Application of loratadine in preparation of medicine for preventing and treating vascular calcification

Abstract

The invention relates to an application of loratadine in preparation of drugs for preventing and treating vascular calcification. The structural formula of the loratadine is as follows: # imgabs0. The loratadine used in the invention can significantly reduce the level of mouse aortic vascular calcification, and the experimental result proves that the loratadine has the effects of preventing and treating vascular calcification. The medicine disclosed by the invention is relatively low in toxicity and relatively high in safety.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine and relates to the application of loratadine in preparing medicines for preventing and treating vascular calcification. Background Art

[0002] Atherosclerosis is one of the main pathological factors of cardiovascular disease. It is a chronic inflammatory disease that seriously endangers human health. Almost all patients with cardiovascular disease have a certain degree of vascular calcification in the later stages. Vascular calcification increases the risk of cardiovascular events and all-cause mortality. Vascular calcification refers to the abnormal deposition of calcium salts in the arterial walls, which is a dynamic process regulated by multiple factors. Depending on the site of vascular calcification, it can be divided into intimal calcification and medial calcification. Intimal calcification, as the name suggests, is calcification that occurs in the vascular endothelium, also called atherosclerotic calcification. It is now believed that intimal calcification is an active and self-regulating process. ] . The occurrence and development of intimal calcification is similar to the multi-step process of bone formation, which is controlled by complex enzyme and cellular pathways. Vascular smooth muscle cells are the main cell type involved in vascular calcification, which have the potential to produce mineralized matrix and undergo osteoblastic differentiation, leading to calcification deposition. At the same time, intimal calcification is often present in advanced atherosclerotic plaques and develops with the development of atherosclerotic lesions. The site of medial calcification is located in the vascular media, which is often accompanied by diseases such as chronic kidney disease (CKD), diabetes and aging, which will reduce vascular compliance and eventually lead to heart failure or hypertension. Vascular medial calcification is also mainly caused by osteoblast-like differentiation of vascular smooth muscle cells. VSMCs are an important component of the vascular wall, providing structural support, regulating vascular tension and vascular remodeling. VSMCs are one of the main sources of osteoblasts involved in the calcification process. VSMCs can dedifferentiate into osteoblast-like cells, acquire the ability to produce mineralized matrix and cause calcium deposition, which is considered to be the initial stage of vascular calcification.

[0003] Histamine is the main mediator of allergic reactions. It is stored in mast cells or basophils until the cells are stimulated by something that allows it to be released from the cell. Allergic reactions occur when histamine interacts with histamine receptors H1, H2, H3 or H4. Among them, drugs targeting H1 receptors are most commonly used to treat allergies. Loratadine is an H1 histamine receptor antagonist and a second-generation non-sedating antihistamine that is often used to treat allergic symptoms. It is used for allergic rhinitis, acute or chronic urticaria, allergic conjunctivitis, hay fever and other allergic skin diseases. Loratadine can also inhibit the immune release of inflammatory mediators.

[0004] The invention patent with publication number CN113209098A discloses the use of desloratadine and its pharmaceutically acceptable salt in the preparation of drugs for treating peripheral neuropathy. In this patent research bureau, the application of desloratadine and its pharmaceutically acceptable salt in treating allergic reactions and peripheral neuropathy caused by paclitaxel is mainly explored. In the application, desloratadine and its pharmaceutically acceptable salt can inhibit the allergic mediators and increased vascular permeability caused by paclitaxel, and at the same time relieve the peripheral nerve pain caused by paclitaxel. We explored the role of loratadine in blood calcium calcification. The main differences are: (1) First, we explored the test of loratadine cell activity in HASMC cells; (2) Loratadine prevents and treats vascular calcification, and we mainly explored HASMC cells and aortic rings; (3) The purpose of the study and the research method are also different. (4) Vascular calcification refers to the pathological phenomenon of calcium salts deposited in the vascular wall, which mainly occurs in arteries. Vascular permeability refers to the ability of the vascular wall to allow water molecules, electrolytes and macromolecules (such as proteins) to pass through. Pathologically increased permeability can lead to edema, inflammation, and other vascular-related lesions. Vascular calcification and vascular permeability are two closely related vascular pathological features with independent physiological mechanisms. Calcification of the vascular media may lead to increased stiffness, weaken the ability of blood vessels to regulate pressure changes, make endothelial cells more susceptible to damage, and increase permeability. The use of loratadine to treat calcification is mainly studied in HASMC. VSMCs can dedifferentiate into osteoblast-like cells, acquire the ability to produce mineralized matrix and cause calcium deposition, which is considered to be the initial stage of vascular calcification.

