Application of SD49-7 in preparation of medicine for preventing and treating cardiac hypertrophy

Abstract

The invention relates to an application of SD49-7 or a pharmaceutically acceptable derivative thereof in preparation of a medicine for preventing and treating myocardial hypertrophy. In-vitro cell experiments and animal experiments show that SD49-7 has the effect of improving cardiac hypertrophy, a new medicine is provided for treatment of cardiac hypertrophy, and the chemical structure of SD49-7 is shown in the specification. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the field of medicine, and particularly to the use of SD49-7 in the preparation of drugs for preventing and treating myocardial hypertrophy. Background Art

[0002] Myocardial hypertrophy is a slow but effective compensatory function of the heart. Due to the increased oxygen consumption of the hypertrophied myocardium, the blood supply of the coronary artery often cannot meet the demand, resulting in myocardial ischemia, and finally leading to the reduction of myocardial contractility, which is a common pathological change in various cardiovascular diseases. Clinically, the main symptoms of myocardial hypertrophy are dyspnea, chest pain, dizziness, palpitation, syncope, etc. Clinically, different drugs can be selected according to the specific symptoms of patients, including β-blockers, calcium antagonists, antiarrhythmic drugs, etc. In severe cases, surgical treatment may be required.

[0003] There are various causes of myocardial hypertrophy clinically, and there is no complete conclusion at present. Clinically, it is generally considered that its main etiological factors include hypertension, aortic valve stenosis, coronary heart disease, endocrine disorders and genetic factors, etc. For example, continuous elevation of blood pressure will cause the myocardium to be in a compensatory state for a long time, resulting in symptoms of myocardial hypertrophy. Myocardial hypertrophy caused by genetic factors mainly refers to hypertrophic cardiomyopathy. The identified pathogenic genes are mainly genes encoding sarcomere proteins, including MYH7, TNNI3, etc. The natural course of hypertrophic cardiomyopathy is very long and shows a benign progression, and obvious changes in the cardiac structure rarely occur. However, in a few patients, the heart cavity gradually enlarges, the ventricular wall thins, and the left ventricular ejection fraction (LVEF) decreases, showing changes similar to those of dilated cardiomyopathy. This end-stage change of hypertrophic cardiomyopathy is called the dilated phase of hypertrophic cardiomyopathy, and the mortality rate is significantly increased at this stage. At present, there is no effective treatment plan for this disease. Therefore, it is very important to clarify the pathogenic mechanism of myocardial hypertrophy and then carry out targeted prevention and treatment.

[0004] KDM4A, also known as JMJD2A, JHDM3A, JMJD2, TDRD14B, and KIAA0677, is a lysine-specific demethylase belonging to the Fe(II)-dependent dioxygenase family. The KDM4 family consists of a total of 6 subtypes, namely KDM4A, KDM4B, KDM4C, KDM4D, KDM4E, and KDM4F. Among them, KDM4C and KDM4A have the highest homology, and the two can form heterodimers and possess regulatory enzyme activity. KDM4D, KDM4E, and KDM4F are approximately half the size of other KDM4 family members, and these KDM4 family members are only homologous to the Jmjc-containing N-terminal demethylase domain. In addition to structural similarity, KDM4A / B / C also have enzymatic specificity for chromatin substrates. Kinetic analysis shows that KDM4A / B / C simultaneously accept H3K9 and H3K36 as substrates; KDM4D / E can catalyze H3K9me2 / 3, and KDM4E can also catalyze H3K56me3. In addition, all these KDM4 family members have been shown to demethylate lysine 26 on histone H1.4 (H1.4K26).

[0005] Basic research shows that Empagliflozin, Losartan, and Metoprolol have an inhibitory effect on drug-induced myocardial hypertrophy. The "Diagnosis and Treatment Guidelines for Hypertrophic Cardiomyopathy in Chinese Adults" list Empagliflozin and Metoprolol as recommended drugs for hypertrophic cardiomyopathy; in addition, the "Diagnosis and Treatment Guidelines for Dilated Cardiomyopathy in China" also list Losartan and Metoprolol as recommended drugs for dilated cardiomyopathy. Therefore, these three drugs can be used as positive control drugs for the myocardial hypertrophy model.

[0006] Chinese Patent CN 113116875A discloses the use of SD49-7 in the preparation of drugs for the treatment of leukemia, but there is currently no relevant report on the use of SD49-7 in the preparation of drugs for improving myocardial hypertrophy.

