Natural product for preventing and treating vascular calcification and application thereof

Abstract

The invention discloses a natural product for preventing and treating vascular calcification and application thereof, and belongs to the technical field of molecular biomedical treatment. According to the present invention, the effect of the target Nesfatin-1 in vascular calcification is determined, the adeno-associated virus (AAV) shRNA interference vector capable of specifically knocking down the vascular smooth muscle Nesfatin-1 gene is provided, and the natural product Cedryl acetal and the like capable of targeting inhibition of the Nesfatin-1 are screened out; the interference vector and the natural product can treat vascular calcification, and a solid research basis is provided for preparation of drugs for preventing and treating vascular calcification.

Description

[0001] This application is a divisional application with application number 202310907855.1, application date: July 24, 2023, and application name: Application of target Nesfatin-1 in the prevention or treatment of vascular calcification. Technical Field

[0002] The invention relates to a natural product for preventing and treating vascular calcification and application thereof, and belongs to the technical field of molecular biomedicine. Background Art

[0003] Vascular calcification is an important feature of the atherosclerotic process, which refers to the deposition of excessive calcium salts in the arterial walls. A large number of studies have shown that there is a close association between vascular calcification and cardiovascular events such as heart attacks and strokes. Calcium salt deposition in the arterial walls exacerbates vascular stenosis and abnormal blood flow, making blood vessels more susceptible to thrombosis and vascular obstruction, leading to cardiovascular events. Effective prevention and treatment methods for vascular calcification have always been an important challenge in the medical field. Current research focuses on the occurrence mechanism and influencing factors of vascular calcification, such as inflammation, oxidative stress, and apoptosis.

[0004] Nesfatin-1 is a polypeptide hormone secreted by the brain and stomach. It was first discovered in rats by Japanese researchers in 2006. The precursor protein of Nesfatin-1 (nesfatin precursor protein) is enzymatically cleaved to generate Nesfatin-1. Nesfatin-1 is believed to play an important role in multiple physiological processes, including appetite regulation, energy metabolism, autonomic nervous system regulation and weight control. Studies have found that serum Nesfatin-1 levels are negatively correlated with the incidence and severity of coronary artery disease, but the specific mechanism is unclear. Although atherosclerosis is the main pathological basis of coronary artery disease and vascular calcification is closely related to atherosclerosis, there is no clear evidence that there is a direct causal relationship between Nesfatin-1 and vascular calcification.

[0005] There are many limitations in the current clinical treatment of vascular calcification, among which the main treatment methods include drug therapy, lifestyle changes (such as diet and exercise), and surgical intervention (such as angioplasty and bypass surgery). However, these methods are not suitable for all patients and have safety risks, uncertain efficacy, high recurrence rate, and large side effects. Therefore, it is of positive significance to find effective treatment methods and drugs for vascular calcification, which can provide new ideas for the treatment and drug development of vascular calcification. Summary of the invention

[0006] In view of the above problems, the present invention uses Nesfatin-1 as a pharmaceutical target for the prevention or treatment of vascular calcification for the first time, designs targeted shRNA based on Nesfatin-1 that has the function of preventing or treating vascular calcification, and screens the natural product cedryl acetate for the prevention or treatment of vascular calcification. The present invention is of breakthrough significance and provides a new idea for finding effective treatment methods and drug preparation for vascular calcification.

[0007] The first object of the present invention is to provide the use of target Nesfatin-1 in the preparation of drugs for preventing or treating vascular calcification.

[0008] The second object of the present invention is to provide a method for studying the function of genes related to vascular calcification, or screening drugs for preventing or treating vascular calcification, or constructing a vascular calcification disease model, or analyzing gene therapy drugs for vascular calcification, the method comprising using an adeno-associated virus (AAV) shRNA interference vector containing a specific knockdown vascular smooth muscle Nesfatin-1 gene to reduce the expression of Nesfatin-1.

[0009] In one embodiment, the use can prevent or treat vascular smooth muscle calcification in in vitro cells, or in in vivo animal models, or in drug screening.

