Drug for treating calcified aortic valve disease based on PARM1 regulation and application thereof

Abstract

The invention relates to a medicine for treating calcified aortic valve diseases based on PARM1 regulation and application thereof. It is found for the first time that PARM1 is remarkably increased in the aortic valve of a patient suffering from calcified aortic valve diseases (CAVD) and plays a key role in the osteogenic differentiation process of valvular interstitial cells (VICs), the osteogenic differentiation and extracellular calcium salt deposition of the VICs can be inhibited by knocking down the expression level of PARM1, aortic valve calcification is relieved, and the action mechanism of PARM1 relates to an MAPK signal channel. The invention provides a new target spot and a treatment thought for the treatment of CAVD, and can be applied to research and development of related medicines and development of treatment methods.

Description

Background Art

[0002] Calcific aortic valve disease (CAVD) is a degenerative disease with high morbidity and mortality in the elderly. The main pathophysiological change is aortic valve calcification, which leads to hemodynamic changes and affects cardiac function. In the past, CAVD was considered to be a degenerative disease associated with aging, which is a process of degeneration and hardening of valve tissue with aging. However, basic research in recent years has shown that CAVD is an active progression 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 the valve tissue mainly include valvular endothelial cells (VECs), valvular interstitial cells, and immune cells. Studies have confirmed that increased calcium salt deposition caused by osteoblastic differentiation of VICs is an important factor in the occurrence and development of valve calcification. At present, there is a lack of effective clinical drug treatment for CAVD, and its main treatment is aortic valve replacement surgery. However, patients who undergo surgical treatment will inevitably bear higher medical risks and economic burdens. Therefore, exploring the specific pathogenesis of CAVD and using non-surgical methods to effectively prevent and / or treat aortic valve calcification are urgent needs in the current clinical treatment of CAVD.

[0003] At present, the treatment of calcific aortic valve disease still has certain challenges, especially in early prevention and treatment. In clinical practice, some drugs have been used to treat this disease, but these drugs still have some defects and shortcomings. Commonly used drugs are lipid-lowering drugs, such as statins. These drugs slow down the process of aortic valve calcification by lowering cholesterol levels, thereby improving the function of the aortic valve to a certain extent. However, current clinical studies on lipid-lowering drugs in aortic valve calcification have confirmed that they cannot prevent or reverse calcification that has already occurred. In addition, long-term use of lipid-lowering drugs may have some adverse reactions, such as muscle pain and muscle damage, elevated liver enzymes or other liver function abnormalities, gastrointestinal discomfort, etc., which require close monitoring. Another class of drugs is anti-calcification drugs, such as antioxidants and anti-inflammatory drugs. The mechanism of action of these drugs is mainly to reduce oxidative stress and inflammatory response, thereby slowing down or inhibiting the process of arterial calcification. However, there is currently a lack of sufficient clinical trial evidence to support the effectiveness 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, there are still many challenges, such as: the pathogenesis of calcific aortic valve disease is not fully understood, resulting in insufficient targeted treatment strategies; 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 originally 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 was significantly upregulated in VICs induced by osteogenic differentiation. However, the function and potential mechanism of PARM1 in osteogenic differentiation and CAVD of VICs have not been reported. Therefore, it is of great significance to comprehensively study the role of PARM1 in aortic valve calcification. Summary of the invention

[0006] The present invention studies the expression of PARM1 (PARM1 gene ID number 25849, and its corresponding human genome reference sequence number is NC_000004.12.) in calcified aortic valves and its regulatory effect on the osteogenic differentiation of human aortic valve interstitial cells, proposes a drug for treating aortic valve calcification based on PARM1 gene regulation and its application, and provides a new non-surgical treatment plan for treating CAVD, that is, by inhibiting the expression of PARM1, reducing the osteogenic differentiation of valve interstitial cells, alleviating aortic valve calcification, thereby treating calcific aortic valve disease.

[0007] The first object of the present invention is to provide a use of a PARM1 inhibitor in preventing or treating calcific aortic valve disease.

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

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

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

[0011] Furthermore, the PARM1 inhibitor is a siRNA for silencing the expression of the PARM1 gene, the nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO.1, and the nucleotide sequence of the antisense strand 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] The second object of the present invention is to provide a drug for preventing or treating calcific aortic valve disease.

