Application of compound Isaridin E or its pharmaceutical salt in the preparation of drugs for anti-vascular calcification and related diseases

Abstract

The application discloses application of a compound Isaridin E or a pharmaceutically acceptable salt thereof in preparation of anti-vascular calcification drugs and related diseases, and relates to the field of medicine and biotechnology.The application adopts a classic high-phosphate-induced smooth muscle cell calcification model and a high-dose vitamin D3 (VitD3)-induced in-vivo vascular calcification model, and finds that the compound Isaridin E has a significant improvement effect on vascular calcification caused by calcium and phosphorus metabolism disorder through in-vitro cell experiments, in-vitro experiments and whole animal experiments.The drug action can significantly inhibit high-phosphate and vitamin D3-induced vascular smooth muscle cell osteogenic differentiation, inhibit vascular calcification, and significantly reduce the formation of calcium and phosphorus crystals in a vascular wall.The application develops a brand-new medical use of the marine compound Isaridin E, and can be used for research and development of drugs for treating vascular calcification and calcification-related diseases.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical biotechnology, specifically to the application of compound Isaridin E or its pharmaceutical salt in the preparation of drugs for anti-vascular calcification and related diseases. Background Technology

[0002] Ninety-nine percent of the body's calcium exists in the form of hydroxyapatite crystals in teeth and bones. When this calcium is abnormally deposited in other tissues and organs, it is called ectopic calcification. When calcium phosphate crystals are abnormally deposited in the cardiovascular system, it is called vascular calcification. Vascular calcification leads to increased stiffness of the blood vessel walls, decreased compliance, vasomotor dysfunction, and hemodynamic instability, thereby increasing the risk of cardiovascular complications such as hypertension, myocardial ischemia, and stroke. It is widely recognized as an independent risk factor for increased morbidity and mortality from cardiovascular events.

[0003] Depending on the location of vascular calcification, it can be classified into intimal calcification, medial calcification, valvular calcification, and uremic arteriolar lesions with calcification. Intimal calcification is commonly seen in atherosclerosis, while medial calcification is frequently observed in patients with chronic kidney disease, diabetes, atherosclerosis, and the elderly, and is relatively common in clinical practice. Smooth muscle cells are the main cellular components of the vascular media and are the main participants in vascular calcification, serving as direct or indirect target cells for numerous factors leading to vascular calcification. Under physiological conditions, vascular smooth muscle cells maintain contractile function and express a large number of contractile proteins. Under the influence of various risk factors (such as high calcium and phosphorus environments, hyperlipidemia, inflammation, and oxidative stress), vascular smooth muscle cells downregulate the expression of contractile proteins, while increasing the expression and secretion of bone formation-related proteins. This promotes the transformation of smooth muscle cells from a contractile phenotype under physiological conditions to an osteogenic phenotype, which is the core link in the development of vascular calcification. Osteoblastic smooth muscle cells secrete matrix vesicles and apoptotic bodies into the extracellular matrix as initial sites for mineral deposition and crystal nucleation. Both have the ability to concentrate calcium and phosphate, and further induce the formation of mineral nodules. Clinical studies have shown that calcification occurs in multiple regions of the aorta during chronic kidney disease. Chest CT scans show that calcification is more likely to occur in the aortic arch throughout the aorta in the early stages, accurately reflecting the developmental stage of vascular calcification. Therefore, aortic arch calcification is a prognostic indicator of cardiovascular disease progression.

[0004] Because hydroxyapatite crystals, the main component of vascular calcification, are highly insoluble, current clinical treatment strategies for vascular calcification remain largely limited to mechanical therapies. For example, mild to moderate coronary artery calcification is treated with percutaneous coronary intervention and balloon angioplasty, while severe calcification is treated with rotational atherectomy or coronary artery bypass grafting. Drug therapy includes regulating calcium and phosphorus levels and interventions directly targeting vascular calcification, such as phosphate sequestrants, pyrophosphates and bisphosphonates, and denosumab. However, drug therapy remains controversial, and there are currently no effective treatments in clinical practice. Therefore, developing novel anti-vascular calcification drugs is of great social significance.

[0005] Isaridin E (ISE) is a cyclic hexapeptide extracted from the marine fungus *Beauveria feline* SYSU-MS7908. Currently, research on the cyclic peptide isaridins mainly focuses on their insecticidal, neutrophil-inhibiting, and antiplatelet thrombotic effects; no reports have been found on their anti-vascular calcification effects. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing technologies and provide the application of compound Isaridin E or its pharmaceutical salt in the preparation of drugs for anti-vascular calcification and related diseases. In the experiment, the inventors used a classic high-phosphate-induced in vitro cell model and a high-dose vitamin D3 (VitD3)-induced in vivo vascular calcification model, using vascular smooth muscle cells and aortic vessels as the observation objects. They found that compound Isaridin E can slow down the occurrence of vascular calcification and can significantly reduce the calcium deposition level in smooth muscle cells and aortic vessels at the administered concentration, inhibiting the histopathological changes caused by ectopic calcium crystals.

