Application of COX-2 and 5-LOX in prevention and treatment of aortic dissection

Abstract

The invention relates to the field of biological medicine, and particularly discloses application of COX-2 and 5-LOX in prevention and treatment of aortic dissection. The invention discovers that the dual inhibitor of COX-2 and 5-LOX, especially Lcofelone, can reduce the incidence rate and / or death rate of aortic dissection, reduce the expansion of aortic diameter and improve the lesion degree of aorta; through qPCR, ELISA and WB, it is further found that dual inhibition of COX-2 and 5-LOX can reduce inflammatory response, improve vascular remodeling, relieve aortic damage and prevent progress of aortic dissection, and a new thought and method are provided for treatment of aortic dissection.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to an application of COX-2 and 5-LOX in preventing and treating aortic dissection. Background Art

[0002] Aortic dissection (AD), as an acute and extremely threatening aortic disease, has always been a major challenge in the field of cardiovascular disease. Its pathological characteristics are mainly manifested in the tearing of the aortic intima, which causes blood to penetrate into the middle layer of the aorta, and then forms two true and false chambers in the vascular wall. This structural change greatly weakens the stability of the aortic wall and increases the risk of rupture. Aortic dissection can be divided into type A and type B according to the anatomical location. Type A dissection is more dangerous because it involves the ascending aorta. For patients with acute type A dissection who are not treated in time, the mortality rate within 24 hours can be as high as 1%-2% per hour, and the mortality rate within 48 hours is more than 50%. Therefore, it is regarded as a cardiovascular emergency that urgently needs emergency medical intervention. Even in the chronic stage, patients with aortic dissection are at long-term risk of aortic aneurysm expansion and rupture, their quality of life is seriously impaired, and their life safety is always threatened.

[0003] Inflammatory mechanisms play a vital role in the occurrence and development of aortic dissection. A large number of inflammatory cells can be seen infiltrating the vascular wall of patients with aortic dissection. These inflammatory cells include macrophages, neutrophils, and T cells, which induce local inflammatory cascade reactions by secreting inflammatory factors such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). This persistent chronic inflammatory environment not only exacerbates the oxidative stress response, but also seriously damages the normal function of vascular smooth muscle cells (VSMCs). As the main maintainer of vascular structure, the abnormal function of VSMCs has a profound impact on the formation and progression of aortic dissection. In aortic dissection, VSMCs show a tendency to apoptosis and phenotypic transformation, from the synthetic type that is originally responsible for maintaining vascular structure and function to the inflammatory phenotype. This change further weakens the strength and integrity of the vascular wall. More seriously, the dysfunction of VSMCs can also promote the abnormal activation of matrix metalloproteinases (MMPs). MMPs are a class of enzymes that can degrade extracellular matrix components. Under normal circumstances, they are involved in vascular remodeling and repair processes. However, under pathological conditions, such as aortic dissection, abnormal activation of MMPs can lead to the degradation of key structural components such as elastic fibers and collagen, thereby significantly increasing the fragility of the aortic wall and making the vascular wall more susceptible to tearing and dissection.

[0004] At present, the clinical treatment of aortic dissection mainly focuses on emergency surgical intervention and drug therapy. Although surgical treatment can quickly solve the problem of vascular wall tearing caused by aortic dissection, the operation has high risks and slow postoperative recovery. For some patients, surgery is not the best choice. Drug treatment mainly reduces the pressure on the aortic wall by controlling physiological indicators such as blood pressure and heart rate, and uses anti-inflammatory, antioxidant and other drugs to reduce inflammatory response and oxidative stress damage. However, the existing drug treatment options have limited effects and have multiple side effects, which are difficult to meet clinical needs.

