Application of LCN2 protein in preparing drugs for treating vascular calcification

Abstract

The present invention relates to the application of lipocalin 2 (LCN2) protein in the preparation of drugs for treating vascular calcification. The present invention respectively provides the application of LCN2 protein in the preparation of drugs for treating vascular calcification, a recombinant adeno-associated virus capable of increasing the expression level of LCN2 protein, and also provides a method for constructing the recombinant adeno-associated virus. Experiments have proved that the expression and secretion of LCN2 protein in vascular smooth muscle cells are inhibited under a high-phosphate environment. By administering exogenous LCN2 protein or transfecting VSMCs with a recombinant adeno-associated virus capable of highly expressing LCN2 protein, fatty acid β-oxidation can be promoted, thereby improving the situation of vascular calcification. Based on the experimental results, it can be known that LCN2 protein can play a role in treating vascular calcification, and therefore has good application prospects in the field of preparing related drugs.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to the application of LCN2 protein in the preparation of drugs for treating vascular calcification. Background Art

[0002] Lipocalin-2 (LCN2), also known as neutrophil gelatinase-associated lipocalin (NGAL), is a 25 kDa secreted protein and a transporter protein for lipophilic small molecules such as steroids, lipopolysaccharides, iron, and fatty acids. LCN2 is expressed in a variety of cells and is involved in the progression of various diseases. For example, LCN2 is a marker of acute kidney injury, and LCN2 is also a natural immune protein associated with acute and chronic inflammation. In addition, LCN2 also plays an important role in the occurrence, development, and metastasis of various cancers. However, whether there are more application fields for LCN2 requires further exploration and research. Summary of the Invention

[0003] The object of the present invention is to disclose the application of LCN2 protein in the preparation of drugs for treating vascular calcification, so as to solve one or more technical problems existing in the prior art, and provide at least one beneficial choice or create conditions.

[0004] The first aspect of the present invention lies in providing the application of Lipocalin-2 (LCN2) protein in the preparation of drugs for treating vascular calcification.

[0005] The second aspect of the present invention lies in providing a recombinant adeno-associated virus capable of increasing the expression level of LCN2 protein.

[0006] The third aspect of the present invention lies in providing a method for constructing the recombinant adeno-associated virus described in the second aspect of the present invention.

[0007] The application described in the first aspect of the present invention means that the LCN2 protein can play an improving role in the face of vascular calcification caused by various reasons, so it has the application prospect of preparing drugs for treating vascular calcification.

[0008] In a further application embodiment, the amino acid sequence of the LCN2 protein is as shown in SEQ ID No: 1.

[0009] In a further application embodiment, the vascular calcification is caused by hypertension, diabetes, chronic kidney disease, or atherosclerosis.

[0010] In a further application embodiment, the dosage form of the drug is one of powder, ointment, gel, drops, suppository, lozenge, granule, capsule, spray, tablet, pill, or solution.

[0011] In a further application embodiment, the drug contains a pharmaceutically acceptable carrier, and the carrier is selected from any one or a combination of fillers, binders, wetting agents, disintegrants, lubricants, and flavoring agents.

[0012] The recombinant adeno-associated virus according to the second aspect of the present invention includes a vector backbone and a target gene fragment, and the target gene fragment encodes the LCN2 protein. Adeno-associated virus (AAV) is a member of the Parvoviridae family. Members of this family are a class of small, non-enveloped, and icosahedral viruses. The diameter of the virus particles is between 20 and 26 nm, and they contain a linear single-stranded DNA genome with a size between 4.7 and 6 kb. Minute viruses have been isolated from insects to humans. AAV viruses belong to the Dependovirus class and were originally discovered as a contaminant in purified adenovirus preparations, hence the name.

[0013] In some application embodiments of the second aspect of the present invention, the vector backbone has a smooth muscle-specific promoter (SM22a) and a fluorescent label (ZsGreen).

[0014] In some application embodiments of the second aspect of the present invention, the nucleotide sequence of the target gene fragment is as shown in SEQ ID No:2.

