Application of miR21-5p / Tpm1 shaft in preparation of medicine for preventing and / or treating diabetic vascular calcification

Abstract

The invention relates to the field of biological pharmacy, and particularly provides application of a miR21-5p / Tpm1 shaft in preparation of a medicine for preventing and / or treating diabetic vascular calcification. The invention discovers that abnormal activation of the miR21-5p / Tpm1 axis exists in a patient with diabetic vascular calcification for the first time, and further discovers a new mechanism that miR21-5p negatively regulates and controls Tpm1 to promote osteogenic differentiation of vascular smooth muscle cells (VSMC). Animal experiments prove that the miR21-5p / Tpm1 inhibitor can effectively inhibit occurrence and development of diabetic vascular calcification, and a new thought and treatment scheme are provided for clinical treatment of diabetic vascular calcification.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular, to an application of a miR21-5p / Tpm1 axis in the preparation of a drug for preventing and / or treating diabetic vascular calcification. Background Art

[0002] Vascular calcification (VC) is a dynamic vascular disease in which calcium phosphate is ectopically deposited in the form of hydroxyphosphate in the vascular wall. It is widely seen in diabetic patients and is the common pathological basis and early vascular damage state of diabetic macrovascular complications. Clinical data show that the risk of cardiovascular events in diabetic patients with severe VC is 10 times that of patients without VC, and the progression and severity of VC are highly correlated with adverse cardiovascular events such as acute coronary syndrome and sudden cardiac death. It is a recognized independent predictor of all-cause mortality and lifelong cardiovascular disease risk in diabetic patients.

[0003] Contrary to the previous understanding that VC is a passive, degenerative vascular disease, in recent years, it has been gradually recognized that VC is an active, multi-gene-regulated biological process. Similar to the osteoblast differentiation process during bone mineralization, it is mainly driven by vascular smooth muscle cells (VSMC). In the vascular tissue of normal adults, VSMC exhibits a contractile phenotype, responding to signals such as acetylcholine or adrenaline by expressing a series of contractile proteins such as α-actin (α-SMA) and SM22α to maintain the normal morphology of blood vessels. In the diabetic state, many hormones and physiological abnormalities associated with hyperglycemia cause highly active pathological phenotypic transformation of VSMC, leading to abnormal deposition of insoluble calcium and phosphate salts in the vascular wall, promoting the formation and development of VC. However, despite the gradual deepening of the understanding of the pathological mechanism of diabetic VC, there is still no effective specific treatment for VSMC osteogenic transformation. Therefore, finding new diabetic VC mechanisms and related drug targets, especially targets that can regulate VSMC osteogenic transformation at the molecular level, is of great significance for improving the clinical symptoms and prognosis of diabetic VC patients. Summary of the invention

[0004] In order to solve the above technical problems, the purpose of the present invention is to study the new molecular mechanism of regulating diabetic VC, especially the high glucose-induced VSMC osteogenic transformation. This study first found that the miR21-5p / Tpm1 axis was significantly activated in patients with diabetic vascular calcification, and further found its regulatory mechanism for promoting VSMC proliferation, migration and osteogenic transformation.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The first aspect of the present invention provides a use of a miR21-5p inhibitor in the preparation of a medicament for preventing and / or treating diabetic vascular calcification, wherein the miR21-5p inhibitor is used to reduce the expression of miR21-5p in vivo or silence the expression of miR21-5p, and the miR21-5p nucleotide sequence is SEQ ID NO.1.

[0006] The second aspect of the present invention is the use of a Tpm1 inhibitor in the preparation of a drug for preventing and / or treating diabetic vascular calcification, wherein the Tpm1 inhibitor is used to reduce the expression of Tpm1 gene in vivo or silence the expression of Tpm1 gene, and the Tpm1 nucleotide sequence is SEQ ID NO.2.

[0007] In one embodiment, the drug is siRNA.

[0008] In one embodiment, the above application includes inhibiting the occurrence and development of vascular calcification in diabetic patients.

[0009] Compared with the prior art, the present invention has the following beneficial effects: The present invention proposes for the first time that the miR21-5p / Tpm1 axis can be used as a target for preventing or treating diabetic vascular calcification. The present invention discovers for the first time that there is activation of the miR21-5p / Tpm1 axis in patients with diabetic vascular calcification, and finds that at the cellular level, inhibiting miR21-5p activation can partially restore the expression level of the target gene Tpm1, thereby effectively reducing the excessive proliferation, migration and osteogenic transformation of VSMC, and playing a role in reducing VC. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 The results of the study on the expression levels of miR21-5p and Tpm1 in patients with diabetic vascular calcification and their correlation with the degree of vascular calcification are shown in Figure 2. Figure 1 A and Figure 1 C is the expression levels of miR21-5p and Tpm1 mRNA in patients with diabetic vascular calcification and non-calcification groups. Figure 1 B and Figure 1 D is the correlation between miR21-5p and Tpm1 mRNA expression levels and the Agatston score of coronary artery calcification. Figure 1 E is the expression of contractile markers αSMA and Tpm1 in the aorta of patients with diabetic coronary heart disease evaluated by immunofluorescence. Compared with the Non-CAC group, # P <0.05, ## P <0.01.

[0011] Figure 2 The effect of miR21-5p on the proliferation and migration of HAVSMCs. Figure 2 A is the expression level of miR21-5p in HAVSMC after transfection with miR21-5p mimic and miR21-5p inhibitor. Figure 2 B is the CCK8 assay to evaluate the proliferation activity of HAVSMCs in different groups. Figure 2 C and Figure 2 D is the evaluation of the migration ability of HAVSMC in different groups by scratch test. ### P <0.001; compared with the HGCM group, P <0.001; compared with mimic / inhibitor NC, & P <0.05, &&& P <0.001.

