Application of Chinese mugwort carbon dots in preparation of medicine for treating aortic valve calcification disease

Abstract

The invention relates to the field of biological medicine, and particularly discloses application of Chinese mugwort carbon dots in preparation of medicine for treating aortic valve calcification. The Chinese mugwort carbon dots are prepared by taking Chinese mugwort leaf powder as a raw material, performing ultrasonic extraction and concentration through an organic solvent, performing hydrothermal reaction at 140-180 DEG C for 4-10 hours, and purifying, and have the particle size of 2.3 + / -0.4 nm. The Chinese mugwort carbon dot provided by the invention can obviously inhibit formation of calcified nodules, is suitable for an aortic valve calcification pathological model induced by beta-sodium glycerophosphate, ascorbic acid and dexamethasone, and provides a new way for relieving symptoms of aortic valve calcification.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the application of Qi Ai carbon dots in the preparation of drugs for treating aortic valve calcification. Background Art

[0002] Traditional Chinese medicine-derived carbon dots not only retain the original pharmacological properties of traditional Chinese medicine but also possess the advantage of fluorescence properties, often demonstrating superior therapeutic efficacy compared to the raw materials themselves. Carbon dots are simple to prepare, and the raw materials are abundant and inexpensive. With diameters typically ranging from 1 nm to 10 nm, carbon dots are readily absorbed by cells and cleared through the kidneys.

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

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

[0005] In order to develop a new solution that can effectively alleviate the progression of CAVD, the present invention provides the use of Qi Ai carbon dots in the preparation of drugs for treating aortic valve calcification. The Qi Ai carbon dots provided by the present invention significantly downregulate calcification-related genes in valve mesenchymal cells. RUNX2 、 ALP 、 BMP2 The results indicate that the drug can inhibit the expression of calcified nodules and is suitable for the aortic valve calcification pathological model induced by sodium β-glycerophosphate, ascorbic acid and dexamethasone, providing a new way to alleviate the symptoms of aortic valve calcification.

[0006] The present invention provides an application of Qi Ai carbon dots in the preparation of a drug for treating aortic valve calcification. The Qi Ai carbon dots are obtained from Qi Ai leaf powder through ultrasonic extraction, filtration, concentration, hydrothermal reaction and purification steps.

[0007] The Qi Ai carbon dots provided by the present invention significantly downregulate calcification-related genes in valve mesenchymal cells RUNX2 、 ALP 、 BMP2 The expression of valvular calcification can be inhibited, thereby inhibiting the formation of calcified nodules and providing a new way to alleviate the symptoms of aortic valve calcification.

[0008] Furthermore, the ultrasonic extraction process is as follows: dissolving the Artemisia argyi leaf powder in an organic solvent, ultrasonically extracting at 33 KHz to 40 KHz for 20 min to 40 min, repeating the extraction 2 to 5 times, and then combining the extracts.

[0009] Furthermore, the concentration temperature is 50°C to 100°C.

[0010] Furthermore, the hydrothermal reaction conditions are 140° C. to 180° C. for 4 h to 10 h.

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

[0012] Furthermore, the particle size of the Qi Ai carbon dots is 2.3±0.4 nm, and the surface functional groups are 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 Qi Ai carbon dots in the medicine is 150 μg / mL~250 μg / mL.

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

[0016] Furthermore, the auxiliary material is any one or more combinations of mannitol, glucose, trehalose and sucrose.

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

[0018] Furthermore, the filtration uses filter paper and microporous filter membrane.

