Use of the miR21-5p / Tpm1 axis in the preparation of a drug for preventing and / or treating diabetic vascular calcification

Abstract

The present invention relates to the field of biopharmaceuticals. Specifically, the present invention provides an application of the miR21-5p / Tpm1 axis in the preparation of a medicament for preventing and / or treating diabetic vascular calcification. The present invention discovers for the first time the abnormal activation of the miR21-5p / Tpm1 axis in patients with diabetic vascular calcification, and further discovers a new mechanism in which miR21-5p negatively regulates Tpm1 to promote the osteogenic differentiation of vascular smooth muscle cells (VSMCs). Animal experiments prove that miR21-5p / Tpm1 inhibitors can effectively inhibit the occurrence and development of diabetic vascular calcification, providing a new idea and treatment plan for the clinical treatment of diabetic vascular calcification.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine. Specifically, the present invention relates to the use of the 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 lesion in which calcium phosphate is ectopically deposited in the vascular wall in the form of hydroxyapatite. It widely occurs 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 lesions are highly correlated with adverse cardiovascular events such as acute coronary syndrome and sudden cardiac death. It is an independent predictor of all-cause mortality and lifetime cardiovascular disease risk in diabetic patients.

[0003] Contrary to the previous understanding of passive and degenerative vascular lesions of VC, in recent years, it has been gradually recognized that VC is an active biological process regulated by multiple genes. Similar to the osteoblast differentiation process in bone mineralization, it is mainly driven by vascular smooth muscle cells (VSMCs). In the vascular tissue of normal adults, VSMCs exhibit a contractile phenotype and respond to signals such as acetylcholine or epinephrine 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 related to hyperglycemia cause a highly active pathological phenotype transformation of VSMCs, resulting in abnormal deposition of insoluble calcium and phosphate salts in the vascular wall, promoting the formation and development of VC. However, despite the increasing understanding of the pathological mechanism of diabetic VC, no effective specific treatment plan targeting the osteogenic transformation of VSMCs has been found yet. Therefore, finding new mechanisms of diabetic VC and related drug targets, especially targets that can regulate the osteogenic transformation of VSMCs 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 object of the present invention is to study a new molecular mechanism for regulating diabetic VC, especially the osteogenic transformation of VSMCs induced by high glucose. This study first found that the miR21-5p / Tpm1 axis was significantly activated in patients with diabetic vascular calcification, and further discovered its regulatory mechanism in promoting the proliferation, migration, and osteogenic transformation of VSMCs.

[0005] In order to achieve the above invention object, the present invention provides the following technical solutions:

[0006] In the first aspect of the present invention, there is provided an application of an miR21-5p inhibitor in the preparation of a drug for preventing and / or treating diabetic vascular calcification, wherein the miR21-5p inhibitor is used to reduce the expression level of miR21-5p in vivo or silence the expression of miR21-5p, and the nucleotide sequence of miR21-5p is SEQ ID NO.1.

[0007] In the second aspect of the present invention, there is provided an application 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 level of the Tpm1 gene in vivo or silence the expression of the Tpm1 gene, and the nucleotide sequence of Tpm1 is SEQ ID NO.2.

[0008] In one embodiment, the above-mentioned drug is siRNA.

[0009] In one embodiment, the above-mentioned application includes inhibiting the occurrence and development of vascular calcification in diabetic patients.

[0010] Compared with the prior art, the present invention has the following beneficial effects:

[0011] The present invention first proposes that the miR21-5p / Tpm1 axis can be used as a target for preventing or treating diabetic vascular calcification diseases. The present invention first discovers the activation of the miR21-5p / Tpm1 axis in patients with diabetic vascular calcification, and also discovers that at the cellular level, inhibiting the activation of miR21-5p can partially restore the expression level of the target gene Tpm1, thereby effectively reducing the excessive proliferation, migration and osteogenic transformation of VSMCs, and playing a role in reducing VC. Description of the Drawings

[0012] Figure 1 It is the research result of the expression levels of miR21-5p and Tpm1 in patients with diabetic vascular calcification and their correlation with the degree of vascular calcification, wherein Figure 1 A and Figure 1 C are the expression levels of miR21-5p and Tpm1 mRNA in the diabetic vascular calcification group and the non-calcification group of patients, Figure 1 B and Figure 1 D is the correlation between the expression levels of miR21-5p and Tpm1 mRNA and the coronary artery calcification Agatston score, Figure 1 E is the evaluation of the expression of the contractile markers αSMA and Tpm1 in the aorta of diabetic coronary heart disease patients by immunofluorescence. Compared with the Non-CAC group, # P <0.05, ## P <0.01.

[0013] Figure 2 The effects of miR21-5p on the proliferation and migration abilities of HAVSMCs, where Figure 2 A shows the expression levels of miR21-5p in HAVSMCs after transfection with miR21-5p mimic and miR21-5p inhibitor, Figure 2 B shows the proliferation activities of HAVSMCs in different groups evaluated by the CCK8 assay, Figure 2 C and Figure 2 D shows the migration abilities of HAVSMCs in different groups evaluated by the scratch assay. 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 3 The effects of miR21-5p overexpression and inhibition on the calcification degree of HAVSMCs, where Figure 3 A shows the formation of calcified nodules in HAVSMCs in different groups evaluated by alizarin red staining, Figure 3 B shows the cellular calcium content of HAVSMCs in different groups detected by colorimetry. 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 4 The effects of miR21-5p overexpression and inhibition on the expression of osteogenic and contractile proteins in HAVSMCs, where Figure 4 A shows the western blot images of osteogenic and contractile related proteins in HAVSMCs in different groups, Figure 4 B-E are Figure 4 the quantitative analysis of the expression of each osteogenic and contractile related protein 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.

