A drug for treating calcific aortic valve disease based on p arm1 regulation and application thereof

Abstract

The present application relates to a kind of drug based on PARM1 regulation treatment calcified aortic valve disease and its application.The present application first finds that PARM1 is significantly increased in the aortic valve of calcified aortic valve disease (CAVD) patient, and plays a key role in the osteogenic differentiation process of valve interstitial cells (VICs), and the expression level of PARM1 is knocked down can inhibit the osteogenic differentiation of VICs and extracellular calcium salt deposition, reduce aortic valve calcification, and its mechanism of action involves MAPK signal pathway.The present application provides a new target and treatment idea for the treatment of CAVD, and can be applied to related drug research and development and treatment method development.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of a PARM1 inhibitor in the treatment of calcific aortic valve disease. Background Technology

[0002] Calcific aortic valve disease (CAVD) is a degenerative disease with high morbidity and mortality rates in the elderly. The main pathophysiological change is aortic valve calcification, leading to hemodynamic changes and affecting cardiac function. Previously, CAVD was considered an age-related degenerative disease, a process of valvular tissue degeneration and hardening with age. However, recent basic research has shown that CAVD is an active progressive process involving complex pathological changes such as endothelial damage, inflammatory cell infiltration, extracellular matrix remodeling, and osteoblastic differentiation of valvular interstitial cells (VICs). The cells in valvular tissue mainly include valvular endothelial cells (VECs), valvular interstitial cells, and immune cells. Studies have confirmed that increased calcium deposition due to osteoblastic differentiation of VICs is a crucial factor in the development and progression of valvular calcification. Currently, there is a lack of effective clinical drug treatment for CAVD, and the main treatment is aortic valve replacement surgery. However, patients undergoing surgery inevitably bear high medical risks and financial burdens. Therefore, exploring the specific pathogenesis of CAVD and using non-surgical methods to effectively prevent and / or treat aortic valve calcification are urgent clinical needs in the treatment of CAVD.

[0003] Currently, the treatment of calcific aortic valve disease remains challenging, especially in early prevention and treatment. Several medications have been used in clinical practice to treat this disease; however, these medications still have some limitations. Commonly used drugs are lipid-lowering drugs, such as statins. These drugs slow the progression of aortic valve calcification by lowering cholesterol levels, thereby improving aortic valve function to some extent. However, current clinical studies on lipid-lowering drugs in aortic valve calcification have confirmed that they cannot prevent or reverse existing calcification. Furthermore, long-term use of lipid-lowering drugs may have adverse reactions, such as muscle pain and muscle injury, elevated liver enzymes or other liver function abnormalities, gastrointestinal discomfort, etc., requiring close monitoring. Another class of drugs are anti-calcification drugs, such as antioxidants and anti-inflammatory drugs. The mechanism of action of these drugs is mainly through reducing oxidative stress and inflammatory responses, thereby slowing or inhibiting the progression of arterial calcification. However, there is currently a lack of sufficient clinical trial evidence to support the efficacy and safety of these drugs in the treatment of calcific aortic valve disease.

[0004] Although some drugs have been tried in the treatment of calcific aortic valve disease, many challenges remain, such as: the pathogenesis of calcific aortic valve disease is not fully understood, leading to insufficient targeted treatment strategies; and existing drugs for CAVD have limited effectiveness in treating early-stage disease and preventing progression.

[0005] Prostate androgen-regulated mucin-like protein 1 (PARM1) encodes a highly glycosylated mucin-like transmembrane protein containing 296 amino acids. PARM1 was initially identified as a highly expressed gene in the prostate of castrated rats. In addition, PARM1 has also been shown to be highly expressed in the heart. Subsequent studies have confirmed that PARM1 is involved in the regulation of cell proliferation, apoptosis, and differentiation in cardiovascular diseases. Importantly, single-cell RNA sequencing analysis showed that PARM1 is significantly upregulated in osteogenic differentiation-induced aortic valve in cells (VICs). However, the function and potential mechanisms of PARM1 in osteogenic differentiation of VICs and cardiovascular arterial disease (CAVD) have not been reported. Therefore, a comprehensive study of the role of PARM1 in aortic valve calcification is of great significance. Summary of the Invention

[0006] This invention investigates the expression of PARM1 (PARM1 gene ID 25849, corresponding to the human genome reference sequence number NC_000004.12.) in calcified aortic valves and its regulatory effect on osteogenic differentiation of human aortic valve interstitial cells. It proposes a PARM1-regulated aortic valve calcification treatment drug and its application, providing a new non-surgical treatment option for CAVD. Specifically, by inhibiting PARM1 expression, osteogenic differentiation of valvular interstitial cells is reduced, aortic valve calcification is alleviated, thereby treating calcified aortic valve disease.

[0007] The first objective of this invention is to provide an application of a PARM1 inhibitor in the prevention or treatment of calcific aortic valve disease.

[0008] Furthermore, the calcific aortic valve disease includes calcific aortic stenosis, calcific aortic insufficiency, or calcific aortic lesions.

[0009] Furthermore, the PARM1 inhibitor inhibits the osteogenic differentiation of valvular interstitial cells (VICs) by suppressing the expression level of the PARM1 nucleotide sequence or the expression level of the PARM1 protein.