[0005] Therefore, finding new drug targets and treatment methods to improve the treatment effect of aortic dissection and reduce mortality and complication rates has become a research hotspot in the field of cardiovascular diseases. In recent years, with the in-depth study of the role of inflammatory mechanisms in aortic dissection and the continuous advancement of drug development technology, cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) as key enzymes for inflammation regulation have gradually entered the field of researchers' field of vision. COX-2 and 5-LOX play an important role in the inflammatory response. They catalyze the biosynthesis of inflammatory mediators such as prostaglandins (PGs) and leukotrienes (LTs), respectively. These inflammatory mediators play a key role in the pathogenesis of aortic dissection. Therefore, inhibiting the activity of COX-2 and 5-LOX and reducing the generation of inflammatory mediators are expected to become a new strategy for the treatment of aortic dissection. Summary of the invention

[0006] In order to solve the defects and shortcomings of the existing technologies for clinical prevention and treatment of aortic dissection, the purpose of the present invention is to provide an application of COX-2 and 5-LOX in the prevention and treatment of aortic dissection. The present invention found that the dual inhibitor of COX-2 and 5-LOX has the effect of significantly improving the symptoms of aortic dissection. The present invention further verified through animal experiments that the dual inhibitor of COX-2 and 5-LOX (Licofelone) can reduce the mortality rate and aortic diameter expansion of mice with aortic dissection, reduce the degree of aortic lesions, reduce the level of inflammatory factors in plasma, and alleviate the phenotypic transformation of aortic smooth muscle.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a use of a dual inhibitor of COX-2 and 5-LOX in the preparation of a medicament for preventing and treating aortic dissection.

[0009] As a further optimized solution of the present invention, the dual inhibitor includes but is not limited to Licofelone.

[0010] As a further optimization scheme of the present invention, the aortic dissection is an aortic dissection induced by BAPN.

[0011] As a further optimization scheme of the present invention, the dual inhibitor can improve the mortality rate, maximum aortic diameter and lesion severity of BAPN-induced aortic dissection.

[0012] As a further optimization scheme of the present invention, the dual inhibitor can reduce the levels of inflammatory factors IL-1B, IL-6, TNF-A and CCL2 in plasma, and can alleviate the phenotypic transformation of aortic smooth muscle.

[0013] In a second aspect, the present invention provides an application of COX-2 and 5-LOX in combination as drug targets in screening drugs for preventing and treating aortic dissection.

[0014] In a third aspect, the present invention provides a use of Licofelone in preparing a product for preventing and treating aortic dissection.

[0015] As a further optimized solution of the present invention, the drug contains the dual inhibitor as an active ingredient and further includes a pharmaceutically acceptable excipient, carrier or diluent.

[0016] As a further optimization scheme of the present invention, the dosage form of the drug includes at least one of a suspension, granules, capsules, powders, tablets, pills, injections, suppositories, aerosols or drops.

[0017] As a further optimization scheme of the present invention, the administration route of the drug includes at least one of intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration or atomization administration.

[0018] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0019] The present invention provides a new method for treating aortic dissection, which administers an effective amount of a dual inhibitor of COX-2 and 5-LOX, particularly Licofelone, to a patient with aortic dissection. The method of the present invention can reduce the incidence and / or mortality of aortic dissection, reduce the expansion of aortic diameter, and improve the degree of aortic lesions; qPCR, ELISA and WB further found that dual inhibition of COX-2 / 5-LOX can reduce inflammatory response and improve vascular remodeling, reduce aortic damage, and prevent the progression of aortic dissection, providing a new idea and method for the treatment of aortic dissection. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The drawings described herein are used to provide a further understanding of the embodiments of the present invention, constitute a part of this application, and do not constitute a limitation of the embodiments of the present invention. In the drawings:

[0021] Figure 1 Licofelone improves the incidence of BAPN-induced aortic dissection in mice. A: Representative gross images after 4 weeks of BAPN drinking water administration. B: Mouse survival curve. C: Statistical graph of the maximum diameter of the mouse aorta. D: H&E and EVG staining of the aorta in the control group and the Licofelone group. * represents p<0.05. Scale bar is 20μm.

