Application of PCN in preparation of medicine for preventing and treating vascular calcification

Abstract

The invention provides application of PCN in preparation of a medicine for preventing and treating vascular calcification, belongs to the technical field of biological medicine, and further provides a medicine for preventing or treating vascular calcification, and the effective component of the medicine comprises PCN. According to the application, PCN is used for treating angiosclerosis, vascular calcification can be effectively improved, and the development process of vascular calcification is inhibited.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to the application of PCN in the preparation of drugs for preventing and treating vascular calcification. Background Art

[0002] Vascular calcification (VC) is a pathological change characterized by calcium deposition in vascular tissue. It primarily includes intimal calcification, medial calcification, and heart valve calcification, with medial calcification being the most common. Vascular calcification is an active, regulated physiological process similar to skeletal development. Its central component is the differentiation of vascular smooth muscle cells (VSMCs) into osteoblasts under the combined effects of hyperphosphatemia and other risk factors. Numerous risk factors contribute to the development and progression of vascular calcification, including both traditional and non-traditional risk factors. Among these, mineral metabolism disorders and elevated serum calcium and phosphorus levels are key factors. Currently, there is no effective treatment for vascular calcification. Therefore, in-depth investigation of the molecular mechanisms of vascular calcification and the development of potential new therapeutic targets are crucial for the clinical treatment of vascular calcification. Summary of the Invention

[0003] In order to obtain a drug for treating vascular calcification, the present invention provides the use of PCN in the preparation of a drug for preventing and treating vascular calcification. This application uses PCN to treat vascular sclerosis, which can effectively improve vascular calcification and inhibit the development of vascular calcification.

[0004] The present invention is achieved through the following technical solutions:

[0005] The present invention provides application of PCN (pregnenolone-16α-nitrile) in preparing drugs for preventing and treating vascular calcification.

[0006] Based on the same inventive concept, the present invention provides a drug for preventing or treating vascular calcification, wherein the active ingredient of the drug includes PCN.

[0007] Based on the same inventive concept, the present invention provides the use of PCN in inhibiting the expression of BMP2 protein and / or OPN protein in vascular smooth muscle cells.

[0008] Based on the same inventive concept, the present invention provides the use of PCN in inhibiting the expression of C4a gene and / or C4b gene in vascular smooth muscle cells.

[0009] Based on the same inventive concept, the present invention provides the use of PCN in preparing an inhibitor of BMP2 protein and / or OPN protein expression in vascular smooth muscle cells.

[0010] Based on the same inventive concept, the present invention provides the use of PCN in preparing an inhibitor of C4a gene and / or C4b gene expression in vascular smooth muscle cells.

[0011] Based on the same inventive concept, the present invention provides the use of C4a gene and / or C4b gene as targets in the development of drugs for preventing and treating vascular calcification.

[0012] Based on the same inventive concept, the present invention also provides the use of a C4b gene expression inhibitor in the preparation of a drug for preventing and treating vascular calcification.

[0013] Based on the same inventive concept, the present invention also provides the use of a complement system inhibitor in the preparation of a drug for preventing and treating vascular calcification.

[0014] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0015] The present invention discloses an application of PCN in the preparation of a drug for preventing and treating vascular calcification. This application uses PCN to treat vascular sclerosis, effectively improving vascular calcification and aortic ring calcification caused by high phosphorus, inhibiting the increase of the osteogenic phenotype protein BMP2 in vascular smooth muscle cells, and inhibiting the transcription level of C4b, thereby achieving the effect of improving VSMC calcification caused by high phosphorus and inhibiting the development of vascular calcification. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following is a brief introduction to the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0017] Figure 1 The results of Alizarin red staining and quantification of VSMCs in Example 1 are shown;

[0018] Figure 2 The figure and quantitative bar graph of Western Blotting results of SM22α and BMP2 in VSMC in Example 1;

[0019] Figure 3 The results of Alizarin red staining and quantification of rat vascular rings in Example 2 are shown;

[0020] Figure 4 The immunohistochemical results of OPN and BMP2 in rat vascular rings and the quantitative bar graph in Example 2 are shown;

[0021] Figure 5 The results of Alizarin red staining and quantification of vascular rings of wild-type mice and PXR knockout mice in Example 3 are shown;