[0015] The construction method according to the third aspect of the present invention includes the following steps:

[0016] A) Construct a recombinant adeno-associated virus vector;

[0017] B) Package adeno-associated virus;

[0018] Among them, the recombinant adeno-associated virus has a target gene fragment.

[0019] In some application embodiments of the third aspect of the present invention, the primer sequences used for amplifying the target gene fragment during the process of constructing the vector in step A) are as shown in SEQ ID No:3 and SEQ ID No:4.

[0020] The present invention provides a new use for the LCN2 protein. Experiments have shown that the expression and secretion of the LCN2 protein in vascular smooth muscle cells (VSMCs) are inhibited under a high-phosphorus environment. By administering exogenous LCN2 protein or transfecting VSMCs with a recombinant adeno-associated virus capable of highly expressing the LCN2 protein, fatty acid β-oxidation can be promoted, thereby improving the situation of vascular calcification. Based on the experimental results, it can be known that the LCN2 protein can play a role in treating vascular calcification, and therefore has good application prospects in the field of preparing related drugs. Description of the Drawings

[0021] Figure 1 Alizarin red staining photograph of VSMCs in Example 1;

[0022] Figure 2 Western Blotting result graph of VSMCs in Example 1;

[0023] Figure 3 Quantitative bar graph of LCN2 protein in VSMCs in Example 1;

[0024] Figure 4 ELISA test result bar graph of VSMCs in Example 1;

[0025] Figure 5 Photograph of LCN2 protein immunohistochemistry of the radial artery in Example 2;

[0026] Figure 6 Calcium score analysis graph of the radial artery in Example 2;

[0027] Figure 7 Alizarin red staining photograph of VSMCs treated with LCN2 protein and high phosphorus medium in Example 3;

[0028] Figure 8 Intracellular calcium content result bar graph of VSMCs treated with LCN2 protein and high phosphorus medium in Example 3;

[0029] Figure 9 Protein expression photograph of SM22α and GAPDH of VSMCs treated with LCN2 protein and high phosphorus medium in Example 3;

[0030] Figure 10 Quantitative bar graph of SM22α protein of VSMCs treated with LCN2 protein and high phosphorus medium in Example 3;

[0031] Figure 11 Bar graph of LCN2 mRNA level of VSMCs treated with LCN2 knockdown in Example 3;

[0032] Figure 12 Alizarin red staining photograph of VSMCs treated with LCN2 knockdown in Example 3;

[0033] Figure 13 Intracellular calcium content result bar graph of VSMCs treated with LCN2 knockdown in Example 3;

[0034] Figure 14 Alizarin red staining photograph of the vascular ring in Example 4;

[0035] Figure 15Quantitative bar graph of alizarin red staining of vascular rings in Example 4;

[0036] Figure 16 Photograph of immunohistochemistry of BMP2 protein in vascular rings in Example 4;

[0037] Figure 17 Quantitative bar graph of BMP2 protein in vascular rings in Example 4;

[0038] Figure 18 Photograph of alizarin red staining of thoracic aorta in Example 6;

[0039] Figure 19 Bar graph of intracellular calcium content results in aorta in Example 6;

[0040] Figure 20 Photographs of protein expression of α-SMA and β-actin in aorta in Example 6;

[0041] Figure 21 Quantitative bar graph of α-SMA protein in aorta in Example 6;

[0042] Figure 22 Bar graph of differential proteins screened by proteomics analysis in Example 7;

[0043] Figure 23 Result graph of protein pathway enrichment in Example 7;

[0044] Figure 24 Bar graph of ATP content in cells in Example 7. Detailed implementation manners

[0045] The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and essence of the present invention, modifications and substitutions made to the methods, steps or conditions of the present invention all fall within the scope of the present invention.

[0046] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0047] For the molecular biology test methods not specifically described in the following examples, they are all referred to "Molecular Cloning: A Laboratory Manual" (Third Edition) or carried out according to the kits and product specifications; the biological materials of the kits, unless otherwise specified, can all be obtained from commercial channels.