[0010] Furthermore, the PARM1 inhibitor used to suppress the expression level of the PARM1 nucleotide sequence is selected from at least one of the following: antisense nucleotides targeting PARM1 mRNA, small interfering RNA (siRNA), short hairpin RNA (shRNA), and microRNA (miRNA).

[0011] Furthermore, the PARM1 inhibitor is an siRNA for silencing PARM1 gene expression, the sense strand nucleotide sequence of the siRNA is shown in SEQ ID NO.1, and the antisense strand nucleotide sequence of the siRNA is shown in SEQ ID NO.2.

[0012] SEQ ID NO.1: 5'-CUGUGUCAGGCAAAGUGAUTT-3';

[0013] SEQ ID NO. 2: 5'-AUCACUUUGCCUGACACAGTT-3'.

[0014] A second objective of this invention is to provide a medicament for the prevention or treatment of calcified aortic valve disease.

[0015] Furthermore, the drug comprises the aforementioned PARM1 inhibitor, as well as a pharmaceutically acceptable carrier, diluent, or excipient, wherein the calcified aortic valve disease is a calcified lesion of the heart valves.

[0016] Furthermore, the PARM1 inhibitor is a small interfering RNA (siRNA) targeting PARM1 mRNA, the sense strand nucleotide sequence of the small interfering RNA (siRNA) is shown in SEQ ID NO.1, and the antisense strand nucleotide sequence of the siRNA is shown in SEQ ID NO.2.

[0017] Furthermore, the dosage forms of the drug include, but are not limited to, oral liquids, injections, tablets, pills, dispersants, capsules, drop pills, granules, suspensions, and emulsions.

[0018] Furthermore, the drug prevents or treats calcific aortic valve disease by regulating the PARM1 signaling pathway and osteogenic differentiation-related proteins.

[0019] A pharmaceutically acceptable carrier may be further comprising an exemplary embodiment of the present invention, wherein the pharmaceutically acceptable carrier may include saline solution, sterile water, Ringer's solution, buffered saline solution, glucose solution, maltodextrin solution, glycerol, ethanol, and mixtures of two or more thereof, which may be used alone or in combination. Other conventional additives, such as antioxidants, buffers, and antibacterial agents, may be added to the pharmaceutically acceptable carrier when necessary. Furthermore, diluents, dispersants, surfactants, binders, and lubricants may be further added to the pharmaceutically acceptable carrier. In this case, the pharmaceutically acceptable carrier may be formulated into an injectable formulation, such as an aqueous solution, suspension or emulsion, pill, capsule, granule, or tablet. Furthermore, the pharmaceutically acceptable carrier may preferably be formulated using appropriate methods known in the relevant art or methods disclosed in Remington's Pharmaceutical Science (latest edition), Mack Publishing Company, Easton, Pa. Examples of pharmaceutically acceptable carriers, formulations of pharmaceutical compositions, and methods for preparing formulations are known in the relevant art.

[0020] Furthermore, the medicament may be a composition comprising the PARM1 inhibitor of the present invention as an active ingredient for administration to a subject to treat aortic valve calcification. This composition may be provided in subjects in need of the effective amount for treating aortic valve calcification, wherein the dose of the PARM1 inhibitor may vary considerably depending on the patient's weight, age, sex, health status, diet, time of administration, method of administration, excretion rate, and severity of disease. In this case, the appropriate dose of the PARM1 inhibitor may optionally be determined by those skilled in the art.

[0021] Furthermore, the drug may be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, intramuscularly, intravascularly, or subcutaneously) as desired for the intended purpose.

[0022] Preferably, the drug is for oral administration. For oral administration, the drug may be provided in the form of capsules, tablets, powder, granules, or suspension; and the drug may include conventional additives such as lactose, mannitol, corn starch, or potato starch, and the binder that may be used in the drug may include crystalline cellulose, cellulose analogues, gum arabic, corn starch, or sodium carboxymethyl cellulose.

[0023] Furthermore, the drug also includes any one or more pharmaceutically acceptable excipients, such as carriers, excipients, disintegrants, lubricants, binders, flavoring agents, absorbents, solubilizers, buffers, penetration enhancers, stabilizers, etc.

[0024] Furthermore, the drug may be prepared into any pharmaceutically acceptable dosage form as needed, having preventive and / or therapeutic effects. Pharmaceutically acceptable dosage forms include liquid and solid dosage forms, suitable for gastrointestinal administration, injection, etc. Liquid dosage forms include, but are not limited to, injections, suspensions, and granules, while solid dosage forms include, but are not limited to, tablets, capsules, powders, and granules.

[0025] Furthermore, the drug can be used alone or in combination with surgical procedures, hormone therapy, drug therapy, and the use of biological response modifiers to prevent or treat valve calcification.

[0026] A third objective of this invention is to provide a method for treating calcified aortic valve disease.

[0027] Furthermore, the method includes administering the aforementioned PARM1 inhibitor or the aforementioned drug to the patient.

[0028] Furthermore, the method inhibits the osteogenic differentiation process of valve interstitial cells by suppressing the expression of PARM1.

[0029] In some embodiments, the method can prevent or treat calcified aortic valve disease in vitro, in in vivo animal models, or in drug screening.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] (1) This invention demonstrates that the expression level of PARM1 gene in human calcified aortic valve tissue is significantly higher than that in normal aortic valve tissue by detecting the expression of PARM1 gene, suggesting that PARM1 can be used as a diagnostic marker for calcified aortic valve disease.