[0022] Figure 6 Statistical graph and heat map of the number of differentially expressed genes after transcriptomic analysis of VSMCs treated with PCN and high-phosphate medium in Example 4;

[0023] Figure 7 This is the result of cluster analysis of differentially expressed genes between the PCN+high phosphorus group and the high phosphorus group in Example 4;

[0024] Figure 8 This is a graph showing the QPCR results of VSMC C4a and C4b treated with PCN and high-phosphate medium in Example 4. DETAILED DESCRIPTION

[0025] The present invention will be described in detail below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific embodiments and examples are for illustrating the present invention, rather than for limiting the present invention.

[0026] Throughout this specification, unless otherwise specified, the terms used herein should be understood as having the same meaning as commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the event of any conflict, the present specification shall take precedence.

[0027] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0028] The overall idea of ​​the present invention is as follows:

[0029] PCN (pregnenolone 16α-carbonitrile, PCN) is an agonist of the pregnane X receptor (PXR). PXR is a member of the nuclear receptor superfamily and a ligand-dependent nuclear receptor subfamily 1, group I, member 2 transcription factor, also known as NR1 I2 (Nuclear Receptor subfamily 1, group I, member 2). PXR is expressed in multiple tissues throughout the body, including the gastrointestinal tract, liver, kidney, vascular smooth muscle, and endothelium. PXR is one of the most important nuclear receptors in the field of metabolism, playing a key role in the metabolism of endogenous and exogenous substances and metabolic homeostasis, exerting significant physiological and pathological effects. Currently, research on PXR has primarily focused on liver regeneration, inflammatory bowel disease, and kidney injury, while studies on vascular calcification have been limited. Furthermore, there are no reports that PCN can treat vascular calcification.

[0030] The present invention proposes the use of PCN in the preparation of a drug for preventing and treating vascular calcification. This application uses PCN to treat vascular sclerosis, can effectively improve vascular calcification, and inhibit the development process of vascular calcification.

[0031] The application of PCN in the preparation of drugs for preventing and treating vascular calcification will be described in detail below with reference to examples and experimental data.

[0032] The specific steps of Western blot, immunohistochemistry, Alizarin red staining and statistical analysis in the present invention are as follows:

[0033] 1.Western blot:

[0034] Total protein from vascular smooth muscle cells was extracted using tissue lysate, and protein concentration was determined using the BCA assay. Twenty micrograms of protein sample was loaded onto a 6% or 10% polyacrylamide gel electrophoresis (SDS-PAGE) gel, followed by wet transfer onto a PVDF membrane at 300 mA for 3 h. The membrane was blocked with 5% BSA for 1 h at room temperature, washed with TBST, and then incubated overnight at 4°C with primary antibodies against GAPDH (Cell Signaling Technology), SM22α (Proteintech), BMP2 (Abcam), and OPN (Proteintech). The membrane was washed three times with TBST for 10 min each, incubated with the corresponding secondary antibody for 1 h at room temperature, and then washed three times with TBST for 10 min each. Antigen-antibody complexes were visualized using enhanced chemiluminescence (ECL), and protein bands were exposed using a ChemiD MP imaging system. Target bands were quantified using Image J software, and the relative expression of the target protein was expressed as the grayscale value of the target band compared to that of the internal control protein GAPDH.

[0035] 2. Immunohistochemistry:

[0036] Tissues were fixed with 4% paraformaldehyde for 48 h, embedded in paraffin, and sectioned, dewaxed, and boiled in sodium citrate (10 mM pH 6.0) in an autoclave for 10 min. The sections were cooled to room temperature and blocked with goat serum for 1 h at room temperature, followed by incubation with BMP2 (Abcam) and OPN (Proteintech) antibodies at 4°C overnight. The sections were washed with PBS three times for 10 min each, stained with DAB, counterstained with hematoxylin, dehydrated, and mounted with neutral gum. The sections were observed under a microscope and images were collected.