[0012] Figure 3 The effects of miR21-5p overexpression and inhibition on HAVSMC calcification. Figure 3 A is the evaluation of the formation of HAVSMC calcification nodules in different groups by Alizarin red staining. Figure 3 B is the detection of cellular calcium content of HAVSMC in different groups by colorimetry. ### P <0.001; compared with the HGCM group, P <0.001; compared with mimic / inhibitor NC, & P <0.05, &&& P <0.001.

[0013] Figure 4 The effects of miR21-5p overexpression and inhibition on the expression of osteogenic and contractile proteins in HAVSMCs. Figure 4 A is the Western blot image of osteogenesis and contraction-related proteins in HAVSMCs of different groups. Figure 4 BE Figure 4 Quantitative analysis of the expression of various osteogenesis- and contraction-related proteins in A. Compared with NG, ### P <0.001; compared with the HGCM group, P <0.001; compared with mimic / inhibitor NC, & P <0.05,&&& P <0.001.

[0014] Figure 5 The effects of miR21-5p overexpression and inhibition on the miR21-5p / Tpm1 axis, Figure 5 A is the expression level of miR21-5p in HAVSMCs of different groups. Figure 5 B is the expression level of Tpm1 mRNA in HAVSMCs of different groups. Figure 5 C is the Western blot image of Tpm1 expression in HAVSMCs of different groups. Figure 5 D is Figure 5 Quantitative analysis of Tpm1 expression in C. Compared with NG, ### P <0.001; compared with the HGCM group, P <0.001; compared with mimic / inhibitor NC, & P <0.05, &&& P <0.001.

[0015] Figure 6 The effects of double silencing of miR21-5p and Tpm1 on the proliferation and migration of HAVSMCs. Figure 6 A is the mRNA expression level of Tpm1 after transfection with Tpm1 siRNA. Figure 6 B is the proliferation activity of HAVSMC in each group detected by CCK-8. Figure 6 C is the scratch test to evaluate the migration ability of HAVSMC in different groups. Figure 6 D is the quantitative analysis of cell migration rate in different groups. Compared with siRNA or NG, ### P <0.001; compared with the HGCM group, P <0.001.

[0016] Figure 7 The effect of double silencing of miR21-5p and Tpm1 on the calcification of HAVSMCs. Figure 7 A is the evaluation of the formation of HAVSMC calcification nodules in different groups by Alizarin red staining. Figure 7 B is the detection of cellular calcium content of HAVSMC in different groups by colorimetry. # P <0.05, ## P <0.01; compared with the HGCM group, P <0.05, P <0.01, P <0.001.

[0017] Figure 8 The effects of double silencing of miR21-5p and Tpm1 on the expression of osteogenic and contractile proteins in HAVSMCs. Figure 8 A is the Western blot image of osteogenesis and contraction-related proteins in HAVSMCs of different groups. Figure 8 BE Figure 8 Quantitative analysis of the expression of various osteogenesis- and contraction-related proteins in A. Compared with NG, # P <0.05, ## P <0.01; compared with the HGCM group, P <0.05, P <0.01, P <0.001.

[0018] Fig. 9 The effect of double silencing of miR21-5p and Tpm1 on the miR21-5p / Tpm1 axis in HAVSMCs. Fig. 9 A is the expression level of miR21-5p in HAVSMCs of different groups. Fig. 9 B is the expression level of Tpm1 mRNA in HAVSMCs of different groups. Fig. 9 C is the Western blot image of Tpm1 expression in HAVSMCs of different groups. Fig. 9 D is Fig. 9 Quantitative analysis of Tpm1 expression in C. Compared with NG, # P <0.05, ## P <0.01; compared with the HGCM group, P <0.05, P <0.01, P <0.001.

[0019] Fig.10 The degree of aortic calcification in rats in each group. Fig.10 A is a representative image of Alizarin red pathological staining of the whole aorta of rats in each group (n=3); Fig.10 B is the quantification of alizarin red staining of the whole aorta (n=3); Fig.10C is a representative image of the aortic arch of rats in each group stained with Alizarin red (n=3, 20×); Fig.10 D is the quantitative analysis of Alizarin red staining of aortic arch of rats in each group (n=3). Fig.10 E is the calcium content of aorta of rats in each group (n=5); Fig.10 F is the ALP activity of the aorta of rats in each group (n=4-5) Note: Compared with the CON group, ### P<0.001; compared with the DVC group, P<0.05, P<0.001.

[0020] Fig.11 The expression of miR21-5p / Tpm1 axis in the aorta of rats in each group. Fig.11 A is the expression level of miR21-5p in the aorta of rats in each group (n=3); Fig.11 B is the level of Tpm1 mRNA in the aorta of rats in each group (n=3); compared with the CON group, ### P<0.001; compared with the DVC group, P<0.05, P<0.001. DETAILED DESCRIPTION The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0022] The experimental methods used in the embodiments of the present invention are described as follows: 1. PBMC extraction: Use EDTA anticoagulant tube to extract 10mL of venous blood from the patient and shake well to avoid blood coagulation. Complete PBMC separation within 2 hours after blood collection. Dilute 10mL of blood with an equal volume of PBS, slowly add to the upper layer of Ficoll-Hypaque lymphocyte separation solution, and centrifuge at 800rpm for 20 minutes (room temperature, speed 1, speed 0). After centrifugation, four layers are formed: plasma layer, mononuclear cell buffy coat layer, separation fluid layer, red blood cells and granulocyte layer. Carefully aspirate the buffy coat layer into a new tube, wash 3 times with PBS (800rpm, 10 minutes), and finally obtain PBMC precipitate for subsequent experiments.

[0023] 2. Cell culture: HAVSMC were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% double antibodies (streptomycin, penicillin) and cultured in a 37°C, 5% CO2 incubator. The cell morphology was observed and the culture medium was replaced every 2 days. When the cell confluence reached 80%-90%, the culture medium was discarded, the cells were washed twice with PBS, and 1 mL of 0.25% trypsin was added for digestion for 1-2 minutes. After observing the cell detachment under a microscope, the trypsin was neutralized with 2-4 times the volume of complete culture medium, centrifuged (10000rpm, 4°C, 5 minutes), resuspended and divided into new culture dishes for continued culture.