[0005] Currently, there is no drug that can effectively prevent or treat vascular calcification in clinical practice. Anticoagulants such as statins and warfarin have the side effect of promoting calcification. Therefore, it is of great significance to find and develop drugs to treat calcification diseases. So far, no studies have shown the role of loratadine in preventing and treating vascular calcification. Summary of the invention

[0006] The purpose of the present invention is to provide an application of an antihistamine loratadine in a medicine for preventing and treating vascular calcification.

[0007] To achieve the above-mentioned purpose and other related purposes, the technical solution provided by the present invention is: use of loratadine in the preparation of a drug for preventing and treating vascular calcification.

[0008] The preferred technical solution is: the structural formula of loratadine is as follows:

[0009]

[0010] To achieve the above-mentioned purpose and other related purposes, the technical solution provided by the present invention is: a pharmaceutical composition for preventing and treating vascular calcification, comprising the above-mentioned loratadine and pharmaceutically acceptable excipients.

[0011] The preferred technical solution is that the dosage form of the drug is oral solution, injection, tablet, pill, dispersant, capsule, pellet, granule, suspension or emulsion.

[0012] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art:

[0013] The loratadine of the present invention can significantly inhibit the calcification of smooth muscle cells and the calcification of mouse aortic rings. The new application of the antihistamine loratadine in treating vascular calcification can expand its research in the field of medical technology and provide a new theoretical direction for clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 These are the results of toxicity test of 0μΜ, 2μΜ, 5μM, 10μM and 20μM loratadine on HASMC cells under normal culture conditions for 24 hours. The results show that loratadine has low damage to cells and has high safety.

[0015] Figure 2 The toxicity test results of 0μM, 2μM, 5μM, 10μM and 20μM loratadine on HASMC cells in calcification medium (high phosphate) show that under the condition of calcification induction for 6 days, loratadine within a concentration of 20μM still has low damage to cells and is relatively safe.

[0016] Figure 3 This is the result of Alizarin red staining of loratadine in the treatment of osteogenic differentiation of smooth muscle cells. The results show that loratadine at 10 μM can have a good inhibitory effect on osteogenic differentiation of smooth muscle cells.

[0017] Figure 4 The results show that loratadine can effectively reduce the expression of BMP2 and RUNX2 proteins in smooth muscle cells. *p<0.05, **p<0.01.

[0018] Figure 5 The results of Alizarin red staining of loratadine on aortic ring calcification in C57BL / 6J mice. The results showed that loratadine at 10 μM can have a good inhibitory effect on aortic ring calcification.

[0019] Figure 6The effect of loratadine on the RUNX2 immunofluorescence intensity of aortic ring calcification in C57BL / 6J mice. The results showed that loratadine could effectively reduce the expression of RUNX2 in phosphate-induced aortic rings. DETAILED DESCRIPTION

[0020] The following is a description of the implementation of the present invention by means of specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0021] See also Figure 1-6 . It should be noted that the structures, proportions, sizes, etc. illustrated in the drawings in this specification are only used to match the contents disclosed in the specification for people familiar with this technology to understand and read, and are not used to limit the limiting conditions for the implementation of the present invention, so they have no substantial technical significance. Any modification of the structure, change in the proportional relationship, or adjustment of the size should still fall within the scope of the technical content disclosed by the present invention without affecting the effects and purposes that can be achieved by the present invention. At the same time, the terms such as "upper", "lower", "left", "right", "middle" and "one" quoted in this specification are only for the convenience of description, and are not used to limit the scope of the implementation of the present invention. Changes or adjustments in their relative relationships should also be regarded as the scope of the implementation of the present invention without substantially changing the technical content.

[0022] Unless otherwise specified, the reagents or materials described in the following examples are commercially available.

[0023] Example 1: Use of loratadine in the preparation of a drug for preventing and treating vascular calcification

[0024] Application of loratadine (CAS No. 79794-75-5) in the preparation of drugs for preventing and treating vascular calcification.

[0025] Through a large number of experiments, the present invention determines that loratadine prevents and treats phosphate-induced vascular calcification of aortic rings and alleviates osteogenic differentiation of vascular smooth muscle cells induced by high phosphate.

[0026] Preferably, loratadine can be used to prepare drugs for treating vascular calcification.

[0027] The drug for treating vascular calcification has loratadine as an effective ingredient.

[0028] A drug for treating vascular calcification contains a pharmaceutically acceptable material of loratadine.