[0015] Furthermore, the drug comprises the above-mentioned PARM1 inhibitor, and a pharmaceutically acceptable carrier, diluent or excipient, and the calcific aortic valve disease is a calcified lesion of the heart valve.

[0016] Furthermore, the PARM1 inhibitor is a small interfering RNA (siRNA) targeting PARM1 mRNA, the positive 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 form of the drug includes but is not limited to oral solution, injection, tablet, pill, dispersion, capsule, pellet, granule, suspension and emulsion.

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

[0019] The medicine of an exemplary embodiment of the present invention may further include a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier may include saline solution, sterile water, Ringer's solution, buffered saline solution, glucose solution, maltodextrin solution, glycerol, ethanol, and a mixture of two or more thereof, which may be used alone or in combination. When necessary, other conventional additives such as antioxidants, buffers and antibacterial agents may be added to the medicine. In addition, diluents, dispersants, surfactants, adhesives and lubricants may be further added to the medicine. In this case, the medicine may be formulated into an injectable preparation such as an aqueous solution, a suspension or an emulsion, a pill, a capsule, a granule or a tablet. In addition, the medicine may preferably be formulated according to individual diseases and components using a suitable method known in the relevant art or a method disclosed in Remington's Pharmaceutical Science (latest edition), Mack Publishing Company, Easton Pa. Examples of pharmaceutically acceptable carriers, preparations of pharmaceutical compositions and methods for preparing preparations are known in the relevant art.

[0020] Further, the drug may be a composition for administering to a subject a composition comprising the PARM1 inhibitor of the present invention as an active ingredient, thereby treating aortic valve calcification, and the composition may be provided in a subject in need thereof in an effective amount for treating aortic valve calcification, wherein the dosage of the PARM1 inhibitor may vary to a great extent according to the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of the disease. In this case, the appropriate dosage of the PARM1 inhibitor may be optionally determined by a technician in the relevant field.

[0021] Further, the drug can be administered orally or parenterally (eg, intravenously, subcutaneously, intraperitoneally, intramuscularly, intravascularly or subcutaneously) according to the desired purpose.

[0022] Preferably, the drug can be administered orally. For oral administration, the drug can be provided in the form of capsules, tablets, powders, granules or suspensions; and the drug can include conventional additives such as lactose, mannitol, corn starch or potato starch, and binders that can be used for the drug can include crystalline cellulose, cellulose analogs, gum arabic, corn starch or sodium carboxymethyl cellulose.

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

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

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

[0026] A third object of the present invention is to provide a method for treating calcific aortic valve disease.

[0027] Furthermore, the method comprises administering the above-mentioned PARM1 inhibitor or the above-mentioned drug to the patient.

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

[0029] In some embodiments, the methods can prevent or treat calcific aortic valve disease in in vitro cells, or 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) The present invention detects the expression of PARM1 gene in human calcified aortic valve tissue and proves that its expression level is significantly higher than that in normal aortic valve tissue, suggesting that PARM1 can be used as a diagnostic marker for calcified aortic valve disease;

[0032] (2) The present 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 is verified by in vitro and in vivo experiments, which provides a theoretical basis and experimental basis for the development of new CAVD therapeutic drugs and methods, and also provides a new target and therapeutic idea for the treatment of calcific aortic valve disease, which helps to solve the problem of limited current CAVD treatment methods. That is, by inhibiting the expression of PARM1, the osteogenic differentiation of VICs can be effectively reduced, the progression of CAVD can be delayed or reversed, and the prognosis of patients can be improved. Therefore, the research results of the present invention lay a foundation for the subsequent development of therapeutic drugs and treatment methods for PARM1, and have broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0034] Figure 2 The expression level of PARM1 in calcified aortic valves: (A) Representative hematoxylin-eosin (H&E), Masson's staining, and von Kossa staining images of non-calcified human aortic valves and calcified aortic valves. (B) Protein immunoblotting (Western blot) was used to determine the expression of PARM1 in aortic valves of patients with CAVD and non-CAVD. (C) The expression of PARM1 was detected in VICs isolated from non-calcified aortic valves and calcified aortic valves. (D) Representative immunofluorescence images of PARM1 localization in VICs in human aortic valve tissue (scale bar: 25 μm). (E) PARM1 protein levels in VICs stimulated with osteogenic medium were determined by Western blot. (F) Low-density lipoprotein receptor gene knockout (Ldlr) fed normal diet (ND) and high-fat diet (HFD) - / - ) Representative immunofluorescence images of PARM1 in mouse aortic valve (scale bar: 25 μm), where values ​​are mean ± SD.