[0048] The specific operation steps of Western blot, immunohistochemistry, immunofluorescence, alizarin red staining and statistical analysis in the present invention are as follows:

[0049] Western blot: Total proteins of vascular tissues / vascular smooth muscle cells were extracted using tissue lysate, and the protein concentration was determined by the BCA method. 20 μg of protein samples were loaded, and 6% or 10% polyacrylamide gel electrophoresis (SDS-PAGE) was performed. The proteins were transferred to a PVDF membrane at 300 mA for 3 hours by wet transfer. The membrane was blocked with 5% BSA blocking solution at room temperature for 1 hour. After washing with TBST, primary antibodies, including LCN2 (Abcam), β-actin (Cell Signaling Technology), GAPDH (Cell Signaling Technology), SM22α (Proteintech), and α-SMA (Abcam), were added and incubated overnight at 4°C. The membrane was washed with TBST 3 times, 10 minutes each time, and then incubated with the corresponding secondary antibody at room temperature for 1 hour. The membrane was washed with TBST 3 times, 10 minutes each time. The antigen-antibody complex was detected by enhanced chemiluminescence (ECL), and the protein bands were exposed using an all-in-one imaging system (ChemiD MP). The target bands were quantitatively analyzed using Image J software, and the relative expression levels of the target proteins were represented by the gray values of the target bands relative to the gray values of the internal reference proteins GAPDH or β-actin.

[0050] Immunohistochemistry: Tissues were fixed with 4% paraformaldehyde for 48 hours, embedded in paraffin, sectioned, and dewaxed to water. Sodium citrate (10 mM, pH 6.0) was boiled in a pressure cooker for 10 minutes. After cooling to room temperature, the sections were blocked with goat serum at room temperature for 1 hour, and then incubated with LCN2 (Abcam) and BMP2 (Abcam) antibodies overnight at 4°C. The sections were washed with PBS 3 times, 10 minutes each time, stained with DAB, counterstained with hematoxylin, dehydrated, and sealed with neutral gum. Images were observed and collected under a microscope.

[0051] Alizarin red staining: Tissues were fixed with 4% paraformaldehyde for 48 hours, embedded in paraffin, sectioned, and dewaxed to water. The sections were baked in an oven at 37°C for 30 minutes, stained with 2% alizarin red staining solution at room temperature for 5 minutes, rinsed with flowing distilled water until the washing solution was almost colorless, dried, and sealed with neutral gum. Images were observed and collected under a microscope.

[0052] Statistical analysis: The experimental data were expressed as mean ± SEM. One-way ANOVA was used for comparison among multiple groups. 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 ****.

[0053] The exogenous LCN2 protein used in the examples was purchased from Cloud-Clone Corp., catalog number: RPB388Ra01.

[0054] Example 1: The expression and secretion of LCN2 protein were inhibited in calcified VSMCs caused by high phosphorus.

[0055] Primary rat aortic VSMCs were cultured and treated with sodium dihydrogen phosphate solution at a final concentration of 3 mM for 3, 5, and 7 days respectively. Alizarin red staining was used to evaluate the degree of cell calcification, Western Blotting was used to detect the expression of LCN2 protein in cells, and ELISA was used to detect the content of LCN2 protein in the culture medium supernatant.

[0056] Figure 1 This is the result of alizarin red staining of VSMCs. It can be seen from the results that the longer the high phosphorus culture time, the more obvious the staining effect, indicating that the more serious the VSMC calcification. Figure 2 This is the result of Western Blotting of VSMCs. The results show that the level of LCN2 protein in VSMC cells decreased significantly after 3 and 5 days of high phosphorus treatment. Figure 3 This is the quantitative graph of LCN2 protein, and the results are Figure 2 consistent with the analysis. Figure 4 This is the result of ELISA detection. The results show that the content of LCN2 protein in the culture medium did not change after 3 days of high phosphorus treatment, but the content of LCN2 protein in the culture medium decreased significantly after 5 and 7 days of high phosphorus treatment. These results suggest that both the expression and secretion of LCN2 protein were inhibited in calcified VSMC cells caused by high phosphorus.

[0057] Example 2: Analyze the expression of LCN2 in the radial artery tissue of hemodialysis patients.

[0058] The radial artery tissues of 17 hemodialysis patients who underwent internal fistula surgery were collected, and the expression of LCN2 was detected by immunohistochemistry. At the same time, the calcification score was performed according to the abdominal lateral radiograph of the patients.