[0016] Figure 5 For the effects of miR21-5p overexpression and inhibition on the miR21-5p / Tpm1 axis, where 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 the 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.

[0017] Figure 6 For the effects of double silencing of miR21-5p and Tpm1 on the proliferation and migration abilities of HAVSMCs, where Figure 6 A is the mRNA expression level of Tpm1 after transfection with Tpm1 siRNA, Figure 6 B is the proliferation activity of HAVSMCs in each group detected by CCK-8, Figure 6 C is the migration ability of HAVSMCs in different groups evaluated by scratch assay, Figure 6 D is the quantitative analysis of the cell migration rate in different groups. Compared with siRNA or NG, ### P <0.001; compared with the HGCM group, P <0.001.

[0018] Figure 7 For the effects of double silencing of miR21-5p and Tpm1 on the calcification degree of HAVSMCs, where Figure 7 A is the formation of calcified nodules in HAVSMCs of different groups evaluated by alizarin red staining, Figure 7 B is the cellular calcium content of HAVSMCs in different groups detected by colorimetry. Compared with NG, # P <0.05, ## P< 0.01; Compared with the HGCM group, P < 0.05, P < 0.01, P < 0.001.

[0019] Figure 8 For the effects of double silencing of miR21-5p and Tpm1 on osteogenic and contractile protein expression in HAVSMC, where Figure 8 A is the Western blot images of osteogenic and contractile related proteins in HAVSMC of different groups, Figure 8 B-E are Figure 8 The quantitative analysis of the expression of each osteogenic and contractile related protein 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.

[0020] Figure 9 For the effects of double silencing of miR21-5p and Tpm1 on the miR21-5p / Tpm1 axis in HAVSMC, where Figure 9 A is the expression level of miR21-5p in HAVSMC of different groups, Figure 9 B is the expression level of Tpm1 mRNA in HAVSMC of different groups, Figure 9 C is the Western blot images of Tpm1 expression in HAVSMC of different groups, Figure 9 D is Figure 9 The 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.

[0021] Figure 10 For the situation of aortic calcification degree in each group of rats, Figure 10 A is the representative image of alizarin red pathological staining of the whole aorta in each group of rats (n = 3); Figure 10B was the quantitative analysis of alizarin red staining of the entire aorta (n = 3); Figure 10 C was the representative image of alizarin red staining of the aortic arch of rats in each group (n = 3, 20×); Figure 10 D was the quantitative analysis of alizarin red staining of the aortic arch of rats in each group (n = 3). Figure 10 E was the calcium content in the aorta of rats in each group (n = 5); Figure 10 F was the ALP activity in 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.

[0022] Figure 11 was the expression of the miR21 - 5p / Tpm1 axis in the aorta of rats in each group, Figure 11 A was the expression level of miR21 - 5p in the aorta of rats in each group (n = 3); Figure 11 B was the Tpm1 mRNA level 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 implementation manners

[0023] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0024] The experimental methods involved in the embodiments of the present invention are described as follows:

[0025] 1. PBMC extraction: 10 mL of venous blood of the patient was drawn using an EDTA anticoagulant tube and shaken well to avoid blood coagulation. PBMC separation was completed within 2 hours after blood collection. 10 mL of blood was diluted with an equal volume of PBS and slowly added to the upper layer of Ficoll - Hypaque lymphocyte separation solution, and centrifuged at 800 rpm for 20 minutes (room temperature, acceleration rate 1, deceleration rate 0). After centrifugation, four layers were formed: plasma layer, mononuclear cell buffy coat layer, separation solution layer, red blood cell and granulocyte layer. The buffy coat layer was carefully aspirated into a new tube and washed 3 times with PBS (800 rpm, 10 minutes), and finally the PBMC precipitate was obtained for subsequent experiments.

[0026] 2. Cell culture: HAVSMCs were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% double antibiotics (streptomycin, penicillin), and placed in an incubator at 37°C and 5% CO2. The cell morphology was observed every 2 days and the medium was changed. When the cell confluence reached 80%-90%, the 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 cell detachment under a microscope, the trypsin was neutralized with 2-4 volumes of complete medium, and the cells were centrifuged (10,000 rpm, 4°C, 5 minutes), resuspended, and transferred to a new culture dish for continued culture.

[0027] 3. Establishment of cell calcification model: When HAVSMCs reached 80% confluence, the normal medium was discarded and replaced with three different media for calcification induction. The control group (NG) used normal DMEM medium, the high-glucose group (HG) used high-glucose medium containing 30 mmol / L glucose, and the high-glucose calcification group (HGCM) used high-glucose calcification medium containing 30 mmol / L glucose, 10 mmol / L β-glycerophosphate, and 3 mmol / L CaCl2. The induction time of the calcification model was 7-10 days. During this period, the cell morphology was observed daily and the medium was changed every 2 days. During the calcification induction process, the cell proliferation rate slowed down significantly and no subculture was required.

[0028] 4. Transfection:

[0029] (1) miR21-5p transfection: Transfection complexes of miR21-5p mimic (final concentration 50 mmol / L, sequence: SEQ ID NO.3-4) and miR21-5p inhibitor (final concentration 100 mmol / L, sequence: SEQ ID NO.5) were prepared. The transfection complexes were added to the cells in a 24-well plate, and the medium was supplemented to 500 μL. After incubation at 37°C for 24 hours, the cells were changed to high-glucose calcification medium and continued to be cultured for 24 hours. (2) Tpm1 siRNA transfection: Tpm1 siRNA complexes (sequence: SEQ ID NO.6-7) were prepared using Lipofectamine 2000 and transfected into HAVSMC cells (culture density 70%-80%). The complete medium was changed 4-6 hours after transfection, and RNA or protein extraction was performed after culturing for 48-72 hours.

[0030] 5. Cell viability detection: The CCK-8 method was used to detect cell viability. Cells in good growth condition were digested and resuspended in serum-free medium, and the cell density was adjusted to 1×10^4 cells / 100 μL. Then, 100 μL of the cell suspension was inoculated into each well of a 96-well plate. After culturing in an incubator at 37°C and 5% CO2 for 24 hours, the medium was discarded, and normal medium (NG), high-glucose medium (HG), and high-glucose calcified medium (HGCM) were added respectively. Each group had 8 replicate wells, and the blank control group used cell-free medium. After continuing to culture for 24 hours, 10 μL of CCK-8 solution was added to each well, and the plate was incubated for 2 hours. The absorbance was measured at a wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0031] 6. Animal culture

[0032] Forty-eight SPF-grade male SD rats, 6 - 7 weeks old and weighing 180 - 200 g, were purchased from Beijing SPF Biotechnology Co., Ltd. (SCXK (Jing) 2019 - 0010). All rats were housed in cages (5 - 6 rats per cage) in an SPF animal facility with a temperature of 22 ± 2°C, a humidity of 55 ± 5%, and a 12 / 12-hour light / dark cycle, along with sterilized water and feed. This animal experiment was approved by the Animal Ethics Committee of Xiyuan Hospital, China Academy of Chinese Medical Sciences (Approval No.: 2024XLC014 - 2).

[0033] 7. Animal model establishment

[0034] (1) Diabetes model: After 1 week of adaptive feeding, SPF-grade male SD rats were intraperitoneally injected with 30 mg / kg STZ (stored on ice and freshly prepared) for 3 consecutive days to induce a diabetes model. Three days later, a blood sample was taken from the rat's tail to measure the fasting blood glucose. A blood glucose value > 11.1 mmol / L was considered successful model establishment. For the remaining rats that did not form the model, they were supplemented with a single intraperitoneal injection of 30 mg / kg STZ and their fasting blood glucose was measured. Rats with a blood glucose value > 11.1 mmol / L were included in the model group for the next experiment.

[0035] (2) Vascular calcification model: Vitamin D3 (Vitamin D3, VitD3) overload was used to induce VC. The established diabetic rats were intraperitoneally injected with a high dose of VitD3 (5.5×10^5 IU / Kg) for 4 consecutive days. Fourteen days after the last injection was considered the calcification model establishment period.

[0036] 8. Adenovirus vector construction and transfection

[0037] (1)Vector construction: The adeno-associated virus (AAV) used in this experiment was constructed, produced, and purified by GenScript Biotech Corporation (ID: VB240116-1098trb, VB010000-9461vph). The specific steps were as follows: The sequences of short hairpin RNA (shRNA) specific to rno-miR21-5p and the control shRNA were designed and inserted into the multiple cloning site downstream of the U6 promoter of the vector pAAV[shRNA]-EGFP-U6 to construct a recombinant plasmid expressing rno-miR21-5p. After the correctness of the vector was verified by restriction enzyme digestion and Sanger sequencing, it was co-transfected with the helper plasmid and the packaging plasmid into HEK293T cells, and the virus was produced using the AAV8 packaging system. After transfection, the cells were cultured for 48-72 hours, and the culture medium and cell lysates were collected. The virus particles were purified by ultracentrifugation, and the virus titer was determined using fluorescence quantitative PCR. 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 fluorescent expression under blue light or ultraviolet light irradiation was observed by fluorescence microscopy or detected by 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 virus was aliquoted and stored at -80°C for subsequent experiments.

[0038] (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 performed a one-time injection of the AAV delivery system into the lateral tail vein 1 week after STZ injection. Wipe the rat tail clean, warm the rat to dilate its blood vessels for easy injection. Before injection, disinfect the injection site with an alcohol cotton ball. The injection position is selected starting from 1 / 3 of the distance from the tip of the tail at the bottom of the tail. Grasp the distal part of the tail and gently turn it to one side to expose the lateral vein. Insert the needle at a small angle and inject the diluted pAAV into the vein. Observe the color of the vein during injection to ensure that the virus is injected into the vein. The stock concentration of pAAV-[shRNA]-EGFP-U6>{rno-miR21-5p} is 2.63×10^13 gene copies / mL, and that of pAAV-[shRNA]-EGFP-U6>Scramble[shRNA#1] is 1.83×10^13 gene copies / mL. Dilute the stock solution to the target concentration with PBS before injection. Referring to previous studies, the injection dose for each rat is 5×10^11 gene copies / mL, and the injection volume is 0.1 mL.

[0039] 9. Animal grouping and intervention methods

[0040] After the model establishment, all the established DVC rats were randomly divided into a model group (DVC group, n = 12), a miR21-5p overexpression control group (miR21-5p NC group, n = 12), a miR21-5p overexpression group (miR21-5p OE group, n = 12), a Dantong Connecting Vessels Formula combined with intermittent fasting + miR21-5p overexpression control group (D+I+miR21-5p NC group, n = 12), a Dantong Connecting Vessels Formula combined with intermittent fasting + miR21-5p overexpression group (D+I+miR21-5p OE group, n = 12). Another 12 blank SD rats were taken as the control group (CON group). The specific drug administration and intervention plans are as follows:

[0041] (1)Control group (CON group): Normal diet, intragastric administration with the same volume of pure water for 4 weeks.

[0042] (2)Model group (DVC group): Normal diet, intragastric administration with the same volume of pure water for 4 weeks.

[0043] (3)miR21-5p overexpression control group (miR21-5p NC group): Normal diet, tail vein injection of miR21-5p NC, intragastric administration with the same volume of pure water for 4 weeks.

[0044] (4) miR21-5p overexpression group (miR21-5p OE group): Normal diet, miR21-5p OE was injected via the tail vein, and an equal volume of pure water was administered by gavage for 4 weeks.

[0045] (5) Dantong Connecting Vessels Formula combined with intermittent fasting + miR21-5p overexpression control group (D+I+miR21-5p NC group): miR21-5p NC was injected via the tail vein, and Dantong Connecting Vessels Formula (0.96 g / kg / d) was administered by gavage for 4 weeks while undergoing intermittent fasting.

[0046] (6) Dantong Connecting Vessels Formula combined with intermittent fasting + miR21-5p overexpression group (D+I+miR21-5p OE group): miR21-5p OE was injected via the tail vein, and Dantong Connecting Vessels Formula (0.96 g / kg / d) was administered by gavage for 4 weeks while undergoing intermittent fasting.

[0047] Note: Dantong Connecting Vessels Formula: An empirical formula developed by Professor Liu Yue of Xiyuan Hospital, China Academy of Chinese Medical Sciences, based on previous research and clinical practice, and has applied for a patent (Patent No.: 202311796613.6). The formula consists of Salvia miltiorrhiza, Coptis chinensis, Polygonum cuspidatum, etc. The cut herbs and extract powder used in the research were identified and processed by the Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences. Each gram of extract powder contains 23.5 grams of crude drug, and it was prepared into a medicinal solution with pure water for use when needed.

[0048] Intermittent fasting adopted a time-restricted feeding strategy. The moment when the light started (8:00 am) was defined as the standard time 0 moment (ZT0). The moment when the light ended (8:00 pm) was defined as the standard time 12 moment (ZT12). The rats were allowed to eat within 8 hours from 10:00 pm (ZT14) to 6:00 am (ZT22), and the feed would be removed at other times until 10:00 pm the next day. Only food intake was restricted during the entire fasting process, and water intake was not restricted.

[0049] 10. Determination of calcium content and ALP

[0050] (1)Calcium content: After the experiment, the calcium content in the aorta was measured by o-cresolphthalein complexone colorimetry. The aorta was minced on ice and rinsed with pre-cooled PBS, then placed in a homogenizer and 200 μL of protein lysate was added. Tissue homogenization was performed at 15000 rpm for 10 s each time, with a 10 s interval, for 3 cycles. After completion, the homogenate was transferred to a centrifuge tube and left standing on ice for 20 minutes. It was centrifuged at 12000 rpm for 15 min in a high-speed centrifuge pre-cooled at 4 °C, and the supernatant was collected for standby. According to the kit requirements, a 96-well plate was taken, and a blank group, a test group, and a standard group were set up. 5 μL of ddH2O, 5 μL of the test supernatant, and 5 μL of the calcium standard were added respectively. Then 200 μL of the calcium ion chromogenic 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-linked immunosorbent assay (ELISA) reader, and the standard curve was plotted to calculate the calcium content in the test sample. The bicinchoninic acid (BCA) method was used to measure the total protein content in the supernatant, and the calcium content was standardized with the protein content (mmol / g protein).

[0051] (2)ALP activity: The steps for obtaining the tissue homogenate were the same as those for calcium content detection. According to the kit requirements, a 96-well plate was taken, and a blank group, a test group, and a standard group were set up. 50 μL of the detection buffer, 50 μL of the test supernatant, and 50 μL of the standard working solution (0.5 mmol / L p-nitrophenol solution) were added to the wells respectively. 50 μL of the chromogenic substrate (prepared freshly before use) was added to the blank group and the test group, and the detection buffer was added to the standard group to make up 100 μL. Incubate at 37 °C for 30 minutes, and then 100 μL of the reaction termination solution was added to all wells to terminate the reaction. The standard wells and the wells with ALP activity would show different shades of yellow. The absorbance value (wavelength 405 nm) was measured using an ELISA reader, and the standard curve was plotted to calculate the ALP activity in the test sample. The BCA method was used to measure the total protein content in the supernatant, and the ALP activity was standardized with the protein content (U / g protein).

[0052] 11. Alizarin red staining

[0053] (1)Cell staining: After HAVSMCs were induced for 7 days in different conditioned media, the medium in the culture dish was aspirated, and the cells were gently rinsed twice with PBS buffer. 4% paraformaldehyde was added and fixed at room temperature for 20 min. After fixation, the cells were washed three times with PBS to remove the excess fixative. Then 3 mL of 2% alizarin red staining solution (pH 4.3) was added dropwise to the culture dish, and the dish was gently shaken to ensure that the staining solution fully covered the cells. Incubate at 37 °C in the dark for 5 min, discard the staining solution, and gently rinse twice with double-distilled water. Dark red calcified nodules could be seen under an inverted microscope and photographed for record.

[0054] (2)Aortic staining: After the experiment, the entire aorta was fixed in 4% PFA overnight. Subsequently, the PFA was discarded, and the aorta was immersed in 2% alizarin red staining solution overnight at room temperature. The aorta was washed with 2% potassium hydroxide solution and photographed using an inverted microscope.

[0055] For the aortic arch, it was fixed in 4% PFA overnight. The vascular tissue was taken out, the excess PFA was washed away, and it was dehydrated with gradient alcohol, then cleared, infiltrated with paraffin, and embedded in paraffin. Proximal sections were cut with a thickness of 5 μm. Baked in an oven at 65 °C for 1 h, the sections were taken out and dewaxed to water by immersing them successively in xylene and gradient alcohol: 10 min each in xylene I and II, 5 min each in absolute ethanol I, absolute ethanol II, 95% ethanol, 90% ethanol, 80% ethanol, and rinsed 3 times with distilled water to remove residual ethanol. The excess water on the sections was shaken off, 2% alizarin red staining solution was added dropwise for staining, the staining solution was washed off after 5 min, and then dehydrated by immersing successively in 80% ethanol, 90% ethanol, 95% ethanol, absolute ethanol I, and absolute ethanol II (5 min each), cleared in xylene I and II (5 min each), and sealed with neutral gum.

[0056] 12. Cell scratch assay: The cell scratch assay was used to detect cell migration ability. After digesting and resuspending 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 cultured in an incubator at 37 °C and 5% CO2 until the cell confluence reached 80%. The supernatant was discarded, washed 2 times with PBS, a straight line was scratched in the center of each well with a 200 μL sterile pipette tip, and the detached cells were washed away with PBS and recorded as 0 h, then photographed under a microscope. The NG group was replaced with normal medium, and the other groups were replaced with high-glucose calcified medium. Observed and photographed after culturing for 24 h, repeated 3 times.

[0057] 13. Immunofluorescence: Human aortic tissue was stained and analyzed by immunofluorescence. After the human aortic specimens were obtained during the operation, they were washed with normal saline, fixed with 4% PFA, dehydrated with gradient alcohol, and embedded in paraffin to prepare 5 μm thick sections. After dewaxing to water, antigen repair was performed, washed with PBS, 0.5% Triton X-100 permeabilization solution was added dropwise, and after washing with PBS, blocking solution was added for blocking. Subsequently, primary antibodies (Anti-aSMA: 1:500; Anti-Tpm1: 1:500) were added dropwise and incubated overnight at 4 °C. After washing with PBS, secondary antibody (1:100) was added, incubated at room temperature, washed with PBS, and stained with DAPI for 10 minutes. After sealing the slices, they were observed using a laser confocal microscope.

[0058] 14. PCR: Total RNA was extracted from aortic tissue, HAVSMC, and PBMC. After treatment with Trizol method, high-purity RNA was obtained through chloroform stratification, isopropanol precipitation, and 75% ethanol washing. The concentration and purity of RNA were detected using NanoDrop. Primers were designed by Primer Primer 5.0 and verified for specificity by Blast, and were synthesized by a professional company. HiScriptIII kit was used for gene cDNA synthesis, and miRNA was reverse-transcribed using the poly(A) tailing method. The operations were carried out strictly according to the kit instructions. 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, 15 seconds at 95°C, and 60 seconds at 60°C for a total of 40 cycles, and melting curve analysis for specificity. The experimental results were calculated by the 2 -△△CT -ΔΔCt method to calculate the relative gene expression level, and 3 replicates were set for each sample to ensure data accuracy.

[0059] 15. WB: Total protein samples of HAVSMC were extracted. After the cell samples were washed and centrifuged with PBS, protein lysate was added for sufficient lysis. After the protein concentration was measured by the BCA method, loading buffer was added and boiled for denaturation to preserve the protein loading solution. After protein separation by SDS-PAGE gel electrophoresis, PVDF membrane was used for protein transfer. After transfer, the membrane was blocked with 5% skim milk powder. Primary antibodies (such as Anti-Runx2, Anti-BMP2, Anti-αSMA, etc.) and secondary antibodies were prepared according to the instructions. The membrane was incubated with the antibodies respectively and washed multiple times with TBST, and then developed with ECL developer. The protein bands were recorded by exposing the film, and finally the gray scale of the bands was analyzed using ImageJ software.

[0060] In the following examples, unless otherwise specified, all are conventional methods. The materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels. In addition, the implementation examples should be understood as illustrative and not limiting the scope of the present invention. The essence and scope of the present invention are only defined by the claims. For those skilled in the art, various changes or modifications to the material components and dosages in these implementation examples without departing from the essence and scope of the present invention also fall within the protection scope of the present invention.

[0061] Example 1 Activation of miR21-5p / Tpm1 axis exists in diabetic vascular calcification

[0062] To comprehensively understand the role of the miR21-5p / Tpm1 axis in the progression of diabetic VC, we enrolled 33 type 2 diabetic patients who visited the outpatient or inpatient departments of Xiyuan Hospital, China Academy of Chinese Medical Sciences (all had signed informed consent forms) to observe the expression levels of the miR21-5p / Tpm1 axis. The results showed that compared with patients in the non-vascular calcification group of diabetes, the level of miR21-5p in PBMC of patients in the diabetic vascular calcification group increased significantly, while the level of Tpm1 mRNA decreased significantly ( Figure 1 A, C, P < 0.05), and the expression of miR21-5p was significantly positively correlated with the coronary VC Agatston score. On the contrary, the expression of Tpm1 was significantly negatively correlated with the coronary VC Agatston score ( Figure 1 B, D, P < 0.01). In addition, we also used immunofluorescence to evaluate the expression of the aortic contraction markers α-SMA and Tpm1 in patients with coronary artery calcification in diabetic coronary heart disease. The results showed that compared with healthy people, the expressions of α-SMA and Tpm1 in the aorta of patients with coronary artery calcification in diabetic coronary heart disease were significantly downregulated ( Figure 1 E).

[0063] The above results indicate that the miR21-5p / Tpm1 axis is involved in regulating the occurrence and development of diabetic VC.

[0064] Example 2 Overexpression of miR21-5p promotes the proliferation and migration of vascular smooth muscle cells

[0065] Enhanced cell proliferation and migration ability are important characteristics of the phenotypic transformation of VSMC. To explore the role of miR21-5p in the proliferation and migration of the in vitro calcification model of HAVSMC induced by high glucose osteogenesis, we transfected miR21-5p mimic, miR21-5p inhibitor and corresponding negative controls, and observed the changes in the proliferation and migration of HAVSMC after overexpression and inhibition of miR21-5p. The PCR results showed that miR21-5p mimic significantly upregulated the expression of miR21-5p in HAVSMC, and miR21-5p inhibitor significantly inhibited the expression of miR21-5p in HAVSMC, indicating successful transfection ( Figure 2 A, P < 0.001). The CCK-8 results showed that compared with the NG group, the cell proliferation activity in the HGCM group was significantly enhanced ( Figure 2 B, P < 0.001). Compared with the cells in the HGCM group, overexpression of miR21-5p further increased the proliferation ability of HAVSMC ( Figure 2 B, P < 0.001), while inhibition of miR21-5p effectively inhibited the proliferation of HAVSMC ( Figure 2 B, P < 0.001).

[0066] The scratch assay was used to detect the changes in the migration ability of HAVSMCs. Compared with the NG group, the migration ability of cells in the HGCM group was significantly enhanced, and the scratch distance within 24 h was significantly shortened ( Figure 2 C-D, P < 0.001). Compared with the HGCM group, overexpression of miR21-5p further increased the migration ability of cells and reduced the scratch area at 24 h, but no significant statistical difference was observed ( Figure 2 C-D, P > 0.05). In contrast, inhibition of miR21-5p could effectively weaken the migration of HAVSMCs and reduce the cell migration rate at 24 h ( Figure 2 C-D, P < 0.001).

[0067] The above results indicate that overexpression of miR21-5p can promote the excessive proliferation and migration of VSMCs, and inhibition of miR21-5p can reverse the above process.

[0068] Example 3 miR21-5p negatively regulates Tpm1 to promote osteogenic transformation of HAVSMCs

[0069] Alizarin red staining was used to observe the effect of miR21-5p on the calcification degree of HAVSMCs. Compared with the NG group, after induction with high-glucose osteogenic medium for 7 days, cells in the HGCM group showed scattered dark red calcified nodules. Compared with the HGCM group, cells in the miR21-5p mimic group showed more severe calcified nodules. However, no significant calcified nodules were observed in cells of the miR21-5p inhibitor group ( Figure 3 A). In addition, the calcium content of HAVSMCs 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 cells in the HGCM group, the calcium content of cells in the miR21-5p mimic group was further increased ( Figure 3 B, P < 0.001), while the calcium content of cells in the miR21-5p inhibitor group was significantly decreased ( 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 the in vitro calcification of HAVSMCs.

[0070] Subsequently, we further detected the expression of contractile and osteogenic proteins in HAVSMCs of each group. The results showed that compared with the NG group, the osteogenic proteins Runx2 and BMP2 in cells of the HGCM group were significantly up-regulated, and the expression of contractile proteins αSMA and SM22α was significantly down-regulated ( Figure 4A-E, P < 0.05, P < 0.001). Compared with the HGCM group, after miR21-5p overexpression, the osteogenic proteins Runx2 and BMP2 were further upregulated, while the expression of the contractile proteins αSMA and SM22α was significantly downregulated ( Figure 4 A-E, P < 0.05, P < 0.001). Inhibiting miR21-5p could effectively reverse the above process, including downregulating the expression of the osteogenic proteins Runx2 and BMP2 and upregulating the expression of the contractile proteins αSMA and SM22α ( Figure 4 A-E, P < 0.05, P < 0.001). These results together indicate that miR21-5p is involved in regulating the osteogenic transformation of HAVSMC in vitro. Activating miR21-5p can promote the osteogenic transformation of HAVSMC, while inhibiting miR21-5p can effectively reverse this process and restore the contractile phenotype of HAVSMC.

[0071] Next, we further observed the effects of miR21-5p overexpression and inhibition on the miR21-5p / Tpm1 axis. The PCR results showed that compared with the NG group, miR21-5p was significantly increased in the HGCM group ( Figure 5 A, P < 0.001). Compared with the HGCM group, miR21-5p was significantly increased in the miR21-5p mimic group, while miR21-5p was significantly decreased in the miR21-5p inhibitor group ( Figure 5 A, P < 0.001), indicating that miR21-5p was correspondingly regulated after transfection with mimic or inhibitor. Subsequently, we detected the changes in the mRNA and protein levels of Tpm1 by PCR and WB. The PCR results showed that compared with the NG group, the level of Tpm1 mRNA was significantly decreased in the HGCM group ( Figure 5 B, P < 0.001). Compared with the HGCM group, the Tpm1 mRNA was further downregulated in the miR21-5p mimic group, while the Tpm1 mRNA was significantly upregulated in the miR21-5p inhibitor group ( Figure 5 B, P < 0.001). The WB results showed the same trend, that is, compared with the NG group, the expression of Tpm1 protein was significantly decreased in the HGCM group. Compared with the HGCM group, the expression of Tpm1 protein was further decreased in the miR21-5p mimic group, while the expression of Tpm1 protein was significantly increased in the miR21-5p inhibitor group ( Figure 5 C-D, P < 0.001).

[0072] The above results together indicate that miR21-5p can negatively regulate Tpm1 to promote the osteogenic transformation of VSMC. Inhibiting miR21-5p can improve the osteogenic transformation of VSMC.

[0073] Example 4 Inhibiting miR21-5p-mediated downregulation of Tpm1 attenuates the excessive proliferation and migration of VSMCs

[0074] To further clarify the relationship between miR21-5p and Tpm1 in the phenotypic transformation of HAVSMCs, we next performed double-silencing experiments on miR21-5p and Tpm1 in HAVSMCs, and used CCK-8 and scratch assays to detect the proliferation and migration abilities of the cells. Transfection with Tpm1 siRNA effectively downregulated the expression of Tpm1 mRNA ( Figure 6 A, P < 0.001). The results of CCK-8 showed that compared with the NG group, the cell proliferation rate in the HGCM group was significantly increased ( Figure 6 B, P < 0.01), while compared with the HGCM group, transfection with miR21-5p inhibitor and control siRNA effectively reduced the cell proliferation rate ( Figure 6 B, P < 0.01). However, after co-transfection with miR21-5p inhibitor and Tpm1 siRNA, this inhibitory effect was reversed, and the cell proliferation rate further increased ( Figure 6 B, P < 0.001). The scratch assay showed that the cell migration rate at 24 h in the HGCM group was significantly higher than that in the NG group ( Figure 6 C-D, P < 0.001). Compared with the HGCM group, transfection with miR21-5p inhibitor and control siRNA effectively reduced the cell migration rate at 24 h ( Figure 6 C-D, P < 0.001), while after co-transfection with miR21-5p inhibitor and Tpm1 siRNA, the inhibitory effect on cell migration rate was reversed ( Figure 6 C-D, P > 0.05).

[0075] The above results indicate that miR21-5p promotes the proliferation and migration of HAVSMCs by downregulating Tpm1. Inhibiting miR21-5p and restoring Tpm1 expression can attenuate the proliferation and migration of HAVSMCs.

[0076] Example 5 Inhibiting miR21-5p-mediated downregulation of Tpm1 improves the osteogenic transformation of VSMCs

[0077] We used alizarin red staining and calcium content determination to observe the effect of double-silencing of miR21-5p and Tpm1 on the osteogenic transformation of HAVSMCs. The results showed that compared with the NG group, the cells in the HGCM group showed significantly dark red calcified nodules after being induced by high-glucose osteogenic medium for 7 days, along with a significant increase in the calcium content of the cells ( Figure 7A - B, P < 0.05). Compared with the HGCM group, after transfection with miR21 - 5p inhibitor and control siRNA, the calcified nodules of HAVSMCs significantly disappeared, and the cellular calcium content was significantly reduced ( Figure 7 A - B, P < 0.001). However, after co - transfection with miR21 - 5p inhibitor and Tpm1 siRNA, the degree of calcification of HAVSMCs was further aggravated, and the cellular calcium content further increased ( Figure 7 A - B, P < 0.001).

[0078] Subsequently, we used WB to detect the effects of double - 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 α - SMA and SM22α were significantly decreased ( Figure 8 A - E, P < 0.05, P < 0.01). Compared with the HGCM group, when transfected with miR21 - 5p inhibitor and control siRNA, the expressions of osteogenic proteins Runx2 and BMP2 in HAVSMCs were significantly decreased, and the expressions of contractile proteins α - SMA and SM22α were increased ( Figure 8 A - E, P < 0.05, P < 0.01, P < 0.001). But when co - transfected with miR21 - 5p inhibitor and Tpm1 siRNA, as expected, the expressions of osteogenic proteins Runx2 and BMP2 in HAVSMCs were restored, and at the same time, the contractile proteins α - SMA and SM22α were significantly down - regulated ( Figure 8 A - E, P > 0.05), and the anti - osteogenic differentiation effect of miR21 - 5p inhibitor was reversed. This indicates that the regulatory effect of miR21 - 5p on the osteogenic differentiation of HAVSMCs depends on Tpm1. Inhibiting the miR21 - 5p - mediated down - regulation of Tpm1 can effectively restore the contractile phenotype of HAVSMCs and inhibit its osteogenic differentiation and calcification.

[0079] Finally, we observed the expression changes of the miR21 - 5p / Tpm1 axis after double - silencing of miR21 - 5p and Tpm1. As expected, the level of miR21 - 5p in the HGCM group was significantly higher than that in the NG group, and at the same time, the Tpm1 mRNA and protein levels were significantly decreased ( Figure 9 A - D, P < 0.001). Compared with the HGCM group, when transfected with miR21 - 5p inhibitor and control siRNA, the miR21 - 5p level in HAVSMCs was significantly decreased, and the Tpm1 mRNA and protein levels increased correspondingly ( Figure 9A-D, P < 0.001). While co-transfecting miR21-5p inhibitor and Tpm1 siRNA, miR21-5p in HAVSMC was restored to a high level, accompanied by a significant down-regulation of Tpm1 mRNA and protein expression.

[0080] The above results together indicate that there is a negative regulatory relationship between miR21-5p and Tpm1. Inhibiting miR21-5p can inhibit the osteogenic transformation of VSMC by restoring Tpm1 expression. It shows that the miR21-5p / Tpm1 axis is a key therapeutic target for VSMC osteogenic transformation and diabetic VC.

[0081] Example 6 Verify the effect of the miR21-5p / Tpm1 axis as a target in the treatment of diabetic vascular calcification through animal experiments

[0082] Based on the diabetic VC rat model, we overexpressed miR21-5p in the rat model through adenovirus transfection to explore the effect of the miR21-5p / Tpm1 axis as a target in the treatment of diabetic vascular calcification. Alizarin red staining was used to observe the calcification of the whole aorta and aortic arch of rats. The staining results showed that there were no obvious dark red calcification nodules in the whole aorta and aortic arch of rats in the CON group. Compared with the CON group, dark red calcification nodules appeared in the whole aorta and aortic arch of rats in the DVC group, mainly concentrated in the middle and inner regions of the blood vessels. The aortic calcification and the area of calcification nodules in the miR21-5p NC group were similar to those in the DVC group. In contrast, the degree of arterial calcification in the miR21-5p OE group was significantly more severe than that in the DVC group and the miR21-5p NC group, manifested as extensive and diffuse dark red calcification nodules in the whole aorta, and large-area calcium salt deposition in the aortic arch ( Figure 10 A-D, P < 0.001). This indicates that overexpressing miR21-5p can exacerbate diabetic VC. After receiving the intervention of Dantong Tongmai Recipe combined with intermittent fasting, compared with the miR21-5p NC group, the aortic calcification nodules in the D+I+miR21-5p NC group were significantly reduced, and the calcification area of the aortic arch was significantly decreased ( Figure 10 A-D, P < 0.001). However, when miR21-5p was overexpressed, compared with the D+I+miR21-5p NC group, the calcification area in the D+I+miR21-5p OE group was significantly increased ( Figure 10 A-D, P < 0.001).

[0083] The calcium content and ALP activity of the aorta of rats in each group were further measured by colorimetry. Compared with the CON group, the calcium content and ALP activity of the aorta of rats in the DVC group were significantly increased ( Figure 10E-F, 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 rats. However, the aortic calcium content and ALP activity in the miR21-5p OE group were significantly higher than those in the DVC group and the miR21-5p NC group ( Figure 10 E-F, P < 0.05, P < 0.01). After receiving the intervention of Dantong Tongmai Recipe combined with intermittent fasting, 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 groups ( Figure 10 E, P > 0.05), and the aortic ALP activity in the D+I+miR21-5p NC group was significantly lower than that in the miR21-5p NC group (Figure Figure 10 E, P < 0.05). After miR21-5p overexpression, compared with the D+I+miR21-5p NC group, both the aortic calcium content and ALP activity in the D+I+miR21-5p OE group were significantly increased ( Figure 10 E-F, P < 0.05). These results indicate that the overexpression of miR21-5p eliminated the anti-calcification effect of Dantong Tongmai Recipe combined with intermittent fasting.

[0084] Subsequently, we further detected the expression of the miR21-5p / Tpm1 axis in the aortas of rats in each group by PCR. The results showed that compared with the CON group rats, the expression of miR21-5p in the aortas of DVC group rats was significantly increased ( Figure 11 A, P < 0.05). Compared with the DVC group rats, there was no obvious change in the expression of miR21-5p in the aortas of miR21-5p NC group rats ( Figure 11 A, P > 0.05), and the expression of miR21-5p in the aortas of miR21-5p OE group rats was significantly increased ( Figure 11 A, P < 0.001), indicating that the adenovirus injection successfully induced the overexpression of miR21-5p. After receiving the intervention of Dantong Tongmai Recipe combined with intermittent fasting, the expression of miR21-5p in the D+I+miR21-5p NC group was significantly lower than that in the miR21-5p NC group ( Figure 11 A, P < 0.001), suggesting that Dantong Tongmai Recipe combined with intermittent fasting can effectively inhibit the activation of miR21-5p. However, after miR21-5p overexpression, the above effect of Dantong Tongmai Recipe combined with intermittent fasting was reversed, manifested as a significant increase in miR21-5p in the D+I+miR21-5p OE group compared with the D+I+miR21-5p NC group ( Figure 11 A, P < 0.001). Compared with the CON group, both the Tpm1 mRNA and protein expressions in the aortas of DVC group rats were significantly decreased ( Figure 11B, 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 ( Figure 11 B, P > 0.05), while the expression of Tpm1 mRNA in the miR21-5p OE group was further decreased ( Figure 11 B, P < 0.001). After receiving the intervention of Dantong Tongmai Prescription combined with intermittent fasting, the expression of Tpm1 in the D+I+miR21-5p NC group was significantly higher than that in the miR21-5p NC group ( Figure 11 B, P < 0.001, P < 0.01), suggesting that Dantong Tongmai Prescription combined with intermittent fasting can effectively restore the expression of Tpm1. However, when miR21-5p was overexpressed, compared with the D+I+miR21-5p NC group, the expression of Tpm1 mRNA in the D+I+miR21-5p OE group was significantly downregulated ( Figure 11 B, P < 0.001). Combining with the previous results, it was jointly proved that the miR21-5p / Tpm1 axis was involved in the regulation of the occurrence and development of diabetic VC, and the treatment method targeting the inhibition of the miR21-5p / Tpm1 axis was a potential drug development strategy for diabetic VC.

[0085] 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 its equivalent technologies, the present invention is also intended to include these changes and modifications.

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 of miR21-5p in vivo or silence the expression of miR21-5p. The miR21-5p inhibitor is the siRNA shown in SEQ ID NO.5.

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