[0035] Figure 3 The following are the analysis diagrams of siRNA and lentivirus intervention efficiency, where (A, B) the silencing efficiency of si-PARM1 was detected by real-time quantitative polymerase chain reaction (qRT-PCR) and protein immunoblot Western blot. (C, D) the overexpression efficiency of oe-PARM1 lentivirus was detected by qRT-PCR and Western blot. Figure 4Figure 1 shows 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-related transcription factor 2 (RUNX2)) were evaluated by real-time quantitative polymerase chain reaction (qRT-PCR) (A) and protein immunoblotting (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 VICs of each group was measured after 14 days of osteogenic medium intervention. (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 evaluated by qRT-PCR (G) and Western blot (H). (I) ALP activity was detected after overexpression of PARM1 in VICs. (J) Calcium deposition in PARM1-overexpressing VICs was detected by Alizarin red staining. Values ​​are mean ± SD.

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

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

[0038] Figure 7 Improvement of Ldlr for PARM1 knockdown - / -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 flow velocity and mean transvalvular pressure gradient in ND+sh-control, HFD+sh-control, and HFD+sh-PARM1 mice. (C) Representative images of H&E, Sirius Red, Masson, and von Koosa staining of aortic valves 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 area, fibrosis area, and total calcification area. (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 ± SD.

[0039] Figure 8 To prove that the MAPK signaling pathway is a downstream pathway of osteogenic differentiation of VICs regulated by PARM1: (A) Gene ontology (GO) analysis of RNA-seq of si-Scr and si-PARM1 groups. (B) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of differentially expressed genes between si-Scr and si-PARM1 groups. (C) Gene set enrichment analysis (GSEA) of differentially expressed genes between si-Scr and si-PARM1 groups. (D) Phosphorylation levels of the MAPK signaling pathway after knockdown of PARM1 were analyzed by Western blot. (E) Protein levels of ALP and RUNX2 were evaluated by Western blot after treatment with P38 inhibitor and ERK inhibitor in VICs. (F) ALP activity was detected after treatment with P38 inhibitor and ERK inhibitor in VICs. (G) Calcium deposition after treatment with P38 inhibitor and ERK inhibitor was detected by Alizarin red staining. Values ​​are mean ± SD.

[0040] Fig. 9 Immunofluorescence staining of p-ERK1 / 2 (A) and p-P38 (B) in VICs under different treatment conditions (scale bar: 100 μm). DETAILED DESCRIPTION

[0041] In order to make the purpose, technical solution and advantages of the present invention more clear, the present invention is further described in detail below. However, it should be understood that the description herein is only used to explain the present invention and is not used to limit the scope of the present invention.

[0042] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention, and the terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The reagents and instruments used herein are all commercially available, and the characterization means involved can refer to the relevant descriptions in the prior art, which will not be repeated herein.

[0043] The English and abbreviations of the main nouns

[0044] Table 1 Main rankings and English abbreviations

[0045]

[0046] 2. Materials and Methods

[0047] 1. Human Aortic Valve Collection

[0048] All aortic valves in this study were obtained from the Second Affiliated Hospital of Zhejiang University School of Medicine. Patients were divided into CAVD group and non-CAVD group according to clinical manifestations, cardiac ultrasound and CT examinations. The inclusion criteria for patients in the CAVD group were as follows: cardiac ultrasound showed degenerative aortic valve calcification with severe stenosis, and postoperative pathological examination revealed obvious calcium nodules in the valve. Bicuspid aortic valve, rheumatic valvular heart disease, myxoid degeneration of the aortic valve, and infective endocarditis were excluded. Aortic valves in the non-CAVD group were obtained from heart transplant patients, normal heart donors, and patients undergoing aortic aneurysm surgery. This study was approved by the Ethics Committee, and each participant provided informed consent.