[0029] A preferred embodiment is that the dosage of loratadine is 2-20 μM.

[0030] A more preferred embodiment is that the dosage of loratadine is 10 μM.

[0031] The loratadine of the present invention can significantly inhibit phosphate-induced vascular calcification. The new application of loratadine in treating vascular calcification can expand its research in the field of medical technology and provide a new theoretical direction for clinical application.

[0032] The use of loratadine in the preparation of a drug for preventing and treating vascular calcification, the loratadine (CAS No. 79794-75-5) used has the following structural formula:

[0033]

[0034] 1. Determination of toxicity of loratadine to cells

[0035] (1) Human aortic smooth muscle cells of passage 2 to 10 were cultured and prepared into a cell suspension using a culture medium containing 10% fetal bovine serum. 5,000 cells were seeded into each well of a 96-well plate, and the volume of the cell suspension in each well was 200 μL.

[0036] (2) Five groups were set up, namely: normal group, loratadine 2 μM, loratadine 5 μM, loratadine 10 μM, and loratadine 20 μM. Six replicate wells were set up for each drug group, and the drug treatment was carried out for 24 hours.

[0037] (3) After the incubation, add 20 μL of MTT solution (5 mg / mL, prepared in double-free medium) to each well. After incubation for 4 hours, terminate the incubation, carefully discard the medium in the wells, add 150 μL of DMSO to each well, and shake on a shaker at room temperature for 15 minutes to allow the crystals to fully dissolve.

[0038] (4) Select 550 nm wavelength, detect the light absorbance of each well on an enzyme-linked immunosorbent assay (ELISA) reader, and record the results.

[0039] 2. Determination of toxicity of loratadine to cells in calcified culture medium

[0040] 1. Preparation of calcification medium

[0041] The corresponding concentration of inorganic phosphate (Na2HPO4 and NaH2PO4 ratio of 1:2) was added to DMEM to induce calcification in smooth muscle cells. The induction process lasted for 6 days, and fresh culture medium was replaced every two days.

[0042] 2. Determination of toxicity of loratadine to cells in calcified culture medium

[0043] (1) Human aortic smooth muscle cells of passage 2 to 10 were cultured and prepared into a cell suspension using a culture medium containing 10% fetal bovine serum. 1000 cells were seeded into each well of a 96-well plate, and the volume of the cell suspension in each well was 200 μL.

[0044] (2) Six groups were set up, namely: normal group, calcification group, calcification loratadine 2 μM, calcification loratadine 5 μM, calcification loratadine 10 μM, and calcification loratadine 20 μM. Six replicate wells were set up for each drug group. The cells were cultured under the same culture conditions for 6 days.

[0045] (3) After the incubation, add 20 μL of MTT solution (5 mg / mL, prepared in double-free medium) to each well. After incubation for 4 hours, terminate the incubation, carefully discard the medium in the wells, add 150 μL of DMSO to each well, and shake on a shaker at room temperature for 15 minutes to allow the crystals to fully dissolve.

[0046] (4) Select 550 nm wavelength, detect the light absorbance of each well on an enzyme-linked immunosorbent assay (ELISA) reader, and record the results.

[0047] III. Alizarin red staining of osteogenic differentiation of smooth muscle cells treated with loratadine

[0048] (1) Add 500 cells to each well of a 12-well plate and allow the cells to adhere to the plate overnight.

[0049] (2) Prepare the calcification medium according to the calcification medium described in the second part. In the calcification medium, treat the cells at 5 or 10 μM loratadine concentrations.

[0050] (3) Place the cells in an incubator and culture them. Change the culture medium every two days for a total of 6 days.

[0051] (4) After incubation, remove the culture medium, add 4% paraformaldehyde to fix the cells for 30 minutes, wash with PBS three times, 5 minutes each time. Then add Alizarin Red dye solution to stain for 30 minutes, and then wash with acidic PBS three times, 5 minutes each time. Take pictures.

[0052] 4. Loratadine reduces the protein expression levels of BMP2 and RUNX2, key calcification proteins in cells

[0053] (1) Protein extraction: The cells were inoculated in a 6-well plate. The cells were treated according to the normal group, calcification group, calcification loratadine 2μM, calcification loratadine 5μM, calcification loratadine 10μM, and calcification loratadine 20μM. Six days later, the cells were washed twice with PBS. After adding protein lysis buffer to fully lyse, the cells were centrifuged at 12000r / min at 4℃ for 10min, and the supernatant was taken and transferred to a -20℃ refrigerator for storage.

