Application of sivelestat in preparation of medicine for treating aortic endothelial cell injury

Abstract

The invention relates to application of sivelestat in preparation of a medicine for treating aortic endothelial cell injury, and belongs to the field of biological medicine. Specifically, the sivelestat reduces the formation of neutrophil extracellular traps (NETs) by inhibiting the activity of neutrophil elastase (NE), thereby reducing the aortic endothelial inflammation injury. In-vitro experiments show that sivelestat can significantly inhibit generation of NETs induced by S100A12 and expression of endothelial inflammatory factors; in-vivo experiments prove that the compound has the effects of reducing the aortic dilatation rate and rupture rate and improving the survival rate on AD model mice induced by beta-aminopropionitrile (BAPN). The invention provides a new treatment strategy for the aortic dissection.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, and particularly relates to application of civelestat in preparation of a drug for treating aortic endothelial cell injury. BACKGROUND

[0002] The aorta is the largest conduit in the human circulatory system, which is divided into various trunks and accessory vessels to transport blood pumped by the heart to various tissues and organs for oxygen and essential nutrient exchange. The aortic wall is structurally composed of three layers: the intima composed of endothelial cells, the media composed of smooth muscle cells, and the adventitia composed of fibroblasts. Endothelial cell dysfunction is the initiating link of various aortic diseases, including inflammation, oxidative stress, aging, etc.

[0003] Aortic endothelial cell inflammation is an important pathophysiological process of aortic diseases, and its core manifestation is a chronic inflammatory response of endothelial cells under metabolic abnormalities, hemodynamic changes or immune stimulation. Endothelial cell inflammation not only affects the integrity of vascular endothelium, but also promotes inflammatory cell infiltration and vascular wall structure destruction through various mechanisms. This process not only drives the occurrence of atherosclerosis, but is also closely related to aortic dissection, aortic aneurysm, hypertension and diabetic vasculopathy. In recent years, with the development of single-cell sequencing and molecular imaging technology, the spatiotemporal dynamic regulation of endothelial inflammation has been better understood.

[0004] Neutrophils, as the first responders of the innate immune system, play a complex and critical role in the occurrence and development of aortic endothelial inflammation. Recent studies have shown that neutrophils not only participate in early endothelial injury through classical inflammatory pathways, but also promote the progression of aortic diseases through neutrophil extracellular traps (NETs), cytokine secretion and interaction with other immune cells.

[0005] The key pathways by which neutrophils exacerbate aortic endothelial inflammation include several aspects. First, adhesion and migration: neutrophils are recruited to the site of inflammation through the leukocyte adhesion cascade. Under the stimulation of inflammation, aortic endothelial cells express adhesion molecules (such as P-selectin, E-selectin, ICAM-1, etc.), and neutrophils adhere firmly to the surface of aortic endothelial cells by binding to these adhesion molecules and migrate to the surface. This process is the initial step of inflammatory cell infiltration, laying the foundation for subsequent inflammatory reactions. Second, release of inflammatory mediators: activated neutrophils release a variety of inflammatory mediators, such as superoxide, tissue factor, neutrophil serine proteases, etc. These substances can directly damage aortic endothelial cells, leading to endothelial dysfunction and further exacerbating inflammation. In addition, neutrophils also recruit more inflammatory cells by releasing cytokines (such as IL-1β, IL-17, etc.) and chemokines (such as CXCL1, CXCL2), forming an inflammatory cascade. Third, oxidative stress. Activated neutrophils initiate an oxidative burst, producing a large amount of reactive oxygen species (ROS). ROS can not only directly damage aortic endothelial cells, but also further induce the secretion of inflammatory cytokines, amplifying the inflammatory response. In addition, oxidative stress also promotes the formation of NETs. Fourth, activation of matrix metalloproteinases: neutrophils can secrete and activate matrix metalloproteinases (such as MMP-8, MMP-9), which can degrade the extracellular matrix of aortic intima and media, leading to the destruction of elastic fibers and collagen fibers. This matrix degradation makes the aortic wall more fragile, increasing the risk of aortic dissection and rupture. Fifth, formation of NETs. NETs can capture and kill pathogens, but at the same time, they also release a large amount of DNA and histone, which have strong pro-inflammatory effects. NETs can also activate platelets, promote thrombus formation, and further aggravate aortic lesions.

