferroptosis induced by the ferroptosis agonists erastin or RSL3 in HAVSMCs. The inventors intervened in HAVSMCs with erastin or RSL3, and then administered NOB. The expression of ferroptosis-related molecules SLC7A11 and GPX4 was detected. The results showed that, compared with the control group, erastin / RSL3 intervention significantly downregulated the expression levels of SLC7A11 and GPX4 proteins in HAVSMCs; NOB improved the changes in SLC7A11 and GPX4 protein expression levels induced by erastin / RSL3 intervention. Figure 19 A and B). The inventors then examined the mitochondrial membrane potential and lipid peroxidation levels of HAVSMCs after NOB and erastin / RSL3 treatment, finding that NOB could improve the erastin / RSL3-induced decrease in HAVSMC mitochondrial membrane potential and lipid peroxidation. Figure 20 and Figure 21 ); intracellular ferrous ions (Fe) 2+ Level detection revealed that NOB can improve erastin / RSL3-induced intracellular Fe in HAVSMCs. 2+ Horizontal change ( Figure 22 Finally, the levels of reactive oxygen species (ROS) in HAVSMCs were examined after NOB and erastin / RSL3 treatment. It was found that NOB could alleviate the erastin / RSL3-induced increase in ROS levels in HAVSMCs. Figure 23 ).

[0061] Figures 19-23 NOB inhibits erastin or RSL3-induced HAVSMC ferroptosis. Figure 19This is a schematic diagram of Western Blot analysis of the expression of ferroptosis-related proteins SLC7A11 and GPX4 in different groups after NOB and erastin / RSL3 treatment, where Figure A shows erastin and Figure B shows RSL3. Figure 20 Immunofluorescence images showing changes in mitochondrial membrane potential in different groups. Figure 21 Immunofluorescence images showing changes in lipid peroxidation levels in different groups. Figure 22 Immunofluorescence images showing the intracellular ferrous ion concentrations in different cell groups. Figure 23 Immunofluorescence images showing changes in total oxidative stress levels in different groups. Figures 19-23 The scale bars are all 50μm.

[0062] Example 8

[0063] This embodiment describes the experimental operation process in this invention.

[0064] Establishment of the mouse BAPN model: Three-week-old male C57BL / 6 mice were selected and acclimatized for 3 days. A 0.3% BAPN solution was prepared using distilled water and given to the mice as drinking water. The 0.3% BAPN solution was replaced every 3 days, and the mouse weight was recorded. After 4 weeks of BAPN drinking water, the mouse aorta was harvested, photographed, and stored for further experiments.

[0065] Western blot: Take an appropriate amount of arterial tissue, place it in a 2 mL grinding tube, add 1 mL of RIPA lysis buffer (containing protease inhibitor and phosphorylated protease inhibitor) and 2-3 grinding beads of different sizes, grind thoroughly in a low-temperature grinder, centrifuge (4℃, 15000g, 10 minutes) after grinding, and transfer the supernatant to a new 1.5 mL EP tube.

[0066] Tissue iron concentration determination: Prepare 0.125 μM Fe 3+ Standard solution. Take approximately 10 mg of aortic tissue, add 100 μL of extraction buffer, homogenize on ice, centrifuge, and collect the supernatant for sample analysis. Prepare iron concentration blank tubes, standard tubes, and test tubes according to the instructions. After thorough mixing, incubate at room temperature for 10 minutes for color development. Set the measurement wavelength to 520 nm using an ELISA reader and measure the OD value.

[0067] Primary aortic vascular smooth muscle cell extraction: After obtaining the aorta, it was placed in an ice bath with PBS and rinsed three times to remove blood cells. The fat and adventitia were carefully dissected, and the endothelial layer was gently scraped away to obtain the medial aorta. After rinsing three times with PBS, the medial aorta was cut into pieces approximately 1 mm in size. 2The small square pieces were transferred to new gelatin-coated culture dishes and incubated in a cell culture incubator for 4 hours to promote tissue adhesion to the surface of the culture dish. Then, complete culture medium was carefully added.