[0007] To address the aforementioned technical problems, this invention provides the application of compound Isaridin E or its pharmaceutical salt in the preparation of anti-vascular calcification drugs.

[0008] In addition, the present invention also provides the use of compound Isaridin E or its pharmaceutical salt in the preparation of drugs for treating vascular calcification-related diseases.

[0009] Preferably, the drug is a drug used to inhibit vascular calcification caused by calcium and phosphorus metabolism disorders.

[0010] Preferably, the drug is a drug for inhibiting abnormal deposition of vascular hydroxyapatite crystals.

[0011] Preferably, the drug is a drug used to inhibit osteogenic differentiation of vascular smooth muscle cells.

[0012] Accordingly, the present invention also provides a medicament for treating vascular calcification and related diseases, the medicament comprising the compound Isaridin E or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier and / or excipient.

[0013] Preferably, the drug is an injectable solution.

[0014] Preferably, the drug is a drug for oral administration.

[0015] Compared with existing drugs, the present invention has the following beneficial effects:

[0016] This invention employs a classic high-phosphate-induced smooth muscle cell calcification model and a high-dose vitamin D3 (VitD3)-induced in vivo vascular calcification model. Through in vitro cell experiments, in vitro experiments, and whole animal experiments, it was found that the compound Isaridin E significantly improves high-phosphate and vitamin D3-induced vascular calcification. This drug significantly inhibits high-phosphate-induced osteogenic differentiation of smooth muscle cells and reduces the production of calcium and phosphorus crystals in a concentration-dependent manner. In vitro experiments on vascular rings also demonstrated that ISE can reduce the level of aortic ring calcification, manifested as a decrease in aortic calcium deposition and a reduced distribution of calcium and phosphorus crystals along elastic fibers in vascular sections. In a high-dose vitamin D3-induced in vivo calcification model, compared to the model group, the calcification foci shown by gross staining of the aorta in the ISE-treated group were significantly inhibited. This drug has low toxicity and high safety, and shows promise for development as a novel anti-vascular calcification drug.

[0017] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and will become apparent from the description or may be learned by practice of the invention. Attached Figure Description

[0018] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this application, do not constitute an undue limitation of the invention. In the drawings:

[0019] Figure 1 A schematic diagram showing the test results of compound Isaridin E inhibiting high phosphorus-induced calcification of mouse aortic smooth muscle cells;

[0020] Figure 2 A schematic diagram showing the test results of the compound Isaridin E inhibiting the expression of osteogenic differentiation marker molecules induced by high phosphorus in mouse aortic smooth muscle cells;

[0021] Figure 3 Isaridin E inhibits high-phosphorus-induced calcification (IC50) in mouse aortic smooth muscle cells. 50 A diagram illustrating the test results;

[0022] Figure 4 A schematic diagram showing the test results of compound Isaridin E inhibiting the level of high phosphorus-induced calcium deposition in the aortic rings of mice;

[0023] Figure 5 A schematic diagram of the staining results of the compound Isaridin E inhibiting high phosphorus-induced aortic ring calcification in mice;

[0024] Figure 6 A schematic diagram showing the test results of compound Isaridin E inhibiting the level of calcium deposition in the aorta of mice induced by high doses of vitamin D3;

[0025] Figure 7 This is a schematic diagram of the test results for the compound Isaridin E inhibiting high-dose vitamin D3-induced macroscopic staining of aortic calcification in mice. Detailed Implementation

[0026] To better understand the technical content of this invention, the invention will be further described and explained below with reference to the accompanying drawings and specific embodiments. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0027] The features, beneficial effects, and advantages of this invention will become apparent to those skilled in the art upon reading the contents of this specification.

[0028] Unless otherwise specified, all percentages, fractions, and ratios are calculated based on the total weight of the compositions of the present invention. The term "weight content" herein may be represented by the symbol "%".

[0029] The terms “comprising,” “including,” “containing,” “having,” “comprising,” or other variations thereof are intended to cover non-closed inclusion, and no distinction is made between these terms. The term “comprising” means that other steps and components may be added without affecting the final result. The term “comprising” also includes the terms “consisting of” and “substantially consisting of”. The compositions and methods / processes of the present invention may comprise, consist of, and substantially consist of the essential elements and limitations described herein, as well as any additional or optional ingredients, components, steps, or limitations described herein.