[0006] It is well known that in the pathological process of aortic dissection, inflammation and matrix degradation work together to damage the structural integrity of the aortic wall, ultimately triggering the formation and development of aortic dissection. There are two possible changes that are considered to be the initiating events of aortic dissection: intimal tear and intramural hematoma formation. These changes are typical and important features of aortic dissection, but the relationship and mechanism between them are not yet clear.

[0007] Under normal circumstances, the media structure of the aorta is intact, and collagen is wrapped in the extracellular matrix, providing the necessary elasticity and strength for the blood vessel. However, in aortic dissection, due to hypertension, genetic diseases (such as Marfan syndrome, Ehlers-Danlos syndrome), aortic valve disease, trauma or other factors causing intimal injury of the aorta, blood can penetrate the intima and enter the media. When the intima is torn, blood flows into the media, causing pressure on the media, leading to further tearing of the media, forming a dissection. This process can cause the extracellular matrix of the media, especially collagen, to be destroyed and exposed. Exposure of collagen can activate platelets and the coagulation cascade, while also attracting inflammatory cells to the injury site, triggering an inflammatory response.

[0008] These two non-negligible responses, inflammation and coagulation, need to be bridged to cross them, making it easier to target and intervene in aortic lesions. Collagen exposure is a key early event in aortic injury. As a damage-associated molecular pattern (DAMP), collagen can activate neutrophils when exposed, triggering the production of reactive oxygen species (ROS) and NETosis. During NETosis, neutrophils release neutrophil extracellular traps (NETs), which can capture coagulation factors and migrating platelets, then induce coagulation. Therefore, NETosis has the potential to become a bridge from inflammation to coagulation in the prevention and treatment of aortic dissection.

[0009] The S100 protein family contains more than 20 members (such as S100A8, S100A9, S100A12, S100B, etc.), all of which have common characteristics, including: 1. Calcium-dependent signal regulation: by binding Ca 2+ Change conformation, activate downstream pathways (such as RAGE, TLR4); 2. Pro-inflammatory function: Most members (such as S100A8 / A9) are damage-associated molecular patterns (DAMPs) involved in innate immune responses. 3. Cell-specific expression: S100A8 / A9 / A12 is mainly highly expressed in neutrophils and monocytes. Among them, S100A12 has been reported to have a direct correlation with NETs. S100A12 and annexin A5 (ANXA5) interact to enhance calcium influx and promote the formation of NETs when myocardial infarction occurs. In addition, S100A12 also triggers NETs in diseases such as sepsis, autoimmune diseases, and lung cancer.

[0010] Sivelestat (C 20 H 22N2O7S) is a selective, competitive, low molecular weight neutrophil elastase synthesis inhibitor. At present, the main approved indications in China are acute lung injury (ALI) / acute respiratory distress syndrome (ARDS) of systemic inflammatory response syndrome. Recent basic research has also confirmed its pharmacological effects in preventing atherosclerosis, kidney injury treatment, and may have a protective effect on the heart. However, there is still no relevant research on the role of sivelestat in AD. Therefore, the present application aims to provide the leukocyte protective use of sivelestat for preventing or treating AD. SUMMARY

[0011] In view of the problems existing in the prior art, the purpose of the present application is to provide sivelestat for reducing the formation of neutrophil NETs in aortic tissue, thereby protecting the aortic endothelial damage induced by inflammation. Aortic dissection (AD) is a specific embodiment.

[0012] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions.

[0013] The present application discloses the application of sivelestat in the preparation of a drug for preventing or treating aortic diseases.

[0014] Further, the aortic disease is aortic dissection.

[0015] Further, the sivelestat plays a role by inhibiting the formation of neutrophil extracellular traps (NETs).