[0037] 3. Alizarin Red Staining:

[0038] Tissues were fixed with 4% paraformaldehyde for 48 h, embedded in paraffin, and sectioned, dewaxed, and then oven-baked at 37°C for 30 min and stained with 2% alizarin red at room temperature for 5 min. The sections were then rinsed with running distilled water until the solution was essentially colorless. The sections were then dried and mounted with neutral gum. The sections were then observed and images were collected using a microscope.

[0039] 4. Statistical Analysis

[0040] All experimental data were expressed as mean ± SEM, and multiple groups were compared using analysis of variance (ANOVA). P < 0.05 was considered statistically significant; p < 0.05 was marked with *; p < 0.01 was marked with **; p < 0.001 was marked with ****.

[0041] Example 1

[0042] PCN improves VSMC cell calcification and phenotypic transformation caused by high phosphorus:

[0043] Primary cultured rat aortic VSMCs were divided into a control group (CTR) (the control group used DMEM culture medium containing 10% fetal bovine serum), a high-phosphate group (Pi), and a high-phosphate + PCN group. The latter two groups were induced to calcify using high-phosphate culture medium (the control group culture medium was supplemented with disodium hydrogen phosphate solution purchased from Sigma to a final phosphorus concentration of 3 mM). The high-phosphate + PCN group was also treated with PCN (400-1000 nM). After 7 days, the degree of cell calcification was assessed by Alizarin red staining, the intracellular calcium content was detected by a kit, and the expression of VSMC contractile phenotype protein SM22α and osteogenic phenotype protein BMP2 was detected by Western blotting. Figure 1 A in the figure is the result of Alizarin Red staining. It can be seen from the results that high phosphorus treatment will cause obvious calcification of VSMC, while 400nM PCN treatment significantly improves the VSMC calcification caused by high phosphorus. Figure 1 B in the figure is the result of intracellular calcium content, which is consistent with Figure 1 The results in Figure A were consistent, high phosphorus led to an increase in intracellular calcium content, and 400nM PCN significantly improved the increase in VSMC calcium content caused by high phosphorus.

[0044] Figure 2 AC are the expression results of SM22α, BMP2 and GAPDH proteins in cells after treatment with high phosphorus and PCN (400-1000nM). According to the results, high phosphorus will lead to a decrease in VSMC contractile phenotype protein SM22α and an increase in osteogenic phenotype protein BMP2. PCN at a concentration of 1000nM can partially alleviate the downregulation of SM22α, and PCN at a concentration of 400nM can inhibit the upregulation of BMP2.

[0045] In summary, these results confirm that PCN can improve VSMC calcification and phenotypic transformation caused by high phosphorus.

[0046] Example 2

[0047] PCN improves high phosphorus-induced aortic ring calcification in rats:

[0048] Rat aortic rings were cultured in vitro and calcified by high-phosphate medium. PCN (800 nM) was also administered. The rings were collected 7 days later, fixed with paraformaldehyde, dehydrated, embedded, and sectioned. Alizarin red staining was used to assess the degree of calcification, and immunohistochemistry was used to detect the expression of osteogenic phenotype proteins BMP2 and OPN. Figure 3 A in the figure is the result of Alizarin red staining of vascular rings. It can be seen from the results that high phosphorus treatment for 7 days will lead to obvious calcification of vascular rings, and treatment with 800nM concentration of PCN can significantly improve the calcification of vascular rings caused by high phosphorus. Figure 3 B in the figure is the quantitative graph of Alizarin Red staining, and its results are similar to Figure 3 The analysis of A is consistent.

[0049] The expression of osteogenic phenotype proteins BMP2 and OPN in the vascular rings was further detected by immunohistochemistry. Figure 4 As shown in the results, the expression of BMP2 and OPN proteins in vascular rings was significantly increased after 7 days of high phosphorus treatment, and PCN (800nM) could effectively inhibit the increase of BMP2 and OPN proteins in vascular rings caused by high phosphorus.

[0050] The above results indicate that PCN can protect the aortic ring calcification caused by high phosphorus and inhibit the upregulation of osteogenic phenotype proteins BMP2 and OPN.