[0007] SD49-7 (CAS NO.: 54009-54-0), the Chinese name is 2-hydroxy-1-naphthaldehyde salicylhydrazone, and the chemical structural formula is shown in (I):

[0008] Summary of the Invention

[0009] In the research of the present invention, it is found that the compound SD49-7 has the effect of improving myocardial hypertrophy.

[0010] Therefore, the present invention claims to protect the following technical solutions:

[0011] Use of SD49-7 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing and treating myocardial hypertrophy, wherein SD49-7 has the following chemical structure:

[0012]

[0013] The use according to the present invention includes inhibiting myocardial cell hypertrophy.

[0014] The use according to the present invention, wherein the medicament is any ingestible medicament.

[0015] The use according to the present invention, wherein the dosage form of the medicament is selected from oral dosage forms or dosage forms for parenteral administration.

[0016] The use according to the present invention, wherein the dosage form of the medicament comprises a therapeutically effective amount of SD49-7 or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers.

[0017] The use according to the present invention, wherein the dosage form of the medicament is an oral dosage form.

[0018] The use according to the present invention, wherein the oral dosage form is selected from tablets, capsules, granules, pills, powders, and oral liquids.

[0019] In some embodiments, through in vitro experiments, the present invention found that SD49-7 has the effect of improving myocardial hypertrophy. An in vitro myocardial hypertrophy cell model was constructed by inducing H9c2 myocardial cells with angiotensin II (Ang II), isoproterenol (ISO), and phenylephrine (PHE). After treatment with the KDM4A small molecule inhibitor SD49-7, it was found that SD49-7 has a significant inhibitory effect on the hypertrophy of H9c2 myocardial cells induced by angiotensin II, isoproterenol, and phenylephrine, and has a significant inhibitory effect on the mRNA expression levels of the myocardial injury marker genes NPPA and NPPB and the pathogenic gene MYH7 of hypertrophic cardiomyopathy, and has no obvious toxic effect on cells under the condition of having the same therapeutic effect as the positive drug. Through in vivo pharmacodynamic experiments, it was found that SD49-7 has an obvious improvement effect on isoproterenol-induced cardiac dysfunction in mice and has an obvious inhibitory effect on isoproterenol-induced myocardial hypertrophy in mice.

[0020] The present invention has the following beneficial effects:

[0021] The present invention provides a new use of SD49-7 in the preparation of a medicament for improving myocardial hypertrophy. Through in vitro cell experiments and in vivo pharmacodynamic experiments, the present invention found that SD49-7 has the effect of improving myocardial hypertrophy, which will provide a new medicament for the treatment of myocardial hypertrophy. Description of the Drawings

[0022] Figure 1Results of phalloidin fluorescence staining for detecting cell area in the H9c2 myocardial hypertrophy model after 48-hour treatment with different concentrations of SD49-7 and 500 nM empagliflozin in Example 3; ****P<0.0001, ns P>0.05.

[0023] Figure 2 Results of phalloidin fluorescence staining for detecting cell area in the H9c2 myocardial hypertrophy model after 48-hour treatment with different concentrations of SD49-7 and 24 μM losartan in Example 3; ****P<0.0001, ns P>0.05.

[0024] Figure 3 Results of phalloidin fluorescence staining for detecting cell area in the H9c2 myocardial hypertrophy model after 48-hour treatment with different concentrations of SD49-7 and 20 μM metoprolol in Example 3; ****P<0.0001, ns P>0.05.

[0025] Figure 4 Representative images of the states of mice after induction with ISO and treatment with different doses of SD49-7 in Example 5;

[0026] Figure 5 Representative images of the heart tissues of mice after induction with ISO and treatment with different doses of SD49-7 in Example 5; *P<0.05, ***P<0.001.

[0027] Figure 6 Hematoxylin-eosin (HE) staining images of the heart tissues of mice after induction with ISO and treatment with different doses of SD49-7 in Example 5; *P<0.05, **P<0.001.

[0028] Figure 7 Wheat germ agglutinin (WGA) staining images of the heart tissues of mice after induction with ISO and treatment with different doses of SD49-7 in Example 5; ****P<0.0001. Detailed implementation manners

[0029] The present invention is further illustrated by the following examples, which should not be construed as limiting the present invention.

[0030] Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0031] The SD49-7 used in the examples of the present invention is a product of Selleck Co., Ltd. (Catalog No.: E1335), and its chemical structural formula is shown as Formula (I):

[0032]

[0033] Example 1. Effect of SD49-7 on the proliferation of H9c2 cells

[0034] Experimental purpose: To detect the effect of SD49-7 on the proliferation of H9c2 cells by MTT method.