[0016] Further, the sivelestat is used for reducing inflammation-induced aortic endothelial cell damage.

[0017] Further, the sivelestat is used for reducing the incidence or rupture rate of aortic dissection.

[0018] The present application also discloses a pharmaceutical composition for preventing or treating aortic diseases, characterized in that it comprises sivelestat as an active ingredient.

[0019] Further, it further comprises a pharmaceutically acceptable excipient.

[0020] Further, the dosage form of the pharmaceutical composition includes a pharmaceutically acceptable dosage form.

[0021] Further, it is characterized in that the composition further comprises an S100A12 protein inhibitor.

[0022] Furthermore, the pharmaceutical composition exerts its effect by inhibiting S100A12-induced formation of extracellular traps (NETs) in neutrophils.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0024] Compared to existing indications, this invention, through extensive experimental findings, demonstrates that cevelexistat can inhibit aortic atrophy (AD) formation. In in vitro cell studies, co-culture of S100A12-stimulated dHL-60 cells and endothelial cells (C166) confirmed significant endothelial cell inflammation during aortic inflammation. The addition of cevelexistat to S100A12-stimulated dHL-60 cells significantly reduced NET formation and inflammation. In vivo animal studies, the use of an Alzet implanted osmotic pump to deliver cevelexistat in BAPN-induced AD model mice significantly reduced AD formation and improved survival rates. These results indicate that cevelexistat plays a crucial regulatory role in the development and progression of aortic inflammation and may be a novel target for protecting against aortic endothelial injury. Attached Figure Description

[0025] Figure 1 Aortic NETs promote endothelial inflammation. Figure A is a schematic diagram of co-culture of neutrophils and endothelial cells. Figure B is a Western blot showing the successful overexpression of S100A12 in neutrophils. Figure C is the Western blot result of NETs stimulating endothelial inflammation.

[0026] Figure 2 In a NETs cell model stimulated by S100A12, administration of cevelex significantly downregulated NE expression. Figure A shows the Western blot results of the SIV concentration gradient, Figure B is the statistical plot of the Western blot, and Figure C shows the SYTOX Green staining at the optimal SIV concentration and time.

[0027] Figure 3 It is for S100A12 TG Transgenic mice given simultaneous administration of SIV and β-aminopropionitrile (BAPN) showed that AD development was inhibited. Figure A shows the method for constructing the transgenic mice; Figure B shows the animal experimental design; Figure C shows the survival curve; Figure D shows the diameter of the thoracic aorta; Figures E and F show the incidence and rupture rate of AD. Figure G shows the HE staining results. Detailed Implementation

[0028] The present invention will be further described in detail below with reference to specific embodiments. However, this should not be construed as limiting the scope of the above-described subject matter of the present invention to the following embodiments; all technologies implemented based on the content of the present invention fall within the scope of the present invention.

[0029] Unless otherwise indicated, reagents and materials used in the present application are commercially available. The present application is all processed by GraphPad Prism 9.5 software package, and p<0.05 is considered to be statistically different.

[0030] Example 1 Aortic NETs promote endothelial inflammation.

[0031] 1. Transfect dHL-60 cells with S100A12 plasmid.

[0032] Using 1.3% DMSO to induce HL-60 cells to differentiate into neutrophils (i.e. dHL-60) for 5 days, after washing with PBS buffer, resuspend the cells in 6cm cell culture dish with 1600μL RPMI 1640 medium containing 10% fetal bovine serum (FBS) at a cell density of about 70%-80%, and use polyplus transfection reagent to transfect ovNC plasmid and ovS100A12 plasmid (S100A12-GFP) into dHL-60 cells, respectively. Incubate in a 37°C, 5% CO2 cell incubator for 6-8h, collect the cells, centrifuge at 2000r for 5min, then resuspend in a 6cm cell culture dish with 2mL RPMI 1640 medium containing 10% FBS, continue to culture for 12h for use in experiments.

[0033] 2. Co-culture of dHL-60 cells and HUVEC cells.