[0068] FerroOrange assay for intracellular ferrous ion content: 24 μg FerroOrange was dissolved in 35 μL DMSO to obtain a 1 mM FerroOrange stock solution; the FerroOrange stock solution was diluted to 1 μM with HBSS solution to obtain the FerroOrange working solution. After cell pretreatment, the supernatant was discarded, and the cells were washed three times with HBSS. FerroOrange working solution was added, and the cells were incubated in a cell culture incubator for 30 minutes. After incubation, the cells were observed and photographed directly under a fluorescence microscope.

[0069] JC-1 Detection of Mitochondrial Membrane Potential Changes: Prepare JC-1 staining working solution and JC-1 staining buffer according to the instructions. After cell pretreatment, wash twice with PBS, then add 1 mL of DMEM / F12 medium and 1 mL of JC-1 staining working solution sequentially. Mix thoroughly and transfer to a cell culture incubator for 20 minutes. After incubation, wash twice with JC-1 staining buffer, add 2 mL of DMEM / F12 medium, and observe and photograph directly under a fluorescence microscope.

[0070] C11 staining was used to detect cellular lipid peroxidation levels. C11 was diluted from 1 mM to 0.5 µM in DMEM / F12 medium to obtain the C11 staining working solution. After cell pretreatment, the supernatant was discarded, and the cells were washed twice with PBS. An appropriate amount of C11 staining working solution was added, and the cells were incubated for 30 minutes. After incubation, the cells were washed three times with DMEM / F12 medium and then observed directly under a fluorescence microscope. C11 staining refers to staining cells using the C11 BODIPY 581 / 591 fluorescent probe, primarily used to detect intracellular lipid peroxidation levels.

[0071] Malondialdehyde (MDA) detection: Approximately 10 mg of arterial specimen was added to RIPA lysis buffer and homogenized thoroughly before centrifugation. The supernatant was collected for detection. Cells were digested, resuspended, and counted, and 1 × 10⁻⁶ cells were collected. 6 Cells were lysed with 100 μL of Western blot and IP lysis buffer, and the cells were sonicated on ice. After centrifugation, the supernatant was collected for sample analysis to prepare the MDA detection working solution. 1M MDA standard was diluted to 1-50 μM. After setting the sample reaction system, the mixture was heated at 100°C for 15 minutes, cooled, and centrifuged at 1000g for 10 minutes at room temperature. 200 μL of the supernatant was transferred to a 96-well plate, and the OD value was measured at 532 nm using a microplate reader.

[0072] Superoxide dismutase (SOD) detection: Approximately 10 mg of arterial specimen was added to 100 μL of SOD sample preparation solution and homogenized at 4°C. After centrifugation, the supernatant was used as the test sample. Cells were washed once with pre-cooled PBS, then 100 μL of SOD sample preparation solution was added, and the cells were thoroughly lysed by pipetting. The supernatant was then collected after centrifugation as the test sample. The protein concentration of the above samples was determined using the BCA method. The test sample, SOD detection buffer, WST-8 / enzyme working solution, and reaction initiation working solution were added sequentially to a 96-well plate, mixed well, and incubated at 37°C for 30 minutes. The OD value was measured at 450 nm using a microplate reader.

[0073] Reduced glutathione (GSH): Approximately 10 mg of arterial sample was ground in liquid nitrogen, and 100 μL of protein removal reagent M was added. After mixing with Vortex, the mixture was incubated at 4°C for 10 minutes. After centrifugation, the supernatant was collected for measurement. Cells were washed, centrifuged, and the supernatant was removed. 30 μL of protein removal reagent M was added, and the mixture was incubated at 4°C for 10 minutes. After centrifugation, the supernatant was diluted with 270 μL of protein removal reagent M. The diluted sample was used for the determination of total GSH. Samples or standards, protein removal reagent M, and GSH detection working solution were added sequentially to a 96-well plate. After incubation at room temperature for 5 minutes, 50 μL of 0.5 mg / mL NADPH was added, mixed, and incubated for 25 minutes. The OD value was measured at 412 nm using a microplate reader.