[0035] Experimental materials:

[0036] H9c2 cells (procell, catalog number CL-0089), RPMI 1640 medium (Gibco, catalog number: 11875101), thiazolyl blue (MTT, Sigma Aldrich, catalog number: 1334GR005), SD49-7 (Selleck, catalog number: E1335)

[0037] Experimental instrument: Multifunctional microplate reader (BioTek, model: SYNERGYH1)

[0038] Experimental procedures:

[0039] 1) Experimental method for detecting the effect of SD49-7 on the proliferation of H9c2 cells by MTT method:

[0040] (1) Resuscitate the cryopreserved H9c2 cells from liquid nitrogen. After taking out the cryopreserved cells from liquid nitrogen, put them into a 37°C water bath. After melting, immediately centrifuge at 1000 rpm for 5 minutes and resuspend with fresh RPMI 1640 medium. Place them in a 37°C cell culture incubator for culture.

[0041] (2) When the cells are in the logarithmic phase, collect a certain amount of cells and adjust the cell concentration to 5×10 4 / ml (i.e., 5×10 3 cell / well / 100 μL). Add 100 μl per well to a 96-well plate, and supplement the edge wells with sterile PBS.

[0042] (3) Dissolve SD49-7 with DMSO and prepare the stock solution, and then dilute SD49-7 with medium containing 2% serum according to 10 preset concentration gradients. Add 100 μl per well to the final concentrations of 50 μM, 25 μM, 10 μM, 5 μM, 2 μM, 1 μM, 0.5 μM, 0.25 μM, and 0.1 μM respectively. Set 8 replicate wells for each concentration. (4) Place the 96-well plate of cells added with SD49-7 in a 37°C cell culture incubator with 5% CO2 for 48 hours.

[0043] (5) Prepare MTT solution (5 mg / ml) with serum-free 1640 medium in advance. Centrifuge the 96-well plate (centrifuge at 1800 rpm for 15 min), carefully discard the supernatant, add 100 μl of serum-free medium containing MTT solution (the final concentration of MTT is 5 μg / ml) to each well, and place the 96-well plate in the cell culture incubator for continued culture for 4 hours.

[0044] (6) Discard the supernatant, add 100 μl of DMSO and incubate for 15 min to fully dissolve the crystals. Place the plate on the microplate reader to detect the absorbance value of each well at a wavelength of 570 nm.

[0045] (7) Calculate the cell survival rate: Cell survival rate = (absorbance value of the experimental group - absorbance value of the control group) / absorbance value of the control group × 100%.

[0046] 2) Result analysis

[0047] As can be seen from Table 1, compared with the control group, the treatment of H9c2 cells with SD49-7 at concentrations of 0.1, 0.25, 0.5, 1, and 2 μM for 48 hours had no significant effect on their survival rate (P > 0.05), while the treatment of H9c2 cells with SD49-7 at concentrations of 5, 10, 25, and 50 μM for 48 hours significantly reduced their survival rate (P < 0.0001). Therefore, it was decided to use SD49-7 at concentrations of 0.25 and 0.5 μM for subsequent experiments, and 48 hours as the treatment time.

[0048] Table 1. Effects of different concentrations of SD49-7 on the survival rate of H9c2 cells (n = 8)

[0049]

[0050] Note: ****P < 0.0001, compared with the control group

[0051] Example 2. Effects of different inducing drugs and positive drugs on the proliferation of H9c2 cells

[0052] Experimental purpose: Use the MTT method to detect the effects of various inducing drugs, positive drugs, and different drug combinations on the proliferation of H9c2 cells.

[0053] Experimental materials:

[0054] H9c2 cells (Procell, catalog number: CL-0089), RPMI 1640 medium (Gibco, catalog number: 11875101), SD49-7 (Selleck, catalog number: E1335), thiazolyl blue (MTT, Sigma Aldrich, catalog number: 1334GR005), isoproterenol (isoproterenol, MCE, catalog number: HY-B0468), norepinephrine (phenylephrine, MCE, catalog number: HY-B0769), angiotensin II (angiotensin II, MCE, catalog number: CSN10313), empagliflozin (MCE, catalog number: HY-15409), losartan (MCE, catalog number: HY-17512), metoprolol (MCE, catalog number: HY-17503)

[0055] Experimental instruments: Multifunctional microplate reader (BioTek, model: SYNERGYH1)

[0056] Experimental procedures:

[0057] (1) Resuscitate the cryopreserved H9c2 cells from liquid nitrogen. After taking out the cryopreserved cells from liquid nitrogen, place them in a 37°C water bath. After thawing, immediately centrifuge at 1000 rpm for 5 minutes and resuspend with fresh RPMI 1640 medium. Place them in a 37°C cell culture incubator for culture.

