Tanshinone and salvianolic acid compound molecular compatibility composition for regulating and controlling DNMTs / TET2 equilibrium relation and application of tanshinone and salvianolic acid compound molecular compatibility composition

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a tanshinone and salvianolic acid compound molecular compatibility composition for regulating and controlling the balance relation of DNMTs / TET2 and application of the tanshinone and salvianolic acid compound molecular compatibility composition. An in-vitro cell model proves that the composition with specific compatibility can synergistically regulate the expression of DNMTs and TET2 and recover the balance relation of DNMTs and TET2, so that the abnormal DNA methylation state is reversed, the normal expression of related genes is recovered, and the composition can be used for preparing the medicine for preventing or treating cardiovascular diseases related to DNA methylation abnormity. The molecular compatibility composition for balance regulation and control of key targets is created for the first time, has the advantages of clear component compatibility, clear action targets and synergistic interaction, and provides a new choice for developing novel epigenetic regulation and control drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to a molecular compatibility composition with a regulating effect on the balance relationship of DNMTs / TET2 and application thereof. BACKGROUND

[0002] DNA methylation is a dynamic process regulated by DNMT and TET enzymes, and its precise balance is crucial for life activities. The balance between DNA methylation and demethylation is a key factor for maintaining genome stability and precise regulation of gene expression. In-depth study of the balance regulation mechanism of the two can help us better understand the molecular mechanisms of biological development, aging and disease development. DNA methylation disorder and DNMTs / TETs imbalance are important pathogenesis and common patterns of chronic diseases such as cardiovascular diseases. After the abnormal methylation of related genes, the expression of related genes will be affected, which directly affects the occurrence and development of diseases. DNA methylation is mainly mediated by three enzymes, including DNMT1, DNMT3a and DNMT3b, among which DNMT1 is mainly responsible for the maintenance of methylation, and DNMT3a and DNMT3b are responsible for the establishment of new methylation. TETs are involved in active DNA demethylation process, mainly including TET1, TET2 and TET3, especially TET2, which is closely related to human cardiovascular diseases. On the one hand, TET2 has been proved to increase angiogenesis, and on the other hand, TET2 regulates gene expression to ultimately lead to cardiomyocyte differentiation. DNMTs and TET2 have theoretical potential as biomarkers in cardiovascular diseases, especially in revealing disease mechanisms and early intervention. However, its clinical application still needs to overcome technical bottlenecks and accumulate more evidence-based medical evidence. In the short term, it may be more suitable for scientific research or auxiliary diagnosis, rather than an independent diagnostic marker. With the development of single-cell epigenomics and liquid biopsy technology, this field is worth continuous attention.

[0003] Traditional Chinese medicine (TCM) is an important component of traditional Chinese medicine, which has the characteristics of multi-component, multi-target and overall regulation. Salvia miltiorrhiza Bunge is a commonly used traditional Chinese medicine for promoting blood circulation and removing blood stasis. Its main active components include tanshinones (such as tanshinone I, tanshinone IIA, cryptotanshinone, etc.) and salvianolic acids (such as salvianolic acid A, salvianolic acid B, protocatechuic aldehyde, etc.). Previous studies have shown that tanshinones and salvianolic acids have significant activities in anti-inflammatory, antioxidant, anti-tumor, and cardiovascular protection. Existing researches are mostly focused on the pharmacological effects of single component, for example, the invention patent with the publication number CN116712425A discloses a total salvianolic acid conversion product from stems and leaves of Salvia miltiorrhiza Bunge and its application, which has antioxidant activity and protects vascular endothelial cells and myocardial cells. For another example, the invention patent with the publication number CN110339199A discloses a composition of tanshinones and salvianolic acids compounds for eNOS / GRK2 regulation and its application. Although it is found that the combination of tanshinones and salvianolic acids in a certain proportion and dosage can increase myocardial energy utilization efficiency and protect myocardium, there is no report on the synergistic effect in the field of cardiovascular diseases. The present invention initiates a molecular combination composition for balanced regulation of key targets, which has the advantages of clear component combination, clear action target and synergistic effect, and provides a new choice for developing new epigenetic regulation drugs. SUMMARY

[0004] In view of the related problems of cardiovascular diseases caused by imbalance of DNMTs / TET2, the present invention provides a molecular combination composition of tanshinones and salvianolic acids compounds for regulating the balance of DNMTs / TET2 and its application.

[0005] In order to achieve the above-mentioned purpose, the technical scheme of the present invention is as follows: The present invention provides a drug for regulating DNMTs / TET2, wherein the active components of the drug comprise tanshinone compounds and salvianolic acid compounds.

[0006] The tanshinone compound is cryptotanshinone, and the salvianolic acid compound is danshensu and protocatechuic acid; The molar ratio of cryptotanshinone to danshensu is 1:2-3, and the molar ratio of cryptotanshinone to protocatechuic acid is 1:1-2; The drug exerts its effect by regulating the balance of DNMTs and TET2; The drug further comprises a pharmaceutically acceptable carrier.

[0007] The combined use concentration of cryptotanshinone and protocatechuic acid in the medicine is 0.416-2.5 μM of cryptotanshinone and 0.416-2.5 μM of protocatechuic acid, and the combined use concentration of cryptotanshinone and tanshinone in the medicine is 0.83-2.5 μM of cryptotanshinone and 0.416-3.75 μM of tanshinone.

[0008] The application provides a preparation method of a DNMTs / TET2 regulating medicine, and the specific steps are as follows: (1) cryptotanshinone, tanshinone and protocatechuic acid are respectively dissolved in an organic solvent, and after dilution, cryptotanshinone mother liquor, tanshinone mother liquor and protocatechuic acid mother liquor are obtained; (2) after dilution, cryptotanshinone medicine liquid, tanshinone medicine liquid and protocatechuic acid medicine liquid with target concentrations are obtained.

[0009] The organic solvent in the step (1) is DMSO.

[0010] The target concentration of the cryptotanshinone medicine liquid in the step (2) is 0.416-10 µM, the target concentration of the protocatechuic acid medicine liquid is 0.416-10 µM, and the target concentration of the tanshinone medicine liquid is 0.416-10 µM.

[0011] The application also provides application of the DNMTs / TET2 regulating medicine in preparation of a medicine for preventing or treating diseases caused by imbalance of DNMTs and TETs. The disease is a cardiovascular disease.