[0059] Figure 5 This is the result of LCN2 immunohistochemistry. It was found that the expression of LCN2 in the radial artery tissue of patients with calcification was significantly lower than that of patients without vascular calcification. Figure 6 This is the correlation analysis result between the quantification of LCN2 immunohistochemistry and the calcification score. It can be seen from the results that the higher the calcification score of the patients, the lower the expression level of LCN2, and there is a negative correlation between the calcification score and the expression of LCN2.

[0060] Example 3: LCN2 protein can improve VSMC calcification caused by high phosphorus.

[0061] Primary cultured rat aortic VSMCs were induced to calcify by high-phosphate medium, and were treated with low concentration (20 ng / mL) and high concentration (200 ng / mL) of LCN2 protein simultaneously. After 7 days, alizarin red staining was used to evaluate the degree of cell calcification, a kit was used to detect the intracellular calcium content, and Western Blotting was used to detect the expression of the VSMC contractile phenotype protein SM22α.

[0062] Figure 7 Shown are the results of alizarin red staining. It can be seen from the results that low-concentration LCN2 protein significantly improved VSMC calcification caused by high phosphate, and the effect of high-concentration exogenous LCN2 protein was more significant. Figure 8 Shown are the results of intracellular calcium content. Consistent with the Figure 7 results, high phosphate led to an increase in intracellular calcium content, and LCN2 protein significantly improved the increase in VSMC calcium content caused by high phosphate. Figure 9 Shown are the protein expression results of cell SM22α and GAPDH. Figure 10 Shown are the quantitative results of cell SM22α protein. These results showed that high phosphate would lead to a decrease in the VSMC contractile phenotype protein SM22α, while LCN2 protein could reverse the decrease of SM22α to a certain extent.

[0063] Conversely, LCN2 siRNA was transfected into VSMCs via liposomes to knockdown LCN2, and then the cells were treated with high-phosphate medium. Alizarin red was used to evaluate the degree of cell calcification, and the calcium content in the cells was measured. Figure 11 Shown is the level of cell LCN2 mRNA detected by qRT-PCR. After transfection with LCN2 siRNA, the cell LCN2 mRNA level decreased significantly, verifying the knockdown efficiency of the siRNA. Figure 12 Shown is that transfection with LCN2 siRNA would exacerbate calcification caused by high phosphate. Figure 13 Shown are the results of intracellular calcium content, which also indicated that transfection with LCN2 siRNA would lead to a further increase in intracellular calcium content.

[0064] In summary, all these results confirmed that LCN2 protein could improve VSMC calcification caused by high phosphate.

[0065] Example 4: LCN2 protein protects against vascular ring calcification caused by high phosphate.

[0066] Rat aortic vascular rings were cultured in vitro and induced to calcify by high-phosphate medium. At the same time, they were treated with low concentration (200 ng / mL) and high concentration (600 ng / mL) of LCN2 protein. After 7 days, the vascular rings were collected, fixed with paraformaldehyde, dehydrated, embedded in paraffin and sectioned. Alizarin red staining was used to evaluate the degree of vascular ring calcification, and immunohistochemistry was used to detect the expression of the osteogenic phenotype protein BMP2.

[0067] Figure 14 This is the result of alizarin red staining. It can be seen from the results that high-phosphate treatment for 7 days can cause obvious calcification of vascular rings, while low-concentration LCN2 protein significantly improves the calcification of vascular rings caused by high phosphate, and the improvement effect of high-concentration LCN2 protein is more significant. Figure 15 This is the quantitative graph of alizarin red staining, and its results are consistent with Figure 14 the analysis. Figure 16 It shows that the expression of BMP2 protein in vascular rings is significantly increased after 7 days of high-phosphate treatment, while interference with high-concentration LCN2 protein can effectively inhibit the increase of BMP2 protein in vascular rings caused by high phosphate. Figure 17 This is the quantitative graph of BMP2 protein. All these results indicate that LCN2 protein can protect against the calcification of vascular rings caused by high phosphate and inhibit the up-regulation of osteogenic phenotype protein BMP2.