[0058] (2) When the cells are in the logarithmic phase, collect a certain amount of cells and adjust the cell concentration to 5×10 4 / ml (i.e., 5×10 3 cell / well / 100 μL). Add 100 μl per well to a 96-well plate, and supplement the edge wells with sterile PBS.

[0059] (3) Add different positive drugs, including 20 μM metoprolol (M), 24 μM losartan (L), 500 nM empagliflozin (E); different induction drugs, including 0.1 μM isoproterenol, 0.5 μM angiotensin II, 50 μM phenylephrine; 0.25 and 0.5 μM SD49-7 are co-administered with isoproterenol, angiotensin II, and phenylephrine respectively. Set 8 replicates for each group.

[0060] (4) Place the 96-well plate with the cells added with drugs in a 37°C cell culture incubator with 5% CO2 for 48 hours.

[0061] (5) Prepare the MTT solution (5 mg / ml) with serum-free 1640 medium in advance. Centrifuge the 96-well plate (centrifuge at 1800 rpm for 15 min), carefully discard the supernatant, add 100 μl of serum-free medium containing the MTT solution (the final concentration of MTT is 5 μg / ml) to each well, and place the 96-well plate in the cell culture incubator for continued culture for 4 hours.

[0062] (6) Discard the supernatant, add 100 μl of DMSO and incubate for 15 min to fully dissolve the crystals. Place the plate on the microplate reader to detect the absorbance value of each well at a wavelength of 570 nm.

[0063] (7) Calculate the cell survival rate: Cell survival rate = (absorbance value of the experimental group - absorbance value of the control group) / absorbance value of the control group × 100%.

[0064] As can be seen from Table 2, treatment of H9c2 cells with metoprolol and empagliflozin alone had no effect on their proliferation (P > 0.05), while treatment of H9c2 cells with losartan alone had a significant inhibitory effect on their proliferation (P < 0.0001); treatment of H9c2 cells with metoprolol combined with isoproterenol had a significant inhibitory effect on their proliferation (P < 0.0001); treatment of H9c2 cells with 0.25 μM and 0.5 μM of SD49-7 combined with isoproterenol had no effect on their proliferation (P > 0.05); treatment of H9c2 cells with losartan combined with angiotensin II had a significant inhibitory effect on their proliferation (P < 0.0001); treatment of H9c2 cells with 0.25 and 0.5 μM of SD49-7 combined with angiotensin II had no effect on their proliferation (P > 0.05); treatment of H9c2 cells with empagliflozin combined with phenylephrine had no effect on their proliferation (P > 0.05); treatment of H9c2 cells with 0.25 and 0.5 μM of SD49-7 combined with phenylephrine had no effect on their proliferation (P > 0.05).

[0065] Table 2. Effects of different induction drugs and positive drugs on the survival rate of H9c2 cells (n = 8)

[0066]

[0067]

[0068] Note: ****P < 0.0001, compared with the control group

[0069] Example 3. Effects of different positive drugs and different drug combinations on H9c2 cell hypertrophy induced by various induction drugs

[0070] Experimental purpose: Use the phalloidin staining method to detect the effects of different positive drugs and different drug combinations on H9c2 cell hypertrophy induced by various induction drugs.

[0071] H9c2 cells (Procell, catalog number CL-0089), DMEM medium (Gibco, catalog number: 11875101), SD49-7 (Selleck, catalog number: E1335), isoproterenol (isoproterenol, MCE, catalog number: HY-B0468), norepinephrine (phenylephrine, MCE, catalog number: HY-B0769), angiotensin II (angiotensin II, MCE, catalog number: CSN10313), empagliflozin (MCE, catalog number: HY-15409), losartan (MCE, catalog number: HY-17512), metoprolol (MCE, catalog number: HY-17503), phalloidin staining solution (Coralaite Plus 488) (Proteintech, catalog number: PF00001)

[0072] Experimental instruments: upright fluorescence microscope (ZEIS, model: Axio Scope A1)

[0073] Experimental procedures:

[0074] 1) Detect the morphology of H9c2 cells by phalloidin staining

[0075] (1) Place a sterile coverslip in a 48-well plate, add H9c2 cells at a concentration of 1×10 5 cells / well, and when the cells are in the logarithmic growth phase, add drugs to each well and incubate for 48 h.