[0034] Resuspend HUVEC cells in a six-well plate with 10% FBS-containing DMEM, and incubate in a 37°C, 5% CO2 cell incubator for 12h after the same time as the start of dHL-60 transfection, with a cell density of 80%, and replace the medium with 10% FBS-containing DMEM 1600μL per well. Place a Transwell (Corning, 3450) with a pore size of 0.4μm in the six-well plate, and place 400μL of ovNC group dHL-60 cells in the upper chamber in one group (3 wells), and place 400μL of ovS100A12 group dHL-60 cells in the upper chamber in another group (3 wells). Co-culture dHL-60 cells and HUVEC cells for 12h.

[0035] 3. Western Blot to detect the effect of NETs on endothelial inflammation.

[0036] (1) Extraction of cell protein. Collect cells from the upper and lower chambers of the two groups of Transwell, centrifuge at 2000r for 5 minutes, then add appropriate amount of RIPA lysis buffer, lyse on ice for 30 min, mix every 5 min. 4°C, 12000g, centrifuge for 20 min, collect the supernatant as total protein. Use BCA colorimetric kit to determine the protein concentration. Use protein lysis buffer, dilute to a final concentration of 2 mg / mL. Then add 4x loading buffer in proportion, mix well, and boil in 100°C water bath for 5 min, centrifuge at 12000g for 30 s at room temperature.

[0037] (2) Western Blot detection. Add 20µg of protein sample to each sample well, turn on the power and start electrophoresis. The voltage and time are as follows: 100V for 30min, 120V for 60min, and turn off the power after the bromophenol blue electrophoresis reaches the bottom of the glass plate. Transfer the sample to the PVDF membrane at a voltage of 90V for 2h. Put the PVDF membrane into 5% milk blocking solution for 1h, then add the primary antibody and incubate at 4°C overnight. S100A12 (Abcam), IL-1β (ABclonal), IL-6 (ABclonal), VCAM1 (HUABIO), and GAPDH (Cell Signaling Technology) were used as primary antibodies, and horseradish peroxidase-labeled goat anti-mouse (or anti-rabbit) antibody (Cell Signaling Technology) was used as secondary antibody. The ECL kit (Amersham) was used for luminescence imaging. The S100A12 antibody is about 15kDa (the exogenous antibody with GFP tag is about 42kDa), the IL-1β antibody is about 17kDa, the IL-6 antibody is about 24kDa, the VCAM1 antibody is about 100kDa, and the GAPDH antibody is about 37kDa. ImageJ 1.54 software was used to measure the gray value of the bands and perform statistical analysis.

[0038] The results show: the mode diagram of co-culture of dHL-60 cells and HUVEC cells using Transwell ( Figure 1 A); Western Blot confirms that S100A12 overexpression in dHL-60 cells of the ovS100S12 group is successful ( Figure 1 B); Western Blot shows that HUVEC cells can cause significant increase in endothelial inflammation indicators after inducing NETs in dHL-60 cells ( Figure 1 C).

[0039] Example 2: The administration of Cibisartan in S100A12 stimulated NETs cell model significantly down-regulated NE expression.

[0040] 1. The successful construction of NETs model by dHL-60 cells with S100A12.

[0041] The dHL-60 cells were resuspended in 6cm cell culture dishes with RPMI 1640 medium containing 20% fetal bovine serum (FBS), and when the cell density reached about 90%, the serum-free and antibiotic-free RPMI 1640 medium was used, and 400 pg / mL of S100A12 was given for 2h to construct the NETs cell model for experiment.

[0042] 2. Western Blot detection of the effect of different concentrations of SIV on NE in NETs cell model.

[0043] (1) Before the construction of NEts cell model by S100A12, 0 µg / mL, 1 µg / mL, 10 µg / mL, and 50 µg / mL of SIV were given for pretreatment, and cultured in a 37℃, 5% CO2 cell incubator for 90 min. According to the preparation condition of SIV dissolved in DMSO, 5 µL of DMSO was added to each group to balance.