[0012] The application has the following beneficial effects: 1. The application finds that cryptotanshinone and tanshinone have a synergistic regulating effect on the activity of DNMTs and TET2 under a specific weight ratio (preferably 1:2-3). Experiments prove that the composition can bidirectionally regulate the dynamic balance of DNA methylation and demethylation, and compared with the use of any component or simple superposition, the effect is more significant and stable, thereby providing a new strategy for realizing more effective disease intervention.

[0013] 2. The application discloses a new mechanism of the synergistic effect of the key active ingredients of Danshen, i.e., the fat-soluble cryptotanshinone and the water-soluble tanshinone (tanshinone and protocatechuic acid), from the molecular level, thereby providing a new mechanism explanation for the excellent curative effect of Danshen and its preparation in preventing and treating cardiovascular diseases and the like, and promoting the modernization process of traditional Chinese medicines. BRIEF DESCRIPTION OF DRAWINGS

[0014] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced. Obviously, the drawings in the following description only constitute some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained without creative labor on the basis of these drawings.

[0015] Figure 1 Effects of different hypoxia / reoxygenation time on H9c2 cell survival rate (compared with Con group, ** P <0.05, ** P <0.01, x̅±s, n=3) Figure 2 Effects of CT, PCA, DSS on normal H9c2 cell viability (compared with Con group, ** P <0.05, ** P <0.01, x̅±s, n=3).

[0016] Figure 3 Effects of CT, PCA, DSS and combined use on H / R damaged H9c2 cell viability; wherein A is CT+PCA; B is CT+DSS; (compared with Con group, ## P <0.01, compared with Mod group, * P <0.05, ** P <0.01, ns, no significant difference, x̅±s, n=4).

[0017] Figure 4 Effects of CT, PCA, DSS and combined use on H / R damaged H9c2 cell SOD, MDA and LDH content; wherein A is SOD content; B is MDA content; C is LDH relative release rate; (compared with Con group, ## P <0.01, compared with Mod group, * P <0.05, ** P <0.01, ns, no significant difference, x̅±s, n=3).

[0018] Figure 5 Effects of different hypoxia / reoxygenation time on HUVEC cell survival rate (compared with Con group, ** P<0.05, **P<0.01, x̅±s, n=3).

[0019] Figure 6 The effects of CT, PCA, and DSS on the viability of normal HUVEC cells.

[0020] Figure 7 The effects of CT, PCA, DSS and their combination on the viability of H / R-damaged HUVEC cells (compared to the Con group), ## P <0.01, compared with the Mod group, ns, no significant difference, * P <0.05, ** P <0.01, x̅±s, n=4).

[0021] Figure 8 The effects of CT, PCA, DSS, and their combinations on SOD, MDA, and LDH levels in H / R-damaged HUVEC cells; where A represents SOD level; B represents MDA level; and C represents relative LDH release rate (compared to the Con group). ## P <0.01, compared to the Mod group, ** P <0.05, ** P <0.01, ns, no significant difference, x̅±s, n=3).

[0022] Figure 9 The combined use of CT and SAs to induce post-contraction vasodilation of the thoracic aortic vascular rings after NE pre-stimulation (compared to the Con group) * P <0.05, ** P <0.01, ① compared to ②, ③ compared to ④, * P <0.05, ** P <0.01, x̅±s, n=3).

[0023] Figure 10 The study investigated the effects of combined CT and SAs on DNMT1 mRNA expression in H9c2 and HUVEC cells after H / R injury. Specifically, A and B represent the effects of CT+DSS and CT+PCA on DNMT1 mRNA expression in H9c2 cells after H / R injury; C and D represent the effects of CT+PCA and CT+DSS on DNMT1 mRNA expression in HUVEC cells after H / R injury (compared to the Con group). ## P <0.01, compared to the Mod group,* P <0.05, ** P <0.01, x̅±s, n=3).

[0024] Figure 11 Effects of CT, SAs combined drug on DNMT3B mRNA expression in H9c2, HUVEC cells after H / R injury; among them, A, B are the effects of CT+DSS and CT+PCA on DNMT3B mRNA expression in H9c2 cells after H / R injury; C, D are the effects of CT+PCA and CT+DSS on DNMT3B mRNA expression in HUVEC cells after H / R injury (compared with Con group, ## P <0.01, compared with Mod group, * P <0.05, ** P <0.01, x̅±s, n=3).

[0025] Figure 12 Effects of CT, SAs combined drug on DNMT3A mRNA expression in H9c2, HUVEC cells after H / R injury; among them, A, B are the effects of CT+PCA and CT+DSS on DNMT3A mRNA expression in H9c2 cells after H / R injury; C, D are the effects of CT+PCA and CT+DSS on DNMT3A mRNA expression in HUVEC cells after H / R injury (compared with Con group, ## P <0.01, compared with Mod group, * P <0.05, ** P <0.01, x̅±s, n=3).

[0026] Figure 13 Effects of CT, SAs combined drug on TET2 mRNA expression in H9c2, HUVEC cells after H / R injury; among them, A, B are the effects of CT+PCA and CT+DSS on TET2 mRNA expression in H9c2 cells after H / R injury; C, D are the effects of CT+PCA and CT+DSS on TET2 mRNA expression in HUVEC cells after H / R injury (compared with Con group, ## P <0.01, compared with Mod group, * P <0.05, ** P<0.01, x̅±s, n=3). DETAILED DESCRIPTION

[0027] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative effort fall within the protection scope of the present application.

[0028] The experimental methods used in the following experimental examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are reagents and materials available through commercial channels unless otherwise specified.

[0029] Example 1 A kind of preparation of tanshinone and salvianolic acid compound combination modulated by DNMTs / TET2, specifically as follows: Respectively, 14.2 mg of cryptotanshinone (CT), 8.3 mg of Danshensu (DSS) and 6.4 mg of protocatechuic acid (PCA) were weighed into an EP tube, and 1.6 mL, 1.397 mL and 1.385 mL of DMSO were added respectively to dissolve them. 100 μL of each was diluted to 1 mL with DMSO to obtain 3 mM of cryptotanshinone (CT) stock solution, 3 mM of Danshensu (DSS) stock solution and 3 mM of protocatechuic acid (PCA) stock solution. 10 μL of CT, DSS and PCA stock solution was added to 3 mL of serum-containing medium to prepare 10 μM drug-containing medium, which was diluted by 1 / 2 to obtain different concentrations of drug solution for subsequent experiments.