[0032] (2) This invention provides a new therapeutic target for CAVD, namely PARM1 and its related MAPK signaling pathway. The therapeutic effect of knocking down PARM1 on CAVD was verified through in vitro and in vivo experiments. This provides a theoretical basis and experimental evidence for the development of new CAVD therapeutic drugs and methods. It also provides a new target and treatment idea for the treatment of calcific aortic valve disease. It helps to solve the problem of limited current CAVD treatment methods. That is, by inhibiting the expression of PARM1, it is possible to effectively reduce the osteogenic differentiation of VICs, delay or reverse the progression of CAVD, and improve the prognosis of patients. Therefore, the research results of this invention lay the foundation for the subsequent development of therapeutic drugs and methods targeting PARM1 and have broad application prospects. Attached Figure Description

[0033] Figure 1Immunofluorescence staining of α-SMA and VE-Cadherin in VICs isolated from human aortic valves (scale bar: 100 μm).

[0034] Figure 2 PARM1 expression levels in calcified aortic valves: (A) Representative hematoxylin-eosin (H&E), Masson's staining, and von Coussa staining images of non-calcified and calcified human aortic valves. (B) Western blot analysis of PARM1 expression in aortic valves from CAVD and non-CAVD patients. (C) Detection of PARM1 expression in VICs isolated from non-calcified and calcified aortic valves. (D) Representative immunofluorescence images of PARM1 localized in VICs from human aortic valve tissue (scale bar: 25 μm). (E) Western blot analysis of PARM1 protein levels in VICs stimulated with osteogenic culture medium. (F) Low-density lipoprotein receptor gene knockout (LDL-DLK) patients fed a normal diet (ND) and a high-fat diet (HFD). - / - Representative immunofluorescence images of PARM1 in the aortic valve of mice (scale bar: 25 μm), where values ​​are mean ± standard deviation.

[0035] Figure 3 The graphs show the efficiency analysis of siRNA and lentivirus intervention. (A, B) show the silencing efficiency of si-PARM1 detected by real-time quantitative polymerase chain reaction (qRT-PCR) and Western blot. (C, D) show the overexpression efficiency of oe-PARM1 lentivirus detected by qRT-PCR and Western blot. Figure 4To illustrate the role of PARM1 in osteogenic differentiation of VICs, this figure shows that knockdown or overexpression of PARM1 can regulate osteogenic differentiation of VICs in vitro: (A, B) After VICs were transfected with si-PARM1, the expression levels of two osteogenic markers (alkaline phosphatase (ALP) and Runx-associated transcription factor 2 (RUNX2)) were assessed by real-time quantitative polymerase chain reaction (qRT-PCR) (A) and Western blot (B). (C) ALP activity was detected after VICs transfected with si-PARM1 or control (si-Scr) were cultured in osteogenic medium. (D) Calcium content in each group of VICs was measured 14 days after intervention in osteogenic medium. (E, F) Calcium deposition in VICs was detected by alizarin red staining. (G, H) After overexpression of PARM1 in VICs, the expression levels of ALP and RUNX2 were assessed by qRT-PCR (G) and Western blot (H). (I) ALP activity was detected after PARM1 overexpression in VICs. (J) Calcium deposition in PARM1-overexpressing VICs was detected by alizarin red staining. Values ​​are mean ± standard deviation.

[0036] Figure 5 A schematic diagram illustrating the establishment of an animal model of aortic valve calcification induced by a high-fat diet and the intervention of PARM1 knockdown.

[0037] Figure 6 Metabolic parameters and cardiac function in mice with high-fat diet-induced aortic valve calcification were assessed. (A) Immunofluorescence staining was used to detect the knockdown efficiency of PARM1 in vivo. (B) Different groups of Ldlr... - / - Mouse body weight. (C) Different groups of Ldlr - / - Blood glucose levels in mice. (D) Different groups of Ldlr - / - Total cholesterol in mice. (E) Ldlr in different groups - / - Low-density lipoprotein in mice. (F) Different groups of Ldlr - / - Triglyceride levels in mice. (G) Ldlr levels in different groups - / - Left ventricular ejection fraction in mice. (H) Different groups of Ldlr - / - Fractional shortening of the left ventricle in mice. Data are presented as mean ± standard deviation.

[0038] Figure 7 To improve Ldlr by knocking down PARM1 - / -High-fat diet-induced aortic valve calcification in mice: (A) Representative echocardiographic images of ND+sh-control, HFD+sh-control, and HFD+sh-PARM1 mice. (B) Quantitative analysis of peak transvalvular velocity and mean transvalvular pressure gradient in ND+sh-control, HFD+sh-control, and HFD+sh-PARM1 mice. (C) Representative images of the aortic valve stained with H&E, Sirius red, Masson's blot, and von Cusa stains in ND+sh-control, HFD+sh-control, and HFD+sh-PARM1 mice (scale bar: 200 μm). (DG) Quantitative analysis of aortic valve thickness, Sirius red-positive areas, fibrotic areas, and total calcification areas. (H) Immunofluorescence images of BMP2 in ND+sh-control, HFD+sh-control, and HFD+sh-PARM1 mice (scale bar: 25 μm). (I) Immunofluorescence images of ALP in ND+sh-control, HFD+sh-control, and HFD+sh-PARM1 mice (scale bar: 25 μm). Values ​​are mean ± standard deviation.