[0024] 3. Establishment of cell calcification model: When HAVSMCs were cultured to 80% confluence, the normal culture medium was discarded and replaced with three different culture media for calcification induction. The control group (NG) used normal DMEM culture medium, the high glucose group (HG) used high glucose culture medium containing 30mmol / L glucose, and the high glucose calcification group (HGCM) used culture medium containing 30mmol / L glucose, 10mmol / L β-glycerophosphate and 3mmol / L CaCl 2 The induction time of the calcification model is 7-10 days. During this period, the cell morphology changes are observed daily and the culture medium is replaced every 2 days. During the calcification induction process, the cell proliferation rate is significantly slowed down and no subculture treatment is required.

[0025] 4. Transfection: (1) miR21-5p transfection: Prepare transfection complexes of miR21-5p mimic (final concentration 50mmol / L, sequence SEQ ID NO.3-4) and miR21-5p inhibitor (final concentration 100mmol / L, sequence SEQ ID NO.5). Add the transfection complexes to the cells in a 24-well plate, add the culture medium to 500μL, incubate at 37℃ for 24 hours, and then switch to high-glucose calcification medium for another 24 hours. (2) Tpm1 siRNA transfection: Use Lipofectamine 2000 to prepare Tpm1 siRNA complexes (sequence SEQ ID NO.6-7) and transfect HAVSMC cells (culture density 70%-80%). Replace the complete culture medium 4-6 hours after transfection, and culture for 48-72 hours for RNA or protein extraction.

[0026] 5. Cell viability detection: CCK-8 method was used for cell viability detection. Well-growing cells were digested and resuspended in serum-free medium, the cell density was adjusted to 1×10^4 cells / 100μL, and inoculated into 96-well plates, 100μL per well. Incubate at 37°C, 5% CO 2After 24 hours of culture in the incubator, the culture medium was discarded and normal culture medium (NG), high glucose culture medium (HG) and high glucose calcification culture medium (HGCM) were added respectively. Eight replicate wells were set for each group, and the blank control group used cell-free culture medium. After 24 hours of continuous culture, 10 μL CCK-8 solution was added to each well, incubated for 2 hours, and the absorbance was measured at a wavelength of 450 nm using an enzyme reader.

[0027] 6. Animal Culture 48 SPF male SD rats, 6-7 weeks old, weighing 180-200 g, were purchased from SCXK (Beijing 2019-0010) of Sibeifu Biotechnology Co., Ltd. All mice were kept in cages (5-6 per cage) and raised in an SPF animal facility (containing sterilized water and feed) with a temperature of 22±2℃, a humidity of 55±5%, and a 12 / 12-hour day and night (light / dark) alternation. This animal experiment has been approved by the Animal Ethics Committee of Xiyuan Hospital, China Academy of Chinese Medical Sciences (approval number: 2024XLC014-2).

[0028] 7. Animal Modeling (1) Diabetes model: After 1 week of adaptive feeding, SPF male SD rats were intraperitoneally injected with 30 mg / kg STZ (stored on ice, prepared immediately before use) for 3 consecutive days to induce a diabetes model. After 3 days, the rats were tail-clipped and blood was collected to test fasting blood glucose. Blood glucose values ​​> 11.1 mmol / L were considered to be successful in modeling. The remaining rats that failed to establish a model were given an additional 30 mg / kg STZ intraperitoneal injection and fasting blood glucose was tested. Those with blood glucose values ​​> 11.1 mmol / L were included in the model group for the next step of the experiment.

[0029] (2) Vascular calcification model: using vitamin D 3 (Vitamin D 3 , VitD 3 ) Overload induced VC. Diabetic rats were intraperitoneally injected with high doses of VitD for 4 consecutive days. 3 (5.5×10^5IU / Kg), 14 days after the last injection was considered the calcification molding period.

[0030] 8. Adenoviral vector construction and transfection (1) Vector construction: The adeno-associated virus (AAV) used in this experiment was constructed, produced and purified by Yunzhou Biotechnology (Guangzhou) Co., Ltd. (ID: VB240116-1098trb, VB010000-9461vph). The specific steps are as follows: design the short hairpin RNA (shRNA) sequence specific to rno-miR21-5p and the control shRNA, insert them into the multiple cloning site downstream of the U6 promoter of the vector pAAV[shRNA]-EGFP-U6, and construct a recombinant plasmid expressing rno-miR21-5p. After the correctness of the vector was verified by restriction digestion and Sanger sequencing, it was co-transfected into HEK293T cells with the auxiliary plasmid and packaging plasmid, and the virus was produced using the AAV8 packaging system. After 48-72 hours of culture after transfection, the culture medium and cell lysate were collected, the virus particles were purified by ultracentrifugation, and the virus titer was determined by fluorescence quantitative PCR. In order to verify the transfection efficiency of the virus and the expression of the target sequence, the enhanced green fluorescent protein (EGFP) carried in the vector was used as a marker, and its green fluorescence expression under blue light or ultraviolet light was detected by fluorescence microscopy or flow cytometry. Finally, the packaged virus structures were miR21-5p OE: pAAV-[shRNA]-EGFP-U6>{rno-miR21-5p} and miR21-5p NC: pAAV-[shRNA]-EGFP-U6>Scramble[shRNA#1]. The purified viruses were aliquoted and stored at -80°C for subsequent experiments.