[0010] In one embodiment, the construction and screening method of the interference vector comprises the following steps:

[0011] (1) Determine the sequence of the Nesfatin-1 gene by gene sequencing technology;

[0012] (2) designing candidate AAV-shRNA sequences, wherein shRNA is a double-stranded RNA molecule composed of two complementary single-stranded RNA sequences, and the designed AAV-shRNA sequence has complementarity with a specific region of the Nesfatin-1 gene;

[0013] (3) Finally, the AAV-shRNA with the most significant inhibitory effect on Nesfatin-1 gene expression level or calcification was selected as the best candidate.

[0014] In one embodiment, the nucleotide sequence of the AAV-shRNA is the sequence shown in SEQ ID NO.1-6, wherein the nucleotide sequence of AAV-shRNA1 is SEQ ID NO.1-2; the nucleotide sequence of AAV-shRNA2 is SEQ ID NO.3-4; and the nucleotide sequence of AAV-shRNA3 is SEQ ID NO.5-6.

[0015] The third object of the present invention is to provide a drug capable of preventing or treating vascular calcification, wherein the drug can inhibit the expression of Nesfatin-1 gene or protein.

[0016] In one embodiment, the drug is a vector for gene therapy, including vectors for gene editing, gene expression regulation or gene delivery; or the drug is a vector for cell therapy, including stem cells, immune cells or repair cells.

[0017] In one embodiment, the medicament prevents or treats vascular calcification by regulating the Nesfatin-1 signaling pathway or downstream effector molecules.

[0018] In one embodiment, the drug comprises one or more of the following: cedrylacetate, ketoisophorone, cotinine, bornylacetate, eugenol, artesunate, estropipate, flavanone, ethisterone. Preferably, the drug contains cedrylacetate.

[0019] In one embodiment, the dosage form of the drug includes oral solution, injection, tablet, pill, dispersion, capsule, pellet, granule, suspension, emulsion.

[0020] The fourth object of the present invention is to provide a use of a natural product in the preparation of a drug for preventing or treating vascular calcification, wherein the natural product comprises one or more of the following: cedryl acetate, ketoisophorone, cotinine, bornyl acetate, eugenol, artesunate, estropipate, flavanone, and ethisterone. Preferably, the drug contains cedryl acetate.

[0021] The fifth object of the present invention is to provide a pharmaceutical composition, which is a pharmaceutical composition for preventing and treating vascular calcification or a drug for inhibiting the expression of Nesfatin-1, and the pharmaceutical composition comprises a pharmaceutically acceptable excipient and one or more of the following: cedryl acetate, ketoisophorone, nicotine, bornyl acetate, eugenol, artesunate, estropipate, flavanone, and ethisterone.

[0022] The present invention constructs a short hairpin RNA (shRNA) specific to vascular smooth muscle cells, screens and obtains shRNA that can effectively reduce the expression level of the nesfatin-1 gene in vascular smooth muscle cells, and verifies its role in vascular calcification. In addition, the present invention conducts large-scale compound screening, finds a natural product that can target and inhibit nesfatin-1, and verifies its preventive and therapeutic effects on vascular calcification.

[0023] Advantages and effects of the present invention:

[0024] (1) The present invention found that the expression level of Nesfatin-1 was significantly increased in both vascular smooth muscle cells treated with high phosphorus and calcified blood vessels, and the more severe the calcification of vascular smooth muscle cells, the higher the expression level of Nesfatin-1.

[0025] (2) The present invention successfully constructed and screened an AAV-shRNA interference vector for specifically knocking down the Nesfatin-1 gene in vascular smooth muscle, and confirmed its role in preventing and treating vascular calcification.

[0026] (3) The present invention screened out natural products that can target and inhibit Nesfatin-1 gene, among which Cedrylacetate had the best inhibition on vascular calcification, reaching 53.6%, followed by Ethisterone, Eugenol, Cotinine, Flavanone, Ketoisophorone, Artesunate, Bornyl acetate and Ethisterone, which inhibited vascular calcification by 45.5%, 43.8%, 41.5%, 41%, 39.5%, 34.9%, 32.3% and 30.8%, respectively. This provides a solid research foundation for the preparation of drugs for the prevention and treatment of vascular calcification. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The expression of Nesfatin-1 in calcified vascular smooth muscle cells and blood vessels. (A) Representative graph of Western blot detection of Nesfatin-1 protein level in vascular smooth muscle cells; (B) Quantitative statistical analysis of Figure A; (C) Nesfatin-1 mRNA level in calcified vascular smooth muscle cells; (D) Representative graph of Western blot detection of Nesfatin-1 protein level in calcified vessels; (E) Quantitative statistical analysis of Figure D; (F) Immunofluorescence detection of Nesfatin-1 protein expression in calcified vessels, blue DAPI is the cell nucleus, red is Nesfatin-1 protein, green α-SMA is a marker of vascular smooth muscle cells, scale bar = 100μm. n = 4-6. **p<0.01 compared with day 0; ***p<0.001 compared with day 0 / control.