[0022] Figure 2 Licofelone alleviates aortic inflammation and smooth muscle phenotypic transformation. A: qPCR was used to detect the expression levels of IL-1B, IL-6, TNF-A and CCL2 in the aortic wall of mice. B: ELISA was used to detect the protein levels of IL-1B, IL-6 and TNF-A in mouse plasma. C: qPCR was used to detect the expression levels of ACTA2, CNN1 and TAGLN in mouse aortic tissue. D: WB was used to detect the expression levels of ACTA2, CNN1 and TAGLN in mouse aortic tissue. DETAILED DESCRIPTION

[0023] The present invention is further described in detail below in conjunction with the accompanying drawings, embodiments and experimental examples. Of course, the protection scope of the present invention is not limited to the following embodiments. Professional and technical personnel in the field can understand that various changes and modifications can be made to the present invention without departing from the spirit of the present invention. The present invention generally and / or specifically describes the materials and test methods used in the experiment. Although many materials and operating methods used to achieve the purpose of the present invention are well known in the art, the present invention is still described as detailed as possible here. The following examples further illustrate the present invention, rather than limiting the present invention. Any equivalent transformation that is only formal and not substantial based on the concept of the present invention should be regarded as the scope of the technical solution of the present invention.

[0024] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0025] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0026] Example 1: COX-2 / 5-LOX dual inhibitor improves morbidity and mortality of BAPN-induced aortic dissection

[0027] Animal modeling: This experiment used 0.5% β-aminopropionitrile (BAPN) to induce aortic dissection in 4-week-old mice. 0.5 g of BAPN was added to 100 mL of drinking water, and the water was changed every 3 days for 28 days. Since BAPN is easily decomposed by light, it is necessary to wrap tin foil outside the water bottle to ensure light protection, thereby ensuring the stability of the drug.

[0028] Methods: We used Licofelone as drug intervention. Physiological saline was used as solvent to dissolve Licofelone. Mice were fed 80 mg / kg / day Licofelone by gavage, that is, 80 mg of pure Licofelone per kilogram of mice per day. Intervention began on the seventh day of modeling until the end of modeling. We set up the following groups on the background of 3-4 week old WT male mice: ① control solution + BAPN; ② 80 mg / kg / day Licofelone + BAPN.

[0029] HE staining method:

[0030] (1) Dewax the sections routinely into water.

[0031] (2) Dewaxed sections were placed in high-definition constant-stain pretreatment for 1 min.

[0032] (3) Place the sections in hematoxylin stain for 5 min, rinse with tap water, differentiate with differentiation solution, rinse with tap water, and bluing solution, rinse with running water.

[0033] (4) Dehydrate the sections in 5% ethanol for 1 min, and then stain them in eosin solution for 15 sec.

[0034] (5) Dehydrate the sections in 75% ethanol for 5 min, 85% ethanol for 5 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, and xylene for 5 min. After dehydration, air-dry the sections and seal them with neutral gum.

[0035] (6) Microscope observation: blue represents the cell nucleus and red represents the cytoplasm.

[0036] EVG staining method:

[0037] In this experiment, EVG staining was performed using the Sewell Bio EVG staining solution kit (G1042).

[0038] (1) Dewax the sections routinely to water.

[0039] (2) Prepare EVG staining solution in advance according to the instructions at a ratio of EVG staining solution A: EVG staining solution B: EVG staining solution C = 5:2:2. Place the sections in the EVG staining solution, stain for 5 minutes, and rinse with tap water.

[0040] (3) Dilute EVG dye solution B by half, place the slice in it, differentiate it slightly, and then rinse it with tap water. Repeat this operation and control the degree of differentiation under a microscope until the elastic fibers appear purple-black and the background appears grayish white or almost colorless.

[0041] (4) According to the instructions, prepare VG dye solution at a ratio of EVG dye solution E: EVG dye solution D = 9:1. Place the slices in it and dye for 2 minutes (the dyeing time depends on the elastic fiber component in the tissue. If the dyeing time is too short, the collagen color will be light, and if the dyeing time is too long, the elastic fiber will fade). Wash quickly with water, and quickly dehydrate in three cylinders of anhydrous ethanol.

[0042] (5) Two cylinders of clean xylene are transparent for 20 seconds and 5 minutes respectively (xylene is dedicated to xylene and should not be shared with other xylenes), and then wet-sealed with neutral gum.

[0043] (6) Microscope observation: Elastic fibers are purple-black, collagen fibers are red, and the background is yellow.

[0044] Results: We found that Licofelone intervention could significantly improve the mortality and maximum aortic diameter ( Figure 1 ). The results of H&E staining and EVG staining indicated that Licofelone could improve the degree of aortic lesions.