[0031] (2) Tail vein injection transfection: The transduction effect of adeno-associated virus in tissues can last for 2-6 months, and the expression peak can generally be reached 2-4 weeks after injection. Therefore, we injected the AAV delivery system once into the lateral tail vein 1 week after STZ injection. Wipe the mouse tail clean, warm the rat to dilate its blood vessels for easy injection, and disinfect the injection site with an alcohol cotton ball before injection. The injection site is selected from the bottom of the tail 1 / 3 away from the tip of the tail. Grasp the distal tail and gently turn it to one side so that the lateral vein can be seen. Insert the needle at a small angle and inject the diluted pAAV into the vein. Pay attention to the color of the vein during injection to ensure that the virus is injected into the vein. The stock solution concentration of pAAV-[shRNA]-EGFP-U6>{rno-miR21-5p} was 2.63×10^13 gene copies / mL, and that of pAAV-[shRNA]-EGFP-U6>Scramble[shRNA#1] was 1.83×10^13 gene copies / mL. The stock solution was diluted to the target concentration with PBS before injection. Referring to previous studies, the injection dose for each rat was 5×10^11 gene copies / mL, and the injection volume was 0.1 mL.

[0032] 9. Animal Grouping and Intervention Methods After the modeling was completed, all DVC rats with modeling were randomly divided into model group (DVC group, n=12), miR21-5p overexpression control group (miR21-5p NC group, n=12), miR21-5p overexpression group (miR21-5p OE group, n=12), Dantongmai prescription combined with intermittent fasting + miR21-5p overexpression control group (D+I+miR21-5p NC group, n=12), Dantongmai prescription combined with intermittent fasting + miR21-5p overexpression group (D+I+miR21-5p OE group, n=12), and 12 blank SD rats were taken as the control group (CON group). The specific drug administration and intervention schemes are as follows: (1) Control group (CON group): Normal diet and intragastric administration of an equal volume of pure water for 4 weeks.

[0033] (2) Model group (DVC group): normal diet and intragastric administration of an equal volume of pure water for 4 weeks.

[0034] (3) miR21-5p overexpression control group (miR21-5p NC group): normal diet, tail vein injection of miR21-5p NC, and oral administration of an equal volume of pure water for 4 weeks.

[0035] (4) miR21-5p overexpression group (miR21-5p OE group): normal diet, miR21-5p OE tail vein injection, and oral gavage of an equal volume of pure water for 4 weeks.

[0036] (5) Dantongmai formula combined with intermittent fasting + miR21-5p overexpression control group (D+I+miR21-5p NC group): miR21-5p NC was injected into the tail vein, and Dantongmai formula (0.96 g / kg / d) was administered orally during intermittent fasting for 4 weeks.

[0037] (6) Dantongmai formula combined with intermittent fasting + miR21-5p overexpression group (D+I+miR21-5p OE group): miR21-5p OE was injected through the tail vein, and Dantongmai formula (0.96 g / kg / d) was administered intragastrically during intermittent fasting for 4 weeks.

[0038] Note: Dantongmai Recipe: Professor Liu Yue of Xiyuan Hospital of China Academy of Chinese Medical Sciences developed an empirical recipe based on previous research and clinical practice, and has applied for an invention patent (patent number: 202311796613.6). The recipe is composed of Danshen, Coptis chinensis, Polygonum cuspidatum, etc. The decoction pieces and extract powder used in the study were identified and processed by the Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences. Each gram of extract powder contains 23.5g of crude drug, and purified water is used to prepare the liquid for use.

[0039] Intermittent fasting uses a time-restricted diet strategy. The time when light starts (8 am) is defined as standard time 0 (ZT0). The time when light ends (8 pm) is defined as standard time 12 (ZT12). Rats are allowed to eat within 8 hours from 10 pm (ZT14) to 6 am (ZT22). The rest of the time, the feed will be withdrawn until 10 pm the next day. The entire fasting process only restricts food intake but not water.

[0040] 10. Calcium content and ALP determination (1) Calcium content: After the experiment, the calcium content of the aorta was determined by the o-phenol peptide complex ketone colorimetric method. The aorta was cut on ice and rinsed with pre-cooled PBS. It was placed in a homogenizer and 200 μL of protein lysis buffer was added. The tissue was homogenized at 15,000 rpm, 10 s / time, and 10 s interval for 3 cycles. After completion, the homogenate was transferred to a centrifuge tube and allowed to stand on ice for 20 minutes. Centrifuge at 12,000 rpm in a high-speed centrifuge pre-cooled at 4°C for 15 minutes, and the supernatant was collected for later use. According to the requirements of the kit, a 96-well plate was taken, and a blank group, a test group, and a standard group were set up. 5 μL ddH2O, 5 μL of the supernatant to be tested, and 5 μL of the calcium standard were added respectively. Then 200 μL of calcium ion colorimetric working solution was added to each well and incubated at room temperature for 10 minutes. The absorbance value (wavelength 610 nm) was measured using an enzyme reader, and a standard curve was drawn to calculate the calcium content in the test sample. The total protein content in the supernatant was determined by the bicinchoninic acid assay (BCA) method, and the protein content was standardized with the calcium content (mmol / g protein).

[0041] (2) ALP activity: The steps for obtaining tissue homogenate are the same as those for calcium content detection. According to the requirements of the kit, a 96-well plate was taken and a blank group, a test group and a standard group were set up. 50 μL of detection buffer, 50 μL of supernatant to be tested, and 50 μL of standard working solution (0.5 mmol / L p-nitrophenol solution) were added to the wells respectively. 50 μL of colorimetric substrate (prepared immediately before use) was added to the blank group and the test group, and 100 μL of detection buffer was added to the standard group. Incubate at 37°C for 30 minutes, and then add 100 μL of reaction stop solution to all wells to terminate the reaction. The standard wells and wells with ALP activity will show different shades of yellow. Use an enzyme reader to measure the absorbance value (wavelength 405 nm), draw a standard curve and calculate the ALP activity in the test sample. The total protein content of the supernatant was determined by the BCA method, and the ALP activity (U / g protein) was standardized with the protein content.