[0028] Figure 2 To construct and screen AAV-shRNA interference vectors for specific knockdown of Nesfatin-1 gene in vascular smooth muscle. (A) Representative diagram of the interference effect of three constructed AAV-shRNA interference vectors on Nesfatin-1 protein expression in vascular smooth muscle cells detected by Western bolt; (B) Quantitative statistical analysis of Figure A.

[0029] Figure 3 This is a diagram showing the preventive and therapeutic effects of AAV-shRNA3 on vascular smooth muscle cell calcification and vascular calcification. (A) The effect of AAV-shRNA3 in the intervention and treatment of vascular smooth muscle cell calcification. The upper picture is a cell culture dish, and the lower picture is an Alizarin Red S staining picture; (B) The results of the ALP enzyme activity assay of vascular smooth muscle cells; (C) The results of the calcium content assay of vascular smooth muscle cells; (D) Representative Alizarin Red S staining of AAV-shRNA3 intervention and treatment of blood calcification; (E) The results of the vascular ALP enzyme activity assay; (F) The results of the vascular calcium content assay. n=4-6. *p<0.05 compared with AAV-Con; **p<0.01 compared with AAV-Con; ***p<0.001 compared with AAV-Con; # p<0.05 compared with AAV-shRNA3.

[0030] Figure 4Screening of natural products for targeted inhibition of Nesfatin-1 and validation experiments. (A) Luciferase reporter gene assay flow chart; (B) RLU values ​​of natural products that can effectively inhibit Nesfatin-1; (C) Ranking of natural products for Nesfatin-1 inhibition, and selection of the natural product Cedryl acetate with the highest Nesfatin-1 inhibition rate; (D) Representative Western bolt diagram of the effect of different concentrations of the natural product Cedryl acetate on the protein level of Nesfatin-1 in calcified vascular smooth muscle cells; (E) Quantitative statistical analysis of Figure D; (F) mRNA level of Nesfatin-1 in calcified vascular smooth muscle cells treated with three different concentrations of natural products; (G) Alizarin red S staining of calcified vascular smooth muscle cells treated with three different concentrations of the natural product Cedrylacetate; (H) Results of vascular ALP enzyme activity assay; (I) Results of vascular calcium content assay; n = 4-6. *p < 0.05 compared with Con; #p < 0.05 compared with High Pi group. DETAILED DESCRIPTION

[0031] 1. Technical solution of the present invention

[0032] 1. Technical solution for specific knockdown of nesfatin-1 gene in vascular smooth muscle cells:

[0033] (1) Design and synthesize nesfatin-1 gene-specific shRNA sequences;

[0034] (2) constructing a shRNA vector including a promoter suitable for vascular smooth muscle cells and a marker gene for screening;

[0035] (3) Clone the synthesized shRNA sequence into the vector and verify the accuracy of the cloning;

[0036] (4) preparing AAV viral vectors with knockdown of nesfatin-1 gene and using them to infect vascular smooth muscle cells;

[0037] (5) Use Western blotting, PCR and other molecular biological methods to verify whether the expression level of the nesfatin-1 gene has been successfully knocked down and evaluate the degree of vascular calcification.

[0038] 2. Technical solution for screening the natural product Cedryl acetate that can target and inhibit the nesfatin-1 gene:

[0039] (1) Design and build compound screening platforms, including screening systems based on cell models and in vitro enzyme reactions;

[0040] (2) Prepare a natural product sample library, including natural product samples from plants, microorganisms or other sources;

[0041] (3) Combine natural product samples with the screening platform to screen out compounds that have inhibitory effects on nesfatin-1 expression;

[0042] (4) Verify the inhibitory effect of the natural product Cedryl acetate obtained by screening, and evaluate its preventive and therapeutic effects on vascular calcification through cell experiments and animal model experiments, so as to promote the next step of drug preparation.

[0043] 2. Specific experimental methods of the present invention

[0044] 1. High Phosphate-Induced Vascular Smooth Muscle Cell Calcification Model

[0045] (1) Cell model selection: Rat vascular smooth muscle cell line A7R5 was selected and cultured in DMEM medium;

[0046] (2) High phosphorus treatment: After 24 h of serum starvation, the DMEM culture medium containing 10% fetal bovine serum was replaced, and 10 mmol / L β-glycerophosphate was added to induce vascular smooth muscle cell calcification. The high phosphorus culture medium was replaced every three days.

[0047] (3) Sample collection: Cell samples were collected after 0, 3, 6, 9, and 12 days of high phosphorus treatment.

[0048] 2. Western blot:

[0049] (1) Sample preparation: a. Collect calcified vascular smooth muscle cells; b. Wash the cell samples with PBS buffer to remove the culture medium or other impurities; c. Add cell lysis buffer containing protease inhibitors to lyse the cells to obtain total protein;

[0050] (2) Protein extraction: a. Centrifugation: Centrifuge the lysed sample to remove solid residues such as cell debris and cell nuclei, and collect the supernatant; b. Protein concentration determination: Use the BCA protein quantification method to determine the protein concentration;

[0051] (3) SDS-PAGE gel electrophoresis: a. Sample preparation: Mix the extracted protein sample with SDS-PAGE sample buffer and heat to denature; b. Gel preparation: Prepare polyacrylamide gel; c. Sample loading: Load the denatured protein sample into the gel wells and load molecular weight markers as references; d. Electrophoresis: Place the gel in an electrophoresis tank, add electrophoresis buffer, and apply an electric field for electrophoresis;

[0052] (4) Transfer: a. Prepare the membrane: transfer the protein from the gel to the protein blotting membrane. Prepare a membrane of appropriate size and pretreat the membrane before transfer; b. Transfer device: stack the gel and membrane in sequence, place them in the protein transfer device, and add appropriate transfer buffer. Make sure there are no bubbles between the gel and the membrane and make them fully contact; c. Transfer: transfer the protein from the gel to the membrane;

[0053] (5) Immunoblotting: a. Blocking: Place the protein blotting membrane in a blocking buffer to block nonspecific binding sites and reduce the nonspecific binding of subsequent antibodies; b. Antibody binding: Add the specific Nesfatin-1 primary antibody to the antibody binding buffer, incubate the membrane with the Nesfatin-1 primary antibody, and allow the Nesfatin-1 primary antibody to bind to the Nesfatin-1 protein on the membrane; c. Washing the membrane: Wash the membrane repeatedly to remove unbound Nesfatin-1 primary antibody and other nonspecific binding substances; d. Secondary antibody binding: Add enzyme-labeled secondary antibody, incubate the membrane with the secondary antibody, and allow the secondary antibody to bind to the Nesfatin-1 primary antibody; e. Washing: Wash the membrane again multiple times to remove unbound secondary antibody and other nonspecific binding substances; f. Signal detection: Use chemiluminescence to detect the signal of the protein on the membrane;

[0054] (6) Data analysis: The protein blot images were quantitatively analyzed using image J image analysis software, graphpad and other tools to obtain the relative expression level of Nesfatin-1 protein and compare the Nesfatin-1 protein levels in each group.

[0055] 3. Reverse transcription polymerase chain reaction (RT-PCR)

[0056] (1) Sample preparation: a. Collect calcified vascular smooth muscle cells; b. Rinse with PBS buffer to remove culture medium or other impurities; c. Add cell lysis buffer containing proteinase K to lyse cells and release DNA;

[0057] (2) PCR reaction: a. preparing a PCR reaction system, including a DNA template, primers, dNTPs, polymerase, buffer and MgCl2; b. mixing the components of the reaction system in a PCR reaction tube and setting up the reaction system; c. performing a PCR reaction, including an initial denaturation step, a cyclic denaturation step, an annealing step, an extension step, and a final extension step;

[0058] (3) Gel electrophoresis analysis: a. Prepare agarose gel and buffer; b. Mix PCR reaction products with DNA markers and load into agarose gel tank; c. Perform gel electrophoresis to separate and visualize PCR amplification products; d. Observe the gel using a protein imaging system and record the size and intensity of the target fragments;

[0059] (4) Data analysis: a. Use ImageJ image analysis software to measure the band position and band intensity of the PCR product; b. Use the relative expression calculation method, i.e., the △△Ct method, to calculate the expression level of the Nesfatin-1 gene relative to the internal reference gene based on the Ct value, and use tools such as GraphPad to compare the Nesfatin-1 gene levels in each group.

[0060] 4. Construction of a mouse model of vascular calcification

[0061] (1) C57BL / 6 mice were selected and raised in a normal environment;

[0062] (2) High-phosphorus diet modeling: a. The mice were divided into two groups, one group was a high-phosphorus diet group and the other group was a control group; b. The mice in the high-phosphorus diet group were given a feed rich in added inorganic phosphate for 6 weeks; c. The mice in the control group continued to receive a standard diet for 6 weeks; d. During the high-phosphorus diet period, the mice were regularly observed and recorded, including body weight, food intake, and general health status;

[0063] (3) Evaluation of calcified lesions: After modeling, the mice were killed and the thoracic aorta was removed for further analysis, such as calcium deposition staining (Alizarin Red S staining) and determination of calcium content, to determine whether the modeling was successful.

[0064] 5. Immunofluorescence of Mouse Thoracic Aorta

[0065] (1) Collecting mouse thoracic aorta tissue: a. Selecting a mouse model of vascular calcification; b. Euthanizing the mouse and performing dissection to quickly remove the thoracic aorta;

[0066] (2) Preparation of arterial annular slices: a. Rinse the obtained thoracic aorta tissue in PBS to remove blood and other contaminants; b. Carefully cut the thoracic aorta tissue with a blade or scissors to obtain arterial annular slices; c. Place the slices in a centrifuge tube containing PBS;

[0067] (3) Fixation and dehydration: a. Fix the slices with 4% paraformaldehyde solution at room temperature for 30 minutes; b. Dehydrate the fixed slices by soaking them in ethanol solutions of different concentrations (70%, 80%, 90%, 95% and 100%) in turn for 5 minutes in each concentration of ethanol solution;

[0068] (4) Immunofluorescence staining: a. Pre-treat the sections in immunofluorescence staining buffer to block nonspecific binding; b. Incubate the sections in Nesfatin-1 and α-SMA antibodies; c. Wash the sections to remove unbound antibodies;

[0069] (5) Microscope observation and imaging: a. Place the stained sections on a glass slide and cover with a mounting medium containing DAPI; b. Observe the sections using a fluorescence microscope and acquire images in the blue channel (DAPI), red channel (Nesfatin-1), and green channel (α-SMA); c. Analyze the images and observe the colocalization.

[0070] 6. Alizarin Red S staining

[0071] (1) Sample preparation: a. including vascular smooth muscle cells and mouse thoracic aorta; b. using 4% formaldehyde solution to fix the tissue for about 30 minutes at room temperature; c. rinsing with PBS to remove the fixative;

[0072] (2) Alizarin Red S staining: a. Prepare Alizarin Red S staining solution; b. Prepare 0.1% Alizarin Red S solution in PBS buffer; c. Place the fixed tissue sample in the Alizarin Red S solution and incubate at room temperature for about 30 minutes to 1 hour to allow it to react with calcium ions; d. Wash the sample with PBS buffer to remove unbound stain;

[0073] (3) Observation and image acquisition: Place the stained sample on a microscope slide and observe the stained sample under a microscope. Alizarin Red S complexes with calcium to produce orange-red to red deposits that can be observed with the naked eye or a microscope.

[0074] 7. Determination of calcium content

[0075] (1) Sample collection: Collect vascular smooth muscle cells and blood vessel samples;

[0076] (2) Extraction of calcium ions: a. Wash the cell sample with Tris-HCl buffer to remove the culture medium and other contaminants; b. Add RIPA buffer cell lysis buffer; c. Place the sample on ice or in a low temperature environment to avoid further loss of calcium ions; d. Centrifuge the cell lysate to obtain the supernatant;

[0077] (3) Determination of calcium content: a. Calcium content was determined using a calcium assay kit; b. The determination method was a colorimetric method, in which the color produced by the reaction of calcium ions with a dye was quantitatively measured; c. According to the steps in the kit instructions, the supernatant was reacted with a calcium assay reagent and the generated signal was measured; d. The absorbance was detected using a spectrophotometer;

[0078] (4) Data analysis: Calcium content data were obtained by using tools such as GraphPad and the differences among the groups were compared.

[0079] 8. Determination of ALP enzyme activity

[0080] (1) Sample collection: Collect vascular smooth muscle cells and blood vessel samples;

[0081] (2) Cell lysis: The cell lysis buffer used is used to lyse the cells to release the enzymes inside the cells;

[0082] (3) Protein content determination: The total protein content in the cell lysate was determined by the BCA method to standardize ALP activity;

[0083] (4) ALP substrate reaction: reacting the lysate with the ALP substrate and catalyzing the hydrolysis reaction of the substrate;

[0084] (5) Reaction termination: The reaction of ALP substrate is terminated by adding stop solution;

[0085] (6) Product detection and data analysis: Use photometry to measure the absorbance of the reaction product, quantify the measurement results based on the standard curve or internal control substances, and perform data analysis and interpretation.

[0086] 9. Luciferase reporter gene assay

[0087] (1) Construction of luciferase reporter gene vector: Using rat vascular smooth muscle cell genomic DNA as a template, the range of 2000 bp before the first exon of Nesfatin-1 was considered as the promoter region. Primer Premier 5.0 software was used to design amplification primers, and forward restriction site KpnⅠ and reverse restriction site HindⅢ and corresponding protective bases were added. The promoter sequence of Nesfatin-1 gene was amplified by PCR. High-fidelity DNA polymerase TransStart FastPfu DNA Polymerase, which can be used for rapid PCR amplification, was used to PCR amplify the target DNA fragment of the promoter region of Nesfatin-1. The above PCR products and vector plasmid pGL3-Enhancer were double-digested with restriction endonucleases KpnI and HindIII. The digested products were subjected to agarose gel electrophoresis, and the target fragment was recovered by gel cutting. The target plasmid was obtained by bacterial liquid amplification, restriction digestion, sequencing and other methods.

[0088] (2) Transfection of target cells: The constructed luciferase reporter gene vector is transfected into vascular smooth muscle cells to ensure that they can stably express luciferase;

[0089] (3) Processing natural product samples: 4160 natural product samples to be screened (10 μM) were added to calcification-induced vascular smooth muscle cells transfected with nesfatin-1 luciferase reporter gene, and co-cultured with the cells for 48 h. Then, the luciferase reporter gene activity of nesfatin-1 was detected using a luciferase reporter gene detection kit;

[0090] (4) Luciferase activity assay: Luciferase activity is measured in the treated cells using a luciferin substrate. After adding the luciferin substrate, luciferase catalyzes the reaction of the substrate to produce a measurable fluorescence signal;

[0091] (5) Fluorescence measurement and data analysis: a. Use a fluorescence microplate reader to measure the luciferase activity of the treated cells. RLU refers to the intensity of the fluorescence signal generated per second, which reflects the level of luciferase activity. b. Comparison and analysis: Compare and analyze the RLU values, perform statistical analysis, and screen for natural products that have an inhibitory effect on Nesfatin-1 activity.

[0092] Example 1: Role of Nesfatin-1 in Vascular Calcification

[0093] The experimental methods involved in Example 1 include: constructing a high phosphorus-induced vascular smooth muscle cell calcification model, constructing a vascular calcification mouse model, Western blot, PCR and immunofluorescence.

[0094] 1. Nesfatin-1 is significantly increased in calcified vascular smooth muscle cells

[0095] The results are as follows Figure 1 As shown, Figure 1 A Detection of Nesfatin-1 protein expression in vascular smooth muscle cells; Figure 1 B is the quantitative statistical analysis of Figure A; Figure 1 C detected the gene expression of Nesfatin-1 in vascular smooth muscle cells. Both Western blot and RT-PCR tests showed that the expression level of Nesfatin-1 was significantly increased in vascular smooth muscle cells treated with high phosphorus, and the more severe the calcification of vascular smooth muscle cells, the higher the expression level of Nesfatin-1.

[0096] 2. Nesfatin-1 is significantly increased in calcified blood vessels

[0097] The results are as follows Figure 1 As shown, Figure 1 D. Detect the protein expression of Nesfatin-1 in blood vessels; Figure 1 E is the quantitative statistical analysis of Figure D. Figure 1F is the protein expression of Nesfatin-1 in calcified blood vessels detected by immunofluorescence. Both Western blot detection and immunofluorescence showed that the expression level of Nesfatin-1 was significantly increased in calcified blood vessels, and the more severe the vascular calcification, the higher the expression level of Nesfatin-1.

[0098] Example 2: Construction and screening of AAV-shRNA interference vectors for vascular smooth muscle-specific knockdown of Nesfatin-1 gene.

[0099] The experimental methods involved in Example 2 include: Western blot, PCR, Alizarin Red S staining, ALP enzyme activity determination, and calcium content determination.

[0100] 1. Successfully constructed an AAV-shRNA interference vector for vascular smooth muscle-specific knockdown of the Nesfatin-1 gene.

[0101] Three AAV-shRNA interference vectors for specifically knocking down the Nesfatin-1 gene were successfully constructed, among which the nucleic acid sequence of AAV-shRNA1 was SEQ ID NO.1-2; the nucleic acid sequence of AAV-shRNA2 was SEQ ID NO.3-4; and the nucleic acid sequence of AAV-shRNA3 was SEQ ID NO.5-6.

[0102] The results are as follows Figure 2 As shown, Figure 2 A shows the protein expression level of Nesfatin-1 in vascular smooth muscle cells after interference with three AAV-shRNAs; Figure 2 B is Figure 2 Quantitative statistical analysis of Figure A. The results showed that the three AAV-shRNA interference vectors constructed could significantly reduce the levels of Nesfatin-1 protein and mRNA in vascular smooth muscle cells, among which AAV-shRNA3 interference vector had the best effect.

[0103] 2. The role of AAV-shRNA3 in the prevention and treatment of vascular calcification.

[0104] The results are as follows Figure 3 As shown, Figure 3 A shows the intervention of AAV-shRNA3 on calcified vascular smooth muscle cells. It can be seen that the calcification of vascular smooth muscle cells was alleviated after the intervention of AAV-shRNA3. Figure 3 B is the result of the ALP enzyme activity assay in vascular smooth muscle cells, which shows that AAV-shRNA3 can significantly reverse the ALP enzyme activity in calcified vascular smooth muscle cells, but has no significant effect on normal vascular smooth muscle cells; Figure 3C is the result of measuring calcium content in vascular smooth muscle cells, which shows that AAV-shRNA3 can significantly reverse the calcium content in calcified vascular smooth muscle cells, but has no significant effect on normal vascular smooth muscle cells; Figure 3 D is a representative picture of Alizarin Red S staining for AAV-shRNA3 intervention and treatment of blood calcification, showing that AAV-shRNA3 intervention can significantly alleviate vascular calcification; Figure 3 E is the result of the vascular ALP enzyme activity assay, which shows that AAV-shRNA3 can significantly reverse the abnormally elevated ALP enzyme activity in calcified blood vessels, but has no significant effect on normal blood vessels; Figure 3 F is the result of vascular calcium content measurement, which shows that AAV-shRNA3 can significantly reverse the abnormally elevated calcium content in calcified blood vessels, while having no significant effect on normal vascular smooth muscle cells. The results show that AAV-shRNA3 can significantly reverse the abnormally elevated ALP enzyme activity and calcium content in calcified vascular smooth muscle cells and calcified blood vessels, while having no significant effect on normal vascular smooth muscle cells and blood vessels.

[0105] Example 3: Screening of the natural product Cedryl acetate for targeted inhibition of Nesfatin-1 and its application in the prevention and treatment of vascular calcification

[0106] The experimental methods involved in Example 3 include: luciferase reporter gene detection, Western bolt, PCR, Alizarin Red S staining, ALP enzyme activity determination, and calcium content determination.

[0107] The results are as follows Figure 4 shown.

[0108] Figure 4 A is the flow chart of luciferase reporter gene assay, which was screened among 4160 natural products;

[0109] Figure 4 B is the RLU value of natural products that can effectively inhibit Nesfatin-1. It can be seen that high phosphorus treatment will significantly increase the RLU value. The natural products Cedryl acetate, Ethisterone, Eugenol, Cotinine, Flavanone, Ketoisophorone, Artesunate, Bornyl acetate and Ethisterone can effectively inhibit the RLU value, among which Cedryl acetate has the best inhibitory effect on the RLU value.

[0110] Figure 4C is the inhibitory effect of some of the natural products screened on Nesfatin-1. The results show that Cedrylacetate has the best inhibition on Nesfatin-1, reaching 53.6%; followed by Ethisterone, Eugenol, Cotinine, Flavanone, Ketoisophorone, Artesunate, Bornyl acetate and Ethisterone, which inhibit Nesfatin-1 by 45.5%, 43.8%, 41.5%, 41%, 39.5%, 34.9%, 32.3% and 30.8%, respectively;

[0111] Figure 4 D is a representative diagram of the effect of different concentrations of the natural product Cedryl acetate on the level of Nesfatin-1 protein in calcified vascular smooth muscle cells in Western bolt;

[0112] Figure 4 E is the quantitative statistical analysis of Figure D, showing that three different concentrations of the natural product Cedryl acetate can significantly inhibit the protein expression of Nesfatin-1 in calcified vascular smooth muscle cells;

[0113] Figure 4 F is the PCR detection of Nesfatin-1 gene expression, which shows that three different concentrations of the natural product Cedrylacetate can significantly reduce the mRNA level of Nesfatin-1 in calcified vascular smooth muscle cells;

[0114] Figure 4 G is an Alizarin Red S staining image of calcified vascular smooth muscle cells and three different concentrations (0μM, 3μM, 10μM) of the natural product Cedrylacetate. The results showed that the calcification of vascular smooth muscle cells was significantly alleviated after treatment with three different concentrations of the natural product Cedryl acetate.

[0115] Figure 4 H is the result of the vascular ALP enzyme activity assay, which shows that three different concentrations (0μM, 3μM, 10μM) of the natural product Cedryl acetate can significantly reverse the abnormally elevated ALP enzyme activity in calcified blood vessels;

[0116] Figure 4 I is the result of measuring vascular calcium content, which shows that three different concentrations (0 μM, 3 μM, 10 μM) of the natural product Cedryl acetate can significantly reverse the abnormally elevated calcium content in calcified blood vessels.

[0117] In this example, Cedrylacetate, which has the highest degree of Nesfatin-1 gene inhibition, was screened out from 4160 natural products, and its inhibition rate on vascular calcification reached 53.6%. Cedryl acetate can significantly reverse the Nesfatin-1 protein and mRNA levels in calcified vascular smooth muscle cells, and reverse the abnormally elevated ALP enzyme activity and calcium content in calcified blood vessels.

[0118] The present invention relates to the sequence:

[0119] SEQ ID NO.1Sense:5'-GTTTGTTTGTGTAATTCTA-3';

[0120] SEQ ID NO.2Antisense:5'-TAGAATTACACAACAAAC-3'

[0121] SEQ ID NO.3Sense:5'-GGTTGGTGCTCCTGTTGAA-3';

[0122] SEQ ID NO.4Antisense:5'-TTCAACAGGAGCACCAACC-3';

[0123] SEQ ID NO.5Sense:5'-GGCTTGCTGTTGAGTTCTA-3';

[0124] SEQ ID NO.6Antisense:5'-TAGAACTCAACAGCAAGCC-3'

[0125] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. Use of a natural product in the preparation of a drug for preventing or treating vascular calcification, characterized in that: The natural products include one or more of the following: cedryl acetate, ketoisophorone, cotinine, bornyl acetate, eugenol, artesunate, estropipate, flavanone, and ethisterone.

2. A pharmaceutical composition for preventing and treating vascular calcification or a pharmaceutical composition for inhibiting the expression of Nesfatin-1, characterized in that: The pharmaceutical composition comprises a pharmaceutically acceptable excipient and one or more of the following: cedryl acetate, ketoisophorone, cotinine, bornylacetate, eugenol, artesunate, estropipate, flavanone, and ethisterone.

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