[0019] Compared with the prior art, the present invention has the following beneficial effects: The Qi Ai carbon dots provided by the present invention can significantly inhibit calcification when the content is 200 μg / mL, and can regulate calcification-related genes in valvular mesenchymal cells by targeting RUNX2 、 ALP 、 BMP2 It can inhibit the formation of calcium nodules and is effective in the pathological calcification model induced by β-sodium glycerophosphate + ascorbic acid + dexamethasone, covering the main causes of CAVD and providing a new way to alleviate CAVD. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0021] Figure 1 Characterization and analysis of Qi Ai carbon dots; In the figure, a is a transmission electron microscope image of Qi Ai carbon dots, where the observation scale of the left image is 50 nm and the observation scale of the right image is 1 nm; b is the diameter distribution diagram of Qi Ai carbon dots; c is the UV-visible spectrum of Qi Ai carbon dots; d is the fluorescence spectrum of Qi Ai carbon dots; e is the Fourier transform infrared spectrum of Qi Ai carbon dots; f is the Raman spectrum of Qi Ai carbon dots; g is the X-ray photoelectron spectrum of Qi Ai carbon dots; h is the C spectrum of the X-ray photoelectron spectrum of Qi Ai carbon dots; i is the O spectrum of the X-ray photoelectron spectrum of Qi Ai carbon dots.

[0022] Figure 2 Qi Ai carbon dots inhibit the expression of genes related to calcification in valvular mesenchymal cells.

[0023] Figure 3 Qi Ai carbon dots inhibit the expression of calcification-related proteins in valvular mesenchymal cells; In the figure, a shows that Qi Ai carbon dots inhibit the expression of calcification-related proteins RUNX2, ALP, and BMP2 in valvular mesenchymal cells; b Statistical analysis of RUNX2 protein expression levels; c Statistical analysis of ALP protein expression levels; d Statistical analysis of BMP2 protein expression levels.

[0024] Figure 4 Qi Ai carbon dots inhibit valvular mesenchymal cell calcification; In the figure, a is Alizarin red staining to characterize the calcification level of valve mesenchymal cells; b Statistical analysis of Alizarin red staining results. DETAILED DESCRIPTION

[0025] 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 by the specific embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial sources unless otherwise specified.

[0026] Example 1: Application of Qi Ai carbon dots in the preparation of drugs for treating aortic valve calcification.

[0027] 1. Test methods 1. Preparation of Qi Ai carbon dots 60 g of Qi Artemisia argyi leaf powder (purchased from Hubei Jingui Chinese Medicine Pieces Co., Ltd.) was dissolved in 800 mL of anhydrous ethanol and ultrasonically extracted at 40 kHz for 30 min using a Kunshan Shumei ultrasonic cleaner (KQ-1000B). This was repeated three times, and the extracts were combined. The extract was filtered through filter paper to remove Qi Artemisia argyi leaf residue, and then vacuum-filtered through a 0.22 μm microporous filter membrane to obtain the Qi Artemisia argyi extract. The extract was concentrated to 5% using a rotary evaporator at 80°C. The concentrated Qi Artemisia argyi extract was placed in a 100 mL hydrothermal reactor, which was placed in an oven at 160°C for 8 h and then cooled to room temperature. The purified Qi Artemisia argyi carbon dots were dialyzed using a 3500 Da dialysis bag with a 1:1 mixture of dichloromethane and methanol as the mobile phase for 48 h. The mobile phase was changed every 4 h. The purified Qi Artemisia argyi carbon dots were stored at -20°C.

[0028] 2. Characterization and analysis of Qi Ai carbon dots The prepared Qi Ai carbon dots were characterized and analyzed using a transmission electron microscope (XPLORE), an ultraviolet spectrophotometer (LAMBDA 1050+), a fluorescence spectrophotometer (RF-6000), a Fourier transform infrared spectrometer (Nicolet iS50), a Raman spectrometer (LabRAM HR Evolution), and an X-ray photoelectron spectrometer (ESCALAB250Xi).

[0029] 3. Cell culture and grouping Human aortic valve tissue was obtained, and after scraping off the endothelial cells, the tissue was cut into small pieces and digested with 1% type I collagenase (purchased from Sigma-Aldrich) for 5 h. The undigested tissue was filtered through a 70 μm cell sieve to prepare a cell suspension to obtain primary human aortic valvular interstitial cells (VICs). Primary VICs cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin at 37°C and 5% CO2 until 80% confluence. They 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 added with 10 mM sodium β-glycerophosphate, 50 μg / mL ascorbic acid, and 100 nM dexamethasone, respectively, to simulate calcification. The ACDs intervention group was additionally added with 200 μg / mL Qi Ai carbon dots (prepared Qi Ai carbon dots were dissolved in DMSO) at a ratio of 1:1000. The control group received no treatment.