[0030] In the following scheme, compound Isaridin E can be abbreviated as compound ISE.

[0031] Example 1

[0032] Effects of compound ISE on calcification levels in mouse aortic smooth muscle cells and its half-maximal inhibitory concentration (IC50)50 Measurement

[0033] (I) Materials and Methods

[0034] 1. Medication preparation

[0035] The compound Isaridin E (chemical formula below) was provided by Professor Liu Lan's research group at the College of Oceanography, Sun Yat-sen University. It is a white crystalline solid and should be stored at room temperature. When needed, it should be ground into powder and dissolved in DMSO to prepare a storage solution (incubation concentrations of 1 μM, 2 μM, 5 μM, 10 μM, and 20 μM, with a controlled addition volume of 2.5 μl), and stored at 4℃ for later use.

[0036] The structural formula of compound Isaridin E is shown below:

[0037]

[0038] Wherein, (A) is a planar structural formula. (B) is a stereo structural formula. Isaridin E is a cyclic hexapeptide compound with PubChem CID 16680915, molecular formula C35H53N5O7, and relative molecular mass 655.8.

[0039] 2. Preparation of mouse aortic smooth muscle cells (MVSMCs)

[0040] Prepare the digestive enzymes in advance (1.4 mg type II collagenase / ml DMEM, sterilized with a 0.22 μm filter). Five 8-week-old male C57BL / 6J mice were anesthetized with pentobarbital and restrained in a mouse restraint device. The abdominal skin and fur of the mice were disinfected with 75% ethanol. In a laminar flow hood, the thoracic cavity was opened with scissors to fully expose the thoracic aorta. The aorta was cut along the body axis above the spine and immediately placed in 10% FBS DMEM high-glucose medium. Blood was gently washed away with ophthalmic forceps, and the peripheral connective tissue and adventitia of the aorta were removed. The vessel was longitudinally flattened with scissors, and the inner side of the vessel was gently scraped to remove the intima. The media was cut into approximately 1 mm pieces with ophthalmic scissors. 2 Small pieces of tissue were separated and placed in 6-well plates. 5 mL of digestive enzyme was added, and the plates were incubated at 37°C for approximately 4 hours. The cells were observed hourly to allow for immediate termination of digestion. Once the tissue pieces had digested into flocculent cell clumps, an appropriate amount of culture medium was added to dilute the cells. The plates were centrifuged at 1000 rpm for 10 minutes, and the supernatant was discarded. The cells were resuspended in high-glucose DMEM containing 20% ​​FBS, mixed thoroughly by pipetting, and then transferred to 25 mm cells. 2 Culture in culture flasks and do not move the flasks for 72 hours. Replace the culture medium with fresh medium after 3 days, observe cell morphology, and passage the cells once they have reached confluence.

[0041] 3. Smooth muscle cell calcification experiment

[0042] Experiments were conducted using healthy cells from passages 3-6. Vascular smooth muscle cells were seeded in small dishes and incubated with different concentrations of Isaridin E (1 μM, 2 μM, 5 μM, 10 μM, and 20 μM) when cell confluence reached 50%-60%, simultaneously receiving 2.6 mmol / L inorganic phosphorus stimulation. The medium was changed to 10% FBS DMEM every two days, with 2.6 mmol / L inorganic phosphorus (Pi) stimulation administered concurrently. The experiment lasted approximately 7-12 days, with the endpoint determined based on cell growth status.

[0043] 4. Alizarin Red staining and quantification of cells

[0044] Aspirate the cell culture medium, wash the cells three times with ice-cold PBS, and aspirate completely dry. Fix the cells with 4% paraformaldehyde for 15 minutes at room temperature. Discard the paraformaldehyde, wash three times with deionized water, and aspirate completely dry. Add 1 ml of 1% Alizarin Red solution (prepared with deionized water) to each well of a 6-well plate and stain for 5 minutes. Aspirate the stain, wash three times with deionized water to remove any excess stain, and observe the cells under a light microscope.

[0045] Remove the alizarin red stained cell culture dishes that have been photographed and dried. Add 1 ml of formic acid solution to each alizarin red stained dish, gently agitate to ensure full contact with the cells, then transfer the formic acid solution to a 1.5 ml centrifuge tube and vortex to mix thoroughly. Add 200 μl of the mixed formic acid solution to each well of a 96-well plate and measure the absorbance of each group at a wavelength of 405 nm.