[0049] 2. Isolation and Culture of VICs

[0050] The isolation process of human VICs is as follows: the leaflets are 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 are wiped with a sterile cotton swab to remove the endothelial cell layer. Subsequently, the leaflets are digested again with 2 mg / ml type II collagenase at 37°C for 6 hours. After the digestion is terminated, the cell suspension is collected for plating. When the cells grow to 80%-90% of the culture area, they are passaged at a ratio of 1:2. The isolated cells are stained with α-smooth muscle actin (α-SMA) and vascular endothelial cadherin (VE-Cadherin), and the α-SMA-positive and VE-Cadherin-negative cells are identified as VICs by fluorescence microscopy (see Figure 1 ).

[0051] Isolated VICs were cultured in high-glucose Dulbecco's modified Eagle's 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). The VICs used in this study were from passages 3-7.

[0052] 3. Cell siRNA transfection experiment

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

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

[0055]

[0056] 4. Cell Lentivirus Infection Experiment

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

[0058] 5. RNA Extraction and qRT-PCR Experiment

[0059] use Total RNA was extracted using the ELISA kit (Invitrogen, Carlsbad, USA). The concentration and quality of RNA samples were measured using 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 480 II (Roche, Switzerland) using the SYBR Green qPCR SuperMix kit (TakaraBio, Kusatsu, Japan). Primers are shown in Table 3. 2 -ΔΔCt The data were analyzed by the PCR method, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal reference.

[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 evaluated. 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). Gene ontology (GO), gene set enrichment analysis (GSEA), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were completed based on differentially expressed genes.

[0064] 7.Western blot

[0065] Proteins were isolated from tissues and cells using RIPA buffer (Biyuntian, Shanghai, China) containing protease and phosphatase inhibitors (Fude Biotechnology, Hangzhou, China). Protein concentration was detected using a BCA protein quantification kit (Fude Biotechnology, Hangzhou, China). Equal amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes (MilliporeSigma, Burlington, USA). After blocking the PVDF membrane with 5% skim milk, it was treated with the corresponding primary antibody and then with the corresponding horseradish peroxidase (HRP)-labeled secondary antibody. Finally, enhanced chemiluminescence reagents (Novozyme, Nanjing, China) and ImageQuant 600 system (Amersham, GE Healthcare) were used for signal detection. Signal strength was analyzed using Image Lab software (BioRad, California, USA), and primary antibody information is shown in Table 4.

[0066] Table 4 Sources of primary antibodies and related information

[0067]

[0068]

[0069]

[0070] 8. Immunofluorescence staining experiment

[0071] Immunofluorescence staining was mainly used to detect protein expression in human and mouse aortic valves. After fixation in 4% paraformaldehyde for 15 minutes, aortic valve cryosections were permeabilized with 0.1% Triton X-100 for 10 minutes and then blocked with 5% bovine serum albumin (BSA) for 30 minutes. Subsequently, these sections were incubated with primary antibodies and then with fluorescently labeled secondary antibodies. Nuclei were counterstained with DAPI (Vector Laboratories, Burlingame, USA). Images were acquired using a Leica fluorescence microscope (Leica, Wetzlar, Germany) and analyzed on ImageJ software.

[0072] 9. ALP activity assay

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

[0074] 10. Calcium content determination experiment

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

[0076] 11. Alizarin red staining experiment

[0077] Aortic valve cryosections were fixed with 4% paraformaldehyde and washed three times with PBS. Sections were stained with 1% Alizarin Red stain (Sigma, Darmstadt, Germany) for 30 min. Images were acquired under a stereomicroscope (Leica, Wetzlar, Germany). After 21 days of incubation in osteogenic differentiation medium, VICs were washed with PBS and fixed in 4% paraformaldehyde for 10 min. They were stained with 1% Alizarin Red staining solution for 10 min and then washed three times with PBS. Calcification areas were evaluated and visualized. To quantify calcification, Alizarin Red-stained calcium nodules were dissolved with 10% acetic acid at room temperature for 30 min, and the absorbance was read at 405 nm.

[0078] 12. Von Koosa staining experiment

[0079] In this study, the von Kussa staining kit (Abcam, Cambridge, UK) was used to detect calcium nodules. Aortic valve sections were fixed with 4% paraformaldehyde for 10 min. A 5% silver nitrate solution was added and irradiated under UV light for 45 min. After discarding the silver nitrate solution, a 5% sodium thiosulfate solution was added and reacted for 2 min. These sections were stained with nuclear red staining solution for 1 min. The calcified areas were evaluated and images were acquired under a microscope (Leica, Wetzlar, Germany).