[0030] 4. RNA Extraction and qRT-PCR Each group of cells were treated for 7 days with TRIzol (purchased from Invitrogen TMTotal cellular RNA was extracted using the RT-PCR method, and the concentration was determined using a NanoDrop microspectrophotometer before reverse transcription into cDNA (Thermo Scientific RevertAid RT Kit, K1691).

[0031] qRT-PCR reaction system: 10 μL SYBR Green Master Mix, 2 μL cDNA, 0.5 μL each primer (10 μM), and ddH2O to 20 μL. Reaction procedure: 95°C initial denaturation for 5 min; 95°C for 10 s, 60°C for 30 s, 40 cycles. Target genes: RUNX2, ALP, BMP2; Reference gene: β-actin. Data analysis: 2 -ΔΔCt The relative gene expression was calculated by the method.

[0032] 5. Western Blot Analysis Cells in each group were treated for 7 days and lysed using RIPA and extracted with buffer (purchased from ThermoScientific TM ) were used to extract total protein, and the concentration was determined by BCA method.

[0033] Loading amount: 30 μg protein / lane, SDS-PAGE electrophoresis (12% separating gel, 5% stacking gel), and transfer to PVDF membrane.

[0034] Blocking: 5% skim milk, room temperature for 1 h.

[0035] The primary antibodies incubation included anti-RUNX2 (1:1000), anti-ALP (1:1000), and anti-BMP2 (1:800). The sections were incubated at 4°C overnight and then washed three times with PBS, 5 min each time.

[0036] Secondary antibody incubation: HRP-conjugated goat anti-rabbit IgG (1:5000) at room temperature for 1 h, followed by PBS washing three times, 5 min each time.

[0037] Development: ECL chemiluminescence method, ImageJ quantification of band gray value.

[0038] 6. Alizarin red staining analysis Cells in each group were seeded in 24-well plates (5 × 10 4 cells / well) and washed three times with PBS (5 min each time) after 21 days of treatment.

[0039] Fixation: 4% paraformaldehyde, room temperature for 15 min.

[0040] Alizarin red staining: stain with 2% alizarin red (pH 4.2) for 10 min and then wash thoroughly with ddH2O.

[0041] Quantitative analysis: The precipitate was dissolved in 10% cetylpyridinium chloride (CPC) and the absorbance was measured at 562 nm.

[0042] 7. Statistical analysis The above experiments were repeated three times, and the data were expressed as mean ± standard deviation. One-way analysis of variance (ANOVA) was performed using SPSS 26.0. *p < 0.05 indicated a significant difference.

[0043] 2. Test results 1. Characterization and analysis of Qi Ai carbon dots The Qi Ai carbon dots (ACDs) were characterized and analyzed. Transmission electron microscopy results showed that the Qi Ai carbon dots were uniform in size with a diameter of 2.3±0.4 nm ( Figure 1 a, b); UV-visible spectrum shows that the absorption peak of Qi Ai carbon dots is 660 nm ( Figure 1 c); Fluorescence spectrum shows that the emission light of Qi Ai carbon dots is at 673 nm ( Figure 1 d); The Fourier transform infrared spectrum of Qi Ai carbon dots shows that the wavelength of 3500 cm -1 The infrared absorption broad peak near 2925 cm corresponds to the OH / NH stretching vibration. -1 The infrared absorption peak near 1735 cm corresponds to the stretching vibration of CH -1 and 1625 cm -1 The infrared absorption peaks near the C=O and C=C stretching vibrations ( Figure 1 e); Raman spectrum of Qi Ai carbon dots shows that 1625 cm -1 The absorption peak at is strong, indicating the presence of carbonization ( Figure 1 f); X-ray photoelectron spectroscopy shows that 284.8 eV and 532.1 eV in Qi Ai carbon dots correspond to C 1s and O 1s ( Figure 1 g~i).

