Application of nuclear receptor ROR alpha agonist in preparation of medicine for resisting aortic valve calcification

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of a nuclear receptor ROR alpha agonist in preparation of a medicine for resisting aortic valve calcification. The invention discloses a nuclear receptor ROR alpha with potential aortic valve calcification resistance, and aortic valve interstitial cell osteogenesis calcification can be effectively inhibited by exciting the nuclear receptor ROR alpha. The agonist of the nuclear receptor ROR alpha is mainly used for inhibiting osteogenic calcification of the interstitial cells of the aortic valve by inhibiting inflammatory response and / or apoptosis mediated by an IL-17 signal channel, so that the aortic valve calcification is inhibited or delayed. The technical problem that at present, only valve replacement operation treatment can be carried out, and an effective medicine treatment means is lacked is solved, an effective non-operation medicine treatment means is provided for the disease, the risk of the valve replacement operation and postoperative complications are avoided, and good application prospects are achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and relates to a cardiovascular drug, and specifically to the use of an agonist of a nuclear receptor RORα in the preparation of a drug for resisting aortic valve calcification. Background Art

[0002] Calcific aortic valve disease (CAVD) is a valvular heart disease characterized by progressive fibrotic remodeling of the valve leaflets, abnormal structure and disorder of extracellular matrix proteins, and ultimately obstruction of the cardiac outflow tract. At present, my country is facing the dual pressures of an aging population and the continued prevalence of cardiovascular disease, and the mortality rate of cardiovascular disease ranks first among the total causes of death among urban and rural residents, 47.74% in rural areas and 44.26% in cities. Among them, "degenerative" valvular disease is the third most common cardiovascular disease, and its prevalence increases with age. In the past decade, the proportion of valve surgery has increased, accounting for more than 20% of all cardiac surgeries, causing a serious disease burden and has become a public health problem that needs to be solved urgently. Aortic stenosis or aortic regurgitation caused by aortic valve calcification has gradually become the main disease with a global cardiovascular disease burden.

[0003] The aortic valve is a three-layer structure consisting of a fibrous layer, a spongy layer, and a ventricular layer, which provide the biomechanical properties required to maintain long-term and repeated cyclic tension. The aortic valve is composed of endothelium, interstitium, and extracellular matrix, among which valvular interstitial cells (VICs) are the main cell type of the valve, and the transformation of VICs to an osteoblastic phenotype leads to fibrosis and calcium deposition, which is the key pathogenesis of valvular calcification. VICs are a heterogeneous cell population, including fibroblasts and smooth muscle cells, that participate in the maintenance of the extracellular matrix and play an important role in the development of CAVD. In the past, valvular calcification was considered to be a "degenerative" process caused by time-dependent wear and passive calcification deposition of the valve. Recent studies have shown that valvular calcification is an active multifactorial disease driven by metabolic and mechanical factors.

[0004] In recent years, although the development of vascular surgery and valve replacement have made great progress in the treatment of CAVD, current clinical lipid-lowering, hemodynamic regulation and other drug treatments cannot delay the progression of valve calcification. Therefore, exploring highly effective and less side-effect anti-aortic valve calcification drugs has become an important direction of basic and clinical research.

[0005] The nuclear receptor RORα (retinoid-related orphan receptor alpha), together with RORβ and RORγ, is a transcription factor belonging to the RORs nuclear receptor superfamily. Due to the regulatory roles of these RORs family members in gene expression, they regulate cardiovascular diseases through key processes such as circadian rhythm, cell proliferation, apoptosis, inflammation, and metabolism. Current studies have shown that ROR activation has been proven to alleviate mitochondrial damage, reduce apoptosis, and inhibit autophagy dysfunction; in osteoblasts, RORs can also regulate metabolic activities related to human osteogenic differentiation. In particular, the activation of RORα has been found to have an effective anti-atherosclerotic effect in atherosclerotic plaques, aortic smooth muscle cells, and endothelial cells; as a negative regulator of atherosclerosis, RORα alleviates autophagy activation, NLRP3 inflammasome, endothelial cell pyroptosis, and vascular endothelial dysfunction. In addition, RORα has an anti-inflammatory effect on endothelial cells and macrophages, and atherosclerosis and inflammation can lead to valve calcification and fragility. Previous reports have shown that RORα agonists have significant metabolic benefits, such as reducing inflammation, reducing fat accumulation, and reversing fibrosis. Given that fat accumulation and fibrosis often coexist during the process of aortic valve calcification, we speculate that RORα may play a key role in the process of aortic valve calcification. Research on nuclear receptors and related drug development are currently at the forefront of domestic and international research. A series of studies have shown the broad prospects of RORα as a drug target in clinical drug development. However, its application in aortic valve calcification has not been explored yet.