[0080] 13. Mouse husbandry and intervention methods

[0081] All animal operations were carried out in strict accordance with the standards set by the Animal Experiment Center of Zhejiang University. Mice were housed in a specific pathogen-free (SPF) environment with a temperature of 22-24°C and a light:dark cycle of 12h:12h. - / - ) mice were purchased from Jicui Yaokang (Nanjing, China). After acclimatization, AAV2-sh-PARM1 or AAV2-sh-control were injected via the tail vein.

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

[0083] A high-fat diet (SYSE Bio-tech, Changzhou, China) was started at 8 weeks of age and fed for 24 weeks. After successful modeling, mice were killed by intraperitoneal injection of an overdose of sodium pentobarbital (0.2 g / kg). Plasma was collected for subsequent tests of total cholesterol, low-density lipoprotein, triglycerides, blood glucose, etc. Mouse organs were collected for subsequent testing.

[0084] 15. Echocardiographic examination method

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

[0086] 16. Histological analysis methods

[0087] After anesthesia, mice in each group were killed by cervical dislocation. The mouse hearts were repeatedly perfused with PBS, placed in 30% sucrose solution for 24 hours, and then embedded in OCT (Sakura Seiki, Japan). The slices were cut perpendicular to the aorta, and the best slice was the one that could observe three aortic valve leaflets at the same time.

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

[0089] Leaflet thickness was assessed by H&E staining and was calculated 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 and appears 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. Aortic valve calcification was assessed in each group of mice using von Kossa staining as described previously.

[0090] 18. Statistical Analysis

[0091] Data are presented as mean ± standard deviation (SD). The normal distribution of variables was assessed using the Shapiro-Wilk normality test. For normally distributed data, t-test was used for comparisons between groups. One-way ANOVA was used for comparisons involving ≥3 groups. For non-normally distributed data, nonparametric rank sum test was used, with p values ​​less than 0.05 considered statistically significant.

[0092] Example 1

[0093] This example demonstrates that the expression level of PARM1 in patients with calcific aortic valve disease is significantly upregulated.

[0094] This example 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 aortic valve peak flow velocity were significantly increased in the CAVD group. There were no statistically significant differences between the two groups in age, sex, hypertension, history of 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. In addition, hematoxylin-eosin (H&E) staining, Masson staining, and Alizarin red staining were performed to verify the pathological characteristics of the aortic valve in the control group and the CAVD group ( 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 calcific aortic valve disease (CAVD) group

[0097]

[0098]

[0099] Subsequently, Western blot was used to identify the expression of PARM1 in the aortic valve. Compared with the normal aortic valve, the expression of PARM1 in the calcified aortic valve was significantly increased ( Figure 2 B). Valvular interstitial cells (VICs) are the most critical pathogenic cells in aortic valve calcification, so we isolated VICs from non-calcified and calcified valves to detect PARM1 expression. Obviously, 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) Figure 2 As shown in E, PARM1 was significantly increased in VICs treated with osteogenic differentiation medium (OM). In addition, the expression of Ldlr in VICs fed normal diet (ND) and high fat diet (HFD) was significantly increased. - / - ) mice were examined by immunofluorescence on the aortic valves, and the results showed that PARM1 expression was higher in the aortic valves of the HFD group mice ( Figure 2 F).

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

[0102] Example 2

[0103] This example studies the process of PARM1 regulating the osteogenic differentiation of VICs in vitro.

[0104] To investigate the role of PARM1 in osteogenic differentiation of VICs, PARM1 was knocked down by 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-related transcription factor 2 (RUNX2) in VICs cultured in OM ( Figure 4 In addition, after PARM1 knockdown, the OM-induced enhancement of ALP activity was significantly alleviated ( Figure 4 C). Consistent with the results of osteogenic marker protein expression, calcium content measurement further confirmed that PARM1 knockdown inhibited extracellular calcium deposition in VICs ( Figure 4 D). In addition, Alizarin red staining confirmed that PARM1 knockdown significantly reduced the formation of extracellular calcium nodules after 21 days of OM induction ( Figure 4 E, 4F). These results suggest that PARM1 plays an important regulatory role in the osteogenic differentiation and extracellular calcium deposition of VICs.