[0039] Figure 8 To demonstrate that the MAPK signaling pathway is a downstream pathway of osteogenic differentiation in VICs regulated by PARM1: (A) Gene Ontology (GO) analysis was performed on RNA-seq data from the si-Scr and si-PARM1 groups. (B) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was performed on differentially expressed genes from the si-Scr and si-PARM1 groups. (C) Gene Set Enrichment Analysis (GSEA) was performed on differentially expressed genes from the si-Scr and si-PARM1 groups. (D) Western blot analysis was used to analyze the phosphorylation level of the MAPK signaling pathway after PARM1 knockdown. (E) ALP and RUNX2 protein levels were assessed by Western blot after treatment with P38 and ERK inhibitors in VICs. (F) ALP activity was detected after treatment with P38 and ERK inhibitors in VICs. (G) Calcium deposition after treatment with P38 and ERK inhibitors was detected by Alizarin Red staining. Values ​​are mean ± standard deviation.

[0040] Figure 9 Immunofluorescence staining of p-ERK1 / 2(A) and p-P38(B) in VICs under different treatment conditions. (Scale bar: 100 μm). Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below. However, it should be understood that the description herein is merely illustrative and not intended to limit the scope of the invention.

[0042] Unless otherwise defined, 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 pertains. The terminology used herein in the specification is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention. All reagents and instruments used herein are commercially available, and the characterization methods involved are described in relevant prior art and will not be repeated herein.

[0043] English translations and abbreviations of major terms

[0044] Table 1. Main Rankings and English Abbreviations

[0045]

[0046] II. Materials and Methods

[0047] 1. Human aortic valve collection

[0048] All aortic valves used in this study were obtained from the Second Affiliated Hospital of Zhejiang University School of Medicine. Patients were divided into a CAVD group and a non-CAVD group based on clinical presentation, echocardiography, and CT scans. The inclusion criteria for the CAVD group were as follows: echocardiography showing degenerative aortic valve calcification with severe stenosis, and postoperative pathological examination revealing significant calcification nodules in the valve. Patients with bicuspid aortic valve, rheumatic valvular heart disease, aortic valve myxoid degeneration, or infective endocarditis were excluded. The aortic valves in the non-CAVD group were all obtained from heart transplant patients, healthy heart donors, and patients who underwent aortic aneurysm surgery. This study was approved by the ethics committee, and informed consent was obtained from each participant.

[0049] 2. Isolation and culture of VICs

[0050] The isolation process for human aortic valve clefts (VICs) was as follows: The valve leaflets were rinsed with sterile phosphate-buffered saline (PBS) and then digested with type II collagenase (2 mg / ml, Sigma-Aldrich, USA) at 37°C for 40 minutes. The aortic valve leaflets were wiped with sterile cotton swabs to remove the endothelial cell layer. Subsequently, the leaflets were digested again with 2 mg / ml type II collagenase at 37°C for 6 hours. After digestion was completed, the cell suspension was collected and plated. When the cells grew to 80%-90% of the culture area, they were passaged at a 1:2 ratio. The isolated cells were stained with α-smooth muscle actin (α-SMA) and vascular endothelial cadherin (VE-Cadherin). Cells that were α-SMA positive and VE-Cadherin negative were identified as VICs by fluorescence microscopy (see [link to relevant documentation]). Figure 1 ).

[0051] Isolated VICs were cultured in high-glucose Dalberg modified Eagle medium (DMEM, Gibco, Thermo Fisher Scientific, USA) containing 10% fetal bovine serum (FBS, Gibco, Thermo Fisher Scientific, USA) and 1% penicillin / streptomycin (Gibco, Thermo Fisher Scientific, USA). Osteogenic differentiation of VICs was induced using osteogenic medium (OM, containing 0.25 mM ascorbic acid, 100 nM dexamethasone, and 10 mM β-glycerophosphate). All VICs used in this study were from generations 3-7.

[0052] 3. Cell siRNA transfection experiment

[0053] VICs were pre-seeded in culture plates, and transfection was performed when the cell confluence reached approximately 60%-70%. siRNAs targeting PARM1 (si-PARM1) and the negative control (si-Scr) were purchased from Qingke Biotechnology Co., Ltd. (Beijing, China). siRNA (50 nM) and Lipofectamine RNAiMAX transfection reagent (Thermo Fisher Scientific, USA) were added to a certain amount of Opti-MEM dilution buffer (Gibco, Thermo Fisher Scientific, USA). The small interfering RNA (siRNA) sequences are shown in Table 2. After thorough mixing, the transfection mixture was added to the cells, and cell status was monitored 6 hours post-transfection. Knockdown efficiency was assessed 24 hours after siRNA transfection.

[0054] Table 2 Small interfering RNA (siRNA) sequences

[0055]

[0056] 4. Cell Lentiviral Infection Experiment

[0057] Lentiviral viruses overexpressing human PARM1 (oe-PARM1) and empty vectors were purchased from Qingke Biotechnology Co., Ltd. (Beijing, China). VICs were placed in culture plates and then infected with the lentivirus at a multiplicity of infection (MOI) of 50 for 6 hours. Infection efficiency was assessed 72 hours after infection based on the expression of green fluorescent protein (GFP) under a fluorescence microscope, and its expression was verified by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot.