[0046] 5. Detection of cellular calcium deposition

[0047] Discard the cell culture medium and wash the cells three times with ice-cold PBS. Add 100 μL of 6% dilute hydrochloric acid (HCl) to each well of the dish, scrape off the cells with a cell scraper, and collect them into a 1.5 mL centrifuge tube. Sonicate on ice for 3 seconds, pause for 3 seconds, repeating a total of 5 times. After sonication, incubate the sample on ice for 30 minutes. Centrifuge at 10,000 rpm at 4°C for 15 minutes, and transfer the supernatant to a new 1.5 mL centrifuge tube for subsequent analysis.

[0048] Prepare calcium standards: The required concentrations, from highest to lowest, are 2.5 mmol / L, 1.25 mmol / L, 0.625 mmol / L, 0.3125 mmol / L, 0.15625 mmol / L, and 0 (i.e., 6% dilute hydrochloric acid). Prepare working solution: Mix equal volumes of solutions R1 and R2 from the calcium assay kit and incubate at room temperature for 5 minutes. Add 10 μL of standard and the sample to be tested to 500 μL of working solution, vortex to mix, incubate at 37°C for 10 minutes, and add 200 μL to a 96-well microplate for detection. Place the 9-well plate in a multi-microplate reader and measure the absorbance at 600 nm. Plot a standard curve using the absorbance of the standards. The calcium content in the sample can be calculated from the standard curve. The calcium content is normalized to the protein concentration to obtain the relative calcium deposition concentration.

[0049] 6. Western blot

[0050] After extracting and quantifying cellular proteins, the samples were added to loading buffer according to the specified ratio, and after protein denaturation at 95-99℃ for 5 min, SDS-PAGE electrophoresis was performed. After electrophoresis, the gel was removed and transferred to a transfer electrophoresis apparatus for membrane transfer. After transfer, the membrane was blocked, incubated with the corresponding specific primary antibody, and shaken overnight at 4℃. The next day, after warming, the membrane was washed three times with TBST for 10 min each time. After incubation with specific secondary antibody for 1-1.5 h, the membrane was washed three times with shaking at room temperature for 10 min each time. The membrane was then developed in a dark room with Chemiluminescent HRP Substrate (Millipore) and imaged on BIO-RAD Molecular Imager ChemiDoc XRS+, and the data were analyzed using Image Lab 4.0.

[0051] 7. Statistical processing

[0052] Data are expressed as mean ± SEM. One-way ANOVA was performed using GraphPad Prism 8.0 software for intergroup analysis, with P < 0.05 considered statistically significant.

[0053] (II) Experimental Results

[0054] 1. Effects of Isaridin E on the level of calcification in high-phosphorus-induced mouse aortic smooth muscle cells

[0055] To investigate the effect of ISE on calcification at the cellular level, the inventors induced calcification by treating cells with 2.6 mmol / L inorganic phosphorus and simultaneously incubating them with different concentrations of Isaridin E for 7-12 days. Alizarin Red staining was then used to assess the level of cellular calcification. Alizarin Red dye chelates with calcium nodules, producing an orange-red compound in an acidic environment. This compound deposits on the surface of calcified cells, reflecting the overall cellular calcification status. The stained area is directly proportional to the calcium salt content; the larger the red area, the more calcium nodules there are, and the more severe the calcification. Figure 1 Staining results showed that Isaridin E (5 μM, 10 μM, 20 μM) inhibited smooth muscle cell calcification in a concentration-dependent manner. Correspondingly, quantitative results showed that Isaridin E treatment significantly inhibited cellular calcium deposition levels compared to the high-phosphorus group, with a statistically significant difference at 5 μM. *P < 0.05, n = 6, compared to the high-phosphorus group.

[0056] 2. Effects of Isaridin E on the expression of osteogenic differentiation markers and contractile markers in high-phosphorus-induced mouse aortic smooth muscle cells

[0057] The core of vascular calcification is the transformation of smooth muscle cells from contractile to osteogenic types. This phenotypic change is accompanied by the loss of contractile markers of vascular smooth muscle cells, such as SM22α and α-SMA, and the increase of osteogenic markers, such as Runt-related transcription factor 2 (Runx2), bone morphogenetic protein-2 (BMP2), and alkaline phosphatase (ALP). Therefore, the inventors used Western blotting experiments to detect the expression of relevant marker molecules after incubation with ISE under high phosphorus stimulation. Figure 2 As shown, high phosphorus stimulation significantly increased the expression of Runx2 and BMP2 proteins in smooth muscle cells, while decreasing the expression of α-SMA, demonstrating that osteogenic phenotypic conversion occurred in smooth muscle cells under high phosphorus conditions. ISE incubation inhibited the protein levels of Runx2 and BMP2 and upregulated the expression of α-SMA protein, indicating that at the molecular level, ISE can inhibit osteogenic phenotypic conversion in smooth muscle cells, thereby reducing the formation of calcium and phosphorus crystals. Compared with the high phosphorus group, *P<0.05, n=6.