[0076] (2) Drug treatment and grouping: Three myocardial hypertrophy models were induced by treatment with 0.5 μM angiotensin II, 0.1 μM isoproterenol, and 50 μM phenylephrine, respectively. Treat the cells with SD49-7 alone at 0.25 and 0.5 μM, or add it simultaneously with the inducing drugs. After 48 h, perform staining detection, and repeat each treatment three times.

[0077] (3) Fix and permeabilize the cells: Pour off the medium from the cultured cell coverslips and wash with PBS. Fix with 4% paraformaldehyde for 30 min, wash with PBS three times, 5 min each time, and add 0.1% TRITON to permeabilize for 20 min.

[0078] (4) Add phalloidin: Discard the liquid in the wells and wash three times with PBS, add 50 - 100 μl phalloidin working solution, incubate at room temperature for 2 h, and wash with PBS three times, 5 min each time.

[0079] (5) Counterstain the cell nuclei with DAPI: Wash the coverslips three times with PBS (pH 7.4), 5 min each time. After removing the PBS, add DAPI staining solution in the circle, incubate in the dark at room temperature for 5 min, and wash three times with PBS.

[0080] (6) Remove the coverslips in the wells, add anti-fluorescence quenching mounting medium, and fix them on glass slides. Place the slides under a fluorescence microscope for photography.

[0081] (7) Use Image J software to statistically analyze the cell areas of each group, and perform normalization using the ratio of the average cell area of each group to that of the control group.

[0082] 2) Result analysis

[0083] As can be seen from Table 3, compared with the control group, 0.25 and 0.5 μM of SD49-7 had no effect on the relative area of H9c2 cells (P>0.05); compared with the "control + phenylephrine group", both 0.25 and 0.5 μM of SD49-7 could significantly inhibit phenylephrine-induced cell hypertrophy (P<0.0001), and 500 nM empagliflozin could significantly inhibit phenylephrine-induced cell hypertrophy (P<0.0001).

[0084] Table 3. Statistical results of the relative area of cells in the phenylephrine-induced H9c2 myocardial hypertrophy model

[0085]

[0086] Note: ****P<0.0001, compared with the "control + phenylephrine group"

[0087] As can be seen from Table 4, compared with the control group, 0.25 and 0.5 μM of SD49-7 had no effect on the relative area of H9c2 cells (P>0.05); compared with the "control + angiotensin II group", both 0.25 and 0.5 μM of SD49-7 could significantly inhibit angiotensin II-induced cell hypertrophy (P<0.0001), and 24 μM losartan could significantly inhibit angiotensin II-induced cell hypertrophy (P<0.0001).

[0088] Table 4. Statistical results of the relative area of cells in the angiotensin II-induced H9c2 myocardial hypertrophy model

[0089]

[0090] Note: ****P<0.0001, compared with the "control + angiotensin II group"

[0091] As can be seen from Table 5, compared with the control group, 0.25 and 0.5 μM of SD49-7 had no effect on the relative area of H9c2 cells (P>0.05); compared with "control + isoproterenol", both 0.25 and 0.5 μM of SD49-7 could significantly inhibit isoproterenol-induced cell hypertrophy (P<0.0001), and 20 μM of metoprolol could significantly inhibit isoproterenol-induced cell hypertrophy (P<0.0001).

[0092] Table 5. Statistical results of the relative area of cells in the H9c2 myocardial hypertrophy model induced by isoproterenol

[0093]

[0094] Example 4. Effects of different drugs on the expression of mRNA of pathogenic genes in H9c2 cell hypertrophy induced by various inducing drugs

[0095] Experimental purpose: To detect the effects of different drugs on the expression of mRNA of pathogenic genes in H9c2 cell hypertrophy induced by various inducing drugs by qPCR method.

[0096] Experimental materials:

[0097] H9c2 cells (Procell, product number CL-0089), 1640 medium (Gibco, product number: 11875101), SD49-7 (Selleck, product number: E1335), RNA extraction reagent (Zomen Biotechnology, product number: BS259A), AceQ SYBR qPCR master mix (Novoprotein, product number: B3221AAB)

[0098] Experimental instruments: PCR instrument (Bio-rad, model: C1000 Touch), fluorescence quantitative PCR instrument (ROCHE, model: LightCycle 96)

[0099] Experimental steps:

[0100] 1) Detect the mRNA expression levels of myocardial injury marker genes NPPA, NPPB and pathogenic gene MYH7 of hypertrophic cardiomyopathy by qPCR

[0101] (1) Seed the cells in a 6-well plate. Treat the cells with the drug. After two days, wash the cells twice with PBS. Add 400 μl of pre-cooled total RNA extraction reagent (Trizol) to each well of the 6-well plate, pipette to mix, transfer to a 1.5 ml centrifuge tube, add chloroform at a volume ratio of 5:1, and mix well by shaking. Centrifuge at 12,000 rpm for 10 min using a 4 °C centrifuge, collect the supernatant into a 1.5 ml centrifuge tube, add an equal volume of isopropanol, and store in a -20 °C refrigerator overnight for precipitation.

