Use of aldh2 in the preparation of a medicament for treating vascular calcification

Abstract

The application belongs to the field of biological medicine, and particularly relates to application of acetaldehyde dehydrogenase 2 (ALDH2) in preparation of a drug for treating vascular calcification. The application provides application of ALDH2 or an inducing agent thereof in preparation of a drug for treating vascular calcification. Through research on ALDH2, the application finds that ALDH2 can regulate vascular smooth muscle cells (VSMCs) in various vascular related diseases, and further finds a new mechanism that ALDH2 reduces transformation of VSMCs from a contraction type to a synthesis type and delays progress of vascular calcification. The application provides a new target for prevention and treatment of vascular calcification, and activation of ALDH2 is expected to be a potential intervention means for vascular calcification. A drug with ALDH2 as an active ingredient is a potential new type of therapeutic reagent for vascular calcification.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the application of ALDH2 in the preparation of drugs for treating vascular calcification. Background Technology

[0002] Vascular calcification refers to the pathological process of hydroxyapatite minerals deposited in the vascular system, and is commonly seen in aging, atherosclerosis, diabetes, and chronic kidney disease.

[0003] Vascular calcification is not simply the passive deposition of calcium phosphate crystals, but an organic, active, complex, cell-regulated osteogenic process involving various cell types, including vascular smooth muscle cells (VSMCs), macrophages, fibroblasts, vascular endothelial cells, and pericytes. Among them, VSMCs play a central role in the vascular calcification process, and their osteogenic differentiation is an important pathological mechanism of vascular calcification. Therefore, osteogenic differentiation of VSMCs may become an important therapeutic target for delaying the progression of vascular calcification.

[0004] However, there are currently no novel drugs that can inhibit the phenotypic transformation of VSMCs and delay vascular calcification. Therefore, it is urgent to develop an effective drug target for the mechanism and progression of vascular calcification in order to achieve the prevention and treatment of vascular calcification. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides the application of aldehyde dehydrogenase 2 (ALDH2) in the preparation of drugs for treating vascular calcification. ALDH2 is a member of the aldehyde dehydrogenase superfamily, expressed in multiple tissues and organs such as the liver, heart, and blood vessels. It is a key enzyme in alcohol metabolism, oxidizing acetaldehyde to acetic acid. This invention investigates the regulatory role of ALDH2 on VSMCs in various vascular-related diseases, thereby reducing the conversion of VSMCs from contractile to anabolic states and delaying the progression of vascular calcification through a novel mechanism.

[0006] Specifically, the technical solution of the present invention is as follows:

[0007] Application of ALDH2 inducers in screening or preparing drugs for the prevention, relief and / or treatment of vascular calcification.

[0008] Furthermore, the ALDH2 inducer includes agents that increase ALDH2 expression (causing ALDH2 overexpression) or activate ALDH2.

[0009] Furthermore, the reagents for activating ALDH2 include agonists that promote ALDH2 protein activity or protein levels, or agonists that promote ALDH2 mRNA levels, the promoting effect of which may be reversible or irreversible.

[0010] Furthermore, the agonist that promotes ALDH2 protein activity or protein level is selected from one or more proteins, peptides, enzymes, natural compounds, synthetic compounds, organic and inorganic substances that promote ALDH2 protein activity or protein level.

[0011] Preferably, ALDH2 inducers include, but are not limited to, Alda-1.

[0012] The use of ALDH2 in screening or preparing drugs for the prevention, relief and / or treatment of vascular calcification.

[0013] Furthermore, the therapeutic conditions for the drugs for treating vascular calcification described in this invention include, but are not limited to, vascular calcification-related diseases such as vitamin D3-induced vascular calcification, vascular calcification in renal insufficiency, arterial vascular calcification (cerebral blood vessels, cardiovascular vessels, etc.), and venous vascular calcification.

[0014] The present invention also provides a medicament for preventing, alleviating and / or treating vascular calcification, comprising a medically acceptable carrier and an effective amount of an active ingredient, said active ingredient comprising ALDH2 or an ALDH2 inducer.

[0015] The "medically acceptable carrier" used in this invention includes one or more of any and all physiologically suitable solvents, dispersion media, coatings, antimicrobial and antifungal agents, isotonic agents, or absorption delay agents. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate-buffered saline, glucose, glycerol, or ethanol, and combinations thereof. In many cases, the composition preferably includes one or more of isotonic agents, such as sugars, mannitol, sorbitol, polyols of sorbitol, or sodium chloride. Pharmaceutically acceptable carriers may also contain small amounts of adjuvants, such as wetting agents or emulsifiers, preservatives, or buffers, which enhance the invention for the preparation of medicaments for treating vascular calcification.

[0016] The present invention also provides a method for screening drugs for treating vascular calcification, which involves detecting the expression level or activity of the ALDH2 gene or ALDH2 protein in subjects before and after drug administration, wherein ALDH2 overexpression or activation after drug administration indicates a candidate drug.

[0017] The drug of the present invention can be administered to humans or other non-human animals.

[0018] The beneficial effects of this invention are as follows:

[0019] This invention is the first to demonstrate that ALDH2 plays a protective role in calcification. ALDH2 inhibits osteoblastic differentiation of VSMCs and reduces the degree of vascular calcification, providing a new target for the prevention and treatment of vascular calcification. Activating ALDH2 is expected to become a potential intervention for vascular calcification, and drugs with ALDH2 as the active ingredient are potential novel therapeutic agents for vascular calcification. Attached Figure Description

[0020] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0021] Figure 1 To investigate the exacerbation of vitamin D3-induced vascular calcification in vivo by ALDH2 knockout. HE, Alizan red, and Von Kossa staining and statistical analysis were performed on aortic tissues (n=10) from (AC) WT and ALDH2-KO mice and vitamin D3-induced aortic tissues. Scale bar = 50 μm. (FH) WT and ALDH2... SMKO HE, Alizan red, and Von Kossa staining and statistical analysis of aortic tissues from WT mice and Vitamin D3-induced aorta (n=10), scale bar = 50 μm. (IJ) WT type and ALDH2 SMKO Immunohistochemical staining and statistical analysis of 4-HNE in aortic tissues of type WT mice and Vitamin D3-induced aorta (n=10), scale bar = 50 μm. (KM)WT type and ALDH2 SMKO Immunofluorescence staining and statistical analysis of RUNX2 and α-SMA in aortic tissues (n=10) from mice and vitamin D3-induced aorta (scale bar = 100 μm). Data are expressed as mean ± standard error (Mean ± SEM). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0022] Figure 2 ALDH2 knockout exacerbates vitamin D3-induced vascular calcification in vitro. (A) β-GP combined with CaCl2 induces WT and ALDH2. SMKO Alizan Red and Von Kossa staining of primary vascular smooth muscle cells from type II mice. (BG)β-GP combined with CaCl2 induces WT and ALDH2. SMKOExpression levels and statistical analysis of RUNX2, BMP2, OPN, MSX2, and α-SMA proteins in primary WT and rs671 mutant human vascular smooth muscle cells induced by β-GP combined with CaCl2. (J) Alizan red and Von Kossa staining in WT and rs671 mutant human primary vascular smooth muscle cells induced by β-GP combined with CaCl2. Expression levels and statistical analysis of RUNX2, BMP2, OPN, MSX2, and α-SMA proteins in WT and rs671 mutant human primary vascular smooth muscle cells induced by β-GP combined with CaCl2. (KP) Data are expressed as mean ± standard error (Mean ± SEM). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0023] Figure 3 To investigate how ALDH2 overexpression or activation can alleviate Vitamin D3-induced vascular calcification in vivo. (AC) Aortic tissues from WT and ALDH2-Tg mice and Vitamin D3-induced calcification (n=10): HE, Alizan red, and Von Kossa staining and statistical analysis, scale bar = 50 μm. (DE) Aortic tissues from WT and ALDH2-Tg mice and Vitamin D3-induced calcification (n=10): 4-HNE immunohistochemical staining and statistical analysis, scale bar = 50 μm. (FH) Aortic tissues from control and Alda-1-treated mice and Vitamin D3-induced calcification (n=10): HE, Alizan red, and Von Kossa staining and statistical analysis, scale bar = 50 μm. (KM) Aortic tissues from WT and ALDH2-Tg mice and Vitamin D3-induced calcification (n=10): RUN X2 and α-SMA immunofluorescence staining and statistical analysis, scale bar = 100 μm. All data are expressed as mean ± standard error (Mean ± SEM). *P<0.05, **P<0.01, ****P<0.0001.

[0024] Figure 4To reduce vitamin D3-induced vascular calcification in vitro by ALDH2 overexpression or activation. (A) Alizan red and Von Kossa staining of primary vascular smooth muscle cells from WT and ALDH2-OE mice induced by β-GP combined with CaCl2. (BG) Expression levels and statistical analysis of RUNX2, BMP2, OPN, MSX2, and α-SMA proteins after β-GP combined with CaCl2 induction of primary vascular smooth muscle cells from WT and ALDH2-OE mice (n=5). (J) Alizan red and Von Kossa staining of primary vascular smooth muscle cells from control and Alda-1 treated mice induced by β-GP combined with CaCl2. (KP) Expression levels and statistical analysis of RUNX2, BMP2, OPN, MSX2, and α-SMA proteins after β-GP combined with CaCl2 induction of primary vascular smooth muscle cells from control and Alda-1 treated mice (n=5). Expression levels of 4-HNE protein in primary vascular smooth muscle cells of mice induced by (QR)β-GP combined with CaCl2 and Alda-1 treatment were statistically analyzed (n=5). Data are expressed as mean ± standard error (Mean ± SEM). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0025] Figure 5 ALDH2 knockout exacerbates vascular calcification in chronic renal failure. HE, Alizan red, and Von Kossa staining and statistical analysis were performed on aortic tissues (n=10) from (AC) WT and ALDH2-KO mice and mice with 5 / 6 nephrectomy combined with a high-phosphorus diet. Scale bar = 50 μm. (FH) WT and ALDH2... SMKO HE, Alizan red, and Von Kossa staining and statistical analysis were performed on aortic tissues (n=10) from mice with nephrectomy and a high-phosphorus diet. Scale bar = 50 μm. Data are expressed as mean ± standard error (Mean ± SEM). *P<0.05, **P<0.01, ****P<0.0001.

[0026] Figure 6To investigate the effect of ALDH2 overexpression or activation on reducing vascular calcification in chronic renal failure in vivo. HE, Alizan red, and Vonkossa staining and statistical analysis were performed on aortic tissues (n=10) from WT and ALDH2-OE mice and mice undergoing 5 / 6 nephrectomy combined with a high-phosphorus diet. Scale bar = 50 μm. HE, Alizan red, and Vonkossa staining and statistical analysis were performed on aortic tissues (n=10) from control and Alda-1-treated mice and mice undergoing 5 / 6 nephrectomy combined with a high-phosphorus diet. Scale bar = 50 μm. Data are expressed as mean ± standard error (Mean ± SEM). *P<0.05, ****P<0.0001. Detailed Implementation

[0027] The present invention will now be described through specific embodiments. Unless otherwise specified, all technical means used in this invention are methods well known to those skilled in the art. Furthermore, the embodiments should be understood as illustrative, not limiting, of the scope of the invention; the essence and scope of the invention are defined only by the claims. For those skilled in the art, various changes or modifications to the material composition and dosage in these embodiments without departing from the essence and scope of the invention also fall within the protection scope of this invention.

[0028] In this invention, we found elevated serum 4-HNE levels in patients with chronic kidney disease, and that 4-HNE accumulates in calcified aortic tissue. Further studies showed that interfering with ALDH2 expression or activity significantly affected the degree of vascular calcification, thus confirming the crucial roles of 4-HNE and ALDH2 in vascular calcification.

[0029] The experimental methods used in the embodiments of the present invention are as follows:

[0030] 1. Construction of animal models

[0031] ALDH2 knockout (ALDH2-KO) mice, ALDH2 vascular smooth muscle cell conditional knockout (ALDH2) SMKO Both the mice and the mice overexpressing ALDH2 (ALDH-Tg) have been bred and cultured in the laboratory to meet the needs of this experiment, and there is no need to purchase them separately.

[0032] (1) Vit D3-induced mouse arterial calcification model: Before model construction, mice were fasted for 24 hours but allowed free access to water. Male mice around 6 weeks old were selected. The calcification model group was subcutaneously injected with Vit D3 injection (500,000 units / kg·day) for 3 consecutive days, while the control group was subcutaneously injected with an equal volume of control solvent for 3 consecutive days. The Alda-1 intervention group was intraperitoneally injected with Alda-1 solution every other day, 20 mg / kg / time. All mice were harvested 6 days after the subcutaneous injection of Vit D3. The experimental animals were randomly divided into 4 groups (n=10): control solvent + ALDH2-WT group; control solvent + ALDH2-KO (or ALDH2SMKO or ALDH2-Tg or Alda-1) group; Vit D3 + ALDH2-WT group; Vit D3 + ALDH2-KO (or ALDH2SMKO or ALDH2-Tg or Alda-1) group.

[0033] (2) Establishment of a mouse model of vascular calcification induced by chronic renal failure: Six- to seven-week-old male ALDH2-Tg, ALDH2-KO, ALDH2SMKO, and corresponding WT mice were selected. They were fasted for 24 hours before surgery but allowed normal water intake. The upper and lower poles of the left kidney were removed under sevoflurane anesthesia. One week later, the right kidney was completely removed. Mice in the sham-operated group underwent the same surgery as those in the 5 / 6 nephrectomy group at the same timeframe, but the renal capsule was removed only after the kidneys were exposed, followed by closure of the abdomen. To accelerate the aortic calcification process, during the study period, animals were fed a high-phosphate diet (0.9% Pi) one week after the 5 / 6 nephrectomy, while the control group was fed a normal diet. A mouse model of uremia was successfully established after 12 weeks. The experimental animals were randomly divided into 4 groups (n=10): Sham+ALDH2-WT group; Sham+ALDH2-KO (or ALDH2SMKO or ALDH2-Tg or Alda-1) group; NR+ALDH2-WT group; NR+ALDH2-KO (or ALDH2SMKO or ALDH2-Tg or Alda-1) group.

[0034] 2. Culture and Experiment of Vascular Smooth Muscle Cells

[0035] 2.1 Extraction of primary mouse vascular smooth muscle cells (mVSMCs):

[0036] Mice were euthanized by dislocation and disinfected by immersion in 75% alcohol for 5 minutes. The mice were then removed and their limbs were fixed. The skin and subcutaneous tissue were cut open from the lower abdomen to the heart. The right atrial appendage was cut open, and a 5mL syringe (containing 5mL PBS) was inserted into the left ventricle to flush out blood from the vessels. The thorax was further cut open to the vicinity of the clavicle, the lungs were removed, and the ribs on both sides were fixed with needles, ensuring a wide field of view without obstructing aortic dissection. Thoracic aortic dissection: The thoracic aorta was cut along its course using ophthalmic scissors. The cut aorta was placed under a stereomicroscope, and the connective tissue around the aorta was cleaned with microforceps. It was then placed in a 1.5ml EP tube and minced using microscissors. The tissue suspension was transferred to a 25cm diameter tube. 2 Spread the culture medium evenly on the bottom of the culture flask, place it in an incubator, incubate upright for 30 minutes, then invert for 1 hour, add culture medium, and slowly invert it back to upright for culture. Once VSMCs have crawled out of the tissue debris and proliferated to a suitable density, they can be passaged. After trypsin digestion and serum neutralization, filter out tissue debris using a 100 μM filter, centrifuge at 800 rpm for 5 minutes, collect the cell pellet, resuspend it, and then passage the cells.

[0037] 2.3 Induction of calcification in vascular smooth muscle cells:

[0038] Preparation of calcification medium: DMEM medium contains 10% fetal bovine serum, 1% penicillin and streptomycin, 10mM β-glycerophosphate, and 1.5mM CaCl2; culture VSMCs in calcification medium, change the medium every other day, and culture continuously for 14 days to form a model.

[0039] 3. Alizarin Red and Von Kossa staining

[0040] Dewaxed aortic tissue sections or vascular smooth muscle cells fixed in 4% formaldehyde at room temperature were incubated in 2% Alizarin Red for 10 min and rinsed with double-distilled water. Positive cells turned red / purple. Alternatively, they were incubated in Von Kossa stain (silver nitrate solution) and irradiated under ultraviolet light for 30 min, then rinsed with double-distilled water. Calcified nodules stained brown to black.

[0041] 4. Western blot experiment

[0042] Tissues or cells lysed by sonication or grinding were placed in RIPA lysis buffer for 30 min, and protein quantification was performed using the BCA method, followed by SDS-PAGE gel electrophoresis. After transfer and blocking, the corresponding primary antibody was added, and the membrane was incubated overnight at 4°C. After washing with TBST, fluorescently labeled secondary antibody was added and incubated at room temperature for 2 h. The Western blot results were then quantitatively analyzed using ImageJ.

[0043] 5. Real-time quantitative PCR

[0044] Total mRNA was extracted from cells using an mRNA extraction kit, and then converted into cDNA via reverse transcription. Using cDNA as a template, primers for the target gene were added, and the target gene was amplified and detected using a real-time quantitative PCR instrument.

[0045] 6. Immunohistochemical staining

[0046] (1) Immunohistofluorescence staining: lung tissue sections were dewaxed and hydrated for antigen retrieval, incubated with primary antibody at 4°C overnight, then incubated with fluorescently labeled secondary antibody, stained with DAPI, mounted with mounting medium, observed and photographed under a fluorescence microscope, and the data were analyzed using Image-Pro Plus software.

[0047] (2) Immunohistochemical staining: lung tissue sections were dewaxed and hydrated for antigen retrieval, incubated with primary antibody at 4°C overnight, then incubated with biotin-labeled IgG, then incubated with SABC, DAB staining under a microscope, counterstained with hematoxylin, mounted, and observed under a microscope. Data were analyzed using Image-Pro Plus software.

[0048] 7. Immunoprecipitation

[0049] Immunoprecipitation was used to detect protein binding. After protein extraction, the protein was incubated sequentially with primary antibody and magnetic beads. After incubation, the supernatant was discarded by centrifugation, and diluted protein loading buffer was added. After boiling the protein, SDS-PAGE gel electrophoresis was performed.

[0050] 8. Dual-luciferase reporter assay

[0051] C2C12 cells were co-transfected with p6xOSE2-luc reporter vector and pcDNA3.0 at a ratio of 5:1. Stably transfected cells were selected in hygromycin B-containing medium, transferred, and passaged. Stable cell clones were screened using the luciferase assay. In transient transfection experiments, the cell density in 6-well plates was 1 × 10⁶ cells / well. 5 / well, transfection dose was 0.5 mg p6xOSE2-luc or 0.5 mg empty vector, transfected with RUNX2 plasmid 24 h later. Luciferase activity was measured using a luciferase reporter assay kit after 12 h of 4-HNE stimulation.

[0052] Example 1

[0053] This embodiment confirms that ALDH2 knockout exacerbates Vitamin D3-induced vascular calcification.

[0054] ALDH2-KO mice were constructed to verify the role of ALDH2 in vitamin D3-induced vascular calcification. Six-week-old male mice were randomly divided into four groups: WT group, ALDH2-KO (KO) group, vitamin D3 (VD) group, and KO+VD group. The vitamin D3 intervention group received subcutaneous injection of vitamin D3 solution (500,000 units / kg·day) for 3 consecutive days, while the control group received an equal volume of control solution subcutaneously for 3 consecutive days. All mice were harvested 6 days after the subcutaneous injection intervention. Mouse aortic tissue was stained with HE, Alizan red, and Von Kossa. The results showed that, compared with the WT group, the VD group had a significantly increased number of Alizan red-positive areas and a significantly increased number of Von Kossa-positive areas in its aortic tissue. Compared with the VD group, the KO+VD group had a significantly increased number of Alizan red-positive areas and a significantly increased number of Von Kossa-positive areas in its aortic tissue. No obvious calcified nodules were found in the aortic tissue of the WT and KO groups (P<0.05). Figure 1 The results showed that calcified nodules were significantly increased in the aortic tissue of mice in the VD group, and that KO+VD could aggravate the calcified nodules induced by VD.

[0055] Similarly, ALDH2SMKO mice were constructed to verify the role of ALDH2 in vitamin D3-induced vascular calcification. Six-week-old male mice were divided into four groups: WT group, ALDH2SMKO (SMKO) group, VD group, and SMKO+VD group. Aortic tissue from the mice was stained with hematoxylin and eosin (HE), Alizan red, and Von Kossa. The results showed that compared with the WT group, the VD group had a significantly increased number of Alizan red-positive areas and a significantly increased number of Von Kossa-positive areas in its aortic tissue. Compared with the VD group, the SMKO+VD group had a significantly increased number of Alizan red-positive areas and a significantly increased number of Von Kossa-positive areas in its aortic tissue. However, no obvious calcified nodules were found in the aortic tissue of the WT and SMKO groups (P<0.05). Figure 1 The presence of FH indicates that calcified nodules were significantly increased in the aortic tissue of mice in the VD group, and that SMKO+VD could exacerbate the calcified nodules induced by VD.

[0056] In vitro experiments were conducted using primary mouse aortic smooth muscle cells. Alizan red and Von Kossa staining results showed that β-GP combined with CaCl2 induced calcification of mVSMCs, and ALDH2SMKO aggravated β-GP combined with CaCl2-induced smooth muscle cell calcification. Figure 2 A). Western blot analysis showed that β-GP combined with CaCl2 increased the expression of RUNX2, BMP2, OPN, and MSX2 proteins and decreased the expression of α-SMA protein. ALDH2SMKO aggravated the β-GP combined with CaCl2-induced increase in RUNX2, BMP2, OPN, and MSX2 protein expression and decrease in α-SMA protein expression. Figure 2 BG).

[0057] Similarly, in vitro experiments were conducted using primary human aortic smooth muscle cells. Alizan red and Von Kossa staining results showed that β-GP combined with CaCl2 induced hVSMC calcification, and the rs671 mutation exacerbated β-GP combined with CaCl2-induced smooth muscle cell calcification. Figure 2 Western blot analysis showed that β-GP combined with CaCl2 increased the expression of RUNX2, BMP2, OPN, and MSX2 proteins and decreased the expression of α-SMA protein. The rs671 mutation exacerbated the β-GP combined with CaCl2-induced increase in RUNX2, BMP2, OPN, and MSX2 protein expression and the decrease in α-SMA protein expression. Figure 2 KP).

[0058] The results above indicate that systemic ALDH2 knockout, conditional knockout of vascular smooth muscle cells, and rs671 mutation can all promote vitamin D3-induced phenotypic transformation of vascular smooth muscle cells and aggravate vascular calcification.

[0059] Example 2

[0060] This embodiment demonstrates that ALDH2 overexpression or activation alleviates Vitamin D3-induced vascular calcification.

[0061] To verify the role of ALDH2 in vitamin D3-induced vascular calcification, 6-week-old male mice were randomly divided into four groups: WT group, ALDH2-Tg (Tg) group, vitamin D3 (VD) group, and Tg+VD group. Aortic tissue from the mice was stained with hematoxylin and eosin (HE), Alizan red, and Vonkossa. The results showed that compared with the WT group, the VD group had a significantly increased number of Alizan red-positive areas and a significantly increased number of Vonkossa-positive areas in the aortic tissue. Compared with the VD group, the Tg+VD group had a significantly decreased number of Alizan red-positive areas and a significantly decreased number of Vonkossa-positive areas in the aortic tissue. No obvious calcified nodules were found in the aortic tissue of the WT and Tg groups (P<0.05). Figure 3This indicates that calcified nodules were significantly increased in the aortic tissue of mice in the VD group, while Tg+VD could alleviate the calcified nodules induced by VD. Immunofluorescence staining results of mouse aortic tissue showed that, compared with the WT group, the VD group had increased RUNX2 expression and decreased α-SMA expression; compared with the VD group, the Tg+VD group had decreased RUNX2 expression and increased α-SMA expression. Figure 3 KM).

[0062] Alda-1 was used to activate ALDH2, and the role of ALDH2 in vitamin D3-induced vascular calcification was verified. Six-week-old male mice were divided into four groups: WT group, Alda-1 group, VD group, and Alda-1+VD group. Aortic tissues from mice were stained with hematoxylin and eosin (HE), Alizanred, and Von Kossa. The results showed that compared with the WT group, the VD group had a significantly increased number of Alizanred-positive areas and a significantly increased number of Von Kossa-positive areas in its aortic tissue. Compared with the VD group, the Alda-1+VD group had a significantly decreased number of Alizanred-positive areas and a significantly decreased number of Von Kossa-positive areas in its aortic tissue. No obvious calcified nodules were found in the aortic tissues of the WT and Alda-1 groups (P<0.05). Figure 3 The presence of FH indicates that calcified nodules were significantly increased in the aortic tissue of mice in the VD group, while Alda-1+VD could alleviate the calcified nodules induced by VD.

[0063] In vitro experiments were conducted using primary smooth muscle cells extracted from the aorta of ALDH2-Tg mice. Alizan red and Von Kossa staining results showed that β-GP combined with CaCl2 induced mVSMC calcification, and ALDH2-Tg alleviated β-GP combined with CaCl2-induced smooth muscle cell calcification. Figure 4 A). Western blot analysis showed that β-GP combined with CaCl2 induced increased expression of RUNX2, BMP2, OPN, and MSX2 proteins and decreased expression of α-SMA proteins. ALDH2-Tg alleviated the β-GP combined with CaCl2-induced increase in RUNX2, BMP2, OPN, and MSX2 protein expression and the decrease in α-SMA protein expression. Figure 4 BG).

[0064] Similarly, in vitro experiments were conducted using primary mouse aortic smooth muscle cells stimulated with Alda-1. Alizanred and Vo n Kossa staining results showed that β-GP combined with CaCl2 induced mVSMC calcification, and Alda-1 alleviated β-GP combined with CaCl2-induced smooth muscle cell calcification. Figure 4 Western blot analysis showed that β-GP combined with CaCl2 induced increased expression of RUNX2, BMP2, OPN, and MSX2 proteins and decreased expression of α-SMA proteins. Alda-1 alleviated the β-GP combined with CaCl2-induced increase in RUNX2, BMP2, OPN, and MSX2 protein expression and decrease in α-SMA protein expression. Figure 4 KP).

[0065] The results above indicate that both overexpression and activation of ALDH2 can improve vitamin D3-induced vascular smooth muscle cell phenotypic transformation and reduce vascular calcification.

[0066] Example 3

[0067] This example confirms that ALDH2 knockout exacerbates vascular calcification in renal insufficiency.

[0068] To further verify the role of ALDH2 in vascular calcification, a mouse model of chronic renal insufficiency with vascular calcification was established using ALDH2-KO mice through 5 / 6 nephrectomy combined with a high-phosphorus diet. Six- to seven-week-old male ALDH2-KO mice were divided into four groups: WT group, KO group, 5 / 6 nephrectomy + high-phosphorus diet (NR) group, and KO + NR group. The surgical group was fasted for 24 hours preoperatively but allowed normal water intake. The upper and lower poles of the left kidney were removed under sevoflurane anesthesia. One week later, the right kidney was completely removed. The sham-operated group underwent the same surgery as the 5 / 6 nephrectomy group at the same timeframe, but the renal capsule was removed only after the kidney was exposed, followed by closure of the abdomen. To accelerate the aortic calcification process, during the study period, animals were fed a high-phosphate diet (0.9% Pi) one week after the 5 / 6 nephrectomy, while the control group was fed a normal diet. Samples were collected after 12 weeks. Mouse aortic tissue was stained with HE, Alizan red, and Von Kossa. Compared with the WT group, the NR group showed a significant increase in Alizan red and Von Kossa positive areas in their aortic tissue. Compared with the NR group, the KO+NR group showed a significant increase in both Alizan red and Von Kossa positive areas in their aortic tissue. No obvious calcified nodules were found in the aortic tissue of the WT and KO groups (P<0.05). Figure 5The results (AC) indicate that calcified nodules were significantly increased in the aortic tissue of mice in the NR group, and that KO+NR could aggravate calcified nodules induced by NR.

[0069] Similarly, a mouse model of vascular calcification in chronic renal failure was established using ALDH2SMKO mice through 5 / 6 nephrectomy combined with a high-phosphorus diet. Six- to seven-week-old male ALDH2SMKO mice were divided into four groups: WT, SMKO, NR, and SMKO+NR. Aortic tissue was collected from the mice and stained with HE, Alizan red, and Von Kossa. Compared with the WT group, the NR group showed a significant increase in Alizan red and Von Kossa-positive areas in its aortic tissue. Compared with the NR group, the SMKO+NR group showed a significant increase in Alizan red and Von Kossa-positive areas in its aortic tissue, while no obvious calcified nodules were found in the aortic tissue of the WT and SMKO groups (P<0.05). Figure 5 The presence of FH indicates that calcified nodules were significantly increased in the aortic tissue of mice in the NR group, and that SMKO+NR could aggravate calcified nodules induced by NR.

[0070] The results above indicate that both systemic knockout of ALDH2 and conditional knockout of vascular smooth muscle cells can promote vascular calcification induced by chronic renal insufficiency.

[0071] Example 4

[0072] This embodiment demonstrates that ALDH2 overexpression or activation reduces vascular calcification in renal insufficiency.

[0073] To further verify the role of ALDH2 in vascular calcification, an ALDH2-overexpressing mouse model of chronic renal insufficiency with vascular calcification was established using 5 / 6 nephrectomy combined with a high-phosphorus diet. Six- to seven-week-old male ALDH2-Tg mice were divided into four groups: WT, Tg, NR, and Tg+NR. Aortic tissue was collected from the mice for HE, Alizan red, and Von Kossa staining. Compared with the WT group, the NR group showed a significant increase in Alizan red and Von Kossa-positive areas in their aortic tissue. Compared with the NR group, the Tg+NR group showed a significant decrease in Alizan red and Von Kossa-positive areas in their aortic tissue. No obvious calcified nodules were observed in the aortic tissue of the WT and Tg groups (P<0.05). Figure 6The results showed that calcified nodules were significantly increased in the aortic tissue of mice in the NR group, and that Tg+NR could alleviate calcified nodules induced by NR.

[0074] Alda-1 was used to activate ALDH2, and the role of ALDH2 in vascular calcification induced by chronic renal failure was verified. Six-week-old male mice were randomly divided into four groups: Sham group, Alda-1 group, NR group, and Alda-1+NR group. Aortic tissues were collected from mice for HE, Alizan red, and Von Kossa staining. Compared with the Sham group, the NR group showed a significant increase in Alizan red and Von Kossa staining in its aortic tissues. Compared with the NR group, the Alda-1+NR group showed a significant decrease in Alizan red and Von Kossa staining in its aortic tissues. No obvious calcified nodules were observed in the aortic tissues of the Sham and Alda-1 groups (P<0.05). Figure 6 The presence of FH indicates that calcified nodules were significantly increased in the aortic tissue of mice in the NR group, and that Alda-1+NR could exacerbate calcified nodules induced by NR.

[0075] The results above indicate that both overexpression and activation of ALDH2 can improve vascular calcification induced by chronic renal insufficiency.

Claims

1. The use of substances that overexpress ALDH2 in the preparation of medicaments for the prevention, relief and / or treatment of vascular calcification; wherein the vascular calcification is induced by vitamin D3 and chronic renal insufficiency.

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