[0058] 3. The half-maximal inhibitory rate (IC50) of Isaridin E on smooth muscle cell calcification 50 Measurement

[0059] Given that ISE significantly inhibits smooth muscle cell calcification, we then determined its half-maximal inhibitory concentration (HMC). The HMC, also known as the half-maximal inhibitory concentration, refers to the concentration of drug or inhibitor required to inhibit a biological process by half. In pharmaceutical science, it is used to characterize the antagonistic ability of antagonists in in vitro experiments; a smaller value indicates a stronger inhibitory effect.

[0060] Since formic acid can dissolve alizarin red bound to calcification sites, and alizarin red is yellow under acidic conditions, and by utilizing the differences in the degree of calcification among groups resulting in varying shades of alizarin red staining, a constant amount of formic acid is added to the culture dish to react with the calcification, producing different shades of yellow formic acid solution. Finally, the absorbance is measured to quantify the alizarin red staining in cells. We used the method of alizarin red staining followed by formic acid quantification to determine the IC50. 50 The relative calcification inhibition rate was calculated using the formula (OD value of high phosphorus group - OD value after treatment with different concentrations of Isaridin E) / OD value of high phosphorus group * 100%, and IC50 was performed using GraphPad Prism. 50 Parametric curve fitting. For example... Figure 3 The curve shows that the half-maximal inhibitory concentration (IC50) of ISE is 4.957 μmol, indicating that ISE has a significant inhibitory effect on vascular calcification even at low doses. n = 5.

[0061] Example 2

[0062] Effect of compound Isaridin E on the level of aortic ring calcification in mice

[0063] (I) Materials and Methods

[0064] 1. Medication preparation

[0065] Based on the results of cell experiments, this part of the experiment used a 20 μM concentration of Isaridin E. A 20 μM stock solution was prepared by dissolving the drug in DMSO and stored at 4°C for later use.

[0066] 2. Aortic ring calcification test

[0067] Eight-week-old male C57BL / 6J mice were anesthetized with pentobarbital, and the skin of the chest and abdomen was thoroughly disinfected by soaking in 75% ethanol for 3 minutes. The thoracic cavity was opened, and approximately 1 cm of the thoracic aorta was cut and placed in sterile PBS buffer. Residual blood was washed away, and the peripheral connective tissue and adventitia of the aorta were carefully dissected, cutting the arterial tissue into arterial rings of approximately 3 mm each. The arterial rings were transferred to 24-well plates, and 1 ml of 10% complete culture medium was added. The next day, the arterial rings were incubated with 20 μM Isaridin E, while simultaneously receiving stimulation with 2.6 mmol / L inorganic phosphorus. The medium was changed every two days. Around day 7, a significant increase in the stiffness of the arterial rings under high phosphorus treatment was observed, indirectly indicating that vascular ring calcification was successfully induced.

[0068] 2. Detection of calcium deposition in vascular tissue

[0069] Add 3-4 3mm ceramic grinding beads to a 1.5mL centrifuge tube, place the vascular tissue on the tube wall, and flash-freeze in liquid nitrogen for 10 seconds. Place the centrifuge tube in a tissue homogenizer pre-cooled in liquid nitrogen and homogenize at 55Hz for 30 seconds. Add 100μL of 6% dilute hydrochloric acid to the homogenized vascular powder, sonicate on ice, with a sonication program of 3 seconds on, 3 seconds off, repeated a total of 5 times. After sonication, let the sample stand on ice for 30 minutes. Centrifuge at 10000rpm at 4℃ for 15 minutes, and transfer the supernatant to a new 1.5mL centrifuge tube for subsequent analysis.

[0070] Prepare calcium standards: The required concentrations, from highest to lowest, are 2.5 mmol / L, 1.25 mmol / L, 0.625 mmol / L, 0.3125 mmol / L, 0.15625 mmol / L, and 0 (i.e., 6% dilute hydrochloric acid). Prepare working solution: Mix equal volumes of solutions R1 and R2 from the calcium assay kit and incubate at room temperature for 5 minutes. Add 10 μL of standard and the sample to be tested to 500 μL of working solution, vortex to mix, incubate at 37°C for 10 minutes, and add 200 μL to a 96-well microplate for detection. Place the 9-well plate in a multi-microplate reader and measure the absorbance at 600 nm. Plot a standard curve using the absorbance of the standards. The calcium content in the sample can be calculated from the standard curve. The calcium content is normalized to the protein concentration to obtain the relative calcium deposition concentration.

[0071] 3. von Kossa staining of blood vessel sections

[0072] After dewaxing paraffin sections, draw circles on the outer side of the vascular tissue using a histochemical pen. Add sufficient 5% AgNO3 solution to the vascular tissue, ensuring complete coverage. Irradiate under UV light for 1-2 hours, observing carefully to prevent drying. Discard the AgNO3 solution, wash with distilled water for 5 minutes, then wash with 5% Na2S2O3 solution for 2 minutes. Stain cell nuclei with hematoxylin for 30 seconds, then rinse with tap water for staining, repeating this process three times for 3 minutes each time. Dehydrate with 70%, 80%, 90%, 95%, 100%, and 100% ethanol sequentially, and clear twice with xylene. Mount with neutral resin and dry in a 37°C incubator.

[0073] 4. Alizarin Red staining of blood vessel sections

[0074] After dewaxing the paraffin sections, draw circles around the vascular tissue using a histochemical pen. Add alizarin red stain to the sections to completely cover the tissue and stain for 5 minutes. The staining time depends on the calcium salt content; observe under a microscope regularly until the calcium salts turn a deep orange-red. Discard the stain and wash with tap water until the slide runs colorless. Counterstain the cell nuclei with hematoxylin and wash with water. Bake the sections in a 65°C oven for 4 hours. Clear the sections in fresh xylene for 5-10 minutes and mount with neutral resin.

[0075] 5. Statistical Analysis Methods

[0076] Data processing was performed using GraphPad Prism 8.0 software. All results are expressed as mean ± SEM. One-way ANOVA was used for statistical analysis, and P < 0.05 was considered statistically significant.

[0077] (II) Experimental Results

[0078] 1. Effect of Isaridin E on the level of aortic ring calcification induced by high phosphorus in mice

[0079] Based on the results of cell experiments, 20 μM Isaridin E showed the strongest inhibitory effect on smooth muscle cell calcification. Therefore, the inventors treated isolated aortic tissue with 20 μM Isaridin E and high phosphorus for 7 days to further evaluate the effect of ISE on vascular calcification. Calcium deposition in the aorta was measured, and the experimental results are as follows: Figure 4 As shown, compared to the solvent group, ISE treatment significantly reduced the level of calcium deposition in the aortic rings. These results are consistent with those obtained at the cellular level.

[0080] Compared with the normal control group, # P<0.05; compared with the solvent group, *P<0.05, n=6.

[0081] 2. Detection of pathological changes in calcification staining of mouse aortic ring sections

[0082] To observe changes in the pathological structure of the aorta, the inventors performed a special staining for calcium salt determination on aortic sections. Alizarin red, an anthraquinone derivative, can chelate with calcium salts in calcium carbonate or calcium phosphate to form an orange-red complex, which can be used to stain small amounts of calcium salt deposits in tissues. It has a good staining effect on some pathological calcifications, such as calcifications of the diseased arterial wall in aortic atherosclerosis and other ectopic calcifications. After staining, the calcium salt deposits appear red or orange-red, with a light red or nearly colorless background. Von Kossa staining is also a classic method for staining mineralized nodules. The basic principle is that silver ions react with insoluble calcium salts in situ to form reducible silver salts in the tissue. Then, under strong light, ultraviolet light, or a very strong reducing agent, the silver salts are reduced to black elemental silver, thus revealing the calcium salts in the tissue. It is suitable for samples with a large amount of calcium salt deposits. After staining, the calcium salt deposit area appears black, the cell nuclei appear blue, and the background is light red. Figure 5 As shown, combining the results of two special staining methods, no calcium salts appeared in the aorta of the control group, while high phosphorus incubation increased the accumulation of calcium salts in the vascular media, causing calcium salts to be distributed along the elastic fibers of the blood vessels; compared with the solvent group, Isaridin E treatment significantly reduced the distribution and deposition of calcium salts in the vascular smooth muscle layer.

[0083] Example 3

[0084] Effects of compound Isaridin E on aortic calcification levels in model mice

[0085] (I) Materials and Methods

[0086] 1. Medication preparation

[0087] The compound Isaridin E was ground into powder and dissolved in a solution system of 4% DMSO, 10% Tween-80, 15% propylene glycol and 71% physiological saline (the concentration ratio of this system was obtained from a preliminary experiment to ensure complete drug dissolution while minimizing solvent toxicity). The drug was then administered by gavage at a predetermined concentration (ISE dosage was 50 mg / kg·d, with a controlled administration volume of 100 μl).

[0088] 2. Experimental animals and vascular calcification models

[0089] Preparation of Vitamin D3 solution: Weigh 66 mg of Vitamin D3 and dissolve it in 200 μl of anhydrous ethanol, then add 1.4 ml of corn oil. Dissolve the mixture thoroughly using a vortex mixer at room temperature and set aside. Weigh 750 mg of glucose and add 18.4 ml of ddH2O to dissolve it completely. Filter the solution to achieve sterility. Add the solution to the previously obtained liquid and vortex again to obtain a uniformly distributed emulsion suspension. In the control group, an equal volume of physiological saline was used instead of Vitamin D3 for preparation; other components were prepared in the same manner as the calcification group.

[0090] Establishment of a vascular calcification model: The skin on the mouse's back was pinched up, and the needle was inserted at a low angle, almost horizontally. After successful skin puncture, the syringe angle was lowered, and the back skin was lifted to inject 400,000 IU / kg cholecalciferol or solvent. After aspirating the syringe and confirming no blood flow, the drug was injected again. A visually observed swelling of the skin on the back indicated successful subcutaneous injection. Mice were subcutaneously injected for three consecutive days and then fed for another week before data collection.

[0091] Wild-type male C57BL / 6J mice (SPF grade, 6-8 weeks old, purchased from Guangdong Yaokang Biotechnology Co., Ltd.) were used in this experiment. They were housed in the Laboratory Animal Center (SPF grade) of Sun Yat-sen University School of Medicine, individually caged in a laminar flow rack, with free access to water and food. The room temperature was controlled at approximately 24℃, with a 12-hour day / night cycle. After one week of acclimatization, all experimental animals were randomly divided into four groups of six mice each. Simultaneous drug administration was then performed on each group: a normal control group (mice were subcutaneously injected with the control solution of VitD3 for three consecutive days), a VitD3 group (mice were subcutaneously injected with VitD3 for three consecutive days), a VitD3+ISE control solvent group (mice were subcutaneously injected with VitD3 for three consecutive days, and simultaneously administered the control solution of ISE by gavage for 10 consecutive days), and a VitD3+ISE group (mice were subcutaneously injected with VitD3 for three consecutive days, and simultaneously administered 50 mg / kg ISE by gavage for 10 consecutive days).

[0092] 3. Alizarin Red staining of mouse aorta

[0093] After euthanizing the experimental animals from which tissue samples were to be collected, the animals were fixed in a supine position on a foam board. The intact blood vessels from the aortic root to the iliac artery branches were washed with histochemical PBS to remove blood contamination and then soaked in 4% paraformaldehyde overnight. The next day, the blood vessel tissue was soaked in 0.004% alizarin red (prepared with 2% potassium hydroxide solution) overnight. The blood vessels were removed and washed twice in 2% potassium hydroxide solution for 5 minutes each time. The gross appearance of the blood vessels was observed and photographed. The blood vessels were then immersed again in 4% paraformaldehyde fixative and stored at room temperature for long-term preservation.

[0094] (II) Experimental Results

[0095] 1. Effect of Isaridin E on vitamin D3-induced aortic calcification levels in mice

[0096] High-dose vitamin D3-induced calcification of the arterial elastic layer has been studied for over 70 years. Vitamin D3 effectively promotes bone resorption, increasing serum calcium levels by more than 30%, leading to calcium and phosphorus metabolism disorders and vascular calcification. It is a commonly used research model for vascular calcification. Figure 6As shown, compared with the control group, vitamin D3 treatment significantly increased aortic calcium deposition, indicating that the vascular calcification model was successfully established; while after ISE gavage, the aortic calcium deposition level in mice was significantly lower than that in the solvent group, indicating that ISE can also significantly inhibit the occurrence and development of vascular calcification in vivo. Compared with the normal control group, # P<0.05; compared with the solvent group, *P<0.05, n=6.

[0097] As mentioned earlier, alizarin red is a classic method for staining calcium salts. It is prepared with 2% potassium hydroxide, causing it to turn purple in an alkaline environment. This purple color combines with calcium salt deposits in the aorta, resulting in a bluish-purple hue, while areas without calcium salts remain milky white. Figure 7 As shown, the blood vessels in the control group did not show calcium salt accumulation and remained milky white, while the aorta after vitamin D3 induction showed a distinct purple color, indicating calcium salt deposition and distribution in the aorta. After ISE gavage, the purple area in the blood vessels was significantly reduced compared to the solvent group, with positive areas mainly concentrated in the aortic arch, abdominal aorta, and common iliac artery, while calcified nodules in the thoracic aorta were significantly fewer. n=6.

[0098] Experimental discussion and evaluation:

[0099] Vascular calcification is an osteogenic process regulated by multiple cell types, with vascular endothelial cells (VSMCs) playing a central role. VSMCs are pluripotent cells found in blood vessels, including both synthetic and contractile phenotypes. Synthetic VSMCs are commonly found in embryonic or developing tissues and organs, and can undergo cell division, proliferation, and differentiation under the influence of various growth factors, playing an important role in vascular wall formation and vascular injury repair. Contractile VSMCs are terminally differentiated cells, maintaining the structural and functional integrity of the arterial wall. Prolonged exposure of VSMCs to a pathological environment with high levels of mineral ions leads to osteo / chondrocyte differentiation, with the highly differentiated contractile VSMCs transforming into dedifferentiated osteoblasts—a core step in vascular calcification. Increased serum calcium and phosphorus levels are significantly correlated with the occurrence of vascular calcification. Disorders of calcium and phosphorus metabolism promote osteogenic transition in smooth muscle cells, leading to an increase in Ca×Pi, which in turn promotes the growth and deposition of hydroxyapatite crystals in the extracellular matrix via thermodynamic mechanisms. This invention uses a high-phosphorus-induced smooth muscle cell calcification model, an isolated aortic ring calcification model, and a vitamin D3-induced mouse aortic calcification model to explore the intervention effect of Isaridin E on vascular calcification.

[0100] Isaridin E (ISE) is a cyclic hexapeptide compound isolated in 2019 from the Beauveria feline SYSU-MS7908, a fungus of the genus Beauveria, sourced from the Xisha Islands in the South China Sea. It has been found to significantly inhibit the release of LPS-induced endothelial inflammatory factors during sepsis and alleviate vascular inflammation and lung damage in mice. Vascular calcification is a pathological phenomenon characterized by the abnormal deposition of calcium and phosphorus crystals in the blood vessel wall. Therefore, the inventors first established a high-phosphorus-induced classic calcification model in cultured mouse aortic smooth muscle cells. Alizarin red staining and cellular calcium deposition levels showed that ISE could, within a certain range, reduce the formation of extracellular matrix calcium and phosphorus crystals in a concentration-dependent manner. Under high-phosphorus conditions, the protein levels of osteogenic markers Runx2 and BMP2 in smooth muscle cells increased, while the protein level of the contractile marker α-SMA decreased, suggesting a phenotypic shift from contractile to osteogenic cells. After administration of Isaridin E, osteogenic markers were downregulated, while contractile markers were upregulated. The inventors then determined the half-maximal inhibitory rate (IC50) of ISE in vascular calcification, showing an IC50 of approximately 5 μmol. These experiments confirmed at the cellular level that ISE has an anti-vascular calcification effect.

[0101] In Example 2, the inventors conducted an in vitro vascular ring experiment, separating mouse aortic vessels and culturing them in vitro. Consistent with the cell experiments, ISE inhibited calcium deposition levels in the aorta; vascular sections showed that ISE inhibited abnormal calcium and phosphorus crystal deposition in the vascular wall induced by high phosphorus levels. In Example 3, the inventors used a classic in vivo vascular calcification model, simulating a mouse aortic calcification model induced by calcium and phosphorus metabolism disorders through subcutaneous injection of high doses of vitamin D3 for three consecutive days, to investigate the effect of Isaridin E on vascular calcification in mice. Consistent with the results of the above experiments, the aortic calcium deposition experiment showed that vitamin D3 significantly increased aortic calcium deposition, while ISE significantly reduced aortic calcification levels. Gross Alizarin Red staining of the aorta showed that vitamin D3 promoted significant aortic calcification, while ISE treatment reduced aortic calcification levels, especially significantly reducing thoracic aortic calcification. The above experiments confirmed the definite protective effect of ISE on vascular calcification in whole animal models.

[0102] In summary, this study demonstrates that ISE or its medicinal salts can inhibit osteogenic differentiation of vascular smooth muscle cells and significantly suppress vascular calcification induced by calcium and phosphorus metabolism disorders, showing promise for development as novel anti-vascular calcification drugs.

[0103] It is worth noting that the anti-vascular calcification drug described in this invention uses Isaridin E as the main active ingredient. However, variations in the formulation system and administration method, derivatives resulting from simple chemical modifications of the compound, and the combined use of multiple active substances are not excluded.

[0104] The technical solutions provided by the embodiments of the present invention have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the embodiments of the present invention. The descriptions of the embodiments above are only for helping to understand the principles of the embodiments of the present invention. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the embodiments of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. The use of compound Isaridin E or its pharmaceutical salt in the preparation of a drug for treating vascular calcification-related diseases, wherein the drug is used to inhibit osteogenic differentiation of vascular smooth muscle cells caused by calcium and phosphorus metabolism disorders; and the disease is chronic kidney disease.

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