[0068] Example 5: Construction of recombinant adeno-associated virus capable of increasing the expression of LCN2 protein.

[0069] A) Preparation of adeno-associated virus vector:

[0070] Select the pHBAAV-SM22a-3flag-T2A-ZsGreen vector (purchased from Hanheng Biotechnology (Shanghai) Co., Ltd., and its nucleotide sequence is shown in SEQ ID No: 5), and the SM22a element in it is a smooth muscle-specific promoter. Design primers for PCR amplification of the LCN2 gene. Forward primer: 3’-AGCCTTGGATCCGCCACCATGGCCCTGAGTGTCAT-5’ (SEQ ID No: 3), reverse primer: 3’-AGTCGTTAATTAAACGCGTGTTGTCAATGCATTGGTCGGT-5’ (SEQ ID No: 4). Select restriction endonucleases BamH I and Mlu I to digest the vector, and recover the purified linearized vector by agarose gel electrophoresis; perform PCR on the target gene fragment according to the designed primers, and recover the target gene fragment of the correct size by agarose gel electrophoresis; ligate the linearized vector and the target gene fragment according to the HB infusion TM one-step cloning ligation system; transform the ligation product into DH5α competent cells, spread the bacterial solution on the plate, and culture for 12 - 16 hours; select monoclonal colonies for colony verification; select positive clones with correct colony verification for sequencing; perform bacterial solution amplification and plasmid extraction and purification on the clones with correct sequencing.

[0071] B) Packaging and quality detection of recombinant adeno-associated virus:

[0072] The virus packaging process is carried out using a three-plasmid adeno-associated virus system; the three-plasmid adeno-associated virus system includes the following plasmids: a vector plasmid carrying the LCN2 gene, a pAAV-RC vector plasmid, and a pHelper vector plasmid; after high-purity endotoxin-free extraction of the three plasmid vectors respectively, the three plasmids are co-transfected into AAV-293 cells using Hanheng's LipofiterTM transfection reagent; 72 hours after transfection, cell pellets are collected; the cells are lysed, cell debris is removed, and the lysate supernatant containing adeno-associated virus particles is collected; purification is carried out using the Biomiga adeno-associated virus purification kit V1469-01, the purified virus solution is collected and stored at -80 °C.

[0073] Example 6: Experiment of transfecting recombinant adeno-associated virus into CKD mice.

[0074] In patients with chronic kidney diseases (CKD), as renal function deteriorates progressively, cardiovascular diseases (CVD) become one of the main causes of death, and vascular calcification is the most common pathological manifestation among them. The mortality rate caused by vascular calcification accounts for about 30% of the total mortality rate of end-stage renal disease. Vascular calcification is a process of calcium phosphate deposition in cardiovascular diseases, which seriously endangers human health. In addition to aging, it is related to the pathological processes of various diseases such as atherosclerosis, hypertension, aortic valve stenosis, coronary artery disease, diabetes, and chronic kidney diseases, and is a powerful predictor of cardiovascular events.

[0075] Establishment of a vascular calcification model in CKD mice: Male C57BL / 6J mice weighing 25 - 27 g are taken and fed with a diet containing 2% high phosphorus and 0.25% adenine for 1 month, and then changed to a diet containing 2% high phosphorus and 0.1% adenine and continued to be fed for 4 months. To further promote the occurrence of vascular calcification, cholecalciferol is administered by intraperitoneal injection (2×10 5 IU / kg body weight), and the treatment is continued for 3 days. Four days later, the mice are sacrificed and tissues such as the aorta are collected.

[0076] The mice are randomly divided into three groups: a control group (CTR), a calcification model group (CKD), and a drug administration group (CKD+LCN2). The mice in the control group are given normal food and injected with a control virus via the tail vein; the mice in the calcification model group are induced to calcify according to the above method and injected with a control virus via the tail vein; the mice in the drug administration group are induced to calcify according to the above method and injected with the recombinant adeno-associated virus prepared in Example 5 via the tail vein.