[0044] (2) Extraction of cell protein. Collect 4 groups of cells, centrifuge at 2000r for 5 minutes, then add appropriate amount of RIPA lysis buffer, lyse on ice for 30 min, mix well every 5 min. 4℃, 12000g, centrifuge for 20 min, collect the supernatant as the total protein of the tissue. The protein concentration was determined by BCA colorimetric kit. Use protein lysis buffer, dilute to a final concentration of 2 mg / mL. Then add 4x loading buffer in proportion, mix well, and boil in 100℃ water bath for 5 min, centrifuge at 12000g for 30 s at room temperature.

[0045] (3) Western Blot detection. Add 20 μg of protein sample to each sample well, turn on the power and start electrophoresis. Perform according to the following voltage and time: 100 V voltage, 30 min, 120 V voltage, 60 min, turn off the power after the bromophenol blue electrophoresis to the bottom of the glass plate. Transfer the sample to the PVDF membrane at a voltage of 90 V for 2 h. After blocking the PVDF membrane in 5% milk for 1 h, add the primary antibody and incubate at 4°C overnight. NE (Abcam Company) and GAPDH (Cell Signaling Technology Company) are used as primary antibodies, and horseradish peroxidase-labeled goat anti-mouse (or anti-rabbit) antibody (Cell Signaling Technology Company) is used as secondary antibody. Use the ECL kit (Amersham Company) for luminescence imaging. The NE antibody is about 19 kDa, and the GAPDH antibody is about 37 kDa. ImageJ 1.54 software is used for gray value measurement of the bands and statistical analysis.

[0046] 3. SYTOX Green staining to detect the effect of SIV on NETs.

[0047] (1) The control group used 1 mL of serum-free and antibiotic-free RPMI 1640 medium containing 400 pg / mL S100A12 to resuspend dHL-60 in a 15 mm confocal dish, and stimulated for 2 h to construct a NETs cell model; the intervention group used 1 mL of serum-free and antibiotic-free RPMI 1640 medium containing 50 μg / mL SIV to resuspend dHL-60 in a 15 mm confocal dish, and pretreated for 90 min, centrifuged at 2000 r for 5 min, and then used 1 mL of serum-free and antibiotic-free RPMI 1640 medium containing 400 pg / mL S100A12 to resuspend dHL-60 in a 15 mm confocal dish, and stimulated for 2 h to construct a NETs cell model.

[0048] (2) Both the control group and the intervention group were centrifuged at 2000 r for 5 min, and then resuspended the cells in a confocal dish with 1 μM SYTOX Green dye (Invitrogen Company) serum-free and antibiotic-free RPMI 1640 medium, 1 mL, respectively, and stained for 30 min in a 37°C, 5% CO2 cell incubator.

[0049] (3) Both the control group and the intervention group were centrifuged at 2000 r for 5 min, the supernatant was discarded, and the cells were washed 1-2 times with 1 mL of PBS, and then centrifuged to remove the supernatant, and then resuspended the cells in a confocal dish with 1 mL of serum-free and antibiotic-free RPMI 1640 medium containing 20 μM Hoechst 33342 dye (MCE Company), and stained for 5 min in a 37°C, 5% CO2 cell incubator.

[0050] (4) Centrifuge at 2000 r for 5 min for the control group and the intervention group respectively, discard the supernatant, wash with PBS 2-3 times, and resuspend the cells in a confocal dish with 1 mL of serum-free and antibiotic-free RPMI 1640 medium. Observe the NETs using a confocal microscope (ZEISS).

[0051] The results showed that Western blot analysis revealed that the inhibitory effect of SIV on NE was concentration-dependent. Figure 2 A, B); Microscopic observation showed that SIV could significantly inhibit the formation of NETs ( Figure 2 C).

[0052] Example 3 is for S100A12 TG Transgenic mice given simultaneous administration of SIV and BAPN showed that AD development was inhibited.

[0053] 1. Construct the Elane-P2A-S100A12 gene knock-in mouse model.