[0102] (2) After overnight, centrifuge to obtain the RNA precipitate. Add 500 μl of 70% ethanol and 100% ethanol in sequence, centrifuge at 12,000 rpm for 10 min using a 4 °C centrifuge, and wash the RNA to remove organic reagents. After washing, air-dry the RNA in a laminar flow hood, then add 20 μl of ddH2O to dissolve the RNA, and measure the RNA concentration using an ultra-micro spectrophotometer.

[0103] (3) Prepare the reverse transcription system premix according to Table 6 for 1 μg of RNA with the calculated concentration.

[0104] Table 6

[0105] Component Dosage 4×gDNA wiper Mix 4 μL Template RNA 1 μg RNase-free ddH2O <![CDATA[16-V RNA μL]]>

[0106] Add 4 μl of 5×HiScript II Select qRT SuperMix II to the sample, centrifuge to mix, incubate at 50 °C for 15 min, and then incubate at 85 °C for 2 min to obtain the corresponding sample cDNA.

[0107] (4) Mix the above-obtained cDNA according to Table 7 for the PCR system.

[0108] Table 7

[0109] Component Dosage (μL) 2×SYBR-GREEN MIX 10 Forward primer 0.5 Reverse primer 0.5 Sterile water 7 cDNA 2

[0110] (5) Use a Roche fluorescence quantitative PCR instrument to amplify the target gene under the conditions shown in Table 8, and the primer sequences used are shown in Table 9.

[0111] Table 8

[0112]

[0113]

[0114] Table 9

[0115]

[0116] 2) Result analysis

[0117] As can be seen from Table 10, compared with the control group, the relative expression level of the NPPA gene in H9c2 cells treated with 0.25 μM and 0.5 μM of SD49-7 alone had no obvious effect; the relative expression level of the NPPA gene in isoproterenol-induced H9c2 cells increased significantly (P < 0.05). 0.25 μM of SD49-7 had no inhibitory effect on the increase in the relative expression level of the NPPA gene induced by isoproterenol (P > 0.05), but 0.5 μM of SD49-7 could significantly inhibit the increase in the relative expression level of the NPPA gene induced by isoproterenol (P < 0.01). 20 μM of metoprolol could significantly inhibit the increase in the relative expression level of the NPPA gene induced by isoproterenol (P < 0.01).

[0118] Table 10. Effects of isoproterenol on the relative mRNA expression level of the NPPA gene detected by qPCR

[0119]

[0120] Note: *P < 0.05, compared with the control group; ##P < 0.01, compared with the isoproterenol group

[0121] As can be seen from Table 11, compared with the control group, the relative expression level of the NPPB gene in H9c2 cells treated with 0.25 μM and 0.5 μM of SD49-7 alone had no obvious effect; the relative expression level of the NPPB gene in isoproterenol-induced H9c2 cells increased significantly (P < 0.0001). Both 0.25 μM and 0.5 μM of SD49-7 could significantly inhibit the increase in the relative expression level of the NPPB gene induced by isoproterenol (P < 0.01). 20 μM of metoprolol could significantly inhibit the increase in the relative expression level of the NPPB gene induced by isoproterenol (P < 0.001).

[0122] Table 11. Effects of isoproterenol on the relative mRNA expression level of the NPPB gene detected by qPCR

[0123]

[0124] Note: ****P < 0.0001, compared with the control group; ##P < 0.01, P < 0.0001, compared with the isoproterenol group

[0125] As can be seen from Table 12, compared with the control group, when H9c2 cells were treated with 0.25 μM and 0.5 μM of SD49-7 alone, there was no significant effect on the relative expression level of the MYH7 gene; the relative expression level of the MYH7 gene in H9c2 cells induced by isoproterenol increased significantly (P < 0.01), and 0.25 μM and 0.5 μM of SD49-7 could significantly inhibit the increase in the relative expression level of the MYH7 gene induced by isoproterenol (P < 0.001; P < 0.0001), and 20 μM of metoprolol could significantly inhibit the increase in the relative expression level of the MYH7 gene induced by isoproterenol (P < 0.0001).

[0126] Table 12. Effect of isoproterenol on the relative mRNA expression level of the MYH7 gene detected by qPCR

[0127]

[0128] Note: **P < 0.01, compared with the control group; P < 0.001, #P < 0.0001, compared with the isoproterenol group

[0129] As can be seen from Table 13, compared with the control group, when H9c2 cells were treated with 0.25 μM and 0.5 μM of SD49-7 alone, there was no significant effect on the relative expression level of the NPPA gene; the relative expression level of the NPPA gene in H9c2 cells induced by phenylephrine increased significantly (P < 0.0001), and 0.25 μM and 0.5 μM of SD49-7 could significantly inhibit the increase in the relative expression level of the NPPA gene induced by phenylephrine (P < 0.001), and 500 nM of empagliflozin could significantly inhibit the increase in the relative expression level of the NPPA gene induced by phenylephrine (P < 0.001).

[0130] Table 13. Effect of phenylephrine on the relative mRNA expression level of the NPPA gene detected by qPCR

[0131]

[0132] Note: ****P < 0.0001, compared with the control group; P < 0.001, compared with the phenylephrine group

[0133] As can be seen from Table 14, compared with the control group, treatment of H9c2 cells with 0.25 μM and 0.5 μM of SD49-7 alone had no significant effect on the relative expression level of the NPPB gene; treatment of H9c2 cells with 0.25 μM and 0.5 μM of SD49-7 alone had no significant effect on the relative expression level of the NPPB gene; the relative expression level of the NPPB gene in H9c2 cells induced by phenylephrine increased significantly (P < 0.01), and 0.25 μM and 0.5 μM of SD49-7 could significantly inhibit the increase in the relative expression level of the NPPB gene induced by phenylephrine (P < 0.01; P < 0.001), and 500 nM empagliflozin could significantly inhibit the increase in the relative expression level of the NPPB gene induced by phenylephrine (P < 0.05).

[0134] Table 14. Effects of phenylephrine on the relative mRNA expression level of the NPPB gene detected by qPCR

[0135]

[0136] Note: **P < 0.01, compared with the control group; #P < 0.05, ##P < 0.01, P < 0.001, compared with the phenylephrine group

[0137] As can be seen from Table 15, compared with the control group, treatment of H9c2 cells with 0.25 μM and 0.5 μM of SD49-7 alone had no significant effect on the relative expression level of the MYH7 gene; the relative expression level of the MYH7 gene in H9c2 cells induced by phenylephrine increased significantly (P < 0.001), and 0.25 μM and 0.5 μM of SD49-7 could significantly inhibit the increase in the relative expression level of the MYH7 gene induced by phenylephrine (P < 0.0001), and 500 nM empagliflozin could significantly inhibit the increase in the relative expression level of the MYH7 gene induced by phenylephrine (P < 0.01).

[0138] Table 15. Effects of phenylephrine on the relative mRNA expression level of the MYH7 gene detected by qPCR

[0139]

[0140] Note: ***P < 0.001, compared with the control group; ##P < 0.01, #P < 0.0001, compared with the phenylephrine group

[0141] As can be seen from Table 16, compared with the control group, the relative expression level of the NPPA gene in H9c2 cells treated with 0.25 μM and 0.5 μM of SD49-7 alone had no obvious effect; the relative expression level of the NPPA gene in H9c2 cells induced by angiotensin II increased significantly (P < 0.01), and 0.25 μM and 0.5 μM of SD49-7 could significantly inhibit the increase in the relative expression level of the NPPA gene induced by angiotensin II (P < 0.01; P < 0.05), and 24 μM of losartan could significantly inhibit the increase in the relative expression level of the NPPA gene induced by angiotensin II (P < 0.001).

[0142] Table 16. Effects of angiotensin II on the relative mRNA expression level of the NPPA gene detected by qPCR

[0143]

[0144] Note: **P < 0.01, compared with the control group; #P < 0.05, ##P < 0.01, compared with the angiotensin II group

[0145] As can be seen from Table 17, compared with the control group, the relative expression level of the NPPB gene in H9c2 cells treated with 0.25 μM and 0.5 μM of SD49-7 alone had no obvious effect; the relative expression level of the NPPB gene in H9c2 cells induced by angiotensin II increased significantly (P < 0.001), 0.25 μM of SD49-7 had no inhibitory effect on the increase in the relative expression level of the NPPB gene induced by angiotensin II (P > 0.05), but 0.5 μM of SD49-7 could significantly inhibit the increase in the relative expression level of the NPPB gene induced by angiotensin II (P < 0.01), and 24 μM of losartan could significantly inhibit the increase in the relative expression level of the NPPB gene induced by angiotensin II (P < 0.0001).

[0146] Table 17. Effects of angiotensin II on the relative mRNA expression level of the NPPB gene detected by qPCR

[0147]

[0148] Note: ***P < 0.001, compared with the control group; ##P < 0.01, #P < 0.0001, compared with the angiotensin II group

[0149] As can be seen from Table 18, compared with the control group, 0.25 μM and 0.5 μM SD49-7 alone treated H9c2 cells had no significant effect on the relative expression level of the MYH7 gene; the relative expression level of the MYH7 gene in H9c2 cells induced by angiotensin II was significantly increased (P<0.0001), 0.25 μM and 0.5 μM SD49-7 could significantly inhibit the increase in the relative expression level of the MYH7 gene induced by angiotensin II (P<0.01; P<0.0001), and 24 μM losartan could significantly inhibit the increase in the relative expression level of the MYH7 gene induced by angiotensin II (P<0.0001).

[0150] Table 18. Effect of angiotensin II on the relative expression of MY7 gene mRNA detected by qPCR

[0151]

[0152] Note: ****P<0.0001, compared with the control group; ##P<0.01, ####P<0.0001, compared with the angiotensin II group

[0153] Example 5: Effect of SD49-7 on ISO-induced myocardial hypertrophy in mice

[0154] Experimental purpose: To construct an ISO-induced myocardial hypertrophy model in mice and to detect the efficacy of SD49-7 on myocardial hypertrophy in mice.

[0155] Experimental Materials:

[0156] SD49-7 (Selleck, Catalog No.: E1335), isoproterenol (MCE, Catalog No.: HY-B0468), isoflurane (Shandong Ante Animal Husbandry Technology Co., Ltd., Batch No.: 2023041501), hematoxylin and eosin stain (biosharp, Catalog No.: BL700B), WGA stain (Sigma aldrich, Catalog No.: L4925), C57BL / 6 mice (Jiangsu Jicui Pharmaceutical Kang Biotechnology Co., Ltd.)

[0157] Experimental instruments: VINNO echocardiography system (VINNO, model: VINNO6), upright fluorescence microscope (ZEIS, model: Axio Scope A1)

[0158] 1) Experimental steps:

[0159] (1) The animal research protocol was approved by the Bioethics Committee of Hefei University of Technology and was strictly operated in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH) during the breeding process. C57BL / 6 mice were fed in a specific pathogen-free (SPF) animal room at about 22 °C and a humidity of about 55%. At the same time, the bedding was changed every three days, and all supplies such as feed water and bedding for breeding animals were used after disinfection and sterilization.

[0160] (2) After the C57BL / 6 mice were adaptively bred, they were divided into a control group, an isoproterenol group, an isoproterenol + SD49-7L group, and an isoproterenol + SD49-7H group, with 12 mice in each group. Among them, the mice in the isoproterenol group were given 5 mg / kg of isoproterenol (subcutaneous injection) every day for the first two weeks, and then 5 mg / kg of isoproterenol (subcutaneous injection) every two days for the next two weeks; the mice in the isoproterenol + SD49-7L group and the isoproterenol + SD49-7H group were given the same treatment as the isoproterenol group, and at the same time, 5 mg / kg and 15 mg / kg of SD49-7 (intraperitoneal injection) were given respectively; the mice in the control group were given the same solvent treatment.

[0161] (3) After 4 weeks of drug administration, the status of the mice was observed and photographed and recorded. Then, echocardiography was used to detect the cardiac function of each group of mice. The specific method is as follows:

[0162] All mice had their chest hair removed, inhaled 1-1.5% isoflurane, and after their breathing was stable and the corneal reflex and rolling movement disappeared, they were placed on a 37 °C thermostatic plate, and M-mode echocardiography was performed using a VINNO6 echocardiogram system. According to the ultrasound results, the left ventricular end-systolic and end-diastolic diameters (LVIDs and LVIDd) were calculated. Calculate the left ventricular end-systolic and end-diastolic diameters, ejection fraction (EF), and fractional shortening (FS): EF (%) = [(LVID3 - LVIDs3) / LVIDd3] × 100; FS (%) = [(LVIDd - LVIDs) / LVIDd] × 100.

[0163] (4) After the mice completed the cardiac echocardiogram detection, euthanasia was used to collect and weigh the tissues and organs of each mouse and store them.

[0164] (5) After separating the mouse heart tissue, paraffin sections of the heart were made. The following steps were used for hematoxylin-eosin staining (HE staining) operation:

[0165] Xylene for 10 min → Xylene for 10 min → 100% ethanol for 5 min