[0077] The aortic tissues of the above grouped mice are collected, and the thoracic aorta part is subjected to alizarin red staining and micro-CT detection to evaluate the degree of vascular calcification. Figure 18This is the result of alizarin red staining of the thoracic aorta. It can be seen from the results that severe calcification was shown in the thoracic aorta of the CKD model group, and transfection with recombinant adeno-associated virus could significantly improve vascular calcification in CKD mice. Proteins were extracted from the abdominal aorta, and calcium content was measured and Western Blotting was performed for detection. Figure 19 This is the result of calcium content measurement. Similarly, the results showed that the calcium content in the aortic tissue of CKD mice was significantly increased, while transfection with recombinant adeno-associated virus significantly decreased the calcium content in the aortic tissue. Figure 20 This is the result of protein detection of α-SMA and β-actin in the aortic tissue. The protein level of α-SMA in the aortic tissue of CKD mice was significantly decreased compared with that of control mice, while transfection with recombinant adeno-associated virus could partially reverse the decrease of α-SMA protein. Figure 21 This is the quantitative graph of α-SMA protein, and the results are consistent with Figure 20 the analysis. These results verified at the animal level that overexpression of LCN2 protein could protect the blood vessels of CKD mice and prevent further calcification.

[0078] Example 7: The mechanism by which LCN2 protein improves calcification of VSMC caused by high phosphorus.

[0079] To further explore the mechanism of action of LCN2 protein in protecting VSMC from calcification, a proteomics experiment was designed to analyze the changes in VSMC cell proteins after treatment with high phosphorus and LCN2 protein. The treatment was similar to that in Example 3. Primary rat aortic VSMCs were cultured, calcification was induced by high-phosphorus medium, and at the same time, the cells were treated with 200 ng / mL of LCN2 protein. After 7 days, the cells were collected for proteomics analysis.

[0080] Figure 22 These are the differentially expressed proteins screened by proteomics analysis. Figure 23 This is the result of enrichment of the protein pathways upregulated in the high phosphorus + LCN2 group compared with the high phosphorus group. It can be seen from the results that after treatment with LCN2 protein, the fatty acid β-oxidation pathway was mainly activated. The products of fatty acid β-oxidation can enter the citric acid cycle to generate energy. Therefore, fatty acid β-oxidation is an important source of cell energy metabolism. Therefore, we detected the ATP content in the cells using a kit, and the results are as Figure 24 shown. High phosphorus led to a significant decrease in the ATP content in VSMC cells, and high-concentration LCN2 protein could significantly increase the ATP content in the cells. These results all suggest that LCN2 protein can improve the calcification of VSMC caused by high phosphorus by enhancing the fatty acid β-oxidation level in VSMC cells and promoting the production of cell ATP.

[0081] It is obvious to those skilled in the art that the present invention is not limited to the details of the above-described exemplary embodiments, and that the present invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the present invention. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, it is intended to embrace all changes that fall within the meaning and scope of the equivalent elements of the claims within the present invention.

Claims

1. The use of Lipocalin-2 protein in the preparation of a drug for treating vascular calcification, characterized in that: The concentration of the Lipocalin-2 protein is 20-600 ng / mL, and the amino acid sequence of the Lipocalin-2 protein is shown in SEQ ID No:

1.

2. The application according to claim 1, characterized in that: The vascular calcification is caused by hypertension, diabetes, chronic kidney disease or atherosclerosis.

3. Use of a recombinant adeno-associated virus for increasing the expression level of Lipocalin-2 protein in the preparation of a drug for treating vascular calcification, characterized in that: The recombinant adeno-associated virus comprises a vector skeleton and a target gene fragment, wherein the vector skeleton has a smooth muscle-specific promoter and a fluorescent marker, and the target gene fragment encodes Lipocalin-2 protein, and the amino acid sequence of the Lipocalin-2 protein is shown in SEQ ID No:

1.

4. The use according to claim 3, characterized in that: The method for constructing the recombinant adeno-associated virus comprises the steps of: A) constructing a recombinant adeno-associated virus vector, wherein the primer sequences used to amplify the target gene fragment during the construction of the vector are shown in SEQ ID No: 3 and SEQ ID No: 4; B) Adeno-associated virus packaging; Wherein, the recombinant adeno-associated virus contains the target gene fragment.

Images