Use of roxburghii polysaccharide in preparing a drug for resisting vascular calcification

Abstract

The invention discloses a use of roxburghii polysaccharide for preparing a drug for resisting vascular calcification. Experimental results show that the roxburghii polysaccharide in the invention has an effect of resisting vascular calcification. Therefore, the roxburghii polysaccharide in the invention has the prospect of being prepared into a drug for resisting vascular calcification, treating diseases caused by vascular calcification or diseases accompanied by vascular calcification, and the diseases accompanied by vascular calcification include atherosclerosis, hypertension and diabetic vascular lesions.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and particularly relates to use of roxburghii polysaccharide in preparing a drug for resisting vascular calcification. Background Art

[0002] Vascular calcification is a systemic vascular disease characterized by the uneven deposition of minerals such as hydroxyapatite in morphology and chemical composition, which leads to decreased elasticity of the vascular wall and damaged vascular structural integrity, thus triggering a series of dynamic vascular lesions. Diseases such as atherosclerosis, hypertension, and diabetic vascular disease are generally accompanied by significant vascular calcification.

[0003] Rosa roxburghii is a plant of the Rosaceae family. It is a precious plant widely distributed in the mountainous areas of southwest and south-central China. Its fruit is called rosa roxburghii. As early as 1640, the "Guizhou Chronicles" written by Tian Wen of the Ming Dynasty recorded rosa roxburghii. Its fruit is similar to pomegranate, but smaller than pomegranate. The "Supplement to Compendium of Materia Medica" describes it as an edible wild fruit with a slightly sour taste that can help digestion. In addition, other parts of rosa roxburghii, such as flowers, leaves, roots, and seeds, can be used as medicine to treat a variety of diseases. Rosa roxburghii fruit is rich in various nutrients, including polysaccharides, ascorbic acid, phenols, and superoxide dismutase.

[0004] Polysaccharides are sugar chains connected by glycosidic bonds, polymerized sugars composed of more than 10 monosaccharides, high molecular carbohydrates, with complex structures and a wide variety of types. Currently, there are hundreds of active polysaccharides that have been discovered, which can be divided into five categories according to their sources: plant polysaccharides, fungal polysaccharides, algae polysaccharides, animal polysaccharides and bacterial polysaccharides.

[0005] The inventors isolated a polysaccharide from Rosa roxburghii (patent application number: 202410729810.4), code-named CLP2-2, which was identified as a homopolymer of galacturonic acid with a structural formula of [→4)α-GalAp-(1→]n and a relative molecular mass of 78.6 kDa.

[0006] The inventors discovered that the roxburghii polysaccharide CLP2-2 has multiple biological activities, and thus proposed the present invention. Summary of the invention

[0007] The object of the present invention is to provide a use of roxburghii polysaccharide for preparing a drug for resisting vascular calcification.

[0008] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0009] A roxburghii polysaccharide with anti-vascular calcification effect, the preparation method comprising the following steps:

[0010] Step S1, polysaccharide extraction: remove the seeds from fresh and ripe roxburghii fruit, rinse it, crush it, air-dry it, and defatted it with an organic solvent to obtain defatted powder; take the defatted powder, add an appropriate amount of water, adjust the pH of the solution to weak acidity, add an appropriate amount of papain, stir and extract it at 50-70° C. for a proper time, and inactivate the enzyme; after cooling, adjust the pH of the solution to weak alkalinity, add an appropriate amount of trypsin, stir and extract it at 30-45° C. for a proper time, and inactivate the enzyme; after cooling, centrifuge to take the supernatant, dialyze for desalination, and freeze-dry to obtain enzyme-extracted roxburghii crude polysaccharide;

[0011] Step S2, polysaccharide purification: taking an appropriate amount of enzyme-extracted roxburghii crude polysaccharide, adding an appropriate amount of water to dissolve, centrifuging to remove insoluble matter, separating the supernatant through a DEAE cellulose anion column, eluting with water, 0.1MNaCl solution and 0.2MNaCl solution in sequence, detecting with sulfuric acid-phenol, collecting and combining the 0.2MNaCl eluate, dialysis desalting, freeze-drying, and obtaining roxburghii polysaccharide CLP2; taking an appropriate amount of roxburghii polysaccharide CLP2, adding an appropriate amount of water to dissolve, purifying through a Bio-Gel P-6Gel gel chromatography column, eluting with water, drawing an elution curve by a sulfuric acid-phenol method, combining the eluates corresponding to the main peak of the elution curve, and freeze-drying to obtain.

[0012] Preferably, the organic solvent in step S1 is anhydrous ethanol.

[0013] Preferably, the DEAE cellulose anion column in step S2 is a Cl-type DEAE cellulose anion column.

