阅读说明：本技术 奥美沙坦在制备延缓血管平滑肌细胞衰老的药物中的应用 (Application of olmesartan in preparation of medicine for delaying vascular smooth muscle cell aging ) 是由 张莉 雷达 张艺 梁庆阳 于 2019-10-18 设计创作，主要内容包括：本发明提供一种奥美沙坦的新用途,具体是指奥美沙坦在制备延缓血管平滑肌细胞衰老的药物中的应用。血管老化影响各种心血管疾病的发生阈值、进展、严重程度和预后,是造成心血管疾病高病死率的重要因素。血管平滑肌细胞是构成血管壁的主要成分之一,是血管老化的重要细胞生物学基础,血管平滑肌细胞的结构和功能在血管老化过程中扮演着重要角色,本发明的奥美沙坦的用途是其能够延缓血管平滑肌细胞衰老,具有积极的临床意义。(The invention provides a new application of olmesartan, and particularly relates to an application of olmesartan in preparation of a medicine for delaying vascular smooth muscle cell aging. Vascular aging affects the occurrence threshold, progression, severity and prognosis of various cardiovascular diseases, and is an important factor contributing to high mortality of cardiovascular diseases. The blood vessel smooth muscle cell is one of main components forming a blood vessel wall, is an important cell biological foundation of blood vessel aging, the structure and the function of the blood vessel smooth muscle cell play an important role in the blood vessel aging process, and the olmesartan has positive clinical significance for delaying the aging of the blood vessel smooth muscle cell.)

1. Application of olmesartan in preparing medicine for delaying vascular smooth muscle cell aging is provided.

2. The use according to claim 1, wherein olmesartan down-regulates miR-665 overexpression delays vascular smooth muscle cell senescence.

Technical Field

The invention relates to a new application of olmesartan, in particular to an application of olmesartan in preparing a medicine for delaying vascular smooth muscle cell aging.

Background

The aging of population is a serious social problem, especially in China. Vascular aging, which is accompanied by molecular and cellular dysfunction, is considered a particular risk factor for cardiovascular disease (CVD), which can lead to atherosclerosis, hypertension, and the like; vascular aging is also one of the targets for intervention in treating CVD. Vascular Smooth Muscle Cells (VSMCs), the most abundant cells inherent to the vessel wall, play a major role in aging-related vascular remodeling and in the pathological course of atherosclerosis. Elucidating the mechanisms associated with vascular aging, especially in VSMC, is of great importance in the prevention and treatment of aging-associated CVD. Micro RNAs (miRNAs) in small RNA are currently the most non-coding RNAs studied in different diseases in different states, including in aging diseases, and alterations in the function of miRNAs can affect aging-related gene expression by regulating key molecular pathways, and participate in the regulation of cellular aging and related signaling pathways.

Angiotensin ii (ang ii) signaling plays an important role in vascular aging-related remodeling. The transcription, translation and activity of angiotensin converting enzyme-1 (ACE-1) in aging VSMCs are all increased significantly. The expression level of Ang II is found to be obviously increased in the aged and thickened intima, and the expression level of the Ang II receptor-AT 1 which is a cleavage product is also up-regulated. Meanwhile, research also finds that Ang II can induce arterial vessel wall reconstruction, including intima-media thickening and VSMCs aseptic inflammation; mediates increased migration/invasion capacity and proliferation capacity of senescence-associated VSMCs and enhanced activity of secreted metallomatrix protease 2(MMP 2). The effect of elevated Ang II levels on vascular remodeling is an important manifestation of vascular aging. Therefore, inhibition of Ang II signaling may be a novel means of inhibiting VSMCs senescence. Olmesartan is an AT1 receptor blocker, and no research on olmesartan-related vascular aging exists AT present.

Disclosure of Invention

The invention aims to provide the application of olmesartan in preparing a medicament for delaying the aging of vascular smooth muscle cells on the basis of research that the olmesartan has the effect of delaying the aging of vascular smooth muscle cells and the effect is realized by regulating miR-665.

Cell senescence model was constructed by inducing human vascular smooth muscle cells (HA-VSMC) with Bleomycin (BLM). SA-beta-Gal staining, NAD⁺Determination of NADH ratio, determination of DNA damage condition by gamma-H2 AX fluorescent staining, and evaluation of cell proliferation capacity index by EdU flow quantitative determinationThe cells are aged. The experiment proves that: olmesartan can delay aging of HA-VSMC.

miR-665 is one of differential expression miRNAs of olmesartan interfering aging vascular smooth muscle cells.