[0054] (2) The total protein concentration was determined by the BCA method, 30 μg of total protein was quantified, and the protein sample loading volume was calculated.

[0055] (3) SDS-polyacrylamide gel electrophoresis was performed at a constant voltage of 124 V for about 90 min.

[0056] (4) Transfer the protein blot to the NC membrane under a constant voltage of 100 V.

[0057] (5) Obtain the target band according to the protein molecular weight and block it with 5% skim milk at room temperature for 1 h.

[0058] (6) After blocking, add the corresponding primary antibody (BMP2, RUNX2) and incubate at 4°C overnight.

[0059] (7) The next day, the primary antibody was recovered, the strips were washed three times with PBST, each time for 8 min, and the strips were incubated in the secondary antibody at room temperature.

[0060] (8) Recover the secondary antibody, wash the strips three times with PBST, each time for 8 min, and detect the immunoblot using an ECL chemiluminescence kit and imaging equipment.

[0061] 5. Alizarin red staining of phosphate-induced aortic rings of C57BL / 6J mice with loratadine

[0062] 1. Induction of arterial ring calcification

[0063] (1) According to experimental needs, experimental mice were killed and the thoracic aorta was dissected.

[0064] (2) The aorta is dissected and trimmed using a surgical microscope.

[0065] (3) The thin straight part of the aorta was cut into pieces (about 5 mm) and placed in a well plate supplemented with complete culture medium overnight.

[0066] (4) According to the experimental needs, the arterial rings were treated experimentally; the groups were set as follows: normal group, calcification group, calcification + loratadine (5 μM), calcification + loratadine (10 μM).

[0067] (4) Add calcification medium and the calcification induction time is 10-15 days.

[0068] (5) After calcification induction, the slices were embedded in OCT and placed in a refrigerator for later use.

[0069] 2. Arterial ring alizarin red staining

[0070] (1) Take the frozen sections out of the refrigerator and thaw them at room temperature for 30 min.

[0071] (2) The sections were soaked in PBS buffer for 30 min and the OCT was removed.

[0072] (3) Soak the sections in Alizarin Red solution for 30 min.

[0073] (4) Wash the sections three times in acidic PBS.

[0074] (5) Observe and take photos under a microscope. The staining results are shown in Figure 5 .

[0075] VI. Immunofluorescence staining of mouse aortic root to detect RUNX2 expression in aortic rings

[0076] (1) Immunofluorescence staining: The frozen sections obtained in the fifth part were taken out of the refrigerator and thawed at room temperature for 30 min, and then soaked in PBS for 5 min.

[0077] (2) Soak in 0.1% Triton-100 for 10 min to break the membrane, and then soak in PBS for 5 min, twice each time.

[0078] (3) Add 2% BSA for blocking, incubate at room temperature for 10 to 15 minutes, and then pour off.

[0079] (4) Add primary antibody and incubate at 4°C overnight. Rinse with PBS three times, 10 min each time.

[0080] (5) Add fluorescently labeled secondary antibody and incubate at 37°C for 45 min. Rinse with PBS for 3 times, 10 min each time.

[0081] (6) Add DAPI staining solution, incubate at room temperature for 15 min, and rinse with PBS three times, each time for 10 min.

[0082] (7) Sealing: Use anti-fluorescence quenching sealing agent to seal the slides, place them horizontally away from light for more than 2 hours, and then take pictures under a fluorescence microscope after drying. The staining results are shown in the table. Figure 6 .

[0083] From the above results, it can be seen that the loratadine of the present invention can significantly treat vascular cell calcification and mouse aortic ring calcification induced by high phosphorus. Therefore, loratadine has a relatively broad application prospect in the prevention and treatment of vascular calcification.

[0084] The above description is only used to explain the preferred embodiments of the present invention, and is not intended to limit the present invention in any form. Therefore, any modifications or changes made to the present invention under the same inventive spirit should still be included in the scope of protection intended by the present invention.

Claims

1. The use of loratadine in the preparation of drugs for preventing and treating vascular calcification.

2. The use of loratadine according to claim 1 in the preparation of a drug for preventing and treating vascular calcification, characterized in that: The structural formula of the loratadine is as follows:

3. A pharmaceutical composition for preventing and treating vascular calcification, characterized in that : comprising the loratadine according to claim 1 or 2 and pharmaceutically acceptable adjuvants.

4. The pharmaceutical composition for preventing and treating vascular calcification according to claim 3, characterized in that: The dosage form of the drug is oral solution, injection, tablet, pill, dispersant, capsule, dripping pill, granule, suspension or emulsion.

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