[0058] 5. RNA extraction and qRT-PCR experiments

[0059] use Total RNA was extracted using reagents (Invitrogen, Carlsbad, USA). RNA concentration and quality were measured using a NanoDrop ND-1000 (Wilmington, USA). RNA reverse transcription was performed using the PrimeScript RT kit (TakaraBio, Kusatsu, Japan). qRT-PCR was performed on a LightCycler 480II (Roche, Switzerland) using the SYBR GreenqPCR SuperMix kit (TakaraBio, Kusatsu, Japan). Primers are listed in Table 3. Two primers were used. -ΔΔCt Data analysis was performed using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control.

[0060] Table 3 Primer sequences for real-time quantitative polymerase chain reaction (qRT-PCR)

[0061]

[0062] 6. RNA sequencing experiment

[0063] The quality, purity, and integrity of the isolated total RNA were assessed. Qualified RNA samples were sent to the DNBSEQ-T7 platform (Shanghai Zhongke New Life Biotechnology Co., Ltd.) for RNA sequencing. Clean reads for subsequent analysis were obtained by removing low-quality reads from the raw data. Differentially expressed genes (DEGs) were identified using R Project (v3.5.1). Based on the differentially expressed genes, gene ontology (GO), gene set enrichment analysis (GSEA), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed.

[0064] 7. Western blot

[0065] Proteins were isolated from tissues and cells using RIPA buffer (Beyotime, Shanghai, China) containing protease and phosphatase inhibitors (Foder Biotech, Hangzhou, China). Protein concentrations were determined using a BCA protein quantification kit (Foder Biotech, Hangzhou, China). Equal volumes of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes (MilliporeSigma, Burlington, USA). After blocking the PVDF membranes with 5% skim milk, they were treated with the appropriate primary antibody and then with the appropriate horseradish peroxidase (HRP)-labeled secondary antibody. Finally, signal detection was performed using an enhanced chemiluminescence reagent (Novizan, Nanjing, China) and an ImageQuant 600 system (Amacia, GE Healthcare). Signal intensity was analyzed using ImageLab software (Bio-Rad Laboratories, California, USA). Primary antibody information is shown in Table 4.

[0066] Table 4. Primary antibody sources and related information

[0067]

[0068]

[0069]

[0070] 8. Immunofluorescence staining experiment

[0071] Immunofluorescence staining is primarily used to detect protein expression in the aortic valves of humans and mice. After fixation in 4% paraformaldehyde for 15 minutes, frozen sections of the aortic valve were permeabilized with 0.1% Triton X-100 for 10 minutes, followed by blocking with 5% bovine serum albumin (BSA) for 30 minutes. These sections were then incubated with primary antibody followed by fluorescently labeled secondary antibody. Nuclear counterstaining was performed using DAPI (Vector Laboratories, Burlingame, USA). Images were acquired using a Leica fluorescence microscope (Leica, Wetzlar, Germany) and analyzed using ImageJ software.

[0072] 9. ALP activity assay

[0073] VICs induced by osteogenic differentiation medium were lysed using RIPA lysis buffer (Beyotime, Shanghai, China) without phosphatase and protease inhibitors. ALP activity was detected according to the protocol of the ALP activity assay kit (Beyotime, Shanghai, China). An equal volume of substrate solution was mixed with 50 μL of protein lysis buffer and incubated at 37 °C for 10 min. After adding stop solution, the optical density (OD) value was read at 405 nm on a spectrophotometer. ALP activity for each group was determined using a standard curve and normalized according to protein concentration.

[0074] 10. Calcium content determination experiment

[0075] After culturing in osteogenic differentiation medium for 14 days, VICs were washed with PBS and incubated with dilute hydrochloric acid at 4°C for 24 hours. The supernatant was collected and centrifuged at 2000g for 5 minutes. The calcium content of each sample was measured using a calcium assay kit from Nanjing Jiancheng Biotechnology Institute, and normalized according to protein concentration.

[0076] 11. Alizarin Red Staining Experiment

[0077] Frozen sections of the aortic valve were fixed with 4% paraformaldehyde and washed three times with PBS. The sections were stained with 1% Alizarin Red (Sigma, Darmstadt, Germany) for 30 minutes. Images were acquired under a stereomicroscope (Leica, Wetzlar, Germany). After incubating VICs in osteogenic differentiation medium for 21 days, they were washed with PBS and fixed in 4% paraformaldehyde for 10 minutes. They were stained with 1% Alizarin Red for 10 minutes and then washed three times with PBS. Calcified areas were assessed and visualized. To quantify calcification, Alizarin Red-stained calcium nodules were dissolved in 10% acetic acid at room temperature for 30 minutes, and absorbance was read at 405 nm.

[0078] 12. Von Cusár staining experiment

[0079] This study used the Von Cusas staining kit (Abcam, Cambridge, UK) to detect calcified nodules. Aortic valve sections were fixed with 4% paraformaldehyde for 10 minutes. 5% silver nitrate solution was added, and the sections were irradiated under UV light for 45 minutes. After discarding the silver nitrate solution, 5% sodium thiosulfate solution was added and reacted for 2 minutes. These sections were then stained with nucleoside red for 1 minute. Calcified areas were assessed, and images were acquired under a microscope (Leica, Wetzlar, Germany).

