Use of PCN in preparation of drugs for preventing and treating blood vessel calcification

Abstract

The application provides application of PCN in preparation of a drug for preventing and treating vascular calcification, belongs to the technical field of biological medicine, and further provides a drug for preventing or treating vascular calcification, wherein an effective component of the drug comprises PCN.The application applies PCN to treatment of vascular sclerosis, and can effectively improve vascular calcification and inhibit the development process of vascular calcification.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of PCN in preparation of a drug for preventing and treating vascular calcification. BACKGROUND

[0002] Vascular calcification (VC) is a pathological change of calcium salt deposition in the vascular wall tissue, mainly including intimal calcification, medial calcification and heart valve calcification, and the medial calcification is the most common. The vascular calcification is an active and controllable physiological process similar to bone development, and the key link is that the vascular smooth muscle cells (VSMC) differentiate into osteoblasts under the combined action of high blood phosphorus and other risk factors. Many risk factors are involved in the occurrence and development of vascular calcification, mainly including traditional risk factors and "non-traditional" risk factors, and the mineral metabolism disorder and the increase of serum calcium and phosphorus levels are key factors. At present, there is no effective treatment for vascular calcification, so it is of great significance to deeply explore the molecular mechanism of vascular calcification and develop possible new therapeutic targets for clinical treatment of vascular calcification. SUMMARY

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

[0004] The application is achieved by the following technical solutions.

[0005] The application provides application of PCN (pregnenolone-16 alpha-nitrile) in preparation of a drug for preventing and treating vascular calcification.

[0006] Based on the same inventive concept, the application provides a drug for preventing or treating vascular calcification, and an effective component of the drug includes PCN.

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

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

[0009] Based on the same inventive concept, the application provides application of PCN in preparation of an inhibitor for inhibiting expression of BMP2 protein and / or OPN protein of vascular smooth muscle cells.

[0010] Based on the same inventive concept, the application provides application of PCN in preparation of a vascular smooth muscle cell C4a gene and / or C4b gene expression inhibitor.

[0011] Based on the same inventive concept, the application provides application of C4a gene and / or C4b gene as a target in development of a vascular calcification prevention and treatment drug.

[0012] Based on the same inventive concept, the application also provides application of a C4b gene expression inhibitor in preparation of a vascular calcification prevention and treatment drug.

[0013] Based on the same inventive concept, the application also provides application of a complement system inhibitor in preparation of a vascular calcification prevention and treatment drug.

[0014] The one or more technical solutions in the embodiments of the application have at least the following technical effects or advantages:

[0015] The application of PCN in preparation of a vascular calcification prevention and treatment drug uses PCN to treat vascular sclerosis, can effectively improve vascular calcification caused by high phosphorus, aortic ring calcification, inhibit the increase of osteogenic phenotype protein BMP2 in vascular smooth muscle cells, and inhibit the transcription level of C4b, so as to improve the VSMC calcification caused by high phosphorus and inhibit the development process of vascular calcification. BRIEF DESCRIPTION OF DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the application, the drawings needed in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are some embodiments of the application, and other drawings can be obtained by those skilled in the art without creative labor.

[0017] Figure 1 The alizarin red staining and quantitative results of VSMC in Example 1;

[0018] Figure 2 The SM22a and BMP2 Western Blotting results chart and quantitative column chart of VSMC in Example 1;

[0019] Figure 3 The alizarin red staining and quantitative results of rat vascular rings in Example 2;

[0020] Figure 4 The OPN and BMP2 immunohistochemical results chart and quantitative column chart of rat vascular rings in Example 2;

[0021] Figure 5 The alizarin red staining and quantitative results of wild-type mouse and PXR knockout mouse vascular rings in Example 3;

[0022] Figure 6 Figure 4A and 4B are the scatter plot and heat map of the number of differential genes after transcriptomic analysis of VSMC treated with PCN and high phosphate medium in Example 4;

[0023] Figure 7 Figure 5 is the result plot of differential gene clustering analysis of PCN+high phosphate group versus high phosphate group in Example 4;

[0024] Figure 8 Figure 6 is the result plot of QPCR of C4a and C4b of VSMC treated with PCN and high phosphate medium in Example 4. DETAILED DESCRIPTION

[0025] The advantages and various effects of the present application will be more clearly apparent from the following detailed description and examples. It should be understood by those skilled in the art that the detailed description and examples are for the purpose of illustration only and not to limit the present application.

[0026] Throughout this specification, unless otherwise specifically indicated otherwise, the terms used herein are understood to have the meanings 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 application belongs. If there is a conflict between the present specification and the definitions of terms, the present specification takes precedence.