[0054] Since mice cannot naturally express S100A12, we used CRISPR-Cas9 technology to edit the Elame gene, integrating the Elane-P2A-S100A12 gene sequence into the transposon, thereby generating transgenic mice. Figure 3 A). The CRISPR / Cas9 system and Donor vector samples were microinjected into mouse zygotes with a C57BL / 6JGpt background; surviving zygotes were then transplanted into pseudopregnant female mice to establish a transgenic (S100A12) model. TG TG mice. Jiangsu Jicui Yaokang Biotechnology Holding Co., Ltd. was responsible for the preparation and breeding of TG mice, which were then preserved in our laboratory under specific pathogen-free conditions. All mice underwent a 12-hour light-dark cycle at a controlled temperature of 22°C, with unrestricted access to food and water.

[0055] 2. Animal experiment design scheme.

[0056] (1) AD model establishment. Four-week-old male S100A12 were used. TG A transgenic mouse model of Alzheimer's disease (AD) was established by administering drinking water containing 0.25% BAPN for 28 days.

[0057] (2) Animal experimental protocol for SIV treatment. Four-week-old male S100A12 animals were used. TGThe 24 transgenic mice were randomly divided into 4 groups, including Ctrl group, 10 mg / kg·d SIV group, 25 mg / kg·d SIV group, and 50 mg / kg·d SIV group. The AD model was established by the above method for 28 days. From the beginning of the experiment, the SIV treatment group was injected intraperitoneally with the corresponding dose of SIV every day Figure 3 B). Survival was recorded.

[0058] 3. Vascular ultrasound was used to observe the blood vessels. On the 28th day of the experiment, vascular ultrasound was performed on the mice under inhalation anesthesia. The diameter of the thoracic aorta was recorded.

[0059] 4. Aorta isolation and extraction. After completing the vascular ultrasound, the mice were sacrificed. The thoracic cavity was exposed, and the aorta was isolated. Using microscissors and forceps, the aorta was isolated from the tail end of the aorta (iliac artery bifurcation) to the aortic arch or from the aortic arch to the iliac artery bifurcation using blunt dissection techniques, and the surrounding fat tissue was cleaned under a microscope to completely expose the aorta. The occurrence and rupture of AD were observed.

[0060] 5. HE staining was used to detect the general morphology of the thoracic aorta. The aorta tissue from the Ctrl group, 10 mg / kg·d SIV group, 25 mg / kg·d SIV group, and 50 mg / kg·d SIV group after 28 days of AD model construction was collected for HE staining.

[0061] The specific steps are as follows.

[0062] (1) Preparation of paraffin sections.

[0063] A. Sample collection: The thoracic aorta of the mouse was placed in a 4% paraformaldehyde solution overnight.

[0064] B. Dehydration: Dehydration was performed with different alcohol concentrations for 2 hours at 70%, 80%, 90%, 95% for 2 hours, 95% for overnight, 100% for 1.5 hours, and 100% for 1.5 hours.

[0065] C. Transparency: The tissue block was immersed in xylene I for 1 hour, and then transferred to xylene II for 1 hour.

[0066] D. Wax immersion: The tissue block was immersed in paraffin I overnight, paraffin II for 1 hour, and paraffin III for 1 hour.

[0067] E. Embedding: The tissue block was embedded with paraffin and placed at room temperature.

[0068] F. Sectioning: The tissue block was sectioned using a paraffin microtome with a thickness of 3 µm, and the sections were attached to glass slides.

[0069] G. Baking and baking: the glass slides were placed in a 60°C slide dryer for 1h, and then the glass slides were placed in a 65°C oven for 48h.

[0070] (2) Dewaxing the section. Place the section in the following reagents for 20 min in xylene I, 20 min in xylene II, 15 min in 95% alcohol I, 15 min in 95% alcohol II, 10 min in 90% alcohol, 5 min in 80% alcohol, 5 min in 70% alcohol, and finally in distilled water for 30 min.

[0071] (3) Nucleus staining.

[0072] A. Immerse the section in hematoxylin solution for 20 min.

[0073] B. Differentiate the paraffin section in 1% hydrochloric acid for 30 s, and rinse with running water.