[0080] 13. Mouse feeding and intervention methods

[0081] All animal procedures were performed strictly in accordance with the standards established by the Animal Experiment Center of Zhejiang University. Mice were housed in a specific pathogen-free (SPF) environment with a temperature of 22-24℃ and a light-dark cycle of 12h:12h. Four-week-old male low-density lipoprotein receptor gene knockout (Ldlr) was also performed. - / - Mice were purchased from Jicui Pharmaceutical (Nanjing, China). After acclimatization, AAV2-sh-PARM1 or AAV2-sh-control was injected via tail vein.

[0082] 14. Construction process of a high-fat diet-induced aortic valve calcification mouse model

[0083] Mice were fed a high-fat diet (SYSE Bio-tech, Changzhou, China) starting at 8 weeks of age and continued for 24 weeks. After successful modeling, mice were euthanized by intraperitoneal injection of an excessive amount of sodium pentobarbital (0.2 g / kg). Plasma was collected for subsequent testing of total cholesterol, LDL cholesterol, triglycerides, and blood glucose. Mouse organs were also collected for further analysis.

[0084] 15. Echocardiography Examination Methods

[0085] Cardiac and aortic valve function were measured under 2.5% isoflurane anesthesia using a VisualSonics Vevo 3100 ultrasound system (Toronto, Canada). Peak aortic flow velocity, mean transvalvular pressure gradient, left ventricular fractional shortening (LVFS), and left ventricular ejection fraction (LVEF) were assessed in each group of mice when their heart rate stabilized at approximately 400–500 beats / minute. All ultrasound data were acquired by the same animal sonographer.

[0086] 16. Histological analysis methods

[0087] After anesthesia, mice in each group were sacrificed by cervical dislocation. The mouse hearts were repeatedly irrigated with PBS, placed in a 30% sucrose solution for 24 hours, and then embedded using OCT (Sakura Seiki, Japan). Sections were prepared perpendicular to the aorta; the optimal section allowed simultaneous observation of all three aortic valve leaflets.

[0088] 17. Special staining to assess leaflet fibrosis and calcification.

[0089] Leaflet thickness was assessed using H&E staining, represented as the average width of the thickest of the three leaflets. Collagen fibers were visualized using Sirius red and Masson staining. Sirius red dye binds to collagen fibers, appearing yellow to orange under polarized light. Masson staining is a method used to stain collagen and muscle fibers, where muscle fibers appear red and collagen fibers appear green or blue. As previously described, aortic valve calcification was assessed in each group of mice using von Cussar staining.

[0090] 18. Statistical Analysis

[0091] Data are expressed as mean ± standard deviation (SD). The normality of the variables was assessed using the Shapiro-Wilke normality test. For normally distributed data, t-tests were used for comparisons between groups. One-way ANOVA was used for comparisons involving ≥3 groups. For non-normally distributed data, a nonparametric rank-sum test was used, where a p-value less than 0.05 was considered statistically significant.

[0092] Example 1

[0093] This embodiment demonstrates that PARM1 expression is significantly upregulated in patients with calcific aortic valve disease.

[0094] This embodiment included 15 patients with calcific aortic valve disease (CAVD) and 15 patients without CAVD. The clinical characteristics of the two groups of patients are listed in Table 5.

[0095] The aortic valve area was significantly decreased, and the peak transvalvular pressure gradient and peak aortic valve velocity were significantly increased in the CAVD group. There were no statistically significant differences between the two groups in terms of age, sex, history of hypertension, diabetes, body mass index (BMI), smoking, coronary artery disease, left ventricular ejection fraction (LVEF), total cholesterol, triglycerides, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol. Furthermore, hematoxylin-eosin (H&E), Masson's staining, and alizarin red staining were performed to verify the pathological features of the aortic valve in the control and CAVD groups. Figure 2 A). The results showed that the calcified aortic valve in the CAVD group was significantly thickened, and the degree of fibrosis and calcium nodule deposition was significantly increased.

[0096] Table 5 Clinical characteristics of patients in the control group and the calcific aortic valve disease (CAVD) group.

[0097]

[0098]

[0099] Subsequently, Western blot was used to identify PARM1 expression in the aortic valve. Compared to the normal aortic valve, PARM1 expression was significantly increased in the calcified aortic valve. Figure 2 B). Valvular interstitial cells (VICs) are the most critical pathogenic cells in aortic valve calcification; therefore, we isolated VICs from non-calcified and calcified valves to detect PARM1 expression. Clearly, PARM1 expression was significantly increased in VICs isolated from calcified aortic valves. Figure 2 C).

[0100] Immunofluorescence staining showed that PARM1 co-localized with vimentin, a marker of VICs in calcified aortic valves. Figure 2 D). As Figure 2 As shown in Figure E, PARM1 was significantly increased in VICs treated with osteogenic differentiation medium (OM). Furthermore, low-density lipoprotein receptor gene knockout (Ldlr) was significantly increased in VICs fed a normal diet (ND) and a high-fat diet (HFD). - / - Immunofluorescence examination of the aortic valves of mice in the HFD group showed that PARM1 expression was higher in the aortic valves of the mice in the HFD group. Figure 2 F).

[0101] In summary, PARM1 is significantly upregulated in CAVD, suggesting that PARM1 plays a key role in regulating the progression of CAVD.

[0102] Example 2

[0103] This embodiment investigates the role of PARM1 in regulating the osteogenic differentiation process of VICs in vitro.