[0074] Immunofluorescence of cells: After preparing cell slides, fix with 4% paraformaldehyde for 15 minutes, permeabilize with TBST for 15 minutes, block with 5% goat serum for 30 minutes, add p-STING, p-TBK1, GPX4 or MMP9 primary antibodies, and incubate overnight at 4°C. Wash three times with PBS, add the corresponding species' fluorescent secondary antibody, and incubate at room temperature for 1 hour. Wash three times with PBS, and add an appropriate amount of DAPI-containing anti-fluorescence attenuation mounting medium to the culture plate; observe and acquire images under a fluorescence microscope.

[0075] In situ fluorescence apoptosis detection: Prepare the YP1 / PI detection working solution according to the experimental requirements, using a ratio of YP1:PI:detection buffer = 1:1:998. After cell pretreatment, wash the cells once with PBS. Add 1 mL of YP1 / PI detection working solution and incubate at 37°C in the dark for 20 minutes. After incubation, observe the cells under a fluorescence microscope as soon as possible.

[0076] Total oxidative stress level determination: DCFH-DA was diluted 1:1000 in DMEM / F12 medium to a final concentration of 10 μM. After pretreatment of cells according to experimental groups, the medium was discarded, and the cells were washed three times with PBS. After thoroughly removing the PBS, 1 mL of diluted DCFH-DA probe was added to each well. The cells were incubated in a cell culture incubator for 20 minutes. The cells were then washed three times with DMEM / F12 medium and observed and photographed under a fluorescence microscope.

[0077] Cellular iron content determination: Prepare 0.5 μM Fe 3+ Standard solution. After cell pretreatment, digest, resuspend, and count the cells, then take 1*102 cells. 6 Cells were centrifuged and the supernatant was discarded. 100 μL of extraction buffer was added and homogenized in an ice bath. After centrifugation, the supernatant was collected for sample analysis. Iron concentration blank tubes, standard tubes, and test tubes were prepared according to the instructions. After thorough mixing, the mixture was allowed to stand at room temperature for 10 minutes for color development. The OD value was measured using a microplate reader with the measurement wavelength set to 510 nm.

[0078] In general, aortic dissection has a high mortality rate, and treatment relies heavily on surgery, which also has an extremely high mortality rate. Currently, there are no effective drugs for the prevention and treatment of aortic dissection in clinical practice. This invention screened and identified noriheptacortine as a drug for the prevention and treatment of aortic dissection, and clarified its molecular mechanism: noriheptacortine targets the ferroptosis-related protein SLC7A11 to improve ferroptosis in vascular smooth muscle cells (VSMCs), thus providing a new target for the treatment of aortic dissection.

[0079] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The use of a compound in the preparation of drugs for preventing and treating aortic dissection, characterized in that, The compound is noriheptacorlin, with the chemical name 3',4',5,6,7,8-hexamethoxyflavone and CAS number 478-01-3. Its molecular structure is as follows: 。 2. The use of a pharmaceutically acceptable salt, ester, solvate, polymorph, or prodrug of the compound as described in claim 1 in the preparation of a medicament for the prevention and / or treatment of aortic dissection.

3. The application according to claim 1 or 2, characterized in that, Its molecular mechanism is that tangeretin targets the ferroptosis-related protein SLC7A11 to improve ferroptosis in vascular smooth muscle cells (VSMCs).

4. The application according to claim 1 or 2, characterized in that, The drug also includes one or more pharmaceutically acceptable carriers, diluents, or excipients.

5. The application according to claim 1 or 2, characterized in that, The dosage form of the drug is an oral preparation, injection, spray, patch, or sustained-release preparation.

6. A pharmaceutical composition, characterized in that, It comprises an effective amount of the compound as described in claim 1 or 2 or a pharmaceutically acceptable salt, ester, solvate, polymorph or prodrug thereof, and one or more pharmaceutically acceptable carriers, the pharmaceutical composition being used for the prevention and / or treatment of aortic dissection.

7. The pharmaceutical composition according to claim 6, characterized in that, The pharmaceutical composition is used in combination with at least one other drug for the treatment of cardiovascular diseases.

8. The pharmaceutical composition according to claim 7, characterized in that, The other drugs used to treat cardiovascular diseases are selected from at least one of antihypertensive drugs, beta-blockers, calcium channel blockers, angiotensin-converting enzyme inhibitors, angiotensin II receptor antagonists, or statins.

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