[0044] 2. qRT-PCR and Western Blot Analysis 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 α-glucan was significantly increased compared with the control group, while the expression level of α-glucan in the ACDs intervention group (OM+ACDs) was significantly increased compared with the control group. RUNX2 、 ALP and BMP2 The expression level of WT group was significantly decreased compared with that in OM group.

[0045] 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 those in the control group, while the expression levels of RUNX2, ALP and BMP2 proteins in OM+ACDs cells were significantly decreased compared with those in the OM group.

[0046] 3. Alizarin red staining analysis The nodules of human aortic valve mesenchymal cells were stained with Alizarin red, and the results showed that ( 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.

[0047] The above results show that the Qi Ai carbon dots (ACDs) provided by the present invention can significantly inhibit calcification-related genes RUNX2 、 ALP and BMP2 The expression of valvular mesenchymal cells can be inhibited, reducing the level of calcified nodules in valve mesenchymal cells and thus alleviating the symptoms of aortic valve calcification.

[0048] Although preferred embodiments of the present invention have been described, additional changes and modifications to these embodiments may occur to those skilled in the art once the basic inventive concepts become known.

[0049] Obviously, those skilled in the art may make various modifications and variations to the present invention without departing from the spirit and scope of the present invention. Thus, if such modifications and variations fall within the scope of the claims and their equivalents, the present invention is intended to include such modifications and variations.

Claims

1. Application of Qi Ai carbon dots in the preparation of a drug for treating aortic valve calcification, characterized in that: The Qi Ai carbon dots are obtained by taking Qi Ai leaf powder as raw material through ultrasonic extraction, filtration, concentration, hydrothermal reaction and purification steps.

2. The use of Qi Ai carbon dots according to claim 1 in preparing a drug for treating aortic valve calcification, characterized in that: The ultrasonic extraction process is as follows: dissolving the Artemisia argyi leaf powder in an organic solvent, ultrasonically extracting at 33 KHz to 40 KHz for 20 min to 40 min, repeating the extraction 2 to 5 times, and then combining the extracts.

3. The use of Qi Ai carbon dots in the preparation of a drug for treating aortic valve calcification according to claim 1, characterized in that: The concentration temperature is 50°C to 100°C.

4. The use of Qi Ai carbon dots in preparing a drug for treating aortic valve calcification according to claim 1, characterized in that: The hydrothermal reaction conditions are 140° C. to 180° C. for 4 h to 10 h.

5. The use of Qi Ai carbon dots in preparing a drug for treating aortic valve calcification according to claim 1, characterized in that: The device used in the purification step is a 3500 Da dialysis bag or a 200-800 mesh silica gel column.

6. The use of Qi Ai carbon dots in preparing a drug for treating aortic valve calcification according to claim 1, characterized in that: The particle size of the Qi Ai carbon dots is 2.3±0.4 nm, and the surface functional groups are OH / NH, CH, C=O and C=C.

7. The use of Qi Ai carbon dots in preparing a drug for treating aortic valve calcification according to claim 1, characterized in that: The drug is used for inhibiting the formation of calcified nodules.

8. The use of Qi Ai carbon dots in preparing a drug for treating aortic valve calcification according to claim 1, characterized in that: The content of Qi Ai carbon dots in the medicine is 150 μg / mL~250 μg / mL.

9. The use of Qi Ai carbon dots in preparing a drug for treating aortic valve calcification according to claim 1, characterized in that: The drug also includes pharmaceutically acceptable excipients.

10. The use of Qi Ai carbon dots according to claim 9 in preparing a drug for treating aortic valve calcification, characterized in that: The auxiliary material is any one or more combinations of mannitol, glucose, trehalose and sucrose.

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