[0104] To investigate the role of PARM1 in osteogenic differentiation of VICs, PARM1 was knocked down using small interfering RNA (siRNA). Figure 3 A, 3B). VICs transfected with si-PARM1 or control (si-Scr) were exposed to OM to induce osteogenic differentiation. Silencing PARM1 reduced the expression of osteogenic markers such as alkaline phosphatase (ALP) and Runx-associated transcription factor 2 (RUNX2) in OM-cultured VICs. Figure 4 A, 4B). Furthermore, PARM1 knockdown significantly alleviated the OM-induced enhancement of ALP activity ( Figure 4 C). Consistent with the results of osteogenic marker protein expression, calcium content assays further confirmed that PARM1 knockdown inhibited extracellular calcium deposition in VICs. Figure 4 D). Furthermore, alizarin red staining confirmed that PARM1 knockdown significantly reduced extracellular calcium nodule formation 21 days after OM induction. Figure 4 These results indicate that PARM1 plays an important regulatory role in osteogenic differentiation and extracellular calcium deposition in VICs.

[0105] To further elucidate the pro-calcification role of PARM1, lentivirus was used to infect VICs to achieve stable overexpression of PARM1. Figure 3 C, 3D). With PARM1 overexpression, ALP and RUNX2 expression further increased on the basis of OM induction. Figure 4 G, 4H). Furthermore, PARM1 overexpression further increased ALP activity after OM induction (G, 4H). Figure 4 I). Alizarin red staining showed that PARM1 overexpression further exacerbated OM-induced calcium nodule deposition ( Figure 4 In summary, based on OM induction, PARM1 overexpression can further promote osteogenic differentiation and extracellular calcium deposition in VICs.

[0106] Example 3

[0107] This embodiment validates that knocking down the PARM1 gene expression level can reduce the aortic valve calcification level induced by a high-fat diet in mice.

[0108] To further explore the role of PARM1 in high-fat diet-induced aortic valve calcification in vivo, PARM1 was knocked down by tail vein injection of AAV2-sh-PARM1. This was performed on 4-week-old Ldlr pups. - / - Mice were divided into three groups: ND+sh-control group, HFD+sh-control group, and HFD+sh-PARM1 group. Figure 5 After 24 weeks of feeding, echocardiography was performed to assess aortic valve function and cardiac function, and then the mice were sacrificed to collect blood samples and valve tissue.

[0109] like Figure 6 As shown in Figure A, compared with the HFD+sh-control group, the expression of PARM1 in the aortic valve was significantly reduced in the HFD+sh-PARM1 group, confirming the knockdown efficiency of PARM1. Furthermore, the HFD+sh-control group showed significantly increased body weight, total cholesterol, triglycerides, low-density lipoprotein, and blood glucose, while PARM1 knockdown had no effect on these indicators. Figure 6 B- Figure 6 F).

[0110] Echocardiographic results showed that, compared with the ND+sh-control group, the peak transvalvular velocity and mean aortic valve pressure gradient were significantly increased in the HFD+sh-control group, indicating that the aortic valve calcification model was successfully established. In contrast, the peak aortic valve velocity and mean aortic valve pressure gradient in the HFD+sh-PARM1 group were significantly lower than those in the HFD+sh-control group. Figure 7 A, 7B). These results indicate that PARM1 knockdown alleviates aortic stenosis induced by hyperlipidemia in mice. Furthermore, LVEF and LVFS levels were similar across the three groups, suggesting that hyperlipidemia and PARM1 knockdown do not affect cardiac function in mice. Figure 7 (G, 7H). This means that the improved transvalvular pressure gradient and flow rate are not caused by changes in cardiac function.

[0111] Further evaluation of aortic valve leaflet morphology, fibrosis, and calcification was conducted. H&E staining revealed aortic valve leaflet thickening in the HFD+sh-control group compared to the ND+sh-control group. However, PARM1 knockdown alleviated the aortic valve leaflet thickening. Figure 7 C Figure 7 D). Valvular fibrosis was assessed using Sirius red and Masson staining. Compared to the HFD+sh-control group, the degree of valvular fibrosis was reduced in the HFD+sh-PARM1 group. Figure 7 C, Figure 7 E, Figure 7 F). Von Cussa staining showed that calcification was significantly higher in the HFD+sh-control group than in the ND+sh-control group, while the degree of calcification was lower in the HFD+sh-PARM1 group than in the HFD+sh-control group. Figure 7 C, Figure 7 G). Immunofluorescence staining showed that the levels of ALP and bone morphogenetic protein 2 (BMP2) in the aortic valve of PARM1 knockdown mice were significantly lower than those in the HFD+sh-control group, indicating reduced osteogenic differentiation of VICs. Figure 7 In summary, PARM1 knockdown can improve high-fat diet-induced aortic valve calcification in vivo.

[0112] Example 4

[0113] This embodiment verifies that PARM1 regulates the osteogenic differentiation level of VICs through the MAPK signaling pathway.