[0105] To further elucidate the calcification-promoting role of PARM1, VICs were infected with lentivirus to achieve stable overexpression of PARM1 ( Figure 3 C, 3D). With the overexpression of PARM1, the expression of ALP and RUNX2 was further increased on the basis of OM induction ( Figure 4 G, 4H). In addition, PARM1 overexpression further increased ALP activity after OM induction ( Figure 4 I). Alizarin red staining showed that PARM1 overexpression further aggravated OM-induced calcium nodule deposition ( Figure 4 J). In conclusion, PARM1 overexpression can further promote osteogenic differentiation and extracellular calcium deposition of VICs based on OM induction.

[0106] Example 3

[0107] This example verifies that knocking down the expression level of the PARM1 gene can reduce the level of aortic valve calcification 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. - / - The mice were divided into three groups, namely, ND+sh-control (sh-control), HFD+sh-control (sh-control), and HFD+sh-PARM1 ( Figure 5 After 24 weeks of feeding, echocardiograms were obtained to assess aortic valve function and heart function, and the mice were then sacrificed to collect blood samples and valve tissue.

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

[0110] The echocardiographic results showed that the peak transvalvular flow velocity and mean aortic valve pressure gradient in the HFD+sh-control group were significantly higher than those in the ND+sh-control group, indicating that the aortic valve calcification model was successfully established. In contrast, the peak transvalvular flow 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 suggest that PARM1 knockdown can alleviate hyperlipidemia-induced aortic stenosis in mice. In addition, the LVEF and LVFS levels of the three groups were similar, indicating that hyperlipidemia and PARM1 knockdown did not affect the cardiac function of 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] Aortic valve leaflet morphology, fibrosis, and calcification were further evaluated. H&E staining showed that the aortic valve leaflets in the HFD+sh-control (sh-control) group were thickened compared with the ND+sh-control (sh-control) group. However, PARM1 knockdown alleviated the thickening of the aortic valve leaflets ( Figure 7 C. Figure 7 D). Sirius red and Masson staining were used to evaluate the degree of valvular fibrosis. Compared with the HFD+sh-control group, the degree of valvular fibrosis in the HFD+sh-PARM1 group was reduced ( Figure 7 C, Figure 7 E, Figure 7 F). Von Kossa staining showed that the calcification deposition in the HFD+sh-control group was significantly higher than that in the ND+sh-control group, while the calcification degree in the HFD+sh-PARM1 group was lower than that 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 (sh-control), indicating that the osteogenic differentiation of VICs was reduced ( Figure 7 H). In conclusion, PARM1 knockdown can improve high-fat diet-induced aortic valve calcification in vivo.

[0112] Example 4

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

[0114] To further investigate the downstream mechanisms, RNA sequencing (RNA-seq) analysis was performed on valvular interstitial cells (VICs) with or 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 cascades ( Figure 8 A). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the DEGs after PARM1 knockdown were mainly enriched in the mitogen-activated protein kinase (MAPK) signaling pathway ( Figure 8 B). In addition, gene set enrichment analysis (GSEA) indicated that the MAPK signaling pathway played an important role in the PARM1-mediated effects ( Figure 8 C). The above bioinformatics analysis indicated that PARM1 may regulate the osteogenic differentiation of VICs through the MAPK signaling pathway. Then, protein immunoblotting (Western blot) analysis confirmed that PARM1 knockdown significantly reduced the phosphorylation levels of P38 and ERK1 / 2 ( Figure 8 D), which 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 ( Fig. 9A, 9B). Therefore, PARM1 may be involved in regulating the osteogenic differentiation of VICs by regulating the ERK1 / 2 and P38 signaling pathways.