[0030] Implementation Effect Example 1 The tanshinone and salvianolic acid compound combination obtained in Example 1 and its application effect in H9c2 cells.

[0031] I. Cell preparation (1) Cell recovery: turn on the constant temperature water bath, and raise the temperature to 37℃. Wipe the operating table with a sterile alcohol cotton ball, turn on the ultraviolet lamp button of the clean bench, and irradiate for 30 min. Take a 10 mL EP tube, add 4 mL of medium into the centrifuge tube. Take the H9c2 cell cryopreservation tube from the -80℃ refrigerator, place it in the 37℃ constant temperature water bath for thawing, and then take the liquid in the cryopreservation tube, add it into the prepared centrifuge tube, centrifuge at 1000 rpm for 3 min, discard the supernatant, add 1 mL of medium to the cell precipitate, mix well by blowing, inoculate into a 6 mm culture dish containing 4 mL of medium, shake well, and culture in a 37℃ CO2 incubator. Change the liquid the next day.

[0032] (2) Cell passage: when the cell growth is about 70%-80% under the microscope, discard the culture medium, add 2 mL PBS buffer for washing, discard the PBS, digest with 0.5 mL trypsin, observe the cell rounding under the microscope, and terminate the digestion by adding 3 times the amount of culture medium. Use a pipette to blow the bottom of the culture dish, then collect the cell suspension into a 5 mL centrifuge tube at 1000 rpm for 3 min; discard the supernatant, add 1 mL of culture medium to the cell pellet, mix well by blowing, and take 0.3 mL to inoculate 3 6 mm culture dishes containing 4 mL of culture medium, and shake well, and culture in a 37℃ CO2 incubator.

[0033] (3) Cell freezing: when the cell growth is about 70%-80%, use trypsin digestion, centrifuge to collect the cell pellet, add 1 mL of cell freezing solution, blow several times, transfer to a freezing tube, label the date, cell type, passage number and operator's name, and store in a -80℃ refrigerator.

[0034] II. Experimental grouping The present application previously investigated the effects of 7.5, 5, 2.5, 1.25, 0.625 µM tanshinone and 7.5, 5, 2.5, 1.25, 0.625 µM salvia acid (DSS, PCA) effective components on the relative cell viability value of normal H9c2 cells. The results showed that CT had a certain cytotoxicity to normal H9c2 cells at a concentration of 5 µM. The results showed that the protective effect of each effective component on H9c2 cells at the same concentration, and the grouping of each effective component is shown in Table 1 and Table 2.

[0035] Table 1 CT, PCA, DSS drug single use experimental concentration grouping Table 2 CT and PCA, DSS combined concentration ratio grouping III. Establishment of H9c2 myocardial cell H / R model 4×10 3 cells per well in a 96-well plate, and cultured for 24 h, then discarded the original culture medium, added 200 µL of DMEM low-sugar medium without FBS, placed the 96-well plate in a sealed bag, and filled N2 into the sealed bag, and then placed it in a 37℃ incubator. After 8 h, replace with DMEM high-sugar medium containing serum, and culture for 4 h to obtain H / R injury model cells.

[0036] The experimental results are as follows Figure 1As shown in the results, when the reoxygenation time was 2 h, the cell viability after hypoxia for 4, 6, 8, and 12 h was 74.12 ± 1.39%, 64.18 ± 0.85%, 59.42 ± 1.01%, and 33.26 ± 1.28%, respectively. The cell survival rate was too low after 12 h of hypoxia, so the hypoxia time was selected as 8 h, and the cell survival rate after reoxygenation for 2, 4, 6, and 8 h was 61.59 ± 1.16%, 48.75 ± 1.64%, 45.97 ± 1.60%, and 39.26 ± 1.93%, respectively. Comprehensive consideration determined the modeling conditions to be: hypoxia for 8 h and reoxygenation for 4 h.

[0037] Four, the effect of CT, SAs on the viability of H9c2 cells (1) Cell plating and pretreatment: H9c2 myocardial cells were inoculated in culture dishes for routine subculture. When the cell density reached 70%-80% confluence, cell counting was performed using a hemocytometer, the suspension concentration was adjusted to 4×10³ cells / well, and inoculation was performed in a 96-well culture plate (200 μL of complete culture medium per well) and placed in a 37°C, 5% CO2 incubator for 24 h.

[0038] (2) Drug treatment: The pre-configured drug-containing medium with different concentrations (200 μL / well) was replaced into the pretreated cell culture system for continuous intervention for 24 h.

[0039] (3) Cell viability detection: After cell adhesion, the original culture medium was discarded, 100 μL of culture medium containing 10% CCK-8 reagent was added to each well (operation in the dark), and incubation was continued for 1.5 h. The absorbance value (OD value) was measured at 450 nm using a microplate reader, and the differences in OD values between the drug groups and the control group were compared to evaluate the effect of the drug on myocardial cell viability.

[0040] The cell viability detection results are shown in Figure 2 The results show that under the foregoing grouping dose concentrations, the cell viability values are all greater than 100%. This indicates that within the above concentration range, tanshinone and salvianolic acid effective components have no cytotoxicity to H9c2 cells.

[0041] Five, the effect of CT, SAs on the viability of H / R injured H9c2 cells H / R injured H9c2 myocardial cells were taken, the original culture medium was discarded, and 200 μL of drug with different concentrations was added to each well. After 24 h of culture, the original culture medium was aspirated, and cell viability detection was performed to determine the protective effect of the drug on H / R injured H9c2 myocardial cells.

[0042] The cell viability detection results are shown in Figure 3 The results show that compared with the normal group, the H9c2 cell viability of the model group decreased significantly (43.53 ± 2.342,P <0.01), the H / R injury model was successfully constructed. Compared with the model group, the cell viability values of each group were increased to different degrees after drug administration, indicating that CT, PCA, DSS and their combinations had certain myocardial protective effects in the measured concentration range.

[0043] For the combined drugs CT+DSS and CT+PCA, significant myocardial protective effects were exhibited, among which in the CP-1 (66.21±1.512, P <0.01), CD-1 (67.59±0.7292, P <0.01), CD-6 (68.83±0.7294, P <0.01) groups, the effects were not only significant compared with the single drug, but also significant compared with other combined drug groups in the same group. Comparing CD-1 and CD-5 at the same DSS concentration, the cell viability was higher in the group with higher CT concentration, indicating that CT played a major role in myocardial cells. The effects of CD-1 and CD-6 were better than those of other groups, but there was no significant difference between them, indicating that the optimal ratio of CT and DSS combination was within the [1:2-1:3] interval.

