Application of carbon dots of moxa in preparation of drugs for treating aortic valve calcification

Abstract

The application relates to the field of biological medicines, and particularly discloses application of aigilCES carbon dots in preparation of a drug for treating aortic valve calcification disease. The aigilCES carbon dots are prepared from aigilCES leaf powder as raw material, through ultrasonic extraction with an organic solvent, concentration, hydrothermal reaction at 140 DEG C to 180 DEG C for 4 h to 10 h, and then purification, and the particle size is 2.3+ / -0.4 nm. The aigilCES carbon dots provided by the application can significantly inhibit the formation of calcified nodules, are suitable for an aortic valve calcification pathological model induced by beta-glyceryl sodium phosphate, ascorbic acid and dexamethasone, and provide a new way for relieving the symptoms of aortic valve calcification disease.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the application of Artemisia argyi carbon dots in the preparation of drugs for treating aortic valve calcification. Background Technology

[0002] Carbon dots derived from traditional Chinese medicine not only retain the original pharmacological properties of the herbs but also possess the advantage of fluorescence, and generally exhibit better therapeutic effects than raw materials. Carbon dots are simple to prepare, and the raw materials are abundant and inexpensive. The diameter of carbon dots is typically between 1 nm and 10 nm, allowing them to be taken up by cells and cleared by the kidneys.

[0003] Calcified aortic valve disease (CAVD) is a common cardiovascular disease characterized by fibrosis, sclerosis, and calcium deposition in the aortic valve and surrounding tissues. Clinical manifestations of CAVD primarily include aortic stenosis and aortic regurgitation; severe cases can lead to heart failure and sudden death.

[0004] Therefore, it is essential to develop a new solution that can effectively alleviate the progression of CAVD. Summary of the Invention

[0005] To develop a novel approach that effectively alleviates the progression of CAVD, this invention provides the application of Artemisia argyi carbon dots in the preparation of drugs for treating aortic valve calcification. The Artemisia argyi carbon dots provided by this invention significantly downregulate calcification-related genes in valvular mesenchymal cells. RUNX2 , ALP , BMP2 The expression of this substance inhibits the formation of calcified nodules and is applicable to aortic valve calcification pathological models induced by β-glycerophosphate sodium, ascorbic acid and dexamethasone, providing a new approach to alleviate the symptoms of aortic valve calcification.

[0006] This invention provides the application of Artemisia argyi carbon dots in the preparation of drugs for treating aortic valve calcification. The Artemisia argyi carbon dots are obtained by using Artemisia argyi leaf powder as raw material, through ultrasonic extraction, filtration, concentration, hydrothermal reaction and purification steps.

[0007] The carbon dots from Artemisia argyi provided by this invention significantly downregulate calcification-related genes in valve mesenchymal cells. RUNX2 , ALP , BMP2 The expression of this substance inhibits the formation of calcified nodules, providing a new approach to alleviate the symptoms of aortic valve calcification.

[0008] Furthermore, the ultrasonic extraction process is as follows: the Artemisia argyi leaf powder is dissolved in an organic solvent, and ultrasonically extracted at 33 kHz to 40 kHz for 20 min to 40 min. The extraction is repeated 2 to 5 times, and the extracts are combined.

[0009] Furthermore, the concentration temperature is 50℃~100℃.

[0010] Furthermore, the hydrothermal reaction conditions are 140℃~180℃ for 4 h~10 h.

[0011] Furthermore, the purification step uses a 3500 Da dialysis bag or a 200-800 mesh silica gel column.

[0012] Furthermore, the carbon dots of the Artemisia argyi have a particle size of 2.3±0.4 nm and surface functional groups of OH / NH, CH, C=O and C=C.

[0013] Furthermore, the drug is used to inhibit the formation of calcified nodules.

[0014] Furthermore, the content of Artemisia argyi carbon dots in the drug is 150 μg / mL to 250 μg / mL.

[0015] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0016] Furthermore, the excipient is any one or a combination of mannitol, glucose, trehalose, and sucrose.

[0017] Furthermore, the organic solvent is methanol, anhydrous ethanol, or acetone.

[0018] Furthermore, the filtration process utilizes filter paper and a microporous membrane.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] The Qi Ai carbon point content provided by this invention can significantly inhibit calcification when it is 200 μg / mL, by targeting and regulating calcification-related genes in valve mesenchymal cells. RUNX2 , ALP , BMP2 It inhibits the formation of calcium nodules and is effective in the pathological calcification model induced by sodium β-glycerophosphate + ascorbic acid + dexamethasone, covering the main causes of CAVD and providing a new way to alleviate CAVD. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 Characterization analysis of carbon dots in Artemisia argyi;

[0023] In the figure, a is a transmission electron microscope image of carbon dots of Artemisia argyi, where the observation scale of the left image is 50 nm and the observation scale of the right image is 1 nm.

[0024] b is the diameter distribution diagram of carbon dots in Artemisia argyi;

[0025] c represents the ultraviolet-visible spectrum of the carbon dots of Artemisia argyi;

[0026] d represents the fluorescence spectrum of the carbon dots of Artemisia argyi;

[0027] e represents the Fourier transform infrared spectrum of the carbon dots of Artemisia argyi;

[0028] f is the Raman spectrum of the carbon dots of Artemisia argyi;

[0029] g represents the X-ray photoelectron spectrum of Artemisia argyi carbon dots;

[0030] h is the C-spectrum of the X-ray photoelectron energy spectrum of Artemisia argyi carbon dots;

[0031] i represents the O spectrum of the X-ray photoelectron energy spectrum of Artemisia argyi carbon dots.

[0032] Figure 2 Artemisia argyi carbon dots inhibit the expression of genes related to calcification of valvular mesenchymal cells.

[0033] Figure 3 Artemisia argyi carbon dots inhibit the expression of valvular mesenchymal cell calcification-related proteins;

[0034] In the figure, a shows that Artemisia argyi carbon dots inhibit the expression of calcification-related proteins RUNX2, ALP, and BMP2 in valvular mesenchymal cells;

[0035] b represents the statistical analysis of RUNX2 protein expression levels;

[0036] c represents the statistical analysis of ALP protein expression levels;

[0037] d represents the statistical analysis of BMP2 protein expression levels.

[0038] Figure 4 Artemisia argyi carbon dots inhibit valvular mesenchymal cell calcification;

[0039] In the figure, a represents the calcification level of valve mesenchymal cells characterized by alizarin red staining;

[0040] b represents the statistical analysis of the alizarin red staining results. Detailed Implementation

[0041] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0042] Example 1: Application of Artemisia argyi carbon dots in the preparation of drugs for treating aortic valve calcification.

[0043] I. Experimental Methods

[0044] 1. Preparation of Artemisia argyi carbon dots

[0045] 60 g of Artemisia argyi leaf powder (purchased from Hubei Jingui Traditional Chinese Medicine Pieces Co., Ltd.) was dissolved in 800 mL of anhydrous ethanol and extracted using a Kunshan Shumei ultrasonic cleaner (KQ-1000B) at 40 kHz for 30 min, repeated 3 times. The extracts were combined. The extract was filtered through filter paper to remove Artemisia argyi leaf residue, and the extract was obtained by vacuum filtration through a 0.22 μm microporous membrane. The extract was concentrated to 5% using a rotary evaporator at 80℃. The concentrated extract was placed in a 100 mL hydrothermal reactor and reacted in an oven at 160℃ for 8 h. After cooling to room temperature, the extract was purified by dialyzing with a 3500 Da dialysis bag using a 1:1 mixture of dichloromethane and methanol as the mobile phase for 48 h, with the mobile phase changed every 4 hours. The purified Artemisia argyi carbon dots were stored at -20℃.

[0046] 2. Characterization analysis of carbon dots in Artemisia argyi

[0047] The carbon dots of Artemisia argyi prepared were characterized and analyzed using transmission electron microscopy (XPLORE), ultraviolet spectrophotometer (LAMBDA 1050+), fluorescence spectrophotometer (RF-6000), Fourier transform infrared spectrometer (Nicolet iS50), Raman spectrometer (LabRAM HR Evolution), and X-ray photoelectron spectrometer (ESCALAB250Xi).

[0048] 3. Cell culture and grouping

[0049] Human aortic valve tissue was harvested, endothelial cells were scraped off, and the tissue was cut into small pieces. It was digested with 1% type I collagenase (purchased from Sigma-Aldrich) for 5 h. Undigested tissue was removed by filtering with a 70 μm cell sieve, and a cell suspension was prepared to obtain primary human aortic valve interstitial cells (VICs). Primary VICs were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin at 37°C and 5% CO2 until 80% confluence. The cells were then divided into a control group, a calcification model group, and an ACDs intervention group. The calcification model group and the ACDs intervention group were respectively treated with 10 mM sodium β-glycerophosphate, 50 μg / mL ascorbic acid, and 100 nM dexamethasone, and then shaken to simulate calcification. The ACDs intervention group was additionally treated with 200 μg / mL Artemisia argyi carbon dots (prepared Artemisia argyi carbon dots dissolved in DMSO), with a ratio of 1:1000 to the culture medium. The control group received no treatment.

[0050] 4. RNA extraction and qRT-PCR

[0051] Each group of cells was treated for 7 days using TRIzol (purchased from Invitrogen). TM Total RNA was extracted from cells using the method described above. After the concentration was measured by NanoDrop micro-spectrophotometer, it was reverse transcribed into cDNA (Thermo Scientific RevertAid RT kit, K1691).

[0052] qRT-PCR reaction system: 10 μL SYBR Green Master Mix, 2 μL cDNA, 0.5 μL each of primers (10 μM), and ddH2O to a final volume of 20 μL. Reaction program: 95℃ pre-denaturation for 5 min; 95℃ for 10 s, 60℃ for 30 s, 40 cycles. Target genes: RUNX2, ALP, BMP2; Internal reference gene: β-actin. Data analysis: 2... -ΔΔCt The method calculates the relative expression level of genes.

[0053] 5. Western Blot Analysis

[0054] Cells in each group were treated for 7 days, and lysis and extraction were performed using RIPA lysis and extraction buffer (purchased from Thermo Scientific). TM Total protein was extracted and its concentration was determined by the BCA method.

[0055] Sample loading: 30 μg protein / lane, SDS-PAGE electrophoresis (12% separating gel, 5% stacking gel), transfer to PVDF membrane.

[0056] Sealing: 5% skim milk, room temperature for 1 hour.

[0057] Primary antibody incubation: anti-RUNX2 (1:1000), anti-ALP (1:1000), anti-BMP2 (1:800), incubated overnight at 4°C, then washed 3 times with PBS, 5 min each time.

[0058] Secondary antibody incubation: HRP-labeled goat anti-rabbit IgG (1:5000), washed 3 times with PBS after 1 h at room temperature, 5 min each time.

[0059] Development: ECL chemiluminescence method, ImageJ quantization of band gray values.

[0060] 6. Alizarin Red Staining Analysis

[0061] Cells from each group were seeded in 24-well plates (5 × 10⁶ cells / well). 4 (cells / well), treated for 21 days, then washed three times with PBS, 5 min each time.

[0062] Fixation: 4% paraformaldehyde, room temperature for 15 min.

[0063] Alizarin Red staining: Stain with 2% alizarin red (pH 4.2) for 10 min, then wash thoroughly with ddH2O.

[0064] Quantitative analysis: The stained precipitate was dissolved in 10% hexadecylpyridine chloride (CPC), and the absorbance was measured at 562 nm.

[0065] 7. Statistical Analysis

[0066] All experiments were repeated three times. Data are expressed as mean ± standard deviation. One-way ANOVA was performed using SPSS 26.0. *p<0.05 was considered statistically significant.

[0067] II. Test Results

[0068] 1. Characterization analysis of carbon dots in Artemisia argyi

[0069] Characterization analysis of the carbon dots (ACDs) of Artemisia argyi was performed. Transmission electron microscopy results showed that the carbon dots were uniform in size, with a diameter of 2.3 ± 0.4 nm. Figure 1 (a, b); UV-Vis spectroscopy shows that the carbon point absorption peak of Artemisia argyi is at 660 nm ( Figure 1 c); Fluorescence spectroscopy showed that the emission light of the carbon dots of Artemisia argyi was at 673 nm ( Figure 1 (d); Fourier transform infrared spectroscopy of carbon dots from Artemisia argyi shows that at 3500 cm⁻¹ -1 The nearby broad infrared absorption peak corresponds to the OH / NH stretching vibration, 2925 cm⁻¹. -1 The nearby infrared absorption peak corresponds to the stretching vibration of CH, at 1735 cm⁻¹.-1 and 1625 cm -1 The nearby infrared absorption peaks correspond to the stretching vibrations of C=O and C=C. Figure 1 (e); Raman spectra of Artemisia argyi carbon dots show that at 1625 cm⁻¹ -1 The strong absorption peak indicates the presence of carbonization. Figure 1 X-ray photoelectron spectroscopy showed that 284.8 eV and 532.1 eV in the carbon dots of Artemisia argyi corresponded to C 1s and O 1s, respectively. Figure 1 g~i).

[0070] 2. qRT-PCR and Western Blot analysis

[0071] The results of qRT-PCR showed that ( Figure 2 Calcification-related genes in calcification model group (OM) cells RUNX2 , ALP and BMP2 The expression level of [a substance] was significantly increased compared to the control group, while the ACDs intervention group with added Artemisia argyi carbon dots (OM+ACDs) showed [significantly higher expression levels]. RUNX2 , ALP and BMP2 The expression level was significantly lower in the group compared to the OM group.

[0072] Western blot results showed that ( Figure 3 (a~d) The expression levels of RUNX2, ALP and BMP2 proteins in OM cells were significantly increased compared with the control group, while the expression levels of RUNX2, ALP and BMP2 proteins in OM+ACDs cells were significantly decreased compared with the OM group.

[0073] 3. Alizarin Red Staining Analysis

[0074] Nodules of human aortic valve mesenchymal cells were stained with alizarin red, and the results showed ( Figure 4 (a, b) The number of calcified nodules in OM was significantly increased compared with the control group, while the number of calcified nodules in OM+ACDs was significantly decreased compared with OM.

[0075] The above results indicate that the Artemisia argyi carbon dots (ACDs) provided by this invention can significantly inhibit calcification-related genes. RUNX2 , ALP and BMP2 The expression of [a substance] reduces the level of calcified nodules in valvular mesenchymal cells, thereby alleviating the symptoms of aortic valve calcification.

[0076] Although preferred embodiments of the invention have been described, those skilled in the art, once they have learned the basic inventive concept, can make other changes and modifications to these embodiments.

[0077] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. The application of Artemisia argyi carbon dots in the preparation of drugs for treating aortic valve calcification, characterized in that, The Qi Ai carbon dots are obtained by using Qi Ai leaf powder as raw material through ultrasonic extraction, filtration, concentration, hydrothermal reaction and purification steps; the particle size of the Qi Ai carbon dots is 2.3±0.4 nm. The ultrasonic extraction process is as follows: dissolve Artemisia argyi powder in anhydrous ethanol, and extract ultrasonically at 33 KHz~40 KHz for 20 min~40 min. Repeat the extraction 2 to 5 times and then combine the extracts. The filtration process uses a 0.22 μm microporous membrane. The concentration temperature is 50℃~100℃; The hydrothermal reaction conditions are 140℃~180℃ for 4 h~10 h; The purification step uses a 3500 Da dialysis bag with a 1:1 mixture of dichloromethane and methanol as the mobile phase.

2. The application of the carbon dots of Artemisia argyi according to claim 1 in the preparation of drugs for treating aortic valve calcification, characterized in that, The surface functional groups of the Artemisia argyi carbon dots are OH / NH, CH, C=O and C=C.

3. The application of the carbonized Artemisia argyi according to claim 1 in the preparation of a drug for treating aortic valve calcification, characterized in that, The drug is used to inhibit the formation of calcified nodules.

4. The application of the carbon dots of Artemisia argyi according to claim 3 in the preparation of drugs for treating aortic valve calcification, characterized in that, The drug is used to inhibit calcification-related genes. RUNX2 , ALP and BMP2 The expression.

5. The application of the carbon dots of Artemisia argyi according to claim 1 in the preparation of a drug for treating aortic valve calcification, characterized in that, The content of Artemisia argyi carbon dots in the drug is 150 μg / mL to 250 μg / mL.

6. The application of the carbonized Artemisia argyi according to claim 1 in the preparation of a drug for treating aortic valve calcification, characterized in that, The drug also includes pharmaceutically acceptable excipients.

7. The application of the carbonized Artemisia argyi according to claim 6 in the preparation of a drug for treating aortic valve calcification, characterized in that, The excipients are any one or more combinations of mannitol, glucose, trehalose, and sucrose.

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