[0014] The roxburghii polysaccharide is used for preparing a drug for resisting vascular calcification.

[0015] The roxburghii polysaccharide is used for preparing medicines for treating diseases caused by vascular calcification.

[0016] The roxburghii polysaccharide is used for preparing medicines for treating diseases accompanied by vascular calcification.

[0017] Preferably, the diseases accompanied by vascular calcification include atherosclerosis, hypertension and diabetic vasculopathy.

[0018] Beneficial effects:

[0019] The experimental results show that the roxburghii polysaccharide in the present invention has an effect of resisting vascular calcification. Therefore, the roxburghii polysaccharide in the present invention has an effect of resisting vascular calcification. Therefore, the roxburghii polysaccharide in the present invention has a prospect of being prepared into a drug for resisting vascular calcification, treating diseases caused by vascular calcification or diseases accompanied by vascular calcification, and the diseases accompanied by vascular calcification include atherosclerosis, hypertension and diabetic vascular lesions, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1This is the IR graph of the roxburghii polysaccharide CLP2-2;

[0021] Figure 2 Rosa roxburghii polysaccharide CLP2-2 13 C NMR spectra (A) and HSQC spectra (B);

[0022] Figure 3 is the expression level of marker genes in RASMCs cells of each group;

[0023] Figure 4 Calcium content and alkaline phosphatase activity in RASMCs cells of each group;

[0024] Figure 5 The results of Alizarin red staining of RASMCs cells in each group;

[0025] Figure 6 is the expression level of SM22α protein in RASMCs cells of each group;

[0026] Figure 7 is the expression level of marker genes in the aorta of mice in each group;

[0027] Figure 8 is the alkaline phosphatase activity in the aorta of mice in each group;

[0028] Fig. 9 is the expression level of SM22α protein in RASMCs cells in each group. DETAILED DESCRIPTION

[0029] The essential contents of the present invention are described in detail below in conjunction with the embodiments, but the protection scope of the present invention is not limited thereto.

[0030] Example 1: Preparation of Rosa roxburghii polysaccharide CLP2-2

[0031] Step S1, polysaccharide extraction: remove the seeds from the fresh and ripe roxburghii fruit, rinse it, crush it and air-dry it, add 10 times the volume (1g:10mL) of anhydrous ethanol after passing through a 100-mesh sieve, and degrease it by hot reflux in a water bath at 70°C for 3h, discard the solvent, and degrease the powder again; dry the defatted powder to obtain defatted powder. Take 25g of defatted powder, add 30 times the volume (1g:30mL) of deionized water, stir, adjust the solution pH to 6 with hydrochloric acid solution, then add 2.5% of the defatted powder by weight of papain (10U / mg), stir and extract at 60°C for 12h, and then inactivate the enzyme at 100°C for 15min. After cooling, adjust the pH to 8 with sodium hydroxide solution, add 2.5% trypsin (≥250N.FU / g) of defatted powder, stir and extract at 37°C for 12h, then inactivate the enzyme at 100°C for 15min, cool, centrifuge to obtain the supernatant, concentrate, dialyze for desalination, concentrate again, and freeze-dry the concentrate to obtain 3.31g of enzyme-extracted Rosa roxburghii crude polysaccharide.

[0032] Step S2, polysaccharide purification: 1 g of the crude polysaccharide extracted from roxburghii roxburghii was dissolved in 30 mL of deionized water, and the insoluble matter was removed by centrifugation. The supernatant was passed through a DEAE cellulose anion column (Cl - Type) was initially separated, eluted with distilled water, 0.1M NaCl solution and 0.2M NaCl solution in turn, detected by sulfuric acid-phenol, and the 0.2M NaCl eluate was collected and combined, concentrated, dialyzed for desalination, and freeze-dried to obtain 125 mg of roxburghii polysaccharide CLP2. 100 mg of roxburghii polysaccharide CLP2 was dissolved in 2 mL of deionized water and centrifuged. After the supernatant was passed through a mixed cellulose membrane, it was purified by a Bio-Gel P-6 Gel gel chromatography column, eluted with deionized water, and the flow rate was 0.3 mL / min. The elution curve was drawn by the sulfuric acid-phenol method, and the eluate corresponding to the main peak of the elution curve was combined (the elution curve had only one main peak), concentrated, and the purification was repeated twice, concentrated and dried to obtain the polysaccharide component CLP2-2.

[0033] Structural analysis of polysaccharide CLP2-2: HPGPC analysis showed that the molecular weight of CLP2-2 was 78.6 kDa. Monosaccharide composition analysis showed that CLP2-2 mainly contained galacturonic acid (83.5%). The infrared spectrum showed that 3279 cm -1 OH stretching vibration absorption peak, 1000-1400cm -1 Nearby are the vibration signals of CO and sugar ring, 1597cm -1 There is an obvious absorption peak at Figure 1 ).