[0042] 11. Alizarin Red Staining (1) Cell staining: After HAVSMC were induced in different conditioned medium for 7 days, the medium in the culture dish was aspirated, and the cells were gently rinsed twice with PBS buffer. 4% paraformaldehyde was added for fixation at room temperature for 20 min. After fixation, the cells were washed three times with PBS to remove excess fixative. Then, 3 mL of 2% Alizarin red stain (pH 4.3) was added to the culture dish. The cells were gently shaken to fully cover the cells. The cells were incubated at 37°C in the dark for 5 min, the stain was discarded, and the cells were gently rinsed twice with double distilled water. Dark red calcified nodules were visible under an inverted microscope, and photographed for record.

[0043] (2) Aortic staining: After the experiment, the whole aorta was fixed in 4% PFA overnight. Then, the PFA was discarded and the aorta was immersed in 2% Alizarin Red staining solution at room temperature overnight. The aorta was washed with 2% potassium hydroxide solution and photographed with an inverted microscope.

[0044] The aortic arch was fixed in 4% PFA overnight. The vascular tissue was removed, the excess PFA was washed off, and it was dehydrated with gradient alcohol, and then transparent, waxed and paraffin-embedded. The proximal section was sliced ​​with a thickness of 5 μm. Bake in a 65° oven for 1 hour, remove the sections, and dewax them in xylene and gradient alcohol in turn: xylene I and II for 10 minutes each, anhydrous ethanol I, anhydrous ethanol II, 95% ethanol, 90% ethanol, 80% ethanol for 5 minutes each, and rinsed with distilled water 3 times to remove residual ethanol. Dry the sections to remove excess water, add 2% alizarin red dye solution to stain, wash off the dye solution after 5 minutes, and immerse in 80% ethanol, 90% ethanol, 95% ethanol, anhydrous ethanol I, and anhydrous ethanol II in turn for dehydration (5 minutes each), permeabilize in xylene I and II (5 minutes each), and seal with neutral gum.

[0045] 12. Cell scratch test: The scratch test was used to detect cell migration ability. After digestion and resuspending of HAVSMC cells under different treatment conditions, the cell density was adjusted to 5×10^5 cells / mL, and 2 mL was inoculated into each well of a 6-well plate and incubated at 37°C and 5% CO 2 Culture in an incubator until the cell confluence reaches 80%. Discard the supernatant, wash twice with PBS, draw a straight line in the center of each well with a 200μL sterilized pipette tip, wash the detached cells with PBS and record it as 0h, and take pictures under a microscope. The NG group was replaced with ordinary culture medium, and the other groups were replaced with high-glucose calcification culture medium. After 24h of culture, observe and take pictures, and repeat 3 times.

[0046] 13. Immunofluorescence: Human aortic tissue was stained and analyzed by immunofluorescence. After the human aortic specimen was obtained during surgery, it was rinsed with physiological saline, fixed with 4% PFA, dehydrated with gradient alcohol, and embedded in paraffin to prepare 5 μm thick sections. After the sections were dewaxed to water, antigen retrieval was performed, 0.5% Triton X-100 permeabilization solution was added after PBS washing, and blocking solution was added after PBS washing. Then the primary antibody (Anti-aSMA: 1:500; Anti-Tpm1: 1:500) was added and incubated at 4°C overnight. After PBS washing, the secondary antibody (1:100) was added, incubated at room temperature, washed with PBS, and stained with DAPI for 10 minutes. After sealing, the sections were observed using a laser confocal microscope.

[0047] 14. PCR: Total RNA was extracted from aortic tissue, HAVSMC and PBMC, and treated with Trizol method, followed by chloroform layering, isopropanol precipitation and 75% ethanol washing to obtain high-purity RNA. NanoDrop was used to detect RNA concentration and purity. Primers were designed using Primer Primer 5.0 and verified for specificity by Blast, and synthesized by a professional company. The HiScriptIII kit was used for gene cDNA synthesis, and the miRNA was reverse transcribed using the Poly(A) tailing method. The operation was performed strictly in accordance with the instructions of the kit. The real-time fluorescence quantitative PCR reaction system used AceQ Universal SYBR qPCR Master Mix. The amplification conditions included pre-denaturation at 95°C for 10 minutes, 95°C for 15 seconds, and 60°C for 60 seconds, for a total of 40 cycles. The melting curve was used to analyze the specificity. The experimental results were analyzed by 2 -△△CT The relative expression level of genes was calculated by the PCR method, and three replicate wells were set for each sample to ensure data accuracy.

[0048] 15.WB: Extract the total protein sample of HAVSMC. After washing and centrifuging the cell samples with PBS, add the protein lysis buffer to fully lyse. After the protein concentration is determined by the BCA method, add the sample buffer and boil it for denaturation, and save the protein loading solution. After separating the protein by SDS-PAGE gel electrophoresis, use PVDF membrane for protein transfer, and block it with 5% skim milk powder after transfer. According to the instructions, configure the primary antibody (such as Anti-Runx2, Anti-BMP2, Anti-αSMA, etc.) and the secondary antibody, incubate the membrane separately and wash it with TBST several times, develop it with ECL developer, record the protein bands by exposing the film, and finally use ImageJ software to analyze the grayscale of the bands.

[0049] In the following examples, unless otherwise specified, all are conventional methods. The materials, reagents, etc. used, unless otherwise specified, can all be obtained from commercial sources. In addition, the embodiments should be understood as illustrative rather than limiting the scope of the present invention, and the essence and scope of the present invention are limited only by the claims. For those skilled in the art, various changes or modifications of the material composition and dosage in these embodiments, without departing from the essence and scope of the present invention, also belong to the protection scope of the present invention.

[0050] Example 1 Activation of miR21-5p / Tpm1 axis in diabetic vascular calcification In order to more fully understand the role of miR21-5p / Tpm1 axis in the progression of diabetic VC, we enrolled 33 patients with type 2 diabetes who were admitted to the outpatient clinic or hospital of Xiyuan Hospital of China Academy of Chinese Medical Sciences (all of whom had signed informed consent) to observe the expression level of miR21-5p / Tpm1 axis. The results showed that compared with the diabetic non-vascular calcification group, the PBMC miR21-5p level of the diabetic vascular calcification group was significantly increased, while the Tpm1 mRNA level was significantly decreased ( Figure 1 A, C, P < 0.05, and miR21-5p expression was significantly positively correlated with coronary VC Agatston score, on the contrary, Tpm1 expression was significantly negatively correlated with coronary VC Agatston score ( Figure 1 B, D, P < 0.01). In addition, we also used immunofluorescence to evaluate the expression of aortic contractile markers α-SMA and Tpm1 in patients with diabetic coronary heart disease and coronary artery calcification. The results showed that compared with healthy controls, the expression of α-SMA and Tpm1 in the aorta of patients with diabetic coronary heart disease and coronary artery calcification was significantly downregulated ( Figure 1 E).

[0051] The above results indicate that the miR21-5p / Tpm1 axis is involved in regulating the occurrence and development of diabetic VC.

[0052] Example 2 Overexpression of miR21-5p promotes proliferation and migration of vascular smooth muscle cells Enhanced cell proliferation and migration are important characteristics of phenotypic transformation of VSMC. In order to explore the role of miR21-5p in the proliferation and migration of HAVSMC in vitro calcification model induced by high glucose osteogenesis, we transfected miR21-5pmimic, miR21-5p inhibitor and corresponding negative controls to observe the changes in HAVSMC proliferation and migration after miR21-5p overexpression and inhibition. PCR results showed that miR21-5p mimic significantly upregulated HAVSMC miR21-5p expression, and miR21-5p inhibitor significantly inhibited HAVSMC miR21-5p expression, indicating that the transfection was successful ( Figure 2 A, P<0.001). CCK-8 results showed that the cell proliferation activity in the HGCM group was significantly enhanced compared with that in the NG group ( Figure 2 B, P<0.001). Compared with the HGCM group cells, miR21-5p overexpression further increased the proliferation capacity of HAVSMCs ( Figure 2 B, P<0.001), while inhibition of miR21-5p effectively inhibited the proliferation of HAVSMCs ( Figure 2 B, P<0.001).

[0053] The scratch test was used to detect the changes in the migration ability of HAVSMC. Compared with the NG group, the migration ability of cells in the HGCM group was significantly enhanced, and the scratch distance within 24 hours was significantly shortened ( Figure 2 CD, P<0.001). Compared with the HGCM group, miR21-5p overexpression further increased the migration ability of cells and reduced the scratch area at 24 h, but no significant statistical difference was observed ( Figure 2 CD, P>0.05). In contrast, inhibition of miR21-5p effectively attenuated the migration of HAVSMCs and reduced the 24h cell migration rate ( Figure 2 CD, P<0.001).

[0054] The above results indicate that overexpression of miR21-5p can promote excessive proliferation and migration of VSMCs, and inhibition of miR21-5p can reverse the above process.

[0055] Example 3: miR21-5p negatively regulates Tpm1 to promote osteogenic transformation of HAVSMC Alizarin red staining was used to observe the effect of miR21-5p on the calcification of HAVSMC. Compared with the NG group, after 7 days of induction with high glucose osteogenic medium, the cells in the HGCM group showed scattered dark red calcified nodules. Compared with the HGCM group, the cells in the miR21-5pmimic group showed more severe calcified nodules. However, no significant calcified nodules appeared in the cells in the miR21-5p inhibitor group ( Figure 3 A). In addition, the calcium content of HAVSMC in each group was detected by colorimetry. The calcium content of cells in the HGCM group was significantly higher than that in the NG group ( Figure 3 B, P<0.001). Compared with the HGCM group, the calcium content of the miR21-5p mimic group cells increased further ( Figure 3 B, P<0.001), the calcium content of cells in the miR21-5p inhibitor group decreased significantly ( Figure 3 B, P<0.001), which was consistent with the results of Alizarin red staining. This indicates that overexpression of miR21-5p can promote calcification of HAVSMC in vitro.

[0056] Subsequently, we further detected the contraction and osteogenic protein expression of HAVSMC in each group. The results showed that compared with the NG group, the osteogenic proteins Runx2 and BMP2 in the HGCM group were significantly upregulated, and the contractile proteins αSMA and SM22α were significantly downregulated ( Figure 4 AE, P <0.05, P <0.001). Compared with the HGCM group, the osteogenic proteins Runx2 and BMP2 were further upregulated after miR21-5p overexpression, while the contractile proteins αSMA and SM22α were significantly downregulated ( Figure 4 AE, P <0.05, P <0.001). Inhibition of miR21-5p can effectively reverse the above process, including down-regulating the expression of osteogenic proteins Runx2 and BMP2, and up-regulating the expression of contractile proteins αSMA and SM22α ( Figure 4 AE, P < 0.05, P < 0.001). These results collectively indicate that miR21-5p is involved in regulating the osteogenic transformation of HAVSMC in vitro. Activation of miR21-5p can promote the osteogenic transformation of HAVSMC, while inhibition of miR21-5p can effectively reverse this process and restore the contractile phenotype of HAVSMC.

[0057] Next, we further observed the effects of miR21-5p overexpression and inhibition on the miR21-5p / Tpm1 axis. PCR results showed that miR21-5p was significantly increased in the HGCM group compared with the NG group ( Figure 5A, P <0.001). Compared with the HGCM group, the miR21-5p level in the miR21-5p mimic group was significantly increased, while that in the miR21-5p inhibitor group was significantly decreased ( Figure 5 A, P <0.001), indicating that miR21-5p was regulated accordingly after transfection of mimic or inhibitor. We then detected the changes in Tpm1 mRNA and protein levels by PCR and WB. PCR results showed that compared with the NG group, the Tpm1 mRNA level in the HGCM group was significantly decreased ( Figure 5 B, P <0.001). Compared with the HGCM group, Tpm1 mRNA was further downregulated in the miR21-5p mimic group, while Tpm1 mRNA was significantly upregulated in the miR21-5p inhibitor group ( Figure 5 B, P <0.001). WB results showed the same trend, that is, compared with the NG group, the expression of Tpm1 protein in the HGCM group was significantly decreased. Compared with the HGCM group, the expression of Tpm1 protein in the miR21-5p mimic group was further decreased, while that in the miR21-5p inhibitor group was significantly increased ( Figure 5 CD, P < 0.001).

[0058] The above results collectively indicate that miR21-5p can negatively regulate Tpm1 to promote the osteogenic transformation of VSMCs. Inhibition of miR21-5p can improve the osteogenic transformation of VSMCs.

[0059] Example 4 Inhibition of miR21-5p-mediated Tpm1 downregulation reduces VSMC hyperproliferation and migration To further clarify the relationship between miR21-5p and Tpm1 in phenotypic transformation of HAVSMC, we then performed a double silencing experiment of miR21-5p and Tpm1 on HAVSMC, and used CCK-8 and scratch assays to detect cell proliferation and migration. Transfection of Tpm1 siRNA can effectively downregulate Tpm1 mRNA expression ( Figure 6 A, P<0.001). CCK-8 results showed that the cell proliferation rate in the HGCM group was significantly increased compared with the NG group ( Figure 6 B, P<0.01), while compared with the HGCM group, transfection of miR21-5pinhibitor and control siRNA effectively reduced the cell proliferation rate ( Figure 6 B, P<0.01), but after co-transfection with miR21-5pinhibitor and Tpm1 siRNA, this inhibitory effect was reversed and the cell proliferation rate was further increased ( Figure 6B, P<0.001). The scratch test showed that the cell migration rate in the HGCM group was significantly higher than that in the NG group at 24 h ( Figure 6 CD, P<0.001). Compared with the HGCM group, transfection of miR21-5p inhibitor and control siRNA effectively reduced the cell migration rate at 24 h ( Figure 6 CD, P<0.001), but after simultaneous transfection of miR21-5p inhibitor and Tpm1 siRNA, the inhibitory effect on cell migration was reversed ( Figure 6 CD, P>0.05).

[0060] These results indicate that miR21-5p promotes the proliferation and migration of HAVSMCs by downregulating Tpm1, and inhibiting miR21-5p and restoring Tpm1 expression can attenuate the proliferation and migration of HAVSMCs.

[0061] Example 5 Inhibition of miR21-5p-mediated Tpm1 downregulation improves osteogenic transformation of VSMCs We used Alizarin red staining and calcium content measurement to observe the effect of double silencing of miR21-5p and Tpm1 on the osteogenic transformation of HAVSMC. The results showed that compared with the NG group, the cells in the HGCM group showed significant dark red calcified nodules after 7 days of induction in high-glucose osteogenic medium, and there was also a significant increase in cellular calcium content ( Figure 7 AB, P<0.05). Compared with the HGCM group, after transfection with miR21-5p inhibitor and control siRNA, the calcified nodules of HAVSMC disappeared significantly, and the cellular calcium content was significantly reduced ( Figure 7 AB, P<0.001). However, after transfection of miR21-5p inhibitor and Tpm1 siRNA, the calcification of HAVSMC was further aggravated, and the cellular calcium content was further increased ( Figure 7 AB, P<0.001).

[0062] Subsequently, we used WB to detect the effects of dual silencing of miR21-5p and Tpm1 on the expression of osteogenic and contractile proteins in HAVSMCs. Compared with the NG group, the levels of osteogenic proteins Runx2 and BMP2 in the HGCM group were significantly increased, and the levels of contractile proteins αSMA and SM22α were significantly decreased ( Figure 8 AE, P<0.05, P <0.01). Compared with the HGCM group, when transfected with miR21-5p inhibitor and control siRNA, the expression of osteogenic proteins Runx2 and BMP2 in HAVSMC was significantly decreased, and the expression of contractile proteins αSMA and SM22α was increased ( Figure 8AE, P<0.05, P <0.01, P <0.001). However, when miR21-5p inhibitor and Tpm1 siRNA were transfected at the same time, as expected, the expression of osteogenic proteins Runx2 and BMP2 in HAVSMC was restored, while the contractile proteins αSMA and SM22α were significantly downregulated ( Figure 8 AE, P>0.05), the anti-osteoblastic differentiation effect of miR21-5p inhibitor was reversed. This indicates that the regulatory effect of miR21-5p on the osteogenic differentiation of HAVSMC depends on Tpm1, and inhibiting miR21-5p-mediated Tpm1 downregulation can effectively restore the contractile phenotype of HAVSMC and inhibit its osteogenic differentiation and calcification.

[0063] Finally, we observed the expression changes of the miR21-5p / Tpm1 axis after dual silencing of miR21-5p and Tpm1. As expected, the miR21-5p level in the HGCM group was significantly increased compared with the NG group, while the Tpm1 mRNA and protein levels were significantly decreased ( Fig. 9 AD, P < 0.001). Compared with the HGCM group, when transfected with miR21-5p inhibitor and control siRNA, the HAVSMC miR21-5p level was significantly decreased, and the Tpm1 mRNA and protein levels were increased accordingly ( Fig. 9 AD, P < 0.001). After transfection of miR21-5 inhibitor and Tpm1 siRNA, HAVSMC miR21-5p was restored to a high level, accompanied by a significant downregulation of Tpm1 mRNA and protein expression.

[0064] The above results indicate that miR21-5p and Tpm1 have a negative regulatory relationship. Inhibition of miR21-5p can inhibit VSMC osteogenic transformation by restoring Tpm1 expression. This indicates that the miR21-5p / Tpm1 axis is a key therapeutic target for VSMC osteogenic transformation and diabetic VC.

