Application of Chipericumin D in the preparation of drugs for treating calcified aortic valve disease

Abstract

This invention belongs to the field of biomedical technology, and particularly relates to the application of Chipericumin D in the preparation of drugs for treating calcific aortic valve disease. The core objective of this invention is to provide a large-scale drug screening method based on dot-blotting to identify drugs that inhibit osteoblast-like differentiation of aortic valve interstitial cells, reduce the expression of osteoblast-specific genes ALP and RUNX2, and decrease the formation of calcified nodules, thereby achieving the goal of preventing or treating calcific aortic valve disease (CAVD).

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of Chipericumin D in the preparation of drugs for treating calcified aortic valve disease. Background Technology

[0002] Calcific aortic valve disease (CAVD) is a serious heart valve disease with a rising global prevalence every year. Currently, transcatheter aortic valve implantation (TAVI) or surgical aortic valve replacement (SAVR) are the only treatment options. However, surgical treatment brings many complications, high medical costs, and a lower quality of life. There are currently no effective drugs to reverse or treat CAVD. Therefore, it is urgent to find drugs to prevent or treat CAVD through large-scale drug screening.

[0003] Studies have shown that the main pathological features of CAVD include endothelial damage, lipid deposition, inflammatory response, and osteoblastic differentiation of valvular interstitial cells, ultimately leading to collagen deposition, extracellular matrix remodeling, and calcified nodule formation. Among these, osteoblastic differentiation of aortic valve interstitial cells plays a crucial role in the pathogenesis of CAVD, accompanied by the upregulation of osteogenic-specific genes ALP and RUNX2. Currently, the application of Chipericumin D, an extract from the traditional Chinese medicine Hypericum perforatum, in CAVD has not been explored.

[0004] Existing technological shortcomings:

[0005] 1. Currently, there are no effective drugs for treating calcific aortic valve disease;

[0006] 2. There is currently no large-scale drug screening targeting osteoblast-like differentiation of aortic valve interstitial cells;

[0007] 3. The application of Chipericumin D in calcific aortic valve disease has not yet been explored. Summary of the Invention

[0008] The core objective of this invention is to provide a large-scale drug screening method based on dot-blotting to find drugs that inhibit osteoblast-like differentiation of aortic valve interstitial cells, reduce the expression of osteoblast-specific genes ALP and RUNX2, and reduce the formation of calcified nodules, thereby achieving the purpose of preventing or treating calcified aortic valve disease (CAVD).

[0009] This invention provides the use of Chipericumin D in the preparation of drugs for the prevention or treatment of calcified aortic valve disease.

[0010] Furthermore, the drug can reduce the expression of osteogenic-related genes ALP and RUNX2.

[0011] Furthermore, the Chipericumin D is used to prepare a drug that inhibits osteoblastic differentiation of VIC.

[0012] Furthermore, Chipericumin D is the sole active ingredient in the drug.

[0013] This invention provides a medicament for the prevention or treatment of calcific aortic valve disease, the medicament containing Chipericumin D.

[0014] Furthermore, the drug treats calcific aortic valve disease by targeting EGFR and inhibiting EGFR phosphorylation activation.

[0015] Furthermore, the dosage forms of the drug include oral liquids, injections, tablets, pills, dispersants, capsules, drop pills, granules, suspensions, and emulsions.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] 1. A large-scale screening of a natural product compound library was conducted using dot-blotting, and candidate compounds were further identified using ALP staining and Alizarin Red staining. ALP staining, Alizarin Red staining, Western blotting, and immunofluorescence confirmed the inhibitory effect of Chipericumin D on osteoblastic differentiation of aortic valve interstitial cells (VICs). Network pharmacology, molecular docking, DARTS, CETSA, SPR analysis, and rescue experiments demonstrated that Chipericumin D inhibits VIC osteoblastic differentiation by targeting EGFR and influencing the downstream PI3K / AKT signaling pathway.

[0018] 2. Treatment of aortic valve interstitial cells with the candidate compound significantly inhibited their osteoblast-like differentiation, including decreased expression of osteoblast-specific genes ALP and RUNX2, and reduced content of ALP-stained and Alizarin Red-stained calcified nodules. Chipericumin D significantly reduced the positive area of ​​ALP-stained and Alizarin Red-stained calcified nodules, significantly reduced the expression levels of osteoblast-specific genes ALP and RUNX2, and significantly reduced the nuclear translocation ratio of the osteoblast-specific transcription factor RUNX2. Chipericumin D inhibits EGFR phosphorylation activation by targeting EGFR; after inhibiting EGFR phosphorylation activation, Chipericumin D inhibits the downstream PI3K / AKT signaling pathway. Network pharmacology and molecular docking revealed the potential target of Chipericumin D; DARTS, CETSA, SPR analysis and rescue experiments confirmed that Chipericumin D targets EGFR.

[0019] 3. It significantly accelerates drug screening and development, enabling rapid drug screening studies in calcified aortic valve disease (CAVD) and its clinical application. Low-dose Chipericumin D can inhibit osteoblastic differentiation of the aortic valve, thus providing a new option for clinical treatment of CAVD. It elucidates the role of the EGFR / PI3K / AKT axis in CAVD, providing a theoretical basis for subsequent drug development; EGFR can be used as an independent target for developing other anti-tumor and osteoblastic differentiation-inhibiting therapies. Attached Figure Description

[0020] Figure 1 The figures shown are the initial screening results from Example 1. Figures A and C represent the results of Dot-blotting and Western blotting to verify the expression level of the osteogenic differentiation-specific gene RUNX2 in VIC at days 1, 3, and 5 after OM induction. Figures B and C are statistical graphs of RUNX2 expression levels. Figure D is a flowchart of drug screening using Dot-blotting. Figure E shows the Dot-blotting screening results, including RUNX2 and β-actin expression results. Figure F is a heatmap of results based on Figure E. Figure G is a scatter plot of results based on Figure E.

[0021] Figure 2 The diagrams shown are from the secondary screening in Example 1. AC represents the anti-calcification effects of the top 10 candidate compounds selected through alizarin red and ALP staining, where B is a statistical graph of alizarin red staining and C is a statistical graph of ALP staining. DF represents the inhibition of osteogenic-specific genes ALP and RUNX2 expression by the top 10 candidate compounds selected through Western blotting, where E is a statistical graph of ALP protein expression and F is a statistical graph of RUNX2 protein expression. G represents the structural formula of Chipericumin D.

[0022] Figure 3 This is a diagram showing the in vitro experimental results of Chipericumin D inhibiting osteoblast-like differentiation of aortic valve interstitial cells in Example 2. Figure A shows the CCK8 assay results, illustrating the IC50 of Chipericumin D. 50The concentration of 27.57 μM is shown in Figure BD. BD represents the results of Western blotting experiments verifying the inhibition of VIC osteogenic differentiation-specific genes ALP and RUNX2 by Chipericumin D, showing that inhibition is achieved at 3 μM. C represents the statistical graph of ALP protein expression, and D represents the statistical graph of RUNX2 protein expression. Figures EF and FE represent the results of PCR verification of the inhibition of VIC osteogenic differentiation-specific genes ALP and RUNX2 transcriptional expression by Chipericumin D. Figure GI represents the results of Western blotting experiments verifying the inhibition of VIC osteogenic differentiation-specific genes ALP and RUNX2 by Chipericumin D, where H represents the statistical graph of ALP protein expression, and I represents the statistical graph of RUNX2 protein expression. Figures JK represent the results of immunofluorescence staining experiments verifying the inhibition of VIC osteogenic differentiation-specific gene RUNX2 nuclear entry ratio by Chipericumin D, where J is a statistical graph. Figure LM represents the results of Alizarin Red staining experiments verifying the inhibition of late calcification by Chipericumin D, where M is a statistical graph. Figure NO represents the results of ALP staining experiments verifying the inhibition of late calcification by Chipericumin D. Figure D shows the experimental results of inhibiting early calcification, where N is a statistical graph;

[0023] Figure 4 A is a Venn diagram of the prediction of potential targets of CAVD and Chipericumin D using network pharmacology in Example 3; Figure 4 B is the network diagram of common targets of CAVD and Chipericumin D in Example 3;

[0024] Figure 5 This is an interaction diagram of the common target sites of CAVD and Chipericumin D in Example 3;

[0025] Figure 6 As in Example 3, based on Figure 5 Diagram of Chipericumin D docking with EGFR molecules;

[0026] Figure 7 As in Example 3, based on Figure 5 Docking diagram of Chipericumin D with NF-KB1 molecules;

[0027] Figure 8 As in Example 3, based on Figure 5 A diagram showing the docking of Chipericumin D with TLR4 molecules;

[0028] Figure 9 As in Example 3, based on Figure 5 Diagram of Chipericumin D docking with STAT3 molecules;

[0029] Figure 10 As in Example 3, based on Figure 5 Docking diagram of Chipericumin D with HIF1A molecules;

[0030] Figure 11 The figure shows the results of the DARTS experiment in Example 3, which verifies the binding of Chipericumin D to the top 5 potential targets. It shows that Chipericumin D binds to the target EGFR. The right side of the figure is a statistical bar chart.

[0031] Figure 12 The figure shows the experimental results of CETSA verification of the binding of Chipericumin D to the target EGFR in Example 3, where the right side of the figure is a statistical scatter plot;

[0032] Figure 13 This is a graph showing the SPR analysis results of Chipericumin D and the target EGFR in Example 3. D The value is 5.11 μM;

[0033] Figure 14 This is a graph showing the results of Chipericumin D inhibiting osteogenic differentiation of aortic valve interstitial cells by inhibiting EGFR phosphorylation in Example 4. AF represents the results of Western blotting experiments verifying whether Chipericumin D affects the EGFR / PI3K / AKT signaling pathway in the presence of the EGFR agonist NSC228155. B represents the p-EGFR / EGFR ratio, C represents the p-PI3K / PI3K ratio, D represents the p-AKT / AKT ratio, E represents ALP expression, and F represents RUNX2 expression. GI represents the results of Alizarin Red and ALP staining experiments verifying whether Chipericumin D affects the EGFR / PI3K / AKT signaling pathway and thus calcification in the presence of the EGFR agonist NSC228155 and the AKT agonist SC79. H represents the Alizarin Red staining bar chart, and I represents the ALP staining bar chart.

[0034] Figure 15 The figures shown are in vivo experimental results of Chipericumin D treatment of a mouse model of calcific aortic valve disease (CAVD) in Example 5. AB shows the aortic valve thickness observed by HE staining in different treatment groups, where A is a representative HE staining image of the mouse aortic valve and B is a statistical graph of the mouse aortic valve thickness. CD shows the calcified area of ​​the mouse aortic valve observed by Alizarin Red staining in different treatment groups, where C is a representative Alizarin Red staining image of the mouse aortic valve and D is a statistical graph of the calcified area of ​​the mouse aortic valve stained with Alizarin Red. Detailed Implementation

[0035] Example 1

[0036] Drug screening and Chipericumin D target validation protocol

[0037] By comparing the differences in Dot-blotting, ALP staining, and Alizarin Red staining between the compound intervention group and the positive control group under osteogenic differentiation medium (OM) culture conditions, the optimal candidate compound for inhibiting osteogenic differentiation of aortic valve interstitial cells (VIC) was determined, such as... Figure 1 , 2 As shown.

[0038] 1. Initial Screening: From a self-built natural product compound library of 88 compounds, the top 10 candidate compounds that inhibit osteogenic differentiation of aortic valve interstitial cells (VICs) were initially screened using the Dot-blotting method; the specific process is as follows:

[0039] The prepared 96-well plate cell lysate was pipetted onto a nitrocellulose membrane, dried at 37°C for 10 minutes, then blocked with 5% skim milk at room temperature for 1 hour, and finally incubated with antibody overnight. The next day, it was incubated with the corresponding species secondary antibody at room temperature for 1 hour for development.

[0040] 2. Secondary screening: The top 10 candidate compounds from the initial screening were subjected to alizarin red staining, ALP staining, and Western blotting; commercial alizarin red staining solution and ALP staining solution were used to evaluate the degree of calcification after compound intervention.

[0041] 3. Grouping and compound intervention:

[0042] Negative control group: Aortic valve interstitial cells (VICs) were cultured in only 2% FBS high glucose medium.

[0043] Positive control group: VIC cultured in osteogenic differentiation medium (OM);

[0044] Compound intervention group: VIC was treated with compounds under OM culture conditions;

[0045] Testing indicators:

[0046] Dot-blotting: to assess changes in the expression of the osteogenic-specific gene RUNX2;

[0047] ALP staining: to assess the degree of early calcification;

[0048] Alizarin red staining: to assess the content of late-stage calcium nodules;

[0049] Experimental results:

[0050] Dot-blotting:

[0051] Negative control group: Low expression of osteogenic specific gene RUNX2;

[0052] Positive control group: High expression level of osteogenic specific gene RUNX2;

[0053] Compound intervention group: The top 10 candidate compounds reduced the expression of the osteogenic-specific gene RUNX2;

[0054] ②ALP staining and Alizarin Red staining:

[0055] Negative control group: negative for ALP staining and alizarin red staining, with few or no calcium nodules;

[0056] Positive control group: positive for ALP staining and alizarin red staining, with high calcium nodule content;

[0057] Compound intervention group: After Chipericumin D intervention, ALP staining and Alizarin Red staining were positive, but significantly lower than those in the positive control group.

[0058] in conclusion:

[0059] The feasibility of large-scale drug screening using dot-blotting was confirmed; through two combined screenings, the natural product Chipericumin D was identified as a candidate compound for inhibiting osteoblast-like differentiation of aortic valve interstitial cells.

[0060] Chipericumin D has a molecular weight of 460.6 and a molecular formula of C. 27 H 40 O6; PubChem CID: 57381512; structural formula as follows Figure 2 As shown in G.

[0061] The targeting mechanism of Chipericumin D:

[0062] Targeting EGFR: Chipericumin D targets and binds to the EGFR protein, forming a hydrogen bond with its Asp-800 amino acid site, thereby inhibiting EGFR phosphorylation;

[0063] Downregulation of the EGFR / PI3K / AKT signaling pathway: Chipericumin D inhibits downstream PI3K / AKT phosphorylation activation by suppressing EGFR phosphorylation, thereby reducing the expression of osteogenic-specific genes ALP and RUNX2 and reducing calcium nodule deposition.

[0064] Example 2

[0065] In vitro experiments showing that Chipericumin D inhibits osteoblast-like differentiation of aortic valve interstitial cells

[0066] We verified that Chipericumin D inhibits osteoblast-like differentiation of aortic valve interstitial cells in vitro, and detected the expression of related genes by qPCR, Western blotting, ALP staining, Alizarin Red staining, and immunofluorescence staining.

[0067] 1. Cell Culture: The aortic valve was obtained from patients with dilated cardiomyopathy. Primary valvular interstitial cells were isolated from the aortic valve and cultured in DMEM medium containing 10% FBS.

[0068] 2. Osteogenic differentiation induction: Aortic valve interstitial cells were divided into 3 groups:

[0069] Negative control group: Aortic valve interstitial cells (VICs) were cultured in only 2% FBS high glucose medium.

[0070] Positive control group: Osteogenic differentiation was induced by VIC culture in osteogenic differentiation medium (OM);

[0071] Chipericumin D intervention group: VIC was treated with Chipericumin D under OM culture conditions;

[0072] 3. Sample collection:

[0073] Cells treated in step 2 were collected, and RNA was extracted using an RNA extraction kit (TIANGEN, A0508A) following the manufacturer's instructions. RNA was then reverse transcribed into cDNA using a reverse transcriptase mixture (Vazyme, R323-01). qRT-PCR was performed using SYBR Green (Vazyme, 7E782J3). VIC cells were lysed using RIPA cell lysis buffer, followed by sonication at 8% power for 1 second, pause for 1 second, for 10 cycles. The cells were then centrifuged at 12,000 rpm for 15 min, and the supernatant was collected. 5x loading buffer was added, and the cells were boiled at 95°C for 5 min to denature proteins. RNA and protein were extracted. Cells were fixed in 4% paraformaldehyde for 10 min, followed by ALP staining, Alizarin Red staining, and immunofluorescence staining.

[0074] 4. qPCR detection: The mRNA expression of osteogenic specific genes (ALP, RUNX2) was detected using the SYBR Green assay;

[0075] 5. Western blot: Detect the expression levels of ALP and RUNX2 proteins;

[0076] 6. ALP staining: to assess the degree of early calcification;

[0077] 7. Alizarin Red staining: to assess the content of late-stage calcium nodules;

[0078] 8. Immunofluorescence staining: to assess the nuclear translocation of the osteogenic-specific gene RUNX2 transcription factor;

[0079] The results are as follows Figure 3 As shown:

[0080] Chipericumin D intervention significantly reduced the mRNA expression of bone-specific genes ALP and RUNX2; significantly reduced the protein expression levels of bone-specific genes ALP and RUNX2; significantly reduced the area of ​​ALP staining in the Chipericumin D intervention group; significantly reduced the content of alizarin red stained calcium nodules in the Chipericumin D intervention group; and significantly reduced the nuclear translocation of the bone-specific gene RUNX2 transcription factor in the Chipericumin D intervention group.

[0081] This indicates that Chipericumin D can significantly inhibit osteogenic differentiation of aortic valve interstitial cells.