[0034] 13CNMR and HSQC spectra showed that the terminal carbon signal at δ98.95 was the C1 signal of galacturonic acid and indicated an α configuration, while δ77.86 indicated that the C4 position of galacturonic acid was substituted, and δ175.29 was the signal of uronic acid substitution at the C6 position ( Figure 2 ), and the C / H hydrogen signals of CLP2-2 were assigned, and the results are shown in Table 1:

[0035] Table 1 CLP2-2 NMR signal attribution

[0036]

[0037] The comprehensive monosaccharide composition, infrared and nuclear magnetic resonance data showed that the roxburghii polysaccharide CLP2-2 was homogalacturonic acid with the structure: [→4)α-GalAp-(1→]n.

[0038] Example 2: Anti-vascular calcification activity of roxburghii polysaccharide CLP2-2 (cellular level)

[0039] 1. Test Materials

[0040] 1. Instruments and reagents

[0041] Instruments: clean bench, centrifuge (Thermo), cell incubator, electrophoresis tank (Biotek), Nano-100 micro-spectrophotometer, gradient PCR instrument, fully automatic fluorescence quantitative PCR instrument, etc.

[0042] Reagents: DMEM high glucose medium (Nanjing Keygen Biotechnology Co., Ltd.), Brazilian fetal bovine serum, trypsin digestion solution (Gibco), rat type I collagenase, CLP2-2 prepared according to the method in Example 1 (purity>98%), alkaline phosphatase activity biochemical kit purchased from Solebao Biotechnology Co., Ltd., and primer synthesis purchased from Sangon Biotechnology Co., Ltd.

[0043] 2. Cells and Animals

[0044] Primary aortic smooth muscle cells (RASMCs) (isolated from 6-8 week old rats).

[0045] 2. Test methods

[0046] 1. Extraction of primary aortic smooth muscle cells (RASMCs)

[0047] A rat weighing about 200 g was euthanized, and then disinfected by immersion in 75% alcohol. The chest cavity was quickly opened to expose the heart. After perfusion with sterile PBS, the aorta was removed in a sterile bench. The aortic adventitia was peeled off under a microscope, and then the aorta was cut open, and the aortic intima layer was gently scraped off with a cotton swab. Primary vascular smooth muscle cells were obtained by the patch method. The aorta was cut into small pieces of about 1 mm × 1 mm with microscissors, placed in a dish containing 0.1% collagenase, and digested for 3-4 hours. After digestion, an equal amount of serum was added to neutralize the collagenase, centrifuged at 2000rpm / min for 5 minutes, the supernatant was discarded, and the precipitate was blown evenly with a culture medium containing 10% fetal bovine serum, added to a dish containing gelatin, and placed in a cell culture incubator. After 2 days of culture, smooth muscle cells can be seen crawling out around the tissue block. After gently changing the medium, continue to culture, and change the medium every two days until the cells are fused to 90% in the dish, and subsequent subculture operations can be performed.

[0048] 2. Cell culture, grouping, modeling and drug administration

[0049] The RASMCs cells were cultured at a rate of 1×10 5 / well were inoculated into 6-well plates and divided into control group (Control), model group (High Pi) and drug-treated group (CLP2-2). When the cells grew and merged to about 60%, the model group (High Pi) was replaced with a medium containing 3mM phosphate (using the high phosphorus method to induce vascular smooth muscle cell calcification), the drug-treated group (CLP2-2) was replaced with a medium containing 3mM phosphate and 25μg / ml or 100μg / ml polysaccharide CLP2-2, and the control group (Control) was replaced with a DMEM high-glucose medium containing only 10% fetal bovine serum, and cultured for 7 days, with the medium changed every 2 days.

[0050] 2. PCR detection of marker gene changes in cells

[0051] PCR experiments were used to detect changes in the mRNA levels of osteogenic transcription factors and bone morphogenetic protein 2, which are marker genes related to calcification of vascular smooth muscle cells. The cells were discarded from the culture medium, washed twice with PBS, and 1 ml of Trizol was added to each well. The cells were blown several times to completely detach. 200 μl of chloroform was added, inverted for 30 seconds, and allowed to stand at room temperature for 5 minutes. Centrifuged at 12,000 rpm for 15 minutes, 500 μl of the upper layer solution was taken and placed in a new Ep tube, and an equal volume of isopropanol was added. The mixture was inverted and mixed, and allowed to stand at 4°C for 2-3 hours. Centrifuged at 4°C, 12,000 rpm for 10 minutes, and the supernatant was carefully aspirated. Be careful not to touch the substrate RNA precipitate. 1 ml of pre-cooled 75% ethanol was added, and the substrate RNA precipitate was gently flicked to wash the RNA. Centrifuged at 4°C, 12,000 rpm for 10 minutes, and the supernatant was discarded again. The Ep tube was inverted at room temperature, and naturally air-dried for 5 minutes. An appropriate amount of DEPC water was added to dissolve the RNA. Pipette 1 μl of dissolved RNA and measure the concentration and purity of RNA using Nano-100 micro-spectrophotometer. Set up the reverse transcription program according to the reverse transcription kit requirements, configure the reverse transcription system, and synthesize cDNA. Store the synthesized cDNA at -20℃.

[0052] Primers were designed using the Gene column of the NCBI website. The primer sequences are as follows:

[0053]

[0054]

[0055] According to the reaction ratio recommended in the reagent manual, the reaction system was prepared: 5μl SYBR Green I Master, 1μl Primer, 1μl cDNA, and 1μl DEPC water per well. After the preparation, the system was added to the PCR plate for RT-qPCR reaction. The data were quantified using the Threshold cycle (Ct) method, with 18s as the internal reference. The expression of the target gene was calculated according to Formula 2 -[(Ct of gene)-(Ct of 18S rRNA)] to calculate.

[0056] 3. Measurement of calcium content in vascular smooth muscle cells

[0057] Discard the culture medium from the cells, wash twice with PBS, add 100 μl of lysis solution containing protease inhibitors to each well, scrape all cells with a cell scraper, put in ice for 30 minutes, centrifuge at 12000 rpm for 15 minutes, and collect the supernatant. Take a small amount of supernatant to determine the protein content according to the BCA protein content kit, and use it to correct the protein concentration of the biochemical kit. According to the instructions of the calcium content kit, take an appropriate amount of protein supernatant for detection.

[0058] 4. Measurement of alkaline phosphatase activity in vascular smooth muscle cells

[0059] Discard the culture medium from the cells, wash twice with PBS, add 100 μl of lysis solution containing protease inhibitors to each well, scrape all the cells with a cell scraper, put it in ice for 30 minutes, centrifuge it at 12000 rpm for 15 minutes, and collect the supernatant. Take a small amount of supernatant to determine the protein content according to the BCA protein content kit, and use it to correct the protein concentration in the biochemical kit. According to the instructions of the alkaline phosphatase activity kit, take an appropriate amount of protein supernatant for detection. High alkaline phosphatase activity indicates an increase in the degree of cell calcification.

[0060] 5. Cell Alizarin Red Staining

[0061] Alizarin red staining of cells is used to characterize the calcium salt content in cells. Alizarin red staining solution can chelate with calcium salts in calcium carbonate or calcium phosphate to form red or orange-red complexes. The darker the color, the more calcium salt content. Remove the cell culture medium, wash the cells twice with PBS, fix the cells with 4% paraformaldehyde at room temperature for 15 minutes, discard the fixative, and wash twice with double distilled water. Completely dry the residual liquid, add alizarin red staining solution (0.2% PH 8.3) and stain at room temperature for 30 minutes, discard the dye, wash 3 times with double distilled water, add a small amount of double distilled water to each well to prevent drying, and observe and take pictures.

[0062] 6. Western blot to observe changes in protein levels

[0063] Western blotting was used to detect the changes in SM22α protein expression levels. The cell culture medium was removed, the cells were washed twice with PBS, and the total protein was extracted with loading buffer or RIPA protein lysis buffer containing protease phosphatase inhibitors. After the total protein was quantified by BCA method, the protein sample was boiled at 99℃ for 5min, and the equal amount of protein sample after boiling and cooling was loaded into the polyacrylamide gel well. After electrophoresis at 80V voltage for 30 minutes in electrophoresis buffer, it was switched to 120V voltage for 1 hour, and then transferred to the membrane at 350mA current for 70 minutes in wet transfer buffer. After the transfer was completed, the membrane was blocked at room temperature for 2h with blocking solution containing 5% skim milk powder. After blocking, the primary antibody (SM22α: Proteintech-60213-1-Ig) was prepared according to the antibody instructions and incubated at 4℃ overnight. The next day, the membrane was washed three times at room temperature with TBST washing solution, each time for 10min. The secondary antibody labeled with horseradish peroxidase diluted in the blocking solution was added and incubated at room temperature for 2 hours. The membrane was washed four times at room temperature with washing solution, each time for 15min. Finally, the film was incubated with developer and then developed.

[0064] 7. Statistical analysis

[0065] The data were analyzed and processed using GraphPad Prism statistical graphics software. The statistics were performed using the One-way analysis of variance (ANOVA) method, and p < 0.05 was considered to be statistically significant.

[0066] 3. Experimental Results

[0067] The expression levels of marker genes in RASMCs cells of each group are as follows Figure 3 As shown in Figure 2, the representative changes of RASMCs cell calcification are the upregulation of osteogenesis-related genes, such as RUNX2 and BMP2. Figure 3 As can be seen from A and B, compared with the control group, the expression levels of RUNX2 and BMP2 in the model group were significantly upregulated, indicating that the high phosphorus-induced smooth muscle cell calcification model was successfully established. Compared with the model group, the expression levels of RUNX2 and BMP2 in the drug-treated group were significantly downregulated, indicating that the roxburghii polysaccharide CLP2-2 significantly improved smooth muscle cell calcification.

[0068] Calcium content and alkaline phosphatase activity in RASMCs cells of each group Figure 4 As shown in Figure 2, the representative changes of RASMCs calcification are increased calcium content and up-regulated alkaline phosphatase activity in cells. Figure 4 As can be seen from A and B, compared with the control group, the calcium content and alkaline phosphatase activity in the model group were significantly upregulated, indicating that the high phosphorus-induced smooth muscle cell calcification model was successfully established. Compared with the model group, the calcium content and alkaline phosphatase activity in the drug administration group were significantly downregulated, indicating that the roxburghii polysaccharide CLP2-2 significantly improved smooth muscle cell calcification.

[0069] Alizarin red staining is a classic staining method for calcification. Figure 5 It can be seen that compared with the control group, the red granule deposition in the model group increased significantly after staining, indicating that the high phosphorus-induced smooth muscle cell calcification model was successfully established and obvious calcium salt deposition appeared in the cells. Compared with the model group, the calcium salt staining particles in the drug administration group were significantly reduced, indicating that the roxburghii polysaccharide CLP2-2 significantly improved the calcification of smooth muscle cells.

[0070] The expression levels of SM22α protein in RASMCs cells in each group were as follows Figure 6 As shown. SM22α is a marker of vascular smooth muscle cell contraction and is strongly expressed in normal RASMCs. Figure 6 It can be seen that compared with the control group, the expression level of SM22α protein in the model group was significantly downregulated, indicating that RASMCs cells underwent phenotypic transformation, induced calcification, decreased normal contractile function of cells, and the high-phosphate-induced smooth muscle cell calcification model was successfully established. Compared with the model group, the expression level of SM22α protein in the drug-treated group was significantly upregulated, indicating that the roxburghii polysaccharide CLP2-2 significantly improved smooth muscle cell calcification.

[0071] The above experimental results show that the roxburghii polysaccharide CLP2-2 has the effect of resisting vascular smooth muscle cell calcification and can effectively improve the contractile phenotype of vascular smooth muscle cells.

[0072] Example 3: Anti-vascular calcification activity of roxburghii polysaccharide CLP2-2 (animal level)

[0073] 1. Test Materials

[0074] 1. Instruments and reagents

[0075] MP120-1 electronic scale (Shanghai Second Balance Instrument Factory), animal surgical instruments, Nano-100 micro-spectrophotometer, gradient PCR instrument, fully automatic fluorescence quantitative PCR instrument, high-throughput tissue homogenizer, high-speed centrifuge.

[0076] 0.9% physiological saline was purchased from Sinopharm Group, CLP2-2 was prepared according to the method in Example 1 (purity>98%), alkaline phosphatase activity biochemical kit was purchased from Solebao Biological Company, primer synthesis was purchased from Sangon Biotechnology Co., Ltd., and tribromoethanol was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0077] 2. Experimental Animals

[0078] Forty-five C57 male mice were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.

[0079] 2. Test methods

[0080] 1. Animal grouping, vascular calcification model establishment and drug administration

[0081] 6-8 week old SPF grade C57BL / 7 male mice were selected and randomly divided into sham control group (Sham), model group (5 / 6Nx), CLP2-2 administration group (5 / 6Nx+CLP2-2, 4mg / kg / d ig), 15 mice in each group. Model group and CLP2-2 administration group: After C57BL / 7 mice were anesthetized, the mice were fixed in the left lateral position, the skin of the left kidney was disinfected with iodine, an incision of about 1 cm long was made in the left kidney, the subcutaneous tissue was bluntly separated, the left kidney was exposed, the kidney capsule was torn off, and the upper and lower 1 / 3 of the kidney were ligated with 4-0 silk thread. After waiting for 5 minutes, the upper and lower poles of the kidney were cut off with sterile scissors, the kidney tissue was returned, and the muscle and skin tissue were sutured layer by layer. One week later, after anesthesia, C57BL / 7 mice were fixed in the right lateral position, and the right kidney skin was disinfected with iodine to expose the renal tissue. The renal artery was ligated with 4-0 silk thread. After waiting for 5 minutes, the right kidney was cut off, and the muscle and skin tissue were sutured layer by layer. The feed of the mice in the model group and the CLP2-2 administration group was changed to a feed containing 1.8% phosphate for 10 weeks. Sham group mice: After anesthesia, C57BL / 7 mice were made with an incision of about 1 cm in length in the kidneys on both sides, without ligating the renal tissue, and the muscle and skin tissue were sutured layer by layer. After surgery, the mice in the Sham group were fed with standard feed for 10 weeks and had free access to water. Administration: Administration began two weeks after recovery after surgery. The mice in the CLP2-2 administration group were orally gavaged with CLP2-2 solution according to the weight of the mice every day, and the dosage was maintained at 4 mg / kg / d; at the same time, the mice in the Sham group and the model group were orally gavaged with the same volume of sterile water as the mice in the CLP2-2 group every day, and the administration time was a total of 8 weeks.

[0082] 2. Measurement of vascular alkaline phosphatase activity in the aorta

[0083] After the mice were anesthetized and blood was taken, they were euthanized. The abdominal cavity was opened immediately after the mice died, and the mouse aorta was removed under a stereo microscope to remove excess fat tissue. Lysis solution containing protease inhibitors was added to the aorta, homogenized in a high-throughput tissue homogenizer for 5 minutes, inserted into ice and allowed to stand for 30 minutes, centrifuged at 12000rpm for 15 minutes, and the supernatant was collected. A small amount of supernatant was taken to determine the protein content according to the BCA protein content kit, and used for the biochemical kit to correct the protein concentration. According to the instructions of the alkaline phosphatase activity kit, an appropriate amount of protein supernatant was taken for detection.

[0084] 3. PCR detection of changes in vascular calcification marker genes

[0085] PCR experiments were used to detect changes in the mRNA levels of osteogenic transcription factors and bone morphogenetic protein 2, marker genes related to calcification of vascular smooth muscle cells. 1 ml of Trizol was added to one aorta, and the aortic tissue was cut as much as possible and homogenized in a high-throughput tissue homogenizer for 5 minutes. 200 μl of chloroform was added, and the tube was inverted for 30 seconds, and then allowed to stand at room temperature for 5 minutes. Centrifuged at 12,000 rpm for 15 minutes, 500 μl of the upper layer solution was taken, placed in a new Ep tube, and an equal volume of isopropanol was added, and the tube was inverted and mixed, and allowed to stand at 4°C for 2-3 hours. Centrifuged at 4°C, 12,000 rpm for 10 minutes, and the supernatant was carefully aspirated, and the substrate RNA precipitate was not touched. 1 ml of pre-cooled 75% ethanol was added, and the substrate RNA precipitate was gently flicked to wash the RNA. Centrifuged at 4°C, 12,000 rpm for 10 minutes, and the supernatant was discarded again. The Ep tube was inverted at room temperature, and naturally air-dried for 5 minutes. An appropriate amount of DEPC water was added to dissolve the RNA. Pipette 1 μl of dissolved RNA and use Nano-100 micro-spectrophotometer to measure the concentration and purity of RNA. Set up the reverse transcription program according to the requirements of the reverse transcription kit, configure the reverse transcription system, and synthesize cDNA. Store the synthesized cDNA at -20℃. Design primers using the Gene column of the NCBI website. The primer sequences are as follows:

[0086]

[0087] According to the reaction ratio recommended in the reagent manual, the reaction system was prepared: 5μl SYBR Green I Master, 1μl Primer, 1μl cDNA, and 1μl DEPC water per well. After the preparation, the system was added to the PCR plate for RT-qPCR reaction. The data were quantified using the Threshold cycle (Ct) method, with 18s as the internal reference. The expression of the target gene was calculated according to Formula 2 -[(Ct of gene)-(Ct of 18S rRNA)] To calculate

[0088] 4. Western blot to observe changes in protein levels

[0089] Western blotting experiments were used to detect changes in the expression levels of α-SMA and SM22α proteins. A whole aorta was isolated and ground with a high-throughput grinder. Total protein was extracted with a loading buffer or RIPA protein lysis buffer containing protease phosphatase inhibitors. After the total protein was quantified by the BCA method, an equal amount of protein sample was taken after boiling and cooling and loaded into the polyacrylamide gel well. After electrophoresis at 80V for 30 minutes in the electrophoresis buffer, it was switched to 120V for 1 hour, and then transferred to the membrane in a wet transfer buffer at 350mA for 70 minutes. After the transfer was completed, the membrane was blocked at room temperature for 2 hours with a blocking solution containing 5% skim milk powder. After the blocking was completed, the primary antibody (NEU1: Proteintech-67032-1-Ig; α-SMA: CST-19245S, SM22α: Proteintech-60213-1-Ig) was prepared according to the antibody instructions and incubated overnight at 4°C. The next day, the membrane was washed three times at room temperature with TBST, each time for 10 minutes. Add horseradish peroxidase-labeled secondary antibody diluted in blocking solution and incubate at room temperature for 2 hours. Wash four times at room temperature with washing solution, each time for 15 minutes. Finally, incubate with developer solution and develop.

[0090] 5. Statistical analysis

[0091] The data were analyzed and processed using GraphPad Prism statistical graphics software. The statistics were performed using the One-way analysis of variance (ANOVA) method, and p < 0.05 was considered to be statistically significant.

[0092] 3. Experimental Results

[0093] The expression levels of marker genes in the aorta of mice in each group are as follows Figure 7 As shown in Figure 2. The representative changes of vascular calcification are the upregulation of osteogenesis-related genes, such as RUNX2 and BMP2. Figure 7 As can be seen from A and B, compared with the control group, the expression levels of RUNX2 and BMP2 in the model group were significantly upregulated, indicating that the vascular calcification model of mice induced by 5 / 6Nx and high-phosphorus diet was successfully established. Compared with the model group, the expression levels of RUNX2 and BMP2 in the drug-treated group were significantly downregulated, indicating that the roxburghii polysaccharide CLP2-2 significantly improved the aortic calcification of mice.

[0094] The alkaline phosphatase activity in the aorta of mice in each group was Figure 8 As shown in Figure 2. The representative change of vascular calcification is the upregulation of alkaline phosphatase activity in blood vessels. Figure 8It can be seen that compared with the control group, the alkaline phosphatase activity in the model group was significantly upregulated, indicating that the vascular calcification model of mice induced by 5 / 6Nx and high-phosphorus diet was successfully established. Compared with the model group, the alkaline phosphatase activity in the drug-treated group was significantly downregulated, indicating that the roxburghii polysaccharide CLP2-2 significantly alleviated smooth muscle cell calcification.

[0095] SM22α is a marker of vascular smooth muscle cell contraction. The expression levels of SM22α protein in RASMCs cells in each group are shown in Fig. 9 As shown. SM22α is strongly expressed in normal blood vessels. Fig. 9 It can be seen that compared with the control group, the expression level of SM22α protein in the model group was significantly downregulated, indicating that the aortic blood vessels of the mice in the model group underwent phenotypic transformation, induced calcification, decreased normal vascular contraction function, and the vascular calcification model of mice induced by 5 / 6Nx and high-phosphorus diet was successfully established. Compared with the model group, the expression level of SM22α protein in the drug-treated group was significantly upregulated, indicating that the roxburghii polysaccharide CLP2-2 significantly improved the aortic calcification of mice induced by 5 / 6Nx and high-phosphorus diet.

[0096] The above experimental results show that the sea buckthorn polysaccharide CLP2-2 has an anti-vascular calcification effect, can effectively improve the aortic calcification of mice induced by 5 / 6Nx and high-phosphorus diet, and can effectively alleviate the weakened vascular contraction function caused by calcification.

[0097] In summary, the roxburghii polysaccharide in the present invention has an effect of resisting vascular calcification. Therefore, the roxburghii polysaccharide in the present invention has an effect of resisting vascular calcification. Therefore, the roxburghii polysaccharide in the present invention has a prospect of being prepared into a drug for resisting vascular calcification, treating diseases caused by vascular calcification or diseases accompanied by vascular calcification, and the diseases accompanied by vascular calcification include atherosclerosis, hypertension and diabetic vascular disease, etc.

[0098] The purpose of the above-mentioned embodiments is to specifically introduce the essential content of the present invention, but those skilled in the art should know that the protection scope of the present invention should not be limited to the specific embodiments.

Claims

1. A use of roxburghii polysaccharide with anti-vascular calcification effect for preparing an anti-vascular calcification drug; wherein: The preparation method of the roxburghii polysaccharide comprises the following steps: Step S1, polysaccharide extraction: remove the seeds from fresh and ripe roxburghii fruit, rinse it, crush it, air-dry it, and defatted it with an organic solvent to obtain defatted powder; take the defatted powder, add water, adjust the pH of the solution to weak acidity, add papain, stir and extract it at 50-70°C, and inactivate the enzyme; after cooling, adjust the pH of the solution to weak alkalinity, add trypsin, stir and extract it at 30-45°C, and inactivate the enzyme; after cooling, centrifuge to take the supernatant, dialyze for desalination, and freeze-dry to obtain enzyme-extracted roxburghii crude polysaccharide; Step S2, polysaccharide purification: take the crude polysaccharide of roxburghii extracted by enzyme, add water to dissolve, centrifuge to remove insoluble matter, separate the supernatant through a DEAE cellulose anion column, elute with water, 0.1M NaCl solution and 0.2M NaCl solution in sequence, detect with sulfuric acid-phenol, collect and combine the 0.2M NaCl eluate, dialyze for desalination, freeze-dry, and obtain roxburghii polysaccharide CLP2; take roxburghii polysaccharide CLP2 and dissolve it in water, purify it through a Bio-Gel P-6 Gel gel chromatography column, elute with water, draw an elution curve by a sulfuric acid-phenol method, combine the eluates corresponding to the main peak of the elution curve, and freeze-dry to obtain.

2. The use according to claim 1, characterized in that: The organic solvent in step S1 is anhydrous ethanol.

3. The use according to claim 1, characterized in that: The DEAE cellulose anion column in step S2 is Cl - Type DEAE cellulose anion column.

4. Use of roxburghii polysaccharide with anti-vascular calcification effect for preparing a drug for treating diseases caused by vascular calcification; wherein: The preparation method of the roxburghii polysaccharide comprises the following steps: Step S1, polysaccharide extraction: remove the seeds from fresh and ripe roxburghii fruit, rinse it, crush it, air-dry it, and defatted it with an organic solvent to obtain defatted powder; take the defatted powder, add water, adjust the pH of the solution to weak acidity, add papain, stir and extract it at 50-70°C, and inactivate the enzyme; after cooling, adjust the pH of the solution to weak alkalinity, add trypsin, stir and extract it at 30-45°C, and inactivate the enzyme; after cooling, centrifuge to take the supernatant, dialyze for desalination, and freeze-dry to obtain enzyme-extracted roxburghii crude polysaccharide; Step S2, polysaccharide purification: take the crude polysaccharide of roxburghii extracted by enzyme, add water to dissolve, centrifuge to remove insoluble matter, separate the supernatant through a DEAE cellulose anion column, elute with water, 0.1M NaCl solution and 0.2M NaCl solution in sequence, detect with sulfuric acid-phenol, collect and combine the 0.2M NaCl eluate, dialyze for desalination, freeze-dry, and obtain roxburghii polysaccharide CLP2; take roxburghii polysaccharide CLP2 and dissolve it in water, purify it through a Bio-Gel P-6 Gel gel chromatography column, elute with water, draw an elution curve by a sulfuric acid-phenol method, combine the eluates corresponding to the main peak of the elution curve, and freeze-dry to obtain.

5. The use according to claim 4, characterized in that: The organic solvent in step S1 is anhydrous ethanol.

6. The use according to claim 4, characterized in that: The DEAE cellulose anion column in step S2 is Cl - Type DEAE cellulose anion column.

7. Use of roxburghii polysaccharide with anti-vascular calcification effect for preparing a drug for treating diseases accompanied by vascular calcification; wherein: The preparation method of the roxburghii polysaccharide comprises the following steps: Step S1, polysaccharide extraction: remove the seeds from fresh and ripe roxburghii fruit, rinse it, crush it, air-dry it, and defatted it with an organic solvent to obtain defatted powder; take the defatted powder, add water, adjust the pH of the solution to weak acidity, add papain, stir and extract it at 50-70°C, and inactivate the enzyme; after cooling, adjust the pH of the solution to weak alkalinity, add trypsin, stir and extract it at 30-45°C, and inactivate the enzyme; after cooling, centrifuge to take the supernatant, dialyze for desalination, and freeze-dry to obtain enzyme-extracted roxburghii crude polysaccharide; Step S2, polysaccharide purification: take the crude polysaccharide of roxburghii extracted by enzyme, add water to dissolve, centrifuge to remove insoluble matter, separate the supernatant through a DEAE cellulose anion column, elute with water, 0.1M NaCl solution and 0.2M NaCl solution in sequence, detect with sulfuric acid-phenol, collect and combine the 0.2M NaCl eluate, dialyze for desalination, freeze-dry, and obtain roxburghii polysaccharide CLP2; take roxburghii polysaccharide CLP2 and dissolve it in water, purify it through a Bio-Gel P-6 Gel gel chromatography column, elute with water, draw an elution curve by a sulfuric acid-phenol method, combine the eluates corresponding to the main peak of the elution curve, and freeze-dry to obtain.

8. The use according to claim 7, characterized in that: The organic solvent in step S1 is anhydrous ethanol.

9. The use according to claim 7, characterized in that: The DEAE cellulose anion column in step S2 is Cl - Type DEAE cellulose anion column.

10. The use according to any one of claims 7 to 9, wherein the diseases accompanied by vascular calcification include atherosclerosis, hypertension and diabetic vascular disease.

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