[0065] Example 6 Animal experiments verify the effect of miR21-5p / Tpm1 axis as a target for the treatment of diabetic vascular calcification Based on the diabetic VC rat model, we overexpressed miR21-5p in the rat model by adenovirus transfection to explore the effect of the miR21-5p / Tpm1 axis as a target for the treatment of diabetic vascular calcification. Alizarin red staining was used to observe the calcification of the entire aorta and aortic arch of the rats. The staining results showed that there were no obvious dark red calcified nodules in the entire aorta and aortic arch of the rats in the CON group. Compared with the CON group, the entire aorta and aortic arch of the rats in the DVC group showed dark red calcified nodules, mainly concentrated in the middle layer and medial area of ​​the blood vessels. The aortic calcification and calcified nodule area of ​​the miR21-5p NC group were similar to those of the DVC group. In contrast, the degree of arterial calcification in the miR21-5p OE group was significantly aggravated compared with the DVC group and the miR21-5p NC group, which was manifested as extensive and diffuse dark red calcified nodules in the entire aorta, and large areas of calcium salt deposition in the aortic arch ( Fig.10 AD, P<0.001) This indicates that overexpression of miR21-5p can aggravate diabetic VC. After intervention with Danliantongmai prescription combined with intermittent fasting, the number of aortic calcification nodules and the area of ​​aortic arch calcification in the D+I+miR21-5p NC group were significantly reduced compared with the miR21-5p NC group ( Fig.10 AD, P<0.001). However, when miR21-5p was overexpressed, the calcification area in the D+I+miR21-5p OE group was significantly increased compared with the D+I+miR21-5p NC group ( Fig.10 AD, P<0.001).

[0066] The colorimetric method was used to further measure the aortic calcium content and ALP activity of rats in each group. Compared with the CON group, the aortic calcium content and ALP activity of rats in the DVC group were significantly increased ( Fig.10 EF, P<0.01, P<0.05). The aortic calcium content and ALP activity in the miR21-5p NC group were comparable to those in the DVC group. However, the aortic calcium content and ALP activity in the miR21-5p OE group were significantly higher than those in the DVC group and miR21-5p NC group ( Fig.10 EF, P<0.05, P<0.01). After receiving Danliantongmai prescription combined with intermittent fasting intervention, the aortic calcium content in the D+I+miR21-5p NC group was slightly lower than that in the miR21-5p NC group, but there was no significant statistical difference between the two groups ( Fig.10 E, P>0.05), the aorta ALP activity in the D+I+miR21-5p NC group was significantly lower than that in the miR21-5p NC group (Fig. Fig.10E, P<0.05). When miR21-5p was overexpressed, the aortic calcium content and ALP activity in the D+I+miR21-5p OE group were significantly increased compared with the D+I+miR21-5p NC group ( Fig.10 EF, P<0.05). These results indicate that overexpression of miR21-5p abolished the anti-calcification effect of Dantongmai recipe combined with intermittent fasting.

[0067] We then used PCR to detect the expression of miR21-5p / Tpm1 axis in the aorta of rats in each group. The results showed that compared with the CON group, the expression of miR21-5p in the aorta of rats in the DVC group was significantly increased ( Fig.11 A, P<0.05). Compared with the rats in the DVC group, there was no significant change in the expression of miR21-5p in the aorta of the rats in the miR21-5p NC group ( Fig.11 A, P>0.05), miR21-5p expression in the aorta of rats in the miR21-5p OE group was significantly increased ( Fig.11 A, P<0.001), indicating that adenovirus injection successfully induced the overexpression of miR21-5p. After receiving Dantongmai prescription combined with intermittent fasting intervention, the expression of miR21-5p in the D+I+miR21-5p NC group was significantly lower than that in the miR21-5p NC group ( Fig.11 A, P<0.001), indicating that Dantongmai prescription combined with intermittent fasting can effectively inhibit the activation of miR21-5p. However, when miR21-5p was overexpressed, the above effect of Dantongmai prescription combined with intermittent fasting was reversed, as shown by a significant increase in miR21-5p in the D+I+miR21-5p OE group compared with the D+I+miR21-5p NC group ( Fig.11 A, P<0.001). Compared with the CON group, the expression of Tpm1 mRNA and protein in the aorta of rats in the DVC group was significantly decreased ( Fig.11 B, P<0.001). Compared with the DVC group, there was no significant change in the expression of Tpm1 mRNA in the miR21-5p NC group ( Fig.11 B, P>0.05), while Tpm1 mRNA was further decreased in the miR21-5p OE group ( Fig.11 B, P<0.001). After receiving the Dantongmai prescription combined with intermittent fasting intervention, the expression of Tpm1 in the D+I+miR21-5p NC group was significantly higher than that in the miR21-5p NC group ( Fig.11B, P<0.001, P<0.01), indicating that Dantongmai prescription combined with intermittent fasting can effectively restore the expression of Tpm1. However, when miR21-5p was overexpressed, the expression of Tpm1 mRNA in the D+I+miR21-5p OE group was significantly downregulated compared with the D+I+miR21-5p NC group ( Fig.11 B, P<0.001). Combined with the previous results, it is proved that the miR21-5p / Tpm1 axis is involved in regulating the occurrence and development of diabetic VC, and the treatment method targeting the inhibition of the miR21-5p / Tpm1 axis is a potential strategy for the development of diabetic VC drugs.

[0068] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Use of a miR21-5p inhibitor in the preparation of a drug for preventing and / or treating diabetic vascular calcification, characterized in that: The miR21-5p inhibitor is used to reduce the expression level of miR21-5p in vivo or silence the expression of miR21-5p.

2. Use of a Tpm1 inhibitor in the preparation of a drug for preventing and / or treating diabetic vascular calcification, characterized in that: The Tpm1 inhibitor is used to reduce the expression level of the Tpm1 gene in vivo or silence the expression of the Tpm1 gene.

3. The use according to claim 1 or 2, characterized in that: The drug is siRNA.

4. The use according to claim 1 or 2, characterized in that: The application includes inhibiting the occurrence and development of vascular calcification in diabetic patients.

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