[0045] Example 2: COX-2 / 5-LOX dual inhibitor significantly alleviates inflammation levels and smooth muscle phenotype transformation

[0046] Methods: We used qPCR to detect the expression levels of IL-1B, IL-6, TNF-A and CCL2 in the mouse aorta wall, and used RK00027, RK00006 and RK00008 ELISA reagents and kits from Ibotek Biotech Co., Ltd. to detect the levels of TNF-α, IL-1β and IL-6 in mouse plasma, respectively. In addition, we used qPCR and WB technology to detect the proteins ACTA2, CNN1 and TAGLN in smooth muscle phenotype transformation. (Table 1).

[0047] ELISA test method:

[0048] 1. Add 100 μL of standards or samples of different concentrations to the blank wells, seal the wells with sealing film, and incubate at 37°C for 2 hours.

[0049] 2. Discard the liquid in the wells, add 350 μL of washing buffer to each well, let it stand for 40 seconds and then discard the liquid. Repeat this step 3 times.

[0050] 3. Add 100 μL of biotinylated antibody working solution to each well, seal the plate with a sealing film, and incubate at 37°C for 1 hour.

[0051] 4. Discard the liquid in the wells, add 350 μL of washing buffer to each well, let it stand for 40 seconds and then discard the liquid. Repeat this step 3 times.

[0052] 5. Add streptavidin-HRP working solution (100 L / well) to each well, cover with a new sealing film, and incubate at 37°C for 30 minutes.

[0053] 6. Discard the liquid in the wells, add 350 μL of washing buffer to each well, let it stand for 40 seconds and then discard the liquid. Repeat this step 3 times.

[0054] 7. Add TMB substrate (100 μL / well) to the wells and incubate at 37°C in the dark for 15-20 minutes.

[0055] 8. Add stop solution (50 pL / well) and immediately place in the microplate reader to measure the OD value of each well at 450 nm within 5 minutes.

[0056] Results: We found that Licofelone could significantly reduce the mRNA levels of IL-1B, IL-6, TNF-A and CCL2 in the aortic wall of mice compared with the control group ( Figure 2 A), and further ELISA revealed that Licofelone could significantly reduce the protein levels of IL-1B, IL-6, and TNF-A in mouse plasma ( Figure 2 B). In addition, the smooth muscle phenotypic transformation markers ACTA2, CNN1 and TAGLN in the mouse aorta wall also decreased significantly, suggesting that Licofelone can reverse the smooth muscle phenotypic transformation ( Figure 2 C-2D).

[0057] Table 1: QPCR primers used in the present invention

[0058]

[0059] In summary, the present invention provides a new method for treating aortic dissection, which has significant therapeutic effects and provides new ideas and methods for the treatment of aortic dissection.

[0060] The above are only preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limiting the present invention, and the protection scope of the present invention should be based on the scope defined by the claims. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. Application of dual inhibitors of COX-2 and 5-LOX in the preparation of drugs for preventing and treating aortic dissection.

2. The use according to claim 1, characterized in that: The dual inhibitors include but are not limited to Licofelone.

3. The use according to claim 1, characterized in that: The aortic dissection is an aortic dissection induced by BAPN.

4. The use according to claim 3, characterized in that: The dual inhibitor can improve the mortality, maximum aortic diameter and lesion severity of BAPN-induced aortic dissection.

5. The use according to claim 1, characterized in that: The dual inhibitor can reduce the levels of inflammatory factors IL-1B, IL-6, TNF-A and CCL2 in plasma, and can also alleviate the phenotypic transformation of aortic smooth muscle.

6. Application of COX-2 and 5-LOX combined as drug targets in screening drugs for the prevention and treatment of aortic dissection.

7. Application of Licofelone in the preparation of products for preventing and treating aortic dissection.

8. The use according to claim 1, 6 or 7, characterized in that: The medicine contains the dual inhibitor as an active ingredient and further comprises a pharmaceutically acceptable excipient, carrier or diluent.

9. The use according to claim 1, 6 or 7, characterized in that: The dosage form of the drug includes at least one of suspension, granules, capsules, powders, tablets, pills, injections, suppositories, aerosols or drops.

10. The use according to claim 1, 6 or 7, characterized in that: The administration route of the drug includes at least one of intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration or atomization administration.

Images