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

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

[0029] PCN (Pregnenolone 16α-carbonitrile, PCN) is an agonist of Pregnane X receptor (PXR). PXR is a member of the nuclear receptor superfamily, belonging to the ligand-dependent nuclear receptor 1 subfamily I transcription factor, also known as NR1 I2 (Nuclear Receptor subfamily 1, group I, member 2). PXR is expressed in multiple tissues throughout the body, such as the gastrointestinal tract, liver, kidney and vascular smooth muscle and endothelium, etc. PXR is one of the most important nuclear receptors in the field of metabolism, plays a key role in the metabolism of endogenous and exogenous substances and the metabolic homeostasis of the body, and produces important physiological and pathological effects. Current research on PXR has focused on liver regeneration, inflammatory bowel disease, kidney injury, etc., but there has been no report on vascular calcification. There has also been no report on PCN treating vascular calcification.

[0030] This invention proposes the application of PCN in the preparation of drugs for the prevention and treatment of vascular calcification. This application uses PCN to treat arteriosclerosis, which can effectively improve vascular calcification and inhibit the development of vascular calcification.

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

[0032] The specific operational steps for Western blot, immunohistochemistry, alizarin red staining, and statistical analysis in this invention are as follows:

[0033] 1. Western blot:

[0034] Total protein was extracted from vascular smooth muscle cells using tissue lysis buffer, and protein concentration was determined using the BCA method. 20 μg of protein sample was loaded onto a PVDF membrane and subjected to 6% or 10% polyacrylamide gel electrophoresis (SDS-PAGE) at 300 mA for 3 hours. The membrane was blocked with 5% BSA at room temperature for 1 hour, eluted with TBST, and then incubated overnight at 4°C with primary antibodies GAPDH (Cell Signaling Technology), SM22α (Proteintech), BMP2 (Abcam), and OPN (Proteintech). The membrane was washed three times with TBST for 10 minutes each time, followed by incubation with the corresponding secondary antibody at room temperature for 1 hour, and then washed three times with TBST for 10 minutes each time. Antigen-antibody complexes were visualized using enhanced chemiluminescence (ECL), and protein bands were exposed using a ChemiDMP imaging system. Quantitative analysis of the target bands was performed using ImageJ software, and the relative expression level of the target protein was expressed as the gray value of the target band compared to the gray value of the internal control protein GAPDH.

[0035] 2. Immunohistochemistry:

[0036] Tissue was fixed with 4% paraformaldehyde for 48 hours, embedded in paraffin, sectioned, and dewaxed to water; boiled in a pressure cooker with sodium citrate (10 mM, pH 6.0) for 10 minutes; cooled to room temperature, blocked with goat serum at room temperature for 1 hour, and then incubated overnight at 4°C with BMP2 (Abcam) and OPN (Proteintech) antibodies; washed three times with PBS for 10 minutes each time, stained with DAB, counterstained with hematoxylin, dehydrated, and mounted with neutral resin; observed under a microscope and images were acquired.

[0037] 3. Alizarin Red staining:

[0038] Tissue was fixed with 4% paraformaldehyde for 48 hours, embedded in paraffin, sectioned, and dewaxed to water; the slides were baked in a 37°C oven for 30 minutes, stained with 2% alizarin red at room temperature for 5 minutes; rinsed with running distilled water until the washing solution was basically colorless; the slides were dried and mounted with neutral resin; the slides were observed under a microscope and images were acquired.

[0039] 4. Statistical analysis:

[0040] All experimental data were expressed as mean ± SEM, and ANOVA was used for multiple comparisons, p < 0.05 was considered statistically significant; p < 0.05 was marked as *; p < 0.01 was marked as **; p < 0.001 was marked as ****.

[0041] Example 1

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

[0043] Primary culture of rat aortic VSMC, divided into control group CTR (control group using DMEM medium, and containing 10% fetal bovine serum), high phosphorus group Pi and high phosphorus + PCN group, the latter two groups by high phosphorus medium (control group medium based on the addition of sodium phosphate solution purchased from sigma company, prepared into 3mM final concentration of phosphorus) induced calcification, high phosphorus + PCN group is given PCN (400-1000nM) treatment, 7 days later, alizarin red staining to evaluate the degree of cell calcification, kit detection of intracellular calcium content, Western Blotting detection of VSMC contractile phenotype protein SM22a and osteogenic phenotype protein BMP2 expression. Figure 1 A in FIG. 1 is the result of alizarin red staining, from the results, it can be seen that high phosphorus treatment will cause VSMC to calcify significantly, and 400nM PCN treatment significantly improves the VSMC calcification caused by high phosphorus. Figure 1 B in FIG. 1 is the result of intracellular calcium content, consistent with the result of A in FIG. 1, high phosphorus leads to an increase in intracellular calcium content, and 400nM PCN significantly improves the increase in VSMC calcium content caused by high phosphorus. Figure 1

[0044] A-C in FIG. 1 are the results of SM22a, BMP2 and GAPDH protein expression after high phosphorus and PCN (400-1000nM) treatment, from the results, high phosphorus will cause VSMC contractile phenotype protein SM22a to decrease and osteogenic phenotype protein BMP2 to increase, 1000nM concentration of PCN can partially alleviate the down-regulation of SM22a, and 400nM concentration of PCN can inhibit the up-regulation of BMP2. Figure 2 In summary, these results confirm that PCN can improve VSMC calcification and phenotypic transformation caused by high phosphorus.