[0074] C. Blue the nucleus back by placing the paraffin section in ammonia water for 30 s, and rinse with running water.

[0075] (4) Cytoplasmic staining: stain the paraffin section in water-soluble eosin solution for 30 min, and rinse with running water.

[0076] (5) Transparency: place the paraffin section in the following reagents according to the procedure, 80% alcohol for 5 min, 90% alcohol for 5 min, 100% alcohol I for 5 min, 100% alcohol II for 5 min, xylene I for 5 min, and xylene II for 5 min.

[0077] (6) Mounting: dry the paraffin section in a fume hood, and mount the section with neutral resin.

[0078] (7) Observe the HE staining results under a microscope and take pictures.

[0079] 6. NE and CD31 immunofluorescence (IF) co-staining.

[0080] Collect the aortic tissues of the Ctrl group, 10 mg / kg·d SIV group, 25 mg / kg·d SIV group, and 50 mg / kg·d SIV group after 28 days of AD model construction, and perform NE and CD31 immunofluorescence staining.

[0081] The specific steps are as follows.

[0082] (1) Section dewaxing: same as HE staining (steps 1 and 2).

[0083] (2) Antigen repair solution repair (China Solerbio Company) for 1h.

[0084] (3) NE and CD31 primary antibody staining: Rabbit anti-NE (Abcam) and mouse anti-CD31 (Santa) were prepared with PBS at a ratio of 1:100, 100µL / slide, and incubated at 37℃ in the dark for 1h, then washed with water.

[0085] (4) Secondary antibody staining: Goat anti-rabbit Alexa Fluor 594 (Thermo Fisher) and goat anti-mouse Alexa Fluor 488 (Thermo Fisher) were prepared with PBS at a ratio of 1:200, 100µL / slide, and incubated at 37℃ in the dark for 1h, then washed with water.

[0086] (5) DAPI staining: 100µL of DAPI staining solution per slide, incubated at 37℃ in the dark for 5 min, then washed with water.

[0087] (6) Sealing: Use anti-quenching sealing medium (Thermo Fisher) to seal the film and let it dry.

[0088] (7) Observe the IF staining results under a microscope and take pictures.

[0089] The results showed that SIV significantly improved the survival rate of BAPN-induced AD models. Figure 3 C), while reducing aortic dilation ( Figure 3 D). In particular, high-dose SIV can significantly reduce the incidence and rupture rate of AD (D). Figure 3 E, F). HE staining showed that SIV could reduce the incidence of AD, and immunofluorescence staining showed that SIV could significantly reduce NE expression and protect endothelial cells (E, F). Figure 3 G).

[0090] The above description is merely a preferred embodiment of the present invention and is not intended to limit the patent scope of the present 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 protection scope of the present invention.

Claims

1. Use of cipamidast for the manufacture of a medicament for preventing or treating aortic disease.

2. Use according to claim 1, wherein The aortic disease is aortic dissection.

3. The use according to claim 1, wherein The cipamidast acts by inhibiting the formation of neutrophil extracellular traps (NETs).

4. The use according to claim 1, wherein The cipamidast is used to reduce inflammation-induced aortic endothelial cell damage.

5. The use according to claim 1, wherein the compound is ###0002### The cipamidast is used to reduce the incidence or rupture rate of aortic dissection.

6. A pharmaceutical composition for preventing or treating aortic disease, characterized by, A pharmaceutical composition comprising cipamidast as an active ingredient.

7. The pharmaceutical composition of claim 6, wherein A pharmaceutical composition further comprising a pharmaceutically acceptable excipient.

8. The pharmaceutical composition of claim 6, wherein The dosage form of the pharmaceutical composition includes a pharmaceutically acceptable dosage form.

9. The pharmaceutical composition of claim 6, wherein characterized in that, The composition further comprises an S100A12 protein inhibitor.

10. The pharmaceutical composition of claim 6, wherein The pharmaceutical composition acts by inhibiting S100A12-induced neutrophil extracellular trap (NETs) formation.

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