[0114] To further investigate downstream mechanisms, RNA sequencing (RNA-seq) analysis was performed on valvular interstitial cells (VICs) with and without PARM1 knockdown. A total of 1103 differentially expressed genes (DEGs) were identified, of which 450 were upregulated and 653 were downregulated. Gene ontology (GO) annotation and enrichment analysis indicated that PARM1 may be involved in the biological processes of the ERK1 and ERK2 cascade. Figure 8 A). Kyoto Encyclopedia of Genetics and Genomes (KEGG) pathway analysis showed that DEGs after PARM1 knockdown were mainly enriched in the mitogen-activated protein kinase (MAPK) signaling pathway. Figure 8 B). Furthermore, gene set enrichment analysis (GSEA) indicated that the MAPK signaling pathway plays a crucial role in the PARM1-mediated effects. Figure 8 C). The above bioinformatics analysis suggests that PARM1 may regulate osteogenic differentiation of VICs through the MAPK signaling pathway. Then, Western blot analysis confirmed that PARM1 knockdown significantly reduced phosphorylation levels of p38 and ERK1 / 2. Figure 8 (D) This indicates that PARM1 plays an important role in mediating the P38 and ERK1 / 2 signaling pathways. To further verify the role of the PARM1-MAPK signaling pathway in CAVD, immunofluorescence staining showed that OM intervention induced an increase in phosphorylated P38 and ERK1 / 2, while PARM1 knockdown attenuated this effect. Figure 9(A, 9B). Therefore, PARM1 may participate in regulating osteogenic differentiation of VICs by modulating the ERK1 / 2 and P38 signaling pathways.

[0115] Subsequently, this example further investigated the function of MAPK activity in the osteogenic process of VICs. The activities of P38 and ERK1 / 2 were inhibited using SB202190 (a P38 phosphorylation inhibitor) and PD0325901 (an ERK1 / 2 phosphorylation inhibitor), respectively. Under osteogenic medium (OM) induction, compared with the PARM1 overexpression group, the mRNA and protein expression of osteogenic markers such as ALP and RUNX2 were significantly reduced in the SB202190 and PD0325901 inhibitor groups. Figure 8 E). Similarly, the increase in ALP activity induced by PARM1 overexpression was inhibited by p38 inhibitors and ERK inhibitors (E). Figure 8 F). Furthermore, alizarin red staining confirmed that blocking the MAPK signaling pathway can counteract the pro-calcification effect of PARM1 in vitro. Figure 8 G).

[0116] In this invention, PARM1 was found to be significantly elevated in the aortic valve of patients with CAVD for the first time. Furthermore, osteogenic differentiation-induced VICs also highly express PARM1. Knockdown of PARM1 expression inhibited osteogenic differentiation and extracellular calcium deposition in VICs, while PARM1 overexpression enhanced these processes. A mouse model of aortic valve calcification was constructed to demonstrate that PARM1 silencing significantly reduced aortic stenosis and calcification. Mechanistically, PARM1 may regulate osteogenic differentiation of VICs by modulating phosphorylation of the MAPK signaling pathway. In summary, PARM1 regulates osteogenic differentiation of VICs in CAVD through the MAPK signaling pathway, providing a potential target for the prevention and treatment of CAVD.

[0117] Those skilled in the art will understand that, for the sake of convenience and brevity, the specific working process and related descriptions of the system described above can be found in the corresponding processes in the foregoing method embodiments, and will not be repeated here.

[0118] Those skilled in the art will understand that, for the sake of convenience and brevity, the specific working process and related descriptions of the storage device and processing device described above can be referred to the corresponding processes in the foregoing method embodiments, and will not be repeated here.

[0119] The term "comprising" or any other similar term is intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus / device that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent in such process, method, article, or apparatus / device.

[0120] The technical solution of the present invention has been described above with reference to the preferred embodiments shown in the accompanying drawings. However, it will be readily understood by those skilled in the art that the scope of protection of the present invention is obviously not limited to these specific embodiments. Without departing from the principles of the present invention, those skilled in the art can make equivalent changes or substitutions to the relevant technical features, and the technical solutions after these changes or substitutions will all fall within the scope of protection of the present invention.

Claims

1. The use of a PARM1 inhibitor in the preparation of a medicament for the prevention or treatment of calcific aortic valve disease, characterized in that, The calcific aortic valve disease includes calcific aortic stenosis, calcific aortic regurgitation, or calcific aortic valve lesions; the PARM1 inhibitor is a small interfering RNA (siRNA), and the sense strand of the nucleotide sequence of the siRNA is SEQ ID NO.1, and the antisense strand is SEQ ID NO.2; SEQ ID NO.1: 5'- CUGUGUCAGGCAAAGUGAUTT -3'; SEQ ID NO. 2: 5'-AUCACUUUGCCUGACACAGTT-3'.

2. The application according to claim 1, characterized in that, The PARM1 inhibitors suppress osteogenic differentiation of valvular interstitial cells (VICs) by inhibiting the expression levels of the PARM1 nucleotide sequence or the PARM1 protein.

3. A drug for the prevention or treatment of calcific aortic valve disease, characterized in that, The drug comprises the PARM1 inhibitor as described in claim 1, and an excipient, wherein the calcified aortic valve disease is a calcified lesion of the heart valve; the PARM1 inhibitor is a small interfering RNA (siRNA) targeting the mRNA of PARM1, the sense strand nucleotide sequence of the small interfering RNA (siRNA) is shown in SEQ ID NO.1, and the antisense strand nucleotide sequence of the siRNA is shown in SEQ ID NO.

2.

4. The drug according to claim 3, characterized in that, The drug also contains a pharmaceutically acceptable diluent.

5. The drug according to claim 3, characterized in that, The dosage forms of the drug include, but are not limited to, oral liquids, injections, tablets, pills, dispersants, capsules, granules, suspensions, and emulsions.

6. The drug according to claim 3, characterized in that, The drug is in the form of drop pills.

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