Application of H-151 in preparation of medicine for preventing and treating calcified aortic valve disease

Abstract

The invention discloses application of H-151 in preparation of drugs for preventing and treating calcified aortic valve diseases, relates to the technical field of biological medicines, and is characterized in that the invention provides application of an STING inhibitor H-151 or pharmaceutically acceptable salts thereof in preparation of drugs for preventing and / or treating calcified aortic valve diseases. The invention reveals for the first time that H-151 can inhibit the increase of the expression level of osteogenic differentiation markers ALP and RUNX2 in aortic valve interstitial cells induced by an osteogenic culture medium in an in-vitro experiment, and inhibit osteogenic differentiation and calcium salt deposition of the aortic valve interstitial cells; in addition, aortic valve thickening, fibrosis and calcium salt deposition of mice induced by an aortic valve injury model are inhibited in vivo. Based on the biological effect, the application of H-151 in developing the medicine for treating the calcified aortic valve disease is provided for the first time, and a new technical path and a new medicine choice are provided for preventing and treating the calcified aortic valve disease.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to the use of H-151 in the preparation of drugs for the prevention and treatment of calcified aortic valve disease. Background Technology

[0002] Calcific aortic valvular disease (CAVD) is the most common valvular disease, characterized by progressively worsening aortic valve fibrosis and calcification, leading to thickening, stenosis, and stiffening of the aortic valve. This ultimately obstructs the left ventricular outflow tract, increasing cardiac afterload and causing heart failure. CAVD is prevalent in developed countries, ranking as the third most common cardiovascular disease after coronary heart disease and hypertension. The incidence of CAVD is greater than 2% in people over 60 years of age. In my country, the incidence of calcific aortic valve disease detected by echocardiography in patients over 45 years of age is 12.8%, and the prevalence increases with age. Furthermore, CAVD has a high mortality rate; the mortality rate within two years of symptom onset is approximately 50%. Because its pathogenesis is not fully understood, there are currently no effective drug interventions to halt the progression and improve the prognosis of CAVD. Lipid-lowering therapy targeting CAVD risk factors also fails to inhibit its progression. Surgical intervention or transcatheter aortic valve replacement (AVR) remains the only treatment option for severe calcified aortic valve disease. Although these treatments have yielded very good results, perioperative risks are unavoidable, including complications such as acute kidney injury, left bundle branch block, and hemorrhage, and even death. Therefore, exploring the pathogenesis of CAVD and finding effective drugs to prevent or treat it are of great significance for prolonging patient life and improving prognosis.

[0003] Mitochondrial damage has been shown to be associated with the progression of CAVD. A key characteristic of mitochondrial damage is the release of mitochondrial mtDNA into the cytoplasm. This released mtDNA can be recognized and bound by cGAS protein, which then catalyzes the synthesis of cyclic GMP-AMP (cGAMP) from ATP and GTP. cGAMP synthesized by cGAS can act as a second messenger, binding to the endoplasmic reticulum membrane linker STING and inducing a conformational change, leading to STING activation. STING activation not only promotes TBK1 phosphorylation, thereby activating IRF3 phosphorylation and allowing it to enter the nucleus to induce downstream inflammatory responses, but also promotes the activation of the kappaB inhibitor kinase (IKK). IKK activation leads to IκB phosphorylation and subsequent degradation of IκB via the ubiquitin-proteasome pathway, resulting in the release of NF-κB into the nucleus, thus inducing downstream inflammatory responses.

[0004] H-151, a STING inhibitor, is a highly selective antagonist that works through covalent modification and has been shown to effectively inhibit the STING signaling pathway in various disease models. Previous studies have reported that H-151 can alleviate ischemia-reperfusion-induced cardiac injury in mice. However, no information has been published regarding the application of H-151 in the prevention or treatment of calcific aortic valve disease. Summary of the Invention

[0005] The purpose of this invention is to provide the application of H-151 in the preparation of drugs for the prevention and treatment of calcified aortic valve disease, and to provide a new solution for the prevention and treatment of calcified aortic valve disease.

[0006] The above-mentioned technical objective of the present invention is achieved through the following technical solution: the application of H-151 in the preparation of a drug for preventing and treating calcified aortic valve disease, wherein the drug achieves the prevention and treatment of calcified aortic valve disease in vitro by inhibiting the abnormal osteogenic differentiation of aortic valve interstitial cells;

[0007] It can prevent and treat calcified aortic valve disease by alleviating pathological thickening, fibrosis and calcium salt deposition in the aortic valve in vivo.

[0008] The present invention is further configured such that the drug is H-151 or a pharmaceutically acceptable salt thereof.

[0009] The present invention is further configured such that the drug is a pharmaceutical composition made of H-151 or a pharmaceutically acceptable salt thereof and a conventional pharmaceutical carrier.

[0010] The present invention is further configured such that the pharmaceutical composition is a capsule, granule, injection, sustained-release tablet, lozenge, or powder for injection.

[0011] In summary, the present invention has the following beneficial effects: The present invention provides the application of H-151 in the preparation of drugs for the prevention and treatment of calcified aortic valve disease, and provides a method that can significantly improve the osteogenic differentiation of aortic valve interstitial cells in vitro after H-151 intervention, and can alleviate aortic valve thickening and calcification in mice in vivo, providing a new potential drug option for calcified aortic valve disease. Attached Figure Description

[0012] Figure 1 This is a schematic diagram illustrating how H-151 can inhibit osteogenic differentiation of aortic valve interstitial cells in Embodiment 1 of the present invention.

[0013] Figure 2 This is a schematic diagram illustrating how H-151 can alleviate the increase in transvalvular velocity, peak aortic valve pressure, and aortic valve area induced by the AVI model in mice, as verified in Example 2 of the present invention.

[0014] Figure 3 This is a schematic diagram illustrating how H-151 can reduce the phosphorylation level of STING in the mouse aortic valve induced by the AVI model in Example 2 of the present invention.

[0015] Figure 4 This is a schematic diagram illustrating how H-151 can alleviate aortic valve thickening, fibrosis, and calcification induced by the AVI model in mice, as verified in Example 2 of this invention.

[0016] Figure 5 This is a schematic diagram illustrating how H-151 can reduce the elevated expression of osteogenic differentiation markers ALP and RUNX2 in mouse valves induced by the AVI model in Example 2 of this invention. Detailed Implementation

[0017] The following is in conjunction with the appendix Figure 1-5 The present invention will be described in further detail below.

[0018] Example 1: Verification of the inhibitory effect of H-151 on abnormal osteogenic differentiation of aortic valve interstitial cells by in vitro culture of human aortic valve interstitial cells

[0019] Primary human aortic valve interstitial cells were isolated from aortic valve tissue obtained after surgical aortic valve replacement. The valve tissue was derived from patients with calcified aortic valve disease. The research protocol was approved by the Ethics Committee of Nanfang Hospital, Southern Medical University, and written informed consent was obtained from all patients.

[0020] 1. Research Methods

[0021] Aortic valve tissue was collected from patients who underwent surgical aortic valve replacement and stored in sterile saline. It was then rapidly transported back to the laboratory in an ice pack. In a laminar flow hood, the aortic valve tissue was first washed with 75% ethanol for approximately 15 seconds, followed immediately by washing with sterile PBS to prevent ethanol damage to the aortic valve interstitial cells. Subsequently, using pre-autoclaved laboratory equipment, the surrounding muscle connective tissue and completely calcified portions of the tissue were carefully removed. The tissue was then minced and transferred to a penicillin bottle equipped with a sterile rotor. Type I collagenase solution (2 mg / ml) was added, and the bottle was placed on a magnetic stirrer (37°C, 150 rpm) for 6 hours of digestion. After complete digestion was observed, the tissue suspension was centrifuged (1000 rpm, room temperature, 5 min) and resuspended in complete culture medium (DMEM medium containing 10% fetal bovine serum and 0.5% penicillin-dextrose antibiotics). The resuspended solution was added to 6 cm culture dishes and incubated at 37°C.

[0022] 2. Result Detection

[0023] (1) Cellular intervention

[0024] After passages of human aortic valve interstitial cells to passages 3-6, the cells were seeded into 6-well plates and cultured until the cell density reached 80%-90% of the plate. At this point, the complete culture medium was replaced with osteogenic medium, and H-151 (1 μM) was added to the H-151+OM group. After culturing for 72 hours, cell proteins were extracted, and changes in relevant osteogenic markers were detected by Western blotting.

[0025] H-151 can inhibit osteogenic responses in valvular interstitial cells induced by osteogenic culture media. For example... Figure 1 As shown, H-151 can reduce the expression levels of osteogenic markers ALP and RUNX2 in aortic valve interstitial cells induced by osteogenic culture medium. Figure 1 (A)

[0026] (2) Alizarin red staining was used to detect calcium nodule deposition in human valve mesenchymal cells.

[0027] Human aortic valve interstitial cells (passages 3-6) were passaged in 6-well plates, with three groups: a negative control group (routine culture), an OM treatment group, and an H-151+OM treatment group. The medium was changed every three days. After 14 days of culture, calcium deposition in the cells was detected using an Alizarin Red staining kit (Saiwell): Cells were fixed with 75% ethanol for 15 min, washed twice with double-distilled water, and stained with 1% Alizarin Red for 10-15 min. The cells were then gently washed with double-distilled water until the outflow was no longer red. Calcium deposition in the aortic valve interstitial cells was then photographed using an inverted microscope. Quantitative analysis of calcium deposition was then performed: 200 μl of 10% acetic acid was added to each well and shaken on a shaker to dissolve the chelate formed by the reaction of calcium salts and Alizarin Red. The supernatant was measured at 450 nm using a spectrophotometer. Calcium salt content was directly proportional to absorbance, allowing for relative quantification of calcium salts in each well. Results are shown below. Figure 1 As shown in Figure B, compared with the negative control group, the calcium salt deposition in the OM group was significantly increased, while the calcium salt deposition in the aortic valve interstitial cells in the H-151+OM group was significantly reduced.

[0028] (3) The activity of alkaline phosphatase in human aortic valve interstitial cells was detected using an alkaline phosphatase activity assay kit.

[0029] Human valvular mesenchymal cells (generations 3-6) were transferred to 6-well plates, with three groups: a standard culture negative control group, an OM treatment group, and an H-151+OM treatment group. The medium was changed every three days. After 14 days of culture, alkaline phosphatase activity was detected using an alkaline phosphatase staining kit (Beyotime). Cells were washed three times with PBS, followed by fixation with 75% ethanol for 15 min. After washing three times with double-distilled water, alkaline phosphatase chromogenic buffer was added, and the cells were incubated overnight at room temperature in the dark. The staining working solution was then washed away with double-distilled water, and images were taken using an inverted microscope. Quantitative analysis of alkaline phosphatase was performed using an alkaline phosphatase assay kit (Beyotime). Cells were lysed with RIPA buffer (purchased from Beyotime Biotechnology Co., Ltd., China), centrifuged at 14,000 g for 30 min, and the supernatant was collected. Protein concentration was determined using the BCA method (purchased from Thermal Fisher Scientific, USA). 50 μL of protein supernatant collected from each group was added to the reaction substrate and incubated at 37°C for 15 minutes. After adding the stop solution, the absorbance at 405 nm was measured using a microplate reader, and the activity of each group was calculated. Results are as follows: Figure 1 As shown in Figure C, the alkaline phosphatase activity in aortic valve interstitial cells was significantly increased after treatment with osteogenic medium compared to the negative control group, while H-151 co-treatment could alleviate the increase in cell alkaline phosphatase activity induced by osteogenic medium.

[0030] Example 2: Animal experiments verifying the in vivo intervention effect of H-151 on calcified aortic valve lesions.

[0031] C57BL / 6J mice were purchased from the Animal Center of Southern Medical University and housed at the Animal Experiment Center of Nanfang Hospital, Southern Medical University. The animals were housed in an SPF environment at 25°C with a 12-hour day-night cycle and were fed a normal diet. This animal experiment was approved by the Ethics Committee of Nanfang Hospital, Southern Medical University (application ID: IACUC-LAC-20220705-0020) and strictly adhered to European guidelines for the care of laboratory animals.

[0032] 1. Research Methods

[0033] (1) Establishing an aortic valve injury (AVI) model:

[0034] Mice were restrained on a wooden board and anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg / kg). Hair removal cream was used to assist in hair removal, and the skin was disinfected with alcohol swabs. The right carotid artery of the mice was exposed using sterile scissors and forceps. The distal end of the carotid artery was ligated with 5-0 sutures, and a slipknot was tied at the proximal end. An incision was made in the carotid artery using ophthalmic scissors, and an FST guidewire (catalog number: FST18000-10, diameter 0.36 mm) was inserted into the carotid artery and secured with sutures. With the assistance of small animal ultrasound (Visualsonics Vevo 2100), the guidewire was moved across the brachiocephalic trunk to the aortic valve orifice. The guidewire was moved up and down 50 times at the valve orifice, followed by 100 rotations. The guidewire was then withdrawn, and the carotid aorta was ligated. Finally, the skin was sutured and disinfected. The sham surgery group (Sham group) also required ligation of the right carotid aorta, but no guidewire was inserted into the aorta. After surgery, the mice were fed a normal diet and kept in the above-mentioned SPF environment.

[0035] (2) Animal intervention

[0036] One day after surgery, the model group mice were randomly divided into two groups: the AVI group (intraperitoneal injection of blank solvent daily) and the H-151+AVI group (750 nmol H-151 was injected intraperitoneally into each mouse daily, and H-151 was dissolved in a solvent prepared with 10% dimethyl sulfoxide, 40% polyethylene glycol 300, 5% Tween 80 and 45% physiological saline).

[0037] 2. Result Detection

[0038] Eight weeks after the surgery, the aortic valve condition of the mice was examined using echocardiography.

[0039] Mouse tissue collection: Mice were anesthetized by injecting an excessive amount of sodium pentobarbital and then fixed in place. The mouse heart was exposed with scissors, and the inferior vena cava was cut open. Physiological saline was then injected through the apex of the heart until the outflowing fluid became colorless. The heart was then removed and immersed in 4% paraformaldehyde. After preparing paraffin sections from the heart specimens, the specimens were stained with hematoxylin and eosin (HE) to evaluate the thickness of the mouse aortic valve, Masson staining to evaluate the degree of aortic valve fibrosis, and Von Kossa and Alizarin Red staining to evaluate the degree of aortic valve calcification. Finally, immunofluorescence staining was performed to detect changes in the expression levels of P-STING and osteogenic markers ALP and RUNX2 in the mouse aortic valve.

[0040] Experimental results: H-151 can inhibit AVI-induced aortic valve lesions in mice. Figure 2As shown, echocardiography at 8 weeks post-surgery revealed that, compared to the Sham group, the AVI group mice exhibited significantly increased peak aortic valve velocity and transvalvular pressure gradient, along with a decreased valve orifice area. H-151 intervention significantly alleviated these functional abnormalities. Subsequently, immunofluorescence assays confirmed that H-151 downregulated the phosphorylation level of STING protein in the valve tissue of AVI model mice, suggesting that its effect is related to the inhibition of the STING signaling pathway. Figure 3 Further histological analysis showed that H-151 treatment could alleviate AVI-induced leaflet thickening (HE staining). Figure 4 (A) and excessive collagen fiber deposition (Masson staining, Figure 4 (Middle B). Von Kossa staining and alizarin red staining results showed that H-151 treatment could also alleviate AVI-induced aortic valve calcification in mice. Figure 4 (CD). Finally, immunofluorescence staining results showed that the expression levels of osteogenic markers ALP and RUNX2 in the aortic valve tissue of mice in the AVI group were significantly increased, while H-151 intervention could reduce the expression levels of ALP and RUNX2. Figure 5 ).

[0041] The above results indicate that inhibiting H-151 can improve aortic valve thickening, fibrosis, and calcification induced by the AVI model to some extent.

[0042] In summary, H-151 can effectively inhibit osteogenic differentiation of aortic valve interstitial cells induced by osteogenic culture medium and alleviate aortic valve lesions induced by the AVI model in mice, providing a new drug option for the prevention and treatment of calcified aortic valve disease.

[0043] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.

Claims

1. The application of H-151 in the preparation of drugs for the prevention and treatment of calcific aortic valve disease, characterized by: The drug achieves prevention and treatment of calcific aortic valve disease in vitro by inhibiting abnormal osteogenic differentiation of aortic valve interstitial cells; It can prevent and treat calcified aortic valve disease by alleviating pathological thickening, fibrosis and calcium salt deposition in the aortic valve in vivo.

2. The use of H-151 according to claim 1 in the preparation of a drug for preventing and treating calcified aortic valve disease, characterized in that: The drug is H-151 or a pharmaceutically acceptable salt thereof.

3. The application of H-151 according to claim 2 in the preparation of a drug for preventing and treating calcified aortic valve disease, characterized in that: The drug is a pharmaceutical composition made from H-151 or a pharmaceutically acceptable salt thereof with a conventional pharmaceutical carrier.

4. The use of H-151 according to claim 3 in the preparation of a drug for preventing and treating calcified aortic valve disease, characterized in that: The pharmaceutical composition is a capsule, granule, injection, sustained-release tablet, lozenge, or powder for injection.

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