[0044] Six, determination of LDH, MDA and SOD contents in H9c2 cells after H / R injury by CT and DSS combination The contents of LDH, MDA and SOD were detected according to the kit instructions. The results are shown in Table 2. Figure 4 As shown in Table 2, compared with the normal group, the MDA content in the model group was significantly increased (P<0.01), the SOD content was significantly decreased (P<0.01), and the relative release amount of LDH was significantly increased (P<0.01). Compared with the model group, drug treatment could reduce the contents of LDH and MDA and increase the content of SOD in H / R injured H9c2 cells (P<0.05), which was consistent with the results of cell viability determination, indicating that each group had a protective effect on H / R injured H9c2 cells. P P <0.01), the SOD content was significantly decreased (13.07±0.5464, P <0.01), and the relative release amount of LDH was significantly increased (P<0.01). Compared with the model group, drug treatment could reduce the contents of LDH and MDA and increase the content of SOD in H / R injured H9c2 cells (P<0.05), which was consistent with the results of cell viability determination, indicating that each group had a protective effect on H / R injured H9c2 cells.

[0045] Compared with the normal group, the SOD content in the model group was significantly decreased (P<0.01), and the H / R injury model was successfully constructed. Compared with the model group, the SOD content in the drug administration group after injury was increased to different degrees, indicating that the drugs had a protective effect on the injured myocardial cells. Compared with the model group, the combined drug groups significantly increased the SOD content in the injured cells, and CP-6 (25.89±0.7071, P <0.01) in the CT and PCA combined drug group was significantly better than its single component and other proportions in the same group, CD-1 (29.72±1.561, P <0.01) and CD-6 (29.72±1.561, P ​<0.01) and CD-6 (30.24±0.866, P <0.01) was significantly better than its monomer composition and other proportions in the same group, but there was no significant difference between the two groups, which also proved that CT played a major role in myocardial cells.

[0046] Compared with the normal group, the MDA (17.07±2.695, P <0.01) was significantly increased, and the H / R injury model was successfully constructed. Compared with the model group, the MDA content in the drug administration group after injury decreased to varying degrees, showing that the drug had a protective effect on the injured myocardial cells. Compared with the model group, the combined drug group significantly reduced the MDA content in the cells after injury, and CP-1 (5.485±0.1496, P <0.01) was significantly better than its monomer composition and other proportions in the same group, suggesting that the best proportion was near the CP-1 group. CD-1 (5.643±0.5204, P<0.01), CD-6 (5.399±0.1633, P <0.01) was significantly better than its monomer composition and other proportions in the same group, suggesting that the best proportion was between [1:2-1:3].

[0047] Compared with the normal group, the relative release amount of LDH in the model group was significantly increased (0.6473±0.02551, P <0.01). Compared with the model group, drug treatment can reduce the release amount of LDH in cells after H / R injury. It shows that each group has a protective effect on H / R injured H9c2 cells. Compared with the model group, the combined drug group significantly reduced the LDH content in the cells after injury, and CP-1 (0.5065±0.02277, P <0.01), CP-6 (0.4999±0.02475, P <0.01) was better than its monomer composition and other proportions in the same group, but there was no significant difference between the two groups, and it was guessed that CT played a major role in myocardial cells. CD-1 (0.5061±0.03716, P <0.01), CD-6 (0.5009±0.02520, P <0.01) was better than its monomer composition and other proportions in the same group, but there was no significant difference between the two groups, and it was guessed that CT played a major role in myocardial cells.

[0048] Implementation Example 2 The application effect of the composition of tanshinones and salvianolic acid compounds obtained by example 1 on HUVEC cells.

[0049] I. Cell preparation (1) Cell recovery: turn on the constant temperature water bath, and raise the temperature to 37°C. Wipe the operating table with a sterile alcohol cotton ball, turn on the ultraclean table UV lamp button, and irradiate for 30 min. Take a 10 mL EP tube, add 4 mL of culture medium into the centrifuge tube for standby. Take the HUVEC cell cryopreservation tube from the -80°C refrigerator, place it in a 37°C constant temperature water bath for thawing, and then suck the liquid in the cryopreservation tube into the prepared centrifuge tube, centrifuge at 1000 rpm for 3 min, discard the supernatant, add 1 mL of culture medium to the cell precipitate, mix well by blowing, inoculate into a 6 mm culture dish containing 4 mL of culture medium, shake well, and culture in a 37°C CO2 incubator. Change the liquid the next day.

[0050] (2) Cell passage: when the cell growth is about 70%-80% under the microscope, discard the culture medium, add 2 mL of PBS buffer for washing, discard the PBS, digest with 0.5 mL of trypsin, and then observe the cell rounding under the microscope. Add 3 times the amount of culture medium to terminate the digestion. Use a pipette to blow the bottom of the culture dish, then collect the cell suspension into a 5 mL centrifuge tube, centrifuge at 1000 rpm for 3 min; discard the supernatant, add 1 mL of culture medium to the cell precipitate, mix well by blowing, take 0.3 mL and inoculate into 3 6 mm culture dishes containing 4 mL of culture medium, shake well, and culture in a 37°C CO2 incubator.

[0051] (3) Cell cryopreservation: when the cell growth is about 70%-80%, use trypsin to digest, centrifuge to collect the cell precipitate, add 1 mL of cell cryopreservation solution, blow several times, transfer to a cryopreservation tube, mark the date, cell type, passage number and operator's name, and store in a -80°C refrigerator.

[0052] II. Establishment of HUVEC cell hypoxia / reoxygenation (H / R) model Inoculate 3×10 3 cells per well in a 96-well plate, culture for one day, discard the original culture medium, add 200 μL of DMEM low-sugar medium without FBS, place the plate in a sealed bag, and fill N2 into the sealed bag, then place it in a 37°C incubator. After 12 h, replace it with DMEM high-sugar medium containing serum, and culture for 4 h to obtain the hypoxia / reoxygenation injury model cells.

[0053] The results are as follows Figure 5It was showed that when the reoxygenation time was 4 h, the cell viability of endothelial cells after hypoxia for 4, 8, 12 and 24 h was 82.73±1.38%, 67.85±1.83%, 55.86±1.14% and 36.7±1.09% respectively. The cell survival rate was too low after hypoxia for 24 h. Therefore, the modeling condition was determined to be hypoxia for 12 h and reoxygenation for 4 h.

[0054] III. Effect of CT and SAs on the viability of HUVEC cells (1) Cell plating and pretreatment: HUVEC cells were inoculated in culture dishes and cultured until the cells grew to 70-80% coverage of the culture dish. Cell counting was performed using a hemocytometer, and the cell concentration was adjusted to 3×10 3 cells / well, and then inoculated in a 96-well culture plate and cultured in a 37°C, 5% CO2 incubator.

[0055] (2) Drug treatment: The pre-configured drug-containing medium with different concentrations (200 μL / well) was replaced into the pretreated cell culture system for continuous culture.

[0056] (3) Cell viability detection: After culture, the absorbance value (OD value) was measured at 450 nm wavelength using a microplate reader to determine the effect on the viability of normal endothelial cells.

[0057] The cell viability detection results are shown in Table 1. Figure 6 The results showed that the cell viability value was greater than 100% under the foregoing grouping dose concentrations. This indicated that CT and SAs had no cytotoxicity to HUVEC cells within the above concentration range.

[0058] IV. Effect of CT and SAs on the viability of H / R damaged HUVEC cells H / R damaged HUVEC cells were taken, and the original culture medium was discarded. 200 μL of drug with different concentrations was added to each well. After 24 h of culture, the original culture medium was removed, and cell viability detection was performed to determine the protective effect on H / R damaged HUVEC cells.

[0059] The cell viability detection results are shown in Table 2. Figure 7 The results showed that compared with the normal group, the HUVEC cell viability of the model group decreased significantly (54.42±3.184 ## , P <0.01), and the H / R damage model was successfully constructed. Compared with the model group, the cell viability decreased after drug administration in the CT2.5 group, which may be due to the decrease in the tolerance of HUVEC cells to CT after damage, resulting in cytotoxicity. The cell viability value increased to different degrees after drug administration in the other groups, indicating that CT, PCA and DSS had certain endothelial protective effect within the measured concentration range.

[0060] The combination of drugs CT+DSS and CT+PCA showed significant endothelial protective effect, among which in CP-1 (69.31±0.8007, P <0.01), CP-4 (72.14±0.5356, P <0.01), CP-6 (75.87±0.7787, P <0.01) groups, the effect was significantly better than that of single drug, but there was no significant difference between groups, it was speculated that SAs played a major protective role in endothelial cells. There was no significant difference between CP-1 and CP-4 in the same PCA concentration, which might be due to the better proportion of CP-1 than CP-4. CD-1 (67.59±0.7292, P <0.01), CD-6 (68.83±0.7294, P <0.01) were significantly better than single drug and other combination drugs in the same group, but there was no significant difference between them, and there was also no significant difference between CD-1 and CD-5 in the same DSS concentration, indicating that the best proportion was between [1:2-1:3].

[0061] Five, determination of LDH, MDA and SOD content in HUVEC cells after CT, DSS combination H / R injury According to the kit instructions, the contents of LDH, MDA and SOD were detected. The results are shown in Figure 8 The results showed that compared with the normal group, the SOD content in the model group was significantly reduced (33.28±1.805, P <0.01), and the H / R injury model was successfully constructed. Compared with the model group, the SOD content in the drug group after injury increased to different degrees, indicating that the drugs had protective effect on injured HUVEC cells. Compared with the model group, the combination drug group significantly increased the SOD content in the injured cells, and CP-6 (61.40±3.858, P <0.01) was significantly better than its monomer composition and other proportions in the same group in the CT and PCA combination drug group, CD-1 (59.12±2.089, P <0.01), CD-6 (65.59±1.561, P <0.01) were significantly better than its monomer composition and other proportions in the same group in the CT and DSS combination drug group, but there was no significant difference between the two groups, which also proved that DSS played a major role in HUVEC cells, and also showed that the best proportion of CT and DSS combination was between [1:2-1:3].

[0062] Compared with the normal group, the MDA content in the model group was (10.78±0.9023, P<0.01) was significantly higher than that in the model group, successfully constructing the H / R injury model. Compared with the model group, the MDA content in the drug administration group after injury was reduced to varying degrees, showing that the drug had a protective effect on the injured HUVEC cells. Compared with the model group, the combined drug group significantly reduced the MDA content in the cells after injury, and CP-6 (4.095±0.09929, P <0.01) was significantly better than its monomer composition and other proportions in the same group in the CT and PCA combined drug group, suggesting that the best proportion is near [1:2]. CD-5 (4.026±0.3790, P <0.01), CD-6 (2.992±0.2215, P <0.01) was significantly better than its monomer composition and other proportions in the same group in the CT and DSS combined drug group, suggesting that the best proportion is near [1:3].

[0063] Compared with the normal group, the relative release amount of LDH in the model group was significantly increased (3.272±0.05963, P <0.01). Compared with the model group, drug treatment can reduce the release amount of LDH in cells after H / R injury. Compared with the model group, the combined drug group significantly reduced the LDH content in the cells after injury, and CP-1 (1.597±0.05213, P <0.01), CP-4 (1.530±0.02589, P <0.01) was better than its monomer composition and other proportions in the same group in the CT and PCA combined drug group, but the above two groups had no significant difference, and it was guessed that PCA played a major role in endothelial cells. CD-5 (1.201±0.1234, P <0.01), CD-6 (1.230±0.0020, P <0.01) was better than its monomer composition and other proportions in the same group in the CT and DSS combined drug group, but the above two groups had no significant difference, and it was guessed that DSS played a major role in myocardial cells.

[0064] Effect Example 3 The tanshinones and salvianolic acid compounds obtained in Example 1 were combined to study the vasodilatory effect of the composition on rat thoracic aortic rings, as follows: (1) Experimental grouping: The vasodilatory effect of the composition on rat thoracic aortic rings was studied according to the grouping shown in Table 3.

[0065] Table 3 Experimental grouping (2) Preparation of isolated rat thoracic aortic rings After euthanizing SD rats by cervical dislocation, the thoracic aorta was rapidly dissected and completely removed. The rats were immediately transferred to a 4°C pre-oxygenated Krebs-Henseleit (KH) buffer solution (95% O2, 5% CO2 mixed gas saturation). Microsurgical instruments were used to meticulously dissect the adventitia, connective tissue, and fat, and 3-5 mm vascular ring samples were prepared by transverse cutting.

[0066] The vascular ring was vertically suspended in a constant-temperature bath at 37.0±0.5℃ (containing 7 mL of oxygen-saturated KH solution), with one end fixed to the bath base support and the other end connected to a high-sensitivity tension sensor (range 0-10 g, resolution 0.1 mg). The vasoconstriction and vasodilation states were recorded in real time using a biosignal acquisition system (sampling rate 1 kHz).

[0067] Tension balance: (1) Initial tension-free equilibrium (15 minutes) to eliminate mechanical stress in the material. (2) Gradually apply the base tension to 0.75 g (equilibrate for 20 minutes) to restore vascular resting tension. (3) Finally adjust to 1.5 g working tension (equilibrate for 60 minutes), and replace with fresh oxygen-saturated KH solution every 15 minutes to maintain ion homeostasis.

[0068] (3) Detection of vascular endothelial function integrity After the vascular rings have achieved the above equilibrium and the tension has stabilized, norepinephrine (NE) is added to the constant temperature bath to achieve a concentration of 1×10⁻ in the KH solution. 6 mol / L. NE can stimulate vascular ring contraction; observe the vascular ring contraction curve until the maximum contractile tension is reached and stabilized. At this point, add sodium nitroprusside to the bath again to achieve a concentration of 1×10⁻⁻⁻⁻⁶. 5 Stimulating the vascular ring with a concentration of mol / L, closely observe the changes in relevant values ​​in the multichannel physiological signal acquisition and analysis system, and calculate the vascular ring relaxation amplitude using the formula: Vascular ring relaxation amplitude = (maximum contraction value - relaxation value after adding sodium nitroprusside) / (maximum contraction value - baseline tension value) × 100%. If the calculated vascular ring relaxation amplitude is greater than 70%, it indicates good vascular ring activity, and the next experiment can be performed; if the vascular ring relaxation amplitude is less than 70%, it indicates significant endothelial damage to the vascular ring, and this vascular ring should be discarded.

[0069] (4) Study on the effect of CT combined with SAs on the thoracic aortic vascular ring in rats After screening the vascular rings in the previous step, selecting those with relatively intact endothelium, the KH solution in the bath was replaced for rinsing and stimulation. This process was repeated three times, and finally, the bath volume was adjusted to 7 mL. Once the vascular rings stabilized again, NE was added to achieve a concentration of 1×10⁻⁶. -6After stimulating vasoconstriction with a concentration of mol / L, and reaching the maximum vasoconstrictive tension value and stabilizing, drug solutions for each group were added separately to achieve the corresponding drug concentrations. Data were recorded after the tension values ​​stabilized, and the diastolic rate for each group was calculated. The diastolic rate of the rat thoracic aorta = diastolic rate (%) = (maximum pre-constriction tension of the vascular ring - tension of the vascular ring at different drug concentrations) / (maximum pre-constriction tension of the vascular ring - baseline equilibrium tension) × 100%.

[0070] The effects of different ratios of CT and SAs on changes in vascular ring tension are as follows: Figure 9 As shown, the results indicated that, compared to the control group, the vasodilation rate was significantly increased in all groups after drug administration. P <0.05).

[0071] (1) In the CT and PCA combined group, CT:PCA = 1:2 (31.16 ± 0.8014, P <0.01) and CT:PCA=1:1 (27.62±0.5587, P The difference of <0.01 indicates a significant difference, suggesting that the combination of CT and PCA at a ratio of 1:2 has the best vasodilatory effect. Therefore, 1:2 is the optimal ratio for the combination of the two.

[0072] (2) In the CT and DSS combined group, CT:DSS = 1:3 (36.94±0.3197, P <0.01) and CT:DSS=1:2 (31.71±1.182, P The difference of <0.01 indicates a significant difference, suggesting that the CT:DSS ratio of 1:3 is optimal for vasodilatory effect when CT and DSS are used in combination. Therefore, 1:3 is the best ratio for the combination of the two.

[0073] Example of implementation effect 4 Effect of the combination of tanshinone and tanshinone acid compounds obtained in Example 1 on DNMTs / TET2 expression I. Drug administration and culture of H9c2 and HUVEC cells Cells were passaged in the T-25 culture system at a seeding density of 4 × 10⁶ cells per flask. 5 Cells were inoculated. A hypoxia / reoxygenation (H / R) model was constructed 24 hours after inoculation, and the drug-containing medium was replaced simultaneously. Drugs were administered according to the experimental groups, with 5 mL of prepared drug-containing medium added to each culture flask. Three replicates were prepared for each concentration group. Subsequent assays were performed after maintaining the culture conditions.

[0074] II. RNA Extraction Extract according to the kit instructions. Specific steps are as follows: (1) After the end of the drug culture, discard the cell supernatant, add 1.5 mL FreeZol Reagent, shake the cell culture dish quickly and lightly to make the FreeZol Reagent fully cover the cell surface, then use a pipette to repeatedly blow the cell bottom to make it fall off, and transfer the lysis solution to a 1.5 mL enzyme-free EP tube.

[0075] (2) Vigorously vortex the suspension until fully lysed, and stand at room temperature for 5 min.

[0076] (3) Add 450 μL Dilution Buffer to the EP tube (add 150 μL Dilution Buffer per 500 μL FreeZol Reagent), tightly cover the centrifuge tube cap, vortex to fully mix the solution, and stand at room temperature for 5 min.

[0077] (4) 11200 rpm (12000 x g ) Centrifuge for 15 min, carefully aspirate the upper solution (RNA) (about 750 μL) to a new enzyme-free EP tube.

[0078] (5) Add an equal volume of isopropanol to the obtained supernatant solution, mix well, and stand at room temperature for 10 min.

[0079] (6) 11200 rpm (12000 x g ) Centrifuge for 10 min, and after centrifugation, a white gelatinous precipitate can be seen on the side and bottom of the tube, carefully discard the supernatant without losing the precipitate.

[0080] (7) Add 1 mL of 75% ethanol (RNase-free ddH2O preparation). Lightly tap the bottom of the tube to suspend the precipitate, and invert several times.

[0081] (8) 9100 rpm (8000 x g ) Centrifuge at room temperature for 3 min, discard the supernatant without losing the precipitate.

[0082] (10) Repeat steps (7) and (8) twice, and discard the supernatant.

[0083] (11) Place at room temperature to dry, add 20 - 100 μL RNase-free ddH2O to dissolve the precipitate, vortex at room temperature for 3 min to fully dissolve the RNA precipitate. The extracted RNA product can be aliquoted and stored at -80°C for long-term storage or at -20°C for short-term storage.

[0084] III. RNA Purity Test RNA concentration and purity were determined using a nucleic acid quantifier, and each sample was measured three times to take the average value.

[0085] Four, RNA reverse transcription to synthesize cDNA (1) The total RNA, 4 × All-in-One Ultra qRT SuperMix and RNase-Free dd H2O were thawed on ice in advance. Each solution was vortexed and centrifuged before use. All operations were performed on ice.

[0086] (2) Take a certain volume of 1 μg RNA, 4 μL 4 × All-in-One Ultra qRT SuperMix, and add a certain volume of RNase-Free dd H2O to make up to 20 μL of reverse transcription system. The reverse transcription conditions are shown in Table 4.

[0087] Table 4 RNA reverse transcription reaction conditions The reverse transcription product was immediately used for the next step experiment.

[0088] Five, Real-time PCR reaction (1) The EP tube containing powdered primers was centrifuged at 4000 rpm for 30 s, and the tube cap was carefully and gently opened. The appropriate amount of RNase-Free dd H2O was added according to the primer synthesis instructions to ensure the primer concentration was 10 μM. The primers were vortexed to dissolve completely, and then centrifuged at 4000 rpm for a short time and stored at -20℃. The sequences of the primers are shown in Table 5.

[0089] Table 5 Real-time PCR reaction primer sequence (2) 2 × Super Real Pre Mix Plus, cDNA template, primers and RNase-Free dd H2O were mixed on ice in the dark. PCR reaction was performed on the machine, and the reaction system and conditions are shown in Tables 6 and 7.

[0090] Table 6 Real-time PCR reaction system Table 7 Real-time PCR reaction conditions Six, the influence of CT, SAs compatibility on DNMTs / TET2 protein expression (1) Sample preparation: Collect pretreated H9c2 and HUVEC cells. Lyse the cells using RIPA buffer pre-cooled with phosphatase inhibitor and benzoyl sulfonate (PMSF) to obtain total protein lysate. Centrifuge (12000 rpm) rpm (4℃, 10 min) to remove cell debris and nuclei. Collect the supernatant into a new centrifuge tube.

[0091] (2) Protein concentration determination: Add the standard and the sample to be tested to a 96-well plate, and add a gradient of protein standards. Use the BCA protein assay kit to determine the total protein concentration in the sample homogenate. Then adjust the protein concentration, add SDS-PAGE loading buffer, and treat at 100°C for 20 minutes to allow the protein to denature fully.

[0092] (3) SDS-PAGE gel electrophoresis: Prepare SDS-PAGE gels of different concentrations and assemble the electrophoresis tank. Load the samples into the gel tank and perform protein electrophoresis separation.

[0093] (4) Transfer: Transfer the protein in the gel to a pretreated PVDF membrane. Use a semi-dry or wet electrotransfer system and follow the equipment instructions.

[0094] (5) Blocking: Use 5% skim milk powder or BSA in Tris-buffered saline Tween (TBST) buffer to reduce nonspecific binding.

[0095] (6) Primary antibody incubation: Incubate the membrane with primary antibody (DNMT1, TET2, etc.) overnight at 4°C.

[0096] (7) Secondary antibody incubation: Wash the membrane to remove unbound primary antibody. Incubate the membrane with the corresponding horseradish peroxidase (HRP)-labeled secondary antibody at room temperature for 2 h.

[0097] (8) Detection: Wash the membrane to remove unbound secondary antibody, then treat the membrane with ECL luminescent solution. Expose the protein bands using an imaging system.

[0098] The results of the analysis on the effect of CT and SAs combination on DNMTs / TET2 protein expression are as follows: Figure 10 The effect of combined CT and SAs administration on DNMT1 mRNA expression levels in H9c2 and HUVEC cells after H / R injury was investigated. Compared to the model group, DNMT1 mRNA expression in H9c2 and HUVEC cells was significantly reduced in all groups after administration of the drugs following H / R injury. P <0.01), and the combined drugs CT+DSS and CT+PCA both showed a significant effect in reducing DNMT1 mRNA expression.

[0099] (1) In H9c2 cells, combined use of CD-6 (0.4983 ± 0.03120, P <0.01), CP-1 (0.6306 ± 0.005763, P <0.01), CP-6 (0.5996 ± 0.02296, P <0.01) not only significantly reduced DNMT1 mRNA expression than single drug, but also showed significant in the same group. But there was no significant difference between CP-1, CP-6 group, it is speculated that the best proportion of CT+PCA combination in the range of [1:1-1:2]. The best proportion of CT+DSS combination is 1:3.

[0100] (2) In HUVEC cells, combined use of CD-6 (0.6151 ± 0.01591, P < 0.01), CP-1 (0.7396 ± 0.01772, P <0.01), CP-6 (0.7573 ± 0.01291, P <0.01) not only significantly reduced DNMT1 mRNA expression than single drug, but also showed significant in the same group. But there was no significant difference between CP-1, CP-6 group, it is speculated that the best proportion of CT+PCA combination in the range of [1:1-1:2]. The best proportion of CT+DSS combination is 1:3.

[0101] Figure 11 The effect of CT, SAs combined drug on the expression level of DNMT3B mRNA in H9c2, HUVEC cells after H / R injury. Compared with the model group, the expression of DNMT3B mRNA in each group after H / R injury was significantly increased (P < 0.01) in H9c2, HUVEC cells, and the combined drug CT+DSS and CT+PCA showed a significant decrease in DNMT3B mRNA expression. P

[0102] (1) In H9c2 cells, combined use of CD-1 (0.5133 ± 0.001714, P <0.01), CD-6 (0.4945 ± 0.01086, P <0.01), CP-1 (0.6406 ± 0.03443, P <0.01), CP-6 (0.5356 ± 0.01566, P ​<0.01), and the same group showed significant performance. But there was no significant difference between CP-1, CP-6, CD-1, CD-6 groups, and it was speculated that the best ratio of CT+PCA combination was in the range of [1:1-1:2]. The best ratio of CT+DSS combination was in the range of [1:2-1:3].

[0103] (2) In HUVEC cells, the combined drug CD-6 (0.6312 ± 0.009728, P <0.01), CP-1 (0.6400 ± 0.008672, P <0.01), and CP-6 (0.6203 ± 0.01348, P <0.01) not only significantly reduced DNMT3A mRNA expression than single drug, but also showed significant performance in the same group. But there was no significant difference between CP-1, CP-6, CD-1, CD-6 groups, and it was speculated that the best ratio of CT+PCA combination was in the range of [1:1-1:2]. The best ratio of CT+DSS combination was 1:3. P

[0104] Figure 12 The effect of CT, SAs combined drug on the expression level of DNMT3A mRNA in H9c2, HUVEC cells after H / R injury. Compared with the model group, the expression of DNMT3A mRNA in each group was significantly increased after H / R injury in H9c2, HUVEC cells (P<0.01), and the combined drug CT+DSS and CT+PCA showed a significant effect on reducing the expression of DNMT3A mRNA. P

[0105] (1) In H9c2 cells, the combined drug CD-6 (0.5713 ± 0.03259, P <0.01), CP-1 (0.6400 ± 0.008672, P <0.01), and CP-6 (0.6203 ± 0.01348, P <0.01) not only significantly reduced DNMT3A mRNA expression than single drug, but also showed significant performance in the same group. But there was no significant difference between CP-1, CP-6, CD-1, CD-6 groups, and it was speculated that the best ratio of CT+PCA combination was in the range of [1:1-1:2]. The best ratio of CT+DSS combination was 1:3.

[0106] (2) In HUVEC cells, the combined drug CD-6 (0.6312 ± 0.009728, P ​​<0.01), CP-6 (0.7403±0.01348, P <0.01), CP-6 (0.7403±0.01348, P <0.01) not only significantly reduced DNMT3A mRNA expression than single drug, but also showed significant in the same group. But CP-1, CP-6 group had no significant difference, speculated CT+PCA combination of the best ratio in [1:1-1:2] range. CT+DSS combination of the best ratio was 1:3.

[0107] Figure 13 The effect of CT, SAs combined drug on TET2 mRNA expression level in H9c2, HUVEC cells after H / R injury. Compared with the model group, TET2 mRNA expression in each group after H / R injury in H9c2, HUVEC cells was significantly reduced (P<0.01) after drug administration. P <0.01), and combined drug CT+DSS and CT+PCA showed a significant increase in TET2 mRNA expression.

[0108] (1) In H9c2 cells, combined drug CD-6 (1.415±0.02620, P <0.01), CP-1 (1.269±0.05131, P <0.01), CP-6 (1.350±0.01587, P <0.01) not only significantly increased TET2 mRNA expression than single drug, but also showed significant in the same group. But CP-1, CP-6, CD-1, CD-6 group had no significant difference, speculated CT+PCA combination of the best ratio in [1:1-1:2] range. CT+DSS combination of the best ratio was 1:3.

[0109] (2) In HUVEC cells, combined drug CD-6 (1.435±0.02620, P <0.01), CP-1 (1.422±0.1065, P <0.01), CP-6 (1.527±0.107, P <0.01) not only significantly increased TET2 mRNA expression than single drug, but also showed significant in the same group. But CP-1, CP-6 group had no significant difference, speculated CT+PCA combination of the best ratio in [1:1-1:2] range. CT+DSS combination of the best ratio was 1:3.

[0110] The results show that the combined use of CT and SAs has a significantly higher effect than single drug use, and can down-regulate DNMT1, DNMT3A and DNMT3B (P<0.05), and up-regulate TET2 expression (P<0.05). It is proved that the combined use of CT and SAs plays an important role in the balanced regulation of DNMTs / TET2, and the best ratio of CT and PCA is 1:2, and the best ratio of CT and DSS is 1:3. From the research results, by comparing the influence of the combined drug on the expression of DNMTs / TET2, it is found that DNMTs and TET2 have the theoretical potential as biomarkers in cardiovascular diseases, especially in revealing the mechanism of the disease, and it has a certain rationality to take them as new indicators of cardiovascular diseases.

[0111] The above merely describes preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A medicament for the modulation of DNMTs / TET2, characterized in that: The active ingredient of the medicine comprises tanshinone compounds and salvianolic acid compounds.

2. The agent modulating DNMTs / TET2 according to claim 1, characterized in that: The tanshinone compounds are cryptotanshinone, and the salvianolic acid compounds are danshensu and protocatechuic acid.

3. The agent modulating DNMTs / TET2 according to claim 2, characterized in that: The molar ratio of cryptotanshinone and danshensu is 1:2-3, and the molar ratio of cryptotanshinone and protocatechuic acid is 1:1-2.

4. The agent modulating DNMTs / TET2 according to claim 3, characterized in that: The medicine plays a role by regulating the balance of DNMTs and TET2.

5. The agent modulating DNMTs / TET2 according to claim 4, characterized in that: The medicine further comprises a pharmaceutically acceptable carrier.

6. The agent modulating DNMTs / TET2 according to claim 5, characterized in that: The combined use concentration of cryptotanshinone and protocatechuic acid in the medicine is 0.416-2.5 μM of cryptotanshinone and 0.416-2.5 μM of protocatechuic acid, and the combined use concentration of cryptotanshinone and danshensu is 0.83-2.5 μM of cryptotanshinone and 0.416-3.75 μM of danshensu.

7. Process for the preparation of a medicament as defined in any one of claims 1 to 6, characterized in that, The specific steps are: (1) Dissolve cryptotanshinone, danshensu and protocatechuic acid in organic solvents respectively, and dilute to obtain cryptotanshinone mother liquor, danshensu mother liquor and protocatechuic acid mother liquor; (2) Dilute the cryptotanshinone mother liquor, danshensu mother liquor and protocatechuic acid mother liquor respectively to obtain cryptotanshinone drug solution, danshensu drug solution and protocatechuic acid drug solution with target concentrations.

8. The method of claim 7, wherein: The organic solvent in step (1) is DMSO.

9. The method of claim 8, wherein: The target concentration of cryptotanshinone drug solution in step (2) is 0.416-10 µM, the target concentration of protocatechuic acid drug solution is 0.416-10 µM, and the target concentration of danshensu drug solution is 0.416-10 µM.

10. Use of the medicament for the modulation of DNMTs / TET2 according to any one of claims 1 to 6 for the preparation of a medicament for the prevention or treatment of a relevant disease caused by an imbalance of DNMTs and TETs, characterized in that: The disease is a cardiovascular disease.

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