[0051] Example 3

[0052] PXR knockout aggravates high phosphorus-induced aortic ring calcification in mice:

[0053] Aortic rings from wild-type mice and PXR knockout mice (PXR whole-body knockout mice purchased from Guangdong Yaokang Biotechnology Co., Ltd., strain number: T01537, strain name: Nr1 i2-KO) were cultured in vitro and given high-phosphate medium to induce calcification. After 7 days, the rings were collected, fixed with paraformaldehyde, dehydrated, embedded, and sectioned. The degree of calcification in the rings was assessed by Alizarin red staining. Figure 5 A in the figure is the result of Alizarin Red staining. It can be seen from the results that high phosphorus treatment for 7 days will cause obvious calcification of the vascular rings of both wild-type mice and PXR knockout mice. Compared with the vascular rings of wild-type mice, the calcification of the vascular rings of PXR knockout mice is more severe. Figure 5 B in the figure is the quantitative graph of Alizarin Red staining, and its results are similar to Figure 4 The analysis of A is consistent.

[0054] The above results indicate that PXR knockout will aggravate aortic ring calcification caused by high phosphorus, and further demonstrate the protective role of PXR in protecting vascular calcification at the level of vascular rings in gene knockout mice.

[0055] Example 4

[0056] PCN may improve VSMC calcification caused by high phosphorus by inhibiting the activation of the complement system:

[0057] To further explore the mechanism of action of PCN in improving vascular calcification, we used transcriptomics to analyze the changes in VSMC cell mRNA after high phosphorus and high phosphorus + PCN (1000nM) treatment.

[0058] The treatment method was similar to that in Example 1. Rat aortic VSMCs were primary cultured and divided into a control group (CTR), a high phosphorus group (Pi), and a high phosphorus + PCN group. The high phosphorus + PCN group was induced to calcify by high phosphorus medium and treated with PCN at the same time. Cells were collected after 7 days for transcriptomic analysis. Figure 6 A in the figure is a statistical chart of the number of differentially expressed genes screened by transcriptomic analysis. The results show that there are 162 differentially expressed genes between the high-phosphorus group and the control group, and 20 differentially expressed genes between the high-phosphorus + PCN group and the high-phosphorus group, of which 10 are overlapping genes. Figure 6 Figures B and C are heat maps of differentially expressed genes between the high phosphorus group and the control group, and between the high phosphorus + PCN group and the high phosphorus group, respectively. C4a and C4b are the most significantly changed genes. The expression of C4a and C4b in the high phosphorus group was significantly increased compared with the control group, while the expression of C4a and C4b in the high phosphorus + PCN group was significantly decreased compared with the high phosphorus group.

[0059] Figure 7 The pathway with the most significant enrichment of differentially expressed genes between the high phosphorus + PCN group and the high phosphorus group was the complement system. C4a and C4b are important components of the complement system. To further verify that PCN may play a protective role in vascular calcification by inhibiting the activation of the complement system caused by high phosphorus, we detected the levels of C4a and C4b mRNA by QPCR experiments. The results showed that high phosphorus treatment led to a significant increase in the levels of C4a and C4b mRNA in VSMCs, while PCN significantly inhibited the transcription level of C4b and had no effect on the transcription level of C4a. Figure 8 AD】.

[0060] In summary, PCN may improve VSMC calcification caused by high phosphorus by inhibiting the activation of C4b-related complement system.

[0061] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements, but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0062] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0063] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. Application of PCN in the preparation of drugs for the prevention and treatment of vascular calcification.

2. A drug for preventing or treating vascular calcification, characterized in that: The active ingredient of the medicine includes PCN.

3. Application of PCN in inhibiting the expression of BMP2 protein and / or OPN protein in vascular smooth muscle cells.

4. Application of PCN in inhibiting the expression of C4a gene and / or C4b gene in vascular smooth muscle cells.

5. Application of PCN in the preparation of inhibitors of BMP2 protein and / or OPN protein expression in vascular smooth muscle cells.

6. Use of PCN in the preparation of inhibitors of C4a gene and / or C4b gene expression in vascular smooth muscle cells.

7. Application of C4a gene and / or C4b gene as targets in the development of drugs for the prevention and treatment of vascular calcification.

8. Application of C4b gene expression inhibitors in the preparation of drugs for the prevention and treatment of vascular calcification.

9. Application of complement system inhibitors in the preparation of drugs for the prevention and treatment of vascular calcification.

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