[0082] Example 3

[0083] Validation of the potential target of Chipericumin D in inhibiting osteogenic differentiation of aortic valve interstitial cells.

[0084] The potential target of Chipericumin D in inhibiting osteogenic differentiation of aortic valve interstitial cells was identified through network pharmacology, molecular docking, DARTS, CETSA, and SPR.

[0085] 1. Network pharmacology: By predicting potential targets of Chipericumin D in the SEA database, Swiss Target Prediction database, and super-prep database, and predicting potential targets of calcific aortic valve disease (CAVD) in the DisGeNET database, GeneCards database, and NCBI database, five core targets were identified after taking the intersection of Chipericumin D targets and CAVD targets and performing protein-protein interaction network analysis in the String database.

[0086] 2. Molecular docking: Predict the binding energy of five core target sites with Chipericumin D through molecular docking;

[0087] 3. DARTS experiment: Chipericumin D (1000 μM) and VIC cell lysis buffer (1 μg / μl, 40 μl) were co-incubated at room temperature for 1 hour. Then, Pronase E (Pronase E to VIC cell lysis buffer protein ratio of 1:200) was added, and the cells were lysed at room temperature for 5 minutes. Then, 5x Loading buffer (10 μl) was added and the protein was denatured at 95°C for 5 minutes. Finally, Western blotting was used to verify the binding of Chipericumin D to the target site.

[0088] 4. CETSA assay: After treating VIC cells with Chipericumin D (3μM) for 24 hours, the VIC cell lysate was divided into two groups. Each group was heated at 42-67℃ (5℃ interval) for 5 minutes, followed by centrifugation at 12,000 rpm / min at 4℃ for 15 minutes. The supernatant was collected, 5x Loading buffer was added, and the protein was denatured at 95℃ for 5 minutes. Finally, Western blotting was used to verify the binding of Chipericumin D to the target EGFR.

[0089] 5. SPR analysis: SPR binding energy analysis was used to determine the binding of Chipericumin D to the target EGFR;

[0090] 6. Detection indicators: Western blotting was used to detect the expression levels of core target proteins.

[0091] The results are as follows Figure 4-13 As shown:

[0092] Network pharmacology analysis revealed that the core targets included EGFR, NF-KB1, TLR4, STAT3, and HIF1A; molecular docking showed that the binding energies of Chipericumin D with EGFR, NF-KB1, TLR4, and STAT3 were all less than -6 kcal / mol; DARTS experiments showed that Chipericumin D binds to EGFR; CETSA experiments further confirmed the binding of Chipericumin D to EGFR; SPR analysis revealed that Chipericumin D directly binds to EGFR, with a binding energy of 5.11 μM.

[0093] This indicates that Chipericumin D binds directly to EGFR.

[0094] Example 4

[0095] Chipericumin D inhibits osteogenic differentiation of aortic valve interstitial cells by inhibiting EGFR phosphorylation.

[0096] The rescue experiment further verified that Chipericumin D targets and binds to EGFR, inhibiting its phosphorylation and downstream PI3K / AKT signaling pathway, thereby inhibiting osteogenic differentiation of aortic valve interstitial cells.

[0097] Negative control group: Aortic valve interstitial cells (VICs) were cultured in only 2% FBS high glucose medium.

[0098] Positive control group: Osteogenic differentiation was induced by VIC culture in osteogenic differentiation medium (OM);

[0099] Chipericumin D intervention group: VIC was treated with Chipericumin D (3 μM) under OM culture conditions;

[0100] NSC 228155 intervention group: VIC was treated with NSC 228155 (100 nM) under OM culture conditions.

[0101] NSC 228155 and Chipericumin D intervention group: VIC was co-treated with NSC 228155 (100 nM) and Chipericumin D (3 μM) under OM culture conditions;

[0102] SC79 intervention group: VIC was treated with SC79 (5 μM) under OM culture conditions;

[0103] SC79 and Chipericumin D intervention group: VIC was co-treated with SC79 (5 μM) and Chipericumin D (3 μM) under OM culture conditions.

[0104] The results are as follows Figure 14 As shown:

[0105] Positive control group: ALP, RUNX2, p-EGFR / EGFR ratio, p-PI3K / PI3K ratio, and p-AKT / AKT ratio were significantly higher than those in the negative control group;

[0106] Chipericumin D intervention group: ALP, RUNX2, p-EGFR / EGFR ratio, p-PI3K / PI3K ratio, and p-AKT / AKT ratio were significantly lower than those in the positive control group;

[0107] The NSC 228155 intervention group showed significantly higher levels of ALP, RUNX2, p-EGFR / EGFR ratio, p-PI3K / PI3K ratio, and p-AKT / AKT ratio compared to the positive control group.