[0006] The present invention discovers a nuclear receptor RORα with potential anti-aortic valve calcification. Activating the nuclear receptor RORα can effectively inhibit the osteogenic calcification of aortic valve interstitial cells and reduce valve calcification. The agonist of the nuclear receptor RORα inhibits the osteogenic calcification of aortic valve interstitial cells by inhibiting the inflammatory response and / or apoptosis mediated by the IL-17 pathway, thereby inhibiting or delaying aortic valve calcification. Based on this, the present invention provides the application of an agonist of the nuclear receptor RORα in the preparation of a drug for anti-aortic valve calcification to solve the technical problem that current clinical drugs for lipid-lowering, regulating hemodynamics, etc. cannot prevent the occurrence of aortic valve calcification or delay its progression. Summary of the Invention

[0007] In view of the deficiencies and actual needs of the prior art, the present invention provides the application of an agonist of the nuclear receptor RORα in the preparation of a drug for anti-aortic valve calcification to effectively prevent and treat the occurrence of aortic valve calcification or delay its progression.

[0008] The present invention provides the application of an agonist of the nuclear receptor RORα in the preparation of a drug for preventing and / or treating aortic valve calcification in an individual.

[0009] The nuclear receptor RORα gene is located on the antisense strand of human chromosome 15, and its locus information is as follows: Homosapiens chromosome 15, GRCh38.p14 Primary Assembly, NC_000015.10(60488284..61229302,complement); its transcript (NM_002943.4) (mRNA) sequence is as shown in SEQ ID NO.1.

[0010] Furthermore, the agonist is SR1078 (chemical name: N-[4-[2,2,2-trifluoro-1-hydroxy-1-(trifluoromethyl)ethyl]phenyl]-4-(trifluoromethyl)benzamide), English name: N-[4-[2,2,2-Trifluoro-1-hydroxy-1-(trifluoromethyl)ethyl]phenyl]-4-(trifluoromethyl)benzamide; CAS number: 1246525-60-9; company: MedChemExpress, and its structural formula is:

[0011]

[0012] Furthermore, the agonist SR1078 of the nuclear receptor RORα inhibits osteogenic calcification of aortic valve interstitial cells by inhibiting the inflammatory response and / or apoptosis mediated by the IL-17 pathway, thereby inhibiting or delaying aortic valve calcification.

[0013] Furthermore, the agonist SR1078 of the nuclear receptor RORα can reduce the expression of inflammatory response factors IL-6, TNF-α, and MCP-1, thereby alleviating the inflammatory response.

[0014] Furthermore, the agonist SR1078 of the nuclear receptor RORα can reduce the expression of Cleaved-Caspase-3 / Caspase-3 and Bax, and simultaneously up-regulate the expression of Bcl2, thereby alleviating apoptosis.

[0015] Furthermore, the effective concentration of the agonist SR1078 of the nuclear receptor RORα is 10 mg / kg, and SR1078 is intraperitoneally injected twice a day for 8 weeks.

[0016] Furthermore, the individual is a mammal.

[0017] Furthermore, the individual is a human.

[0018] Furthermore, the drug also contains a pharmaceutically acceptable carrier.

[0019] The beneficial effects of the present invention are as follows: The present invention provides the application of the nuclear receptor RORα agonist SR1078 in the preparation of a drug for preventing and / or treating aortic valve calcification in an individual. It can effectively activate the activity of the nuclear receptor RORα, and can inhibit valvular interstitial cell osteogenic calcification and reduce calcium salt deposition by inhibiting the inflammatory response and / or apoptosis mediated by the IL-17 pathway, thereby effectively inhibiting or delaying aortic valve calcification. It solves the technical problem that currently, only valve replacement surgery can be used for treatment, and lipid-lowering and hemodynamic regulation drugs cannot prevent the occurrence of aortic valve calcification or delay its progression, that is, there is a lack of effective drug treatment means. It provides an effective drug treatment means for this disease, avoids the risks and postoperative complications of valve replacement surgery, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 : Expression of RORα in the aortic valve of mice. Figure A shows the protein expression levels of RORα and calcification-related indicators in ApoE - / - group and ApoE - / - +WI group of mice; Figure B shows the fluorescence expression of RORα in the aortic valve of ApoE - / - group and ApoE - / - +WI group of mice.

[0021] Figure 2 : Effects of RORα mutation on the morphology and function of the aortic valve. Figure A shows the aortic valve morphology and function diagrams of ApoE - / - group and sg / sg, ApoE - / - group of mice under wire injury (WI) and non-wire injury conditions, and Figure B shows the quantitative analysis results of peak aortic valve blood flow, valve orifice separation, and aortic valve area of ApoE - / - group and sg / sg, ApoE - / - group of mice under wire injury (WI) and non-wire injury conditions.

[0022] Figure 3 : Enrichment of calcification signaling pathways by RNA-seq analysis of RORα mutant mice. Figure A shows the transcriptome sequencing analysis of the aortic valve of mice, and Figure B shows the quantitative results of RNA-seq analysis of the aortic valve tissue of ApoE - / - group and ApoE - / - +WI group of mice after wire injury (WI).

[0023] Figure 4: Effects of the RORα agonist SR1078 on osteogenic calcification of aortic valve interstitial cells. A shows the quantitative analysis results of the calcium deposition of aortic valve interstitial cells verified by in vitro experiments with the RORα agonist SR1078, and B shows the alizarin red staining of aortic valve interstitial cells verified by in vitro experiments with the RORα agonist SR1078.

[0024] Figure 5 : Effects of the RORα agonist SR1078 on aortic valve inflammatory response, apoptosis and valve calcification. A shows the immunohistochemistry and ELISA detection of IL-6, TNF-α and MCP-1, B shows the protein expression results of apoptosis indexes in the aortic valve tissues of mice after caudal vein injection of normal saline and SR1078 after wire injury, and C and D show the functional evaluation results of the aortic valves of mice. Detailed implementation manners

[0025] The following further details the specific implementation manners and technical solutions of the present invention in combination with the accompanying drawings and specific embodiments. The specific embodiments give preferred implementation manners. In the present invention, unless otherwise specified, the raw materials can be purchased from conventional commercially available products, and the equipment and testing methods are conventional equipment and methods in the art.

[0026] Experimental animals: RORα heterozygous mice (RORα + / sg ) with a C57BL / 6J genetic background bred by Shanghai Model Organisms Center, Inc., were crossed with ApoE - / - mice to generate RORα-deficient chimeric mice (sg / sg, ApoE - / - ). Mice carrying ApoE - / - were used as the control group. sg / sg, ApoE - / - mice and their ApoE - / - littermate mice were all induced to establish a calcific aortic valve disease (CAVD) model by wire injury combined with 8-week high-fat diet (HFD). All experimental procedures complied with the guidelines stipulated in the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH publication, 8th edition, 2011). The study was approved by the Animal Ethics Committee of Shanghai Jiao Tong University, approval number: RJ2018-1018. Adult male and female mice aged 8 to 12 weeks were housed under standardized conditions. The breeding environment was strictly controlled at 24 ± 2 °C and humidity 40 ± 5% to ensure the health of the animals. The animals were housed in a pathogen-free environment with a 12-hour light / dark cycle. They had unrestricted access to food and water.

[0027] Molecular biochemical reagents: β-phosphoglycerate, dexamethasone, ascorbic acid, protease and phosphatase inhibitors, collagenase II type II, etc. were purchased from Sigma, USA; BCA quantitative kit was purchased from Thermo Scientific, USA; Tri-Buffered Saline (TBS), Tween-20, Phosphate-Buffered Saline (PBS), RORα primers and DAPI staining solution were purchased from Sangon Biotechnology (China); RORα, Vimentin, TNF-α, IL-6, MCP-1, Caspase-3, Cleaved-Caspase-3, Bax, Bcl2 and other antibodies were purchased from abcam, USA; GAPDH antibody was purchased from Cell Signaling Technology, USA; chemiluminescent HRP substrate ECL luminescent solution was purchased from Millipore, USA; goat anti-mouse Ig G-HRP and goat anti-rabbit Ig G-HRP were purchased from Santa Clara, CA. cruz, USA, isoflurane was purchased from Runanbet, China, ELISA kits were purchased from R&Dsystem, USA, and RORα agonist SR1078 was purchased from MCE, USA.

[0028] Example 1: Establishment of mouse aortic valve calcification model.

[0029] Take 8-12 weeks old sg / sg, ApoE - / - Mice and their littermates ApoE - / - In mice, aortic valve injury was induced using a wire transcarotid artery insertion technique. Specifically, the mice were anesthetized with isoflurane (flow rate 1.0 L / min, isoflurane concentration approximately 1.5%). Blunt dissection exposed the right carotid artery, and a spring wire (diameter 0.36 mm) bent at a 15-degree angle was inserted into the aorta under the guidance of echocardiography. The aortic valve was gently scratched 20 times with the spring wire, and rotated 100 times on the aortic valve to cause aortic injury. Subsequently, the neck wound was rinsed with saline and then sutured. The right carotid artery was punctured through the left ventricle, and no spring wire was inserted as a control group. Aortic valve tissue was collected after 8 weeks of high-fat diet (HFD) after surgery.

[0030] Example 2: Establishment of an in vitro model of osteocalcification of aortic valve interstitial cells.

[0031] Human primary aortic valve interstitial cells (h-VICs) were isolated from the aortic valves of patients. Specifically, in aortic valve replacement surgery, the patient's aortic valve was first removed and immediately immersed in cold phosphate-buffered saline (PBS), and then digested with 0.25% type II collagenase (Worthington, B1067, USA) at 37°C for 30 minutes. After preliminary digestion, the endothelium on the aortic and ventricular surfaces of the leaflets was gently scraped off with a cotton swab. Subsequently, the leaflets were cut into 1-2 mm slices and incubated in a 1 mg / ml collagenase solution at 37°C for 4-6 hours. Subsequently, the suspension was centrifuged at 1000 rpm for 10 minutes to form h-VICs into clusters, and then the tissue clusters were broken by repeated aspiration. h-VICs were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco) supplemented with 1% penicillin / streptomycin and 10% fetal bovine serum. The culture conditions were maintained in a humid environment containing 5% carbon dioxide at 37°C. The cells were identified by immunofluorescence staining. h-VICs were induced with osteogenic medium (OM) from passage 2 to passage 4. The OM medium was prepared by adding 10 mM β-glycerophosphate, 10 nM dexamethasone, and 50 μg / mL ascorbic acid to DMEM respectively. After 14 days of OM induction, an in vitro model of osteogenic calcification of aortic valve interstitial cells was prepared.

[0032] Example 3: Expression of RORα in human and mouse aortic valves.

[0033] Total RNA was extracted from valve tissues and cells using TRizol and reverse transcribed into cDNA using the PrimeScript RT kit (Takara, Japan) according to the manufacturer's instructions. Quantitative expression of mRNA was performed using SYBR Premix Ex Taq MasterMix (2×) (Takara, Japan) in a 480 RT-PCR system (Roche Applied Science). GAPDH was used as an internal control to normalize sample differences. Figure 1 For the expression level of RORα in mouse aortic valves, Figure 1 Figures 1A and 1B show that compared with the ApoE- / - group of mice, the protein expression of RORα was decreased in the aortic valves of the ApoE- / - + WI group of mice.

[0034] Example 4: Effects of RORα mutation on the morphology and function of aortic valves.

[0035] To observe the morphological and functional characteristics of the in vivo aortic valve, transthoracic echocardiography was performed using an 18-38 MHz phased array probe connected to a Vevo 1100 imaging system. Each mouse was evaluated under 2.5% isoflurane anesthesia according to a predefined protocol. The parasternal short-axis view at the level of the papillary muscles was used for left ventricular M-mode imaging. The left ventricular end-diastolic (LVEDd) and end-systolic (LVEDs) dimensions were measured, and the left ventricular fractional shortening (LVFS) was calculated using the formula: (LVEDd - LVEDs) / LVEDd × 100%. In addition, the interventricular septum diameter (IVSd) and posterior wall diameter (PWd) were measured simultaneously with M-mode tracing. The left ventricular volume and ejection fraction (EF) were determined using the Teichholz formula and quinidine preparations, respectively. The left ventricular outflow tract (LVOT) diameter was measured using magnified parasternal long-axis imaging, and the LVOT cross-sectional area (CSALVOT) was calculated as π(D / 2) 2 . The LVOT blood flow velocity was evaluated using pulsed-wave Doppler in the apical 5-chamber view. The aortic transvalvular blood flow velocity was recorded using continuous-wave Doppler. Color Doppler aligned the Doppler beam along the axis formed by the LVOT and the aortic root to minimize the interrogation angle of continuous-wave Doppler. The peak transvalvular jet velocity was evaluated from the continuously recorded Doppler spectrum, and the mean transvalvular pressure gradient was calculated using the Bernoulli formula: (mean transvalvular pressure gradient = ∑4 × flow velocity 2 ). As previously described, the aortic valve area (AVA) was determined using the continuity equation method: 0.785 × [(CSALVOT 2 × VLVOT) / peak transvalvular jet velocity]. Global longitudinal strain (GLS) analysis of the left ventricle was performed using a standard long-axis view. GLS was measured at the end of systole and averaged over three cardiac cycles. M-mode analysis of the aortic and mitral valves helped to determine the end-systolic and end-diastolic times. In the specified end-systolic frame, in the long-axis view, the left ventricular endocardial border was manually traced from one end of the mitral annulus, across the entire endocardium, to the junction of the aortic annulus and the left ventricular myocardium. Multimodal imaging techniques such as magnetic resonance imaging (MRI) and micro-ultrasound imaging (MUI) were used to measure key parameters of left ventricular function and the aortic valve. Measurements were made using a Vevo770 system from Visualsonics, Inc., Ontario, Canada, and a 7.0T small animal MRI system from Bruker BioSpin MRI GmbH, Ettlingen, Germany, with ParaVision 5.1 software. Specifically, the left ventricular diameter, peak transvalvular jet velocity, and orifice area were evaluated. These imaging evaluations were performed 8 weeks after a high-fat diet (HFD) following surgery Figure 2 A In the absence of mouse aortic valve injury, sg / sg, ApoE - / - mice and ApoE - / -There was no significant difference in the morphology and function of the aortic valves in mice; under wire injury (WI), echocardiography and magnetic resonance imaging showed that compared with ApoE - / - mice, the peak blood flow through the aortic valve in sg / sg, ApoE - / - mice increased significantly over time; the valve orifice separation was significantly narrowed; and the aortic valve area was significantly reduced. Figure 2 Figure B shows the analysis results of the peak blood flow through the aortic valve, valve orifice separation, and aortic valve area. The above results indicate that RORα mutation exacerbates aortic valve calcification stenosis induced by wire injury (WI).

[0036] Example 5: RNA-seq analysis of RORα mutant mice shows enrichment of the calcification signaling pathway.

[0037] Figure 3 Figure A shows the transcriptome sequencing analysis of the aortic valve in mice. After extracting the aortic valve tissue of mice, it was quickly frozen in liquid nitrogen to maintain the integrity of cellular RNA. Total RNA was detected using TRIzol reagent from Qiagen. The extracted RNA was quantified and its quality was evaluated to ensure its suitability for cDNA synthesis. cDNA was amplified from the isolated RNA and an RNA-seq library was established for high-throughput sequencing. RNA sequencing was performed using the Solexa sequencing technology provided by OE Biotech in Shanghai, China, according to the manufacturer's protocol. The gene expression levels were determined by quantifying the number of mapped reads for each gene using the HiSeq 2500 system of Illumina, Inc. (San Diego, CA, USA). Finally, the original RNA-seq data was stored in the public repository Gene Expression Omnibus (GEO) for further analysis and access by the scientific community. Figure 3 Figure B shows that after wire injury (WI) in sg / sg, ApoE - / - mice and ApoE - / - mice, aortic valve tissue was taken for RNA-seq analysis. Total RNA was extracted from the valve tissue and cells using TRIzol and reverse transcribed into cDNA using the PrimeScript RT reagent kit (Takara, Japan) according to the manufacturer's instructions. The mRNA expression was quantified using SYBR Premix Ex Taq MasterMix (2×) (Takara, Japan) in a 480 RT-PCR system (Roche Applied Science). GAPDH was used as an internal control to normalize sample differences. The results are as Figure 3 shown, as Figure 3Analysis of the transcriptome sequencing of the aortic valves of mice showed that in sg / sg, ApoE mice, - / - gene differences in the aortic valves of mice were enriched in ossification signals, osteoblast differentiation signals, and endochondral ossification signaling pathways, further indicating that RORα mutation activates the osteogenic calcification signal of the aortic valve induced by wire injury (WI).

[0038] Example 6: In vitro and in vivo CAVD intervention experiments with the RORα agonist SR1078.

[0039] Figure 4 A is the analysis of the in vitro experiment to verify the effect of the RORα agonist SR1078 on calcium deposition in aortic valve interstitial cells. Calcium content determination was performed according to the established method. The o-cresolphthalein complexone colorimetric method was used to measure the calcium concentration in the aortic valve cusp extract and in vitro cultured human valve interstitial cells (h-VICs). An aortic valve interstitial cell extract was prepared with 0.1 mol / L hydrochloric acid. The calcium content was quantified and expressed as micrograms per milligram of weight. The results were normalized to the weight of the aortic valve cusp or the total amount of cellular protein. This normalization allows for a comparative assessment of the calcium content between samples. To further confirm the relationship between RORα and the aortic valve, Figure 4 B is the analysis of the in vitro experiment to verify the alizarin red staining results of the RORα agonist SR1078 on aortic valve interstitial cells. When valve interstitial cells (VICs) reached 80% confluence, they were placed in the conditioning medium for the designated intervention. 10 mM β-glycerophosphate, 50 μg / mL ascorbic acid, and 10 nM dexamethasone were added to the medium and maintained for 14 days, with the medium renewed every 3 days. To evaluate calcium deposition, alizarin red staining was performed according to the established protocol. Briefly, after rinsing twice with phosphate-buffered saline (PBS), the VIC monolayer was fixed in 4% paraformaldehyde for 15 minutes. The cells were incubated with 0.2% alizarin red solution (pH 4.2) for 30 minutes. Unbound dye was removed by washing with distilled water. To quantify calcium deposition, the alizarin red-stained samples were stained with 10% acetic acid at 75 °C. The supernatant was analyzed by spectrophotometry at 450 nm to determine the amount of calcium deposition. The results are as Figure 4 shown. The results showed that SR1078 treatment alleviated calcium deposition in cells stimulated by OM.

[0040] Example 7: In vivo experiment to analyze the effects of SR1078 on the inflammatory response, apoptosis, and morphological function of the aortic valve tissue in mice after wire injury (WI).

[0041] Figure 5A is the immunohistochemical and ELISA detection of IL-6, TNF-α, and MCP-1. For immunohistochemical staining, after a similar process of dewaxing, rehydration, and antigen retrieval, 6-μm paraffin-embedded sections were incubated with primary antibodies against IL-6 (1:50, ab233706, Abcam), TNF-α (1:50, ab183218, Abcam), and MCP-1 (1:50, ab214819, Abcam), appropriately diluted and stored overnight at 4°C. The sections were incubated with biotin-conjugated secondary antibodies and stained signals were detected using the Avidin-Biotin Complex peroxidase system. Hematoxylin counterstaining was performed for nuclear identification and images were captured using a fluorescence microscope. As previously described, the fluorescence intensity was quantified using Image-Pro Plus 6.0 software. For enzyme-linked immunosorbent assay (ELISA), aortic valve tissues and human valve interstitial cells (h-VICs) were homogenized in 180 μl of PBS and then centrifuged at 4°C at a relative centrifugal force of 15,000 × g for 15 minutes according to the manufacturer's instructions. The resulting supernatant was collected for further analysis. The levels of cytokines and chemokines, including interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and monocyte chemoattractant protein-1 (MCP-1), were detected using a detection kit from MultiSciences, China. All measurements were performed according to the manufacturer's protocol. The absorbance of the samples was quantified at 450 nm using a MultiSkan FC reader from ThermoFisher, USA. It was found that SR1078 reduced the expression of IL-6, TNF-α, and MCP-1 induced by wire injury (WI) and alleviated the inflammatory response. Figure 5Group B shows the expression of apoptotic proteins in the aortic valve tissues of mice after tail vein injection of normal saline and SR1078 following detection of guide wire injury. Total proteins were extracted from aortic valve tissues or h-vic using RIPA buffer for protein extraction and immunoprotein experiments. Then, the protein samples were subjected to Western blot analysis using 10% SDS-PAGE. For Western blot, the membrane was first blocked in 5% skim milk and then incubated overnight at 4°C with a series of primary antibodies. These antibodies target a variety of proteins, including Caspase-3 (1:1000, 9662, Cell Signaling Technology), Cleaved-caspase-3 (1:1000, 9661, Cell Signaling Technology), Bcl2 (1:1000, 3498, Cell Signaling Technology), and Bax (1:1000, 5023, Cell Signaling Technology). After incubation with the primary antibodies, they were treated with the corresponding secondary antibodies diluted at a ratio of 1:10000. The immune complexes were visualized using an enhanced chemiluminescence (ECL) kit (Millipore, WBKLS0500, USA), and the band density was analyzed using Image J software. It was found that SR1078 reduced Cleaved-Caspase-3 / Caspase-3 and Bax induced by guide wire injury (WI), upregulated the expression of Bcl2, and alleviated apoptosis. Figure 5 Figures C / D show the results of the functional evaluation of the aortic valves of mice. It was found that SR1078 alleviated the peak blood flow through the aortic valve induced by guide wire injury (WI), alleviated the valve orifice separation, and increased the active valve area. The above results indicate that SR1078 alleviated the functional changes of the aortic valves of mice induced by guide wire injury (WI).

Claims

1. Use of an agonist of nuclear receptor RORα in the preparation of a drug for preventing and / or treating aortic valve calcification.

2. The use according to claim 1, characterized in that: The nuclear receptor RORα gene is located on the antisense strand of human chromosome 15, and its site information is Homo sapiens chromosome 15, GRCh38.p14 Primary Assembly, NC_000015.10 (60488284..61229302, complement), and its transcript NM_002943.4 sequence is shown in SEQ ID NO.

1.

3. The use according to claim 2, characterized in that: The agonist of the nuclear receptor RORα is SR1078, the chemical name of which is N-[4-[2,2,2-trifluoro-1-hydroxy-1-(trifluoromethyl)ethyl]phenyl]-4-(trifluoromethyl)benzamide), and the CAS registration number is 1246525-60-9.

4. The use according to claim 3, characterized in that: The nuclear receptor RORα agonist SR1078 inhibits osteogenic calcification of aortic valve interstitial cells by inhibiting inflammatory response and / or cell apoptosis mediated by the IL-17 pathway, thereby inhibiting or delaying aortic valve calcification.

5. The use according to claim 4, characterized in that: The nuclear receptor RORα agonist SR1078 can reduce the expression of inflammatory response factors IL-6, TNF-α, and MCP-1, thereby alleviating the inflammatory response.

6. The use according to claim 4, characterized in that: The nuclear receptor RORα agonist SR1078 can reduce the expression of Cleaved-Caspase-3 / Caspase-3 and Bax, and simultaneously upregulate the expression of Bcl2, thereby alleviating cell apoptosis.

7. The use according to claim 5 or 6, characterized in that: The effective concentration of the nuclear receptor RORα agonist SR1078 is 10 mg / kg, and SR1078 is intraperitoneally injected twice a day for 8 weeks.

8. The use according to claim 1, characterized in that: The medicine also contains a pharmaceutically acceptable carrier.

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