[0115] Subsequently, this example further investigated the function of MAPK activity in the osteogenesis of VICs. SB202190 (a P38 phosphorylation inhibitor) and PD0325901 (an ERK1 / 2 phosphorylation inhibitor) were used to inhibit the activity of P38 and ERK1 / 2, respectively. Under osteogenic medium (OM) induction, the mRNA and protein expressions of osteogenic markers such as ALP and RUNX2 in the SB202190 and PD0325901 inhibitor groups were significantly reduced compared with the PARM1 overexpression group ( Figure 8 E). Similarly, the increase in ALP activity caused by PARM1 overexpression was suppressed by P38 inhibitor and ERK inhibitor ( Figure 8 F). In addition, Alizarin red staining confirmed that blocking the MAPK signaling pathway could counteract the calcification-promoting effect of PARM1 in vitro ( Figure 8 G).

[0116] In the present invention, it was found for the first time that PARM1 was significantly elevated in the aortic valve of CAVD patients. In addition, VICs induced by osteogenic differentiation also highly expressed PARM1. Knocking down the expression level of PARM1 inhibited the osteogenic differentiation and extracellular calcium deposition of VICs, while PARM1 overexpression enhanced these processes. An aortic valve calcification mouse model was constructed to demonstrate that PARM1 silencing can significantly reduce aortic valve stenosis and calcification. In terms of mechanism, PARM1 may regulate the osteogenic differentiation of VICs by regulating the phosphorylation of the MAPK signaling pathway. In summary, PARM1 regulates the osteogenic differentiation of VICs in CAVD through the MAPK signaling pathway, which provides a potential target for the prevention and treatment of CAVD.

[0117] Those skilled in the art can clearly understand that, for the convenience and brevity of description, the specific working process and related instructions of the system described above can refer to the corresponding process in the aforementioned method embodiment, and will not be repeated here.

[0118] Those skilled in the art can clearly understand that, for the convenience and brevity of description, the specific working process and related instructions of the storage device and processing device described above can refer to the corresponding process in the aforementioned method embodiment and will not be repeated here.

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

[0120] So far, the technical solutions of the present invention have been described in conjunction with the preferred embodiments shown in the accompanying drawings. However, it is easy for those skilled in the art to understand that the protection scope of the present invention is obviously not limited to these specific embodiments. Without departing from the principle 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 fall within the protection scope of the present invention.

Claims

1. A use of a PARM1 inhibitor in preventing or treating calcific aortic valve disease, characterized in that: The calcific aortic valve disease includes calcific aortic valve stenosis or calcific aortic valve insufficiency or calcific aortic valve lesions.

2. The use according to claim 1, characterized in that: The PARM1 inhibitor inhibits the osteogenic differentiation process of valvular interstitial cells (VICs) by inhibiting the expression level of the PARM1 nucleotide sequence or the expression level of the PARM1 protein.

3. The use according to claim 2, characterized in that: The PARM1 inhibitor that inhibits the expression level of the PARM1 nucleotide sequence is selected from at least one of the following: antisense nucleotides, small interfering RNA (siRNA), short hairpin RNA (shRNA) and micro RNA (miRNA) against PARM1 mRNA.

4. The use according to claim 3, characterized in that: The sense strand and antisense strand of the nucleotide sequence of the small interfering RNA (siRNA) are as follows: Sense strand nucleotide sequence SEQ ID NO.1: 5'-CUGUGUCAGGCAAAGUGAUTT-3'; The antisense strand nucleotide sequence is SEQ ID NO.2: 5'-AUCACUUUGCCUGACACAGTT-3'.

5. A drug for preventing or treating calcific aortic valve disease, characterized in that: The drug comprises the PARM1 inhibitor according to claim 1, and a pharmaceutically acceptable carrier, diluent or excipient, and the calcific aortic valve disease is a calcified heart valve lesion.

6. The drug according to claim 5, characterized in that The dosage form of the drug includes, but is not limited to, oral solution, injection, tablet, pill, dispersion, capsule, pellet, granule, suspension and emulsion.

7. The drug according to claim 5, characterized in that The PARM1 inhibitor is a small interfering RNA (siRNA) targeting PARM1 mRNA, the positive 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.

8. A method for treating calcific aortic valve disease, characterized in that: The method comprises administering to the patient an effective amount of the medicament of any one of claims 5-7.

9. The method according to claim 8, characterized in that The method inhibits the osteogenic differentiation process of valve interstitial cells by inhibiting the expression or function of PARM1.

10. The method according to claim 8, characterized in that The method can prevent or treat calcific aortic valve disease in in vitro cells, in in vivo animal models, or in drug screening.

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