[0045] Example 2

[0046] PCN improves rat aortic ring calcification caused by high phosphorus:

[0047]

[0048] ​Rat aortic vascular rings were cultured in vitro and calcified by high-phosphorus medium. They were also treated with PCN (800 nM). After 7 days, the vascular rings were collected, fixed with paraformaldehyde, dehydrated, embedded, and sectioned. The degree of calcification of the vascular rings was assessed by alizarin red staining, and the expression of osteogenic phenotype proteins BMP2 and OPN was detected by immunohistochemistry. Figure 3 A in the figure represents the results of Alizarin Red staining of the vascular rings. The results show that high phosphorus treatment for 7 days leads to significant calcification of the vascular rings, while treatment with 800 nM PCN significantly improves the calcification of the vascular rings caused by high phosphorus. Figure 3 B in the diagram represents the quantitative staining map of Alizarin Red, and its results are consistent with... Figure 3 This is consistent with analysis A in the previous analysis.

[0049] Further immunohistochemical detection of the expression of osteogenic phenotype proteins BMP2 and OPN in the vascular ring yielded the following results: Figure 4 As shown, after 7 days of high phosphorus treatment, the expression of BMP2 and OPN proteins in the vascular rings was significantly increased, while PCN (800 nM) could effectively inhibit the increase of BMP2 and OPN proteins in the vascular rings caused by high phosphorus.

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

[0051] Example 3

[0052] Knocking out PXR exacerbates aortic ring calcification in mice induced by high phosphorus levels.

[0053] Aortic vascular rings of 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 calcified by high-phosphorus medium. After 7 days, the vascular rings were collected, fixed with paraformaldehyde, dehydrated, embedded, sectioned, and stained with alizarin red to assess the degree of vascular ring calcification. Figure 5 A in the figure represents the results of Alizarin Red staining. The results show that high phosphorus treatment for 7 days leads to significant calcification of the vascular rings in both wild-type mice and PXR knockout mice. Compared with the vascular rings of wild-type mice, the vascular rings of PXR knockout mice show more severe calcification. Figure 5 B in the diagram represents the quantitative staining map of Alizarin Red, and its results are consistent with... Figure 4 This is consistent with analysis A in the previous analysis.

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

[0055] Example 4

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

[0057] To further explore the mechanism of PCN in improving vascular calcification, we analyzed the changes in VSMC mRNA after high phosphorus and high phosphorus + PCN (1000 nM) treatment by transcriptome analysis.

[0058] The treatment method is similar to Example 1. Primary cultured rat aortic VSMCs were divided into control group CTR, high phosphorus group Pi, and high phosphorus + PCN group. The high phosphorus + PCN group was induced to calcify by high phosphorus medium and treated with PCN. After 7 days, the cells were collected for transcriptome analysis. Figure 6 Figure A in the figure is a histogram of the number of differential genes screened by transcriptome analysis. The results show that there are 162 differential genes between the high phosphorus group and the control group, and 20 differential genes between the high phosphorus + PCN group and the high phosphorus group, of which there are 10 intersection genes. Figure 6 Figures B and C are heat maps of differential genes between the high phosphorus group and the control group, and the high phosphorus + PCN group and the high phosphorus group, respectively. C4a and C4b are the most significant genes. Compared with the control group, the expression of C4a and C4b in the high phosphorus group was significantly increased. Compared with the high phosphorus group, the expression of C4a and C4b in the high phosphorus + PCN group was significantly decreased.

[0059] Figure 7 Figure D shows that the most significant pathway enriched by differential genes between the high phosphorus + PCN group and the high phosphorus group is the complement system. C4a and C4b are important components of the complement system. To further verify that PCN can inhibit the activation of the complement system caused by high phosphorus to play a protective role in vascular calcification, we detected the mRNA levels of C4a and C4b by QPCR experiment. The results show that high phosphorus treatment can significantly increase the mRNA levels of C4a and C4b in VSMCs, while PCN can significantly inhibit the transcription level of C4b, but has no effect on the transcription level of C4a. Figure 8 A-D.

[0060] In summary, PCN can inhibit the activation of the complement system related to C4b, thereby improving the calcification of VSMC caused by high phosphorus.

[0061] Finally, it should be noted that the terms "include", "contain" or any other variant thereof are intended to cover non-exclusive inclusion, so that the process, method, article or equipment including a series of elements not only includes those elements, but also includes other elements not explicitly listed or inherent to such process, method, article or equipment.

[0062] While the preferred embodiments of the application have been described, additional variations and modifications can be made to these embodiments by those skilled in the art once they have the benefit of the present disclosure without departing from the spirit and scope of the application. Accordingly, it is intended that the appended claims include all such modifications and variations as fall within the scope of the present application.

[0063] It is apparent that those skilled in the art can make various changes and modifications to the application without departing from the spirit and scope of the application. It is therefore intended that the present application cover all such changes and modifications that are within its scope.

Claims

1. Application of PCN in the preparation of drugs for the prevention and treatment of vascular calcification, wherein PCN is pregnenolone-16α-nitrile.

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