[0108] In the NSC 228155 and Chipericumin D intervention group, ALP, RUNX2, p-EGFR / EGFR ratio, p-PI3K / PI3K ratio, and p-AKT / AKT ratio were significantly lower than those in the NSC 228155 intervention group.

[0109] The SC79 intervention group showed significantly higher levels of ALP, RUNX2, p-EGFR / EGFR ratio, p-PI3K / PI3K ratio, and p-AKT / AKT ratio compared to the positive control group.

[0110] In the SC79 and Chipericumin D intervention groups, ALP, RUNX2, p-EGFR / EGFR ratio, p-PI3K / PI3K ratio, and p-AKT / AKT ratio were significantly lower than those in the SC79 intervention group.

[0111] This indicates that Chipericumin D targets and binds to EGFR, inhibiting its phosphorylation and downstream PI3K / AKT signaling pathway, thereby suppressing osteogenic differentiation of aortic valve interstitial cells.

[0112] Example 5

[0113] In vivo experiments of Chipericumin D in a mouse model of calcified aortic valve disease (CAVD)

[0114] Through ApoE - / - Knockout mice (starting from 8 weeks old, divided into a negative control group, a positive control group, and a chipericumin D injection group; 12 mice in each group) were fed a high-fat diet (HFD, containing 0.25% cholesterol) to establish a CAVD disease model. At the same time, in vivo drug treatment was carried out by intraperitoneal injection of chipericumin D to verify whether chipericumin D can treat calcific aortic valve disease (CAVD) in mice.

[0115] Negative control group: Wild-type c57 mice were fed a normal diet (ND) and injected intraperitoneally with DMSO (0.1 mg / kg) every 3 days. They did not receive intraperitoneal injection of Chipericumin D.

[0116] Positive control group: ApoE - / - Mice were fed a high-fat diet (HFD, containing 0.25% cholesterol) and were intraperitoneally injected with DMSO (0.1 mg / kg) every 3 days. No intraperitoneal injection of Chipericumin D was given.

[0117] Chipericumin D spraying group: ApoE - / -Mice were fed a high-fat diet (HFD, containing 0.25% cholesterol) and injected intraperitoneally every 3 days with Chipericumin D diluted in DMSO solution (0.1 mg / kg).

[0118] The above process lasts for 6 months.

[0119] The results are as follows Figure 15 As shown:

[0120] Negative control group: HE and alizarin red staining of mouse aortic valve sections showed that the aortic valve was thin and no obvious calcified nodules were observed.

[0121] Positive control group: HE and alizarin red staining of mouse aortic valve sections showed that the aortic valve was significantly thickened, with obvious calcified nodules and an increased calcification area.

[0122] Chipericumin D injection group: HE and alizarin red staining of mouse aortic valve sections showed that the aortic valve thickness was thinner than that of the positive control group, and a small number of calcified nodules were visible, with a smaller calcified area.

[0123] This indicates that Chipericumin D can treat calcified aortic valve disease (CAVD) in mice in vivo.

[0124] This invention addresses the current lack of large-scale drug screening targeting osteoblastic differentiation of aortic valve interstitial cells (VICs) through dot-blotting, and identifies Chipericumin D as a candidate compound that inhibits VIC osteoblastic differentiation, thus initially solving the core problem of the lack of effective therapeutic drugs for calcific aortic valve disease (CAVD).

[0125] Experimental data show that low-dose chipericumin D significantly inhibits osteoblastic differentiation of the aortic valve by targeting EGFR and inhibiting its phosphorylation and activation of the downstream PI3K / AKT signaling pathway. This comprehensive strategy not only provides a new drug screening method for calcific aortic valve disease, but also offers a new strategy for the treatment of other vascular diseases.

[0126] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Application of Chipericumin D in the preparation of drugs for the prevention or treatment of calcified aortic valve disease.

2. The application according to claim 1, characterized in that, The drug can reduce the expression of osteogenic genes ALP and RUNX2.

3. The application according to claim 1, characterized in that, Chipericumin D is used to prepare a drug that inhibits osteoblastic differentiation of VIC.

4. The application according to claim 1, characterized in that, Chipericumin D is the only active ingredient in the drug.

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