HA-VSMCs of a control group, a bleomycin induced aging group and an olmesartan intervention group are subjected to gene chip detection, differential miRNAs are analyzed for expression, and Top 8 differential expression miRNAs are selected. qRT-PCR confirmed: miR-665 expression is up-regulated in senescent cells, while miR-193a-3p, miR-3133, miR-4517 and miR-942-3p expression is down-regulated; olmesartan, however, reversed the expression of miR-665 and miR-3133 only. Notably, miR-665 produced more significant changes than miR-3133, and therefore miR-665 was selected for further study. The olmesartan medoxomil has the effect of delaying the aging of vascular smooth muscle cells by regulating miR-665.

The technical scheme of the invention has the following beneficial effects:

vascular aging affects the occurrence threshold, progression, severity and prognosis of various cardiovascular diseases, and is an important factor contributing to high mortality of cardiovascular diseases. The blood vessel smooth muscle cell is one of main components forming a blood vessel wall, is an important cell biological foundation of blood vessel aging, the structure and the function of the blood vessel smooth muscle cell play an important role in the blood vessel aging process, and the olmesartan has positive clinical significance for delaying the aging of the blood vessel smooth muscle cell.

Drawings

FIG. 1 is a graph showing SA- β -Gal staining patterns and mean value measurement results of vascular smooth muscle cells in a control group, a bleomycin-induced aging group and an olmesartan intervention group;

FIG. 2 is the NAD of vascular smooth muscle cells of control group, bleomycin-induced aging group and olmesartan-pretreated group⁺A graph of the results of NADH ratio determination;

FIG. 3 is a graph showing the staining of vascular smooth muscle cells by gamma-H2 AX in the control group, bleomycin-induced aging group and olmesartan-pretreated group;

FIG. 4 is a graph showing the results of EdU flow quantification and mean value measurement of vascular smooth muscle cells in a control group, a bleomycin-induced aging group and an olmesartan intervention group;

FIG. 5 is a graph showing gene chip detection and clustering of miRNAs differentially expressed by Top 8 in the control group, bleomycin-induced aging group and olmesartan pretreatment group;

FIG. 6 shows the verification of the miRNAs with Top 8 differential expression in the control group, bleomycin-induced aging group and olmesartan pretreatment group by qRT-PCR;

FIG. 7 is a method for detecting the methylation state of miR-665in a control group and a bleomycin-induced aging group by a sulfuration sequencing PCR method;

FIG. 8 shows that the TargetScan software predicts the presence of miR-665 binding domain to the SDC 13' UTR (untranslated region);

FIG. 9 is a dual luciferase report assay to detect the direct complementary pairing of miR-665 and the 3' UTR (untranslated region) of SDC 1;

FIG. 10 shows the expression of SDC1 in the control and bleomycin-induced aging groups as determined by qRT-PCR and immunoblotting;

fig. 11 is a SA- β -Gal staining pattern and mean value measurement result pattern of vascular smooth muscle cells of a control group, a bleomycin-induced aging group, an olmesartan intervention group, and an olmesartan intervention combined miR-665 overexpression or SDC1 overexpression/interference group;

FIG. 12 shows NAD of vascular smooth muscle cells in control group, bleomycin-induced aging group, olmesartan intervention group and olmesartan intervention combined miR-665 overexpression or SDC1 overexpression/interference group⁺A graph of the results of NADH ratio determination;

fig. 13 is a gamma-H2 AX staining pattern of vascular smooth muscle cells of a control group, a bleomycin-induced aging group, an olmesartan intervention group and an olmesartan intervention combined miR-665 overexpression or SDC1 overexpression/interference group;

fig. 14 is a graph showing EdU flow quantification and mean value measurement results of vascular smooth muscle cells in a control group, a bleomycin-induced aging group, an olmesartan intervention group and an olmesartan intervention combined miR-665 overexpression or SDC1 overexpression/interference group;

fig. 15 is a schematic view of olmesartan slowing vascular smooth muscle cell aging.

Detailed Description

In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, the following detailed description is given with reference to specific embodiments.

1. Olmesartan for delaying vascular smooth muscle cell aging

Grouping experiments:

control group: HA-VSMCs (human vascular smooth muscle cells) without intervention;

② bleomycin aging-induced group: intervention of HA-VSMCs for 48h by bleomycin;

③ olmesartan dry pre-group: HA-VSMCs are pretreated for 2h by olmesartan and then intervened for 48h in combination with bleomycin.

The 3 experimental groups described above were used to construct a model of cellular senescence. The experimental method comprises the following steps: staining with SA-beta-Gal, NAD⁺Measuring the ratio of NADH, measuring the DNA damage condition by gamma-H2 AX fluorescent staining, and quantitatively measuring the cell proliferation capacity index by EdU flow to evaluate the cell senescence.

The results are shown in FIGS. 1-4: the intervention of bleomycin can increase SA-beta-Gal blue-staining positive cells; NAD (nicotinamide adenine dinucleotide)⁺Increased NADH ratio; staining with γ -H2AX indicated increased DNA damage; EdU flow quantification indicated a decrease in cell proliferation capacity, suggesting that bleomycin intervention may induce VSMC senescence. And the combined dry olmesartan reduces SA-beta-Gal blue-staining positive cells; NAD (nicotinamide adenine dinucleotide)⁺decreased/NADH ratio; staining with γ -H2AX showed reduced DNA damage; the EdU flow quantification indicates that the cell proliferation capacity is enhanced, suggesting that olmesartan may delay VSMC senescence. Note: BLM, bleomycin; OMST, olmesartan; SA- β -Gal, senescence-associated β galactosidase; p<0.05。

The experimental procedures involved in this section are as follows:

1.1 cell culture

Human aortic vascular smooth muscle cells (HA-VSMCs) were purchased from ATCC (CRL-1999). The cells were cultured in F-12K medium (ATCC) containing 10% fetal bovine serum (Gibco). The medium was supplemented with 0.05mg/mL ascorbic acid, 0.01mg/mL insulin, 0.01mg/mL transferrin, 10ng/mL sodium selenite, 0.03mg/mL Endothelial Cell Growth Supplement (ECGS), 10mM hydroxyethylpiperazine ethanesulfonic acid (HEPES), and 10mM tris (hydroxymethyl) -methylamino-ethanesulfonic acid (TES). HA-VSMCs were cultured, passaged (less than 3-5 passages), and treated as follows for each experimental group: treating HA-VSMCs with bleomycin (0.5 μ g/ml) for 48 hr to induce aging; in olmesartan dry pretreatment group, adding HA-VSMCs into olmesartan (2mM) for pretreatment for 2h before the intervention of bleomycin; as a control group, HA-VSMCs were cultured normally. The medium was changed every 2 days.

1.2 senescence-associated beta galactosidase (SA-beta-gal) staining

SA- β -gal is a biological marker for identifying senescent cells. 3-5 generation 5 x 10⁵HA-VSMCs were inoculated into 6-well plates, washed twice with PBS when 50% of the cells had grown, fixed for 5min with 4% paraformaldehyde, and stained with SA- β -gal staining solution (C0602, Byunnan Biotech). 4 fields are randomly selected and observed under a microscope, the percentage of cells (SA-beta-gal positive cells) with blue staining substances in cytoplasm is counted, and the condition of cell aging is judged.

1.3NAD⁺Determination of the NADH ratio

NAD⁺NADH is a coenzyme essential for cellular energy metabolism, and its level directly affects important vital processes such as cell rhythm, aging, canceration and death. NAD in senescent cells⁺The ratio NADH increases. WST-8 Farad NAD⁺Determination of the NADH ratio (NAD)⁺NADH detection kit, Shanghai Biyuntian Biotech Co.). 3-5 generation 1x 10⁶HA-VSMCs were seeded in 6-well plates and 200. mu.l NAD was added to HA-VSMCs after various interventions⁺NADH extracting solution, 12,000g, centrifuging at 4 ℃ for 5-10min, and taking supernatant as a sample to be detected. Calculation of NAD in cell samples according to the Standard Curve⁺And the concentration of NADH.

1.4 Gamma-H2 AX immunofluorescence assay

The persistent DNA damage response in senescent cells leads to the accumulation of senescent DNA damage, a process that can be identified by co-localization of gamma-H2 AX. 3-5 generation 5 x 10⁵And inoculating HA-VSMCs into a 6-pore plate provided with a climbing sheet, fixing the HA-VSMCs with 4% paraformaldehyde after intervention, and sealing 5% BSA working solution at the temperature for 2h after 0.2% TritonX-100 permeation. Cells were incubated with gamma-H2 AX antibody (1: 200) overnight at 4 ℃ and FITC-labeled secondary antibody incubated for 2H at room temperature in the dark, followed by DAPI staining for 10min at room temperature. Dropping anti-fluorescence quenching sealTablets, nail polish seals, confocal microscopy (dark).

1.5EdU cell proliferation flow assay

Senescent cells increased AMP/ADP: ATP and NAD⁺The ratio/NADH activates AMPK, which in turn enhances the G1 phase arrest of the cell cycle, accompanied by a reduced cell proliferation capacity. 3-5 generation 5 x 10⁵Individual HA-VSMCs were plated in 6-well plates and the HA-VSMCs were subjected to various interventions. The cell culture solution is changed into a prepared EdU culture medium per well, and the incubation is carried out for 2 h. Cells were collected into a flow tube, fixed with 4% paraformaldehyde, neutralized with 2mg/ml glycine, permeabilized with 0.5% TritonX-100 and incubated. After addition of the 1XApollo staining reaction solution, the reaction solution was again infiltrated with 0.5% Triton X-100 and immediately subjected to flow cytometry (BDbiosciences) detection.

2. miR-665 is one of differential expression miRNAs of olmesartan interfering aging vascular smooth muscle cells

The HA-VSMCs line gene chip detection and parallel cluster analysis of the control group, the bleomycin induced aging group and the olmesartan intervention group are performed, and the result is shown in FIG. 5: and a plurality of miRNAs are differentially expressed in a pairwise comparison manner, and Top 8 is selected to differentially express the miRNAs, wherein the miRNAs comprise has-miR-1267, has-miR-665, has-miR-4742-3p, has-miR-193a-3p, has-miR-5579-3p, has-miR-3133, has-miR-4517 and has-miR-942-3 p. 8 differentially expressed miRNAs were verified using qRT-PCR as shown in figure 6: miR-665 expression is up-regulated in senescent cells, while miR-193a-3p, miR-3133, miR-4517 and miR-942-3p expression is down-regulated; olmesartan, however, reversed the expression of miR-665 and miR-3133 only. Notably, miR-665 produced more significant changes than miR-3133, so we chose miR-665 for further study. Note: BLM, bleomycin; OMST, olmesartan; comparison with control group P<0.05, P compared to control group<0.01，^#P compared with bleomycin-induced aging group<0.05，^##P compared with bleomycin-induced aging group<0.01。

The experimental procedures involved in this section are as follows:

2.1miRNA biochips

Extracting total RNA from HA-VSMCs with TRIzol, and further extractingRNeasy mini kit (Qiagen) was purified efficiently. The RNA concentration was measured on an ultraviolet spectrophotometer. According to miRCURY^TMLNA Array (Exiqon, Denmark) microRNAs detection method using by mircurY^TMThe Power labeling kit labels the total RNA of the cell sample, and the labeled sample RNA is labeled in the mircurY containing the miRNAs probe^TMHybridization was performed in LNA Array. After hybridization, the raw signal intensities of the chip images were read for data analysis using a GenePix4000B chip scanner (Axon, USA).

2.2miRNA chip data analysis

The results of the chip were scanned using a GeneChip Scanner 3000(Affymetrix) and the raw data were read using a Command transducer software (version 4.0; Affymetrix). After normalization, screening for differentially expressed miRNAs between the two groups was performed using the Fold-change (Fold difference of expression >2.0) and T-test (p <0.05) statistical methods. And visually and graphically displaying the screened differentially expressed miRNAs between different groups through a cluster map. TargetScan database software (http:// TargetScan. org /) was used to predict the target genes for differentially expressed miRNAs.

2.3 real-time quantitative PCR

Detecting the expression level of the miRNAs with differential expression, collecting the cultured HA-VSMCs, and extracting the total RNA by sequentially utilizing the lysate and the prepared buffer solution. The RNA concentration was measured on an ultraviolet spectrophotometer and 500ng of RNA was reverse transcribed into cDNA using the first strand synthesis kit. The Real-time quantitative PCR analysis was performed using ABI Prism 7900 Real-time quantitative PCR system (applied biosystems) using dye method (SYBR Green I), U6 was used as an internal reference. All primers were designed by primer design software 3.0(Applied Biosystems) and synthesized by Invitrogen corporation.

3. Abnormal methylation is the reason for the difference of miR-665 expression

In order to verify whether miR-665 highly expressed in bleomycin-induced aging HA-VSMCs is related to abnormal methylation, a sulfuration sequencing PCR method is used for detecting abnormal methylation of miR-665 promoter regions CpG islands of a control group and a bleomycin-induced aging group. As shown in fig. 7: miR-665 methylation status was reduced in aging HA-VSMC (35.38% vs. 69.23%) compared to control. Suggesting abnormal methylation (increased level of demethylation) of miR-665in aging HA-VSMCs. Note: BLM, bleomycin.

The experimental procedures involved in this section are as follows:

sequencing By Sulfide PCR (BSP)

Genomic DNA was first extracted from HA-VSMCs using TRIzol and then subjected to bisulfite treatment using EZ DNA methylation-Gold kit (Zymo Research). Specific miR-665 methylation primers were designed for amplification using "MethPrimer" (http:// www.urogene.org/cgi-bin/MethPrimer. cgi), the amplified fragments were cloned into Pgemt Easy vector (Promega), and 5 clones were randomly selected for sequencing. Percentage methylation was calculated using QUMA (http:// QUMA. cdb. riken. jp/top/index. html).

4. Olmesartan can delay vascular smooth muscle cell aging through miR-665/SDC1 signal path

This section includes the following two aspects:

syndecano-1 (SDC1) is a miR-665 downstream target gene, and the expression of the gene is reduced in bleomycin-induced aging HA-VSMCs

First, bioinformatics (TargetScan) was applied to predict miR-665 downstream target genes, multiple candidate target genes were present, studies have demonstrated that SDC1 is associated with aging and cellular senescence, and SDC1 was therefore selected as the putative target gene for subsequent studies. FIG. 8 shows: miR-665 presents a binding domain to the SDC 13' UTR (untranslated region). Secondly, a dual-luciferase report experiment is applied to prove that miR-665 can be directly complementarily paired with 3' UTR of SDC 1. miR-665 imic/inhibitor and wild type/mutant SDC1 reporter plasmids were co-transfected in HEK293 cells, with the results shown in figure 9: in a wild-type report plasmid, miR-665mimic (overexpression) reduces luciferase activity, and miR-665inhibitor (interference) increases luciferase activity; in the mutant report plasmid, the miR-665 micic/inhibitor does not change the activity of the luciferase. Finally, fig. 10 shows: qRT-PCR and immunoblotting confirmed that bleomycin induced the down-regulation of SDC1 expression in aging HA-VSMCs compared to controls. Note: BLM, bleomycin; p < 0.05.

② olmesartan for delaying HA-VSMC senescence through miR-665/SDC1 signal path

Respectively pass throughMiR-665 overexpression, SDC1 overexpression/interference and combined dry prognosis olmesartan-treated bleomycin-induced aging HA-VSMC, staining with SA-beta-Gal, NAD⁺Measuring a ratio of NADH, measuring DNA damage condition by gamma-H2 AX fluorescent staining, measuring cell proliferation capacity index by EdU flow type quantitative measurement to evaluate cell senescence, and observing a mechanism of retarding HA-VSMC senescence by olmesartan. The results are shown in FIGS. 11-14: miR-665 overexpression and SDC1 interference can inhibit the effect of olmesartan on delaying HA-VSMC aging; and the SDC1 can reverse the function of miR-665in the over-expression for inhibiting olmesartan and delaying HA-VSMC aging. Note: BLM, bleomycin; OMST, olmesartan; p<0.05。

The experimental procedures involved in this section are as follows:

4.1 immunoblotting

And detecting the expression level of the SDC1 protein of the target gene downstream of the miR-665. Collecting cells by using a protein extracting solution, carrying out 4-20% SDS-PAGE electrophoresis on 15-30 mu g of protein sample, transferring 1h to a nitrocellulose film at 100V, and putting the nitrocellulose film into a confining liquid for confining 1h at 37 ℃; primary antibody (SDC 11: 2000, abcam) was allowed to stand overnight at 4 ℃. After repeated washing of the membrane, the membrane was incubated with horseradish peroxide-conjugated secondary antibody, gently shaken at room temperature for 1h, and after washing, observed with an immunoblot image analyzer (model GS-700, Bio-Rad Laboratories, Inc.), and absorbance values of the respective bands were measured with an image analysis software for quantitative analysis. Beta-actin is used as an internal reference.

4.2. Real-time quantitative PCR

SDC1 expression level is detected, cultured HA-VSMCs are collected, and total RNA is extracted by using lysate and configured buffer solution in sequence. The RNA concentration was measured on an ultraviolet spectrophotometer and 500ng of RNA was reverse transcribed into cDNA using the first strand synthesis kit. Real-time quantitative PCR analysis was performed using ABI Prism 7900 Real-time quantitative PCR system (Applied Biosystems) using dye method (SYBR Green I), GAPDH was used as an internal reference.

4.3. Dual luciferase reporter

A target gene SDC 13 '-UTR fragment containing a miR-665 binding site is designed and synthesized, and the target gene SDC 13' -UTR fragment is inserted into a pmirGLO vector (Promega Corporation) containing a fluorescein reporter gene to construct a reporter plasmid (a wild type and a mutant type). 3-5 generation 2 x 10⁴Transfection of HA-VSMCsThe cells were passaged to 24-well plates the day before, the cells grew about 50% -60% the next day, and were transfected with 50nM miR-665mimic/miR-665inhibitor (control set) in combination with 0.5g of wild-type or mutant fluorescent reporter plasmid 3' -UTR using Lipofectamin 2000. After 48h, analysis was performed using the dual fluorescein reporter system (Promega, Corporation).

4.4. Cell transfection

Cell-borne staining included miR-665 overexpression plasmid, SDC1 small interfering RNA (siSDC1) and negative control (miR-NC or sinC) (Chongzhou acute Bo Biotech Co., Ltd.), and SDC1 overexpression plasmid (pcDNA3.1/SDC1, Beijing OriGene Biotech Co., Ltd.). 3-5 generation 2 x 10⁵One day before transfection of HA-VSMCs, the cells were passaged to 6-well plates, and the next day, the cells were grown to about 50% -60%, and combined transfection was performed with Lipofectamin 2000 alone or in various combinations, and controls were set up.

4.5. Senescence-associated beta-galactosidase (SA-beta-gal) staining

3-5 generation 5 x 10⁵HA-VSMCs were inoculated into 6-well plates, washed twice with PBS when 50% of the cells had grown, fixed for 5min with 4% paraformaldehyde, and stained with SA- β -gal staining solution (C0602, Byunnan Biotech). 4 fields are randomly selected and observed under a microscope, the percentage of cells (SA-beta-gal positive cells) with blue staining substances in cytoplasm is counted, and the condition of cell aging is judged.

4.6.NAD⁺Determination of the NADH ratio

WST-8 Farad NAD⁺Determination of the NADH ratio (NAD)⁺NADH detection kit, Shanghai Biyuntian Biotech Co.). 3-5 generation 1x 10⁶HA-VSMCs were seeded in 6-well plates and 200. mu.l NAD was added to HA-VSMCs after various interventions⁺NADH extracting solution, 12,000g, centrifuging at 4 ℃ for 5-10min, and taking supernatant as a sample to be detected. Calculation of NAD in cell samples according to the Standard Curve⁺And the concentration of NADH.

4.7. Gamma-H2 AX immunofluorescence assay

3-5 generation 5 x 10⁵And inoculating HA-VSMCs into a 6-pore plate provided with a climbing sheet, fixing the HA-VSMCs with 4% paraformaldehyde after intervention, and sealing 5% BSA working solution at the temperature for 2h after 0.2% TritonX-100 permeation. Gamma-H2 AX antibody (1: 200)Cells were incubated overnight at 4 ℃ and FITC-labeled secondary antibody incubated 2h away from light at room temperature and stained with DAPI for 10min at room temperature. Dropping the anti-fluorescence quenching sealing tablet, sealing nail polish, and observing under a confocal microscope (keeping out of the sun).

EdU cell proliferation flow assay

3-5 generation 5 x 10⁵Individual HA-VSMCs were plated in 6-well plates and the HA-VSMCs were subjected to various interventions. The cell culture solution is changed into a prepared EdU culture medium per well, and the incubation is carried out for 2 h. Cells were collected into a flow tube, fixed with 4% paraformaldehyde, neutralized with 2mg/ml glycine, permeabilized with 0.5% TritonX-100 and incubated. After addition of the 1XApollo staining reaction solution, the reaction solution was again infiltrated with 0.5% Triton X-100, and immediately subjected to flow cytometry (BD Biosciences).

Fig. 15 is a schematic view of olmesartan slowing vascular smooth muscle cell aging. The up-regulation of miR-665 expression and abnormal methylation thereof are mechanisms for inducing HA-VSMC to age by bleomycin; olmesartan can delay the aging of HA-VSMC by reducing the expression of miR-665.

While the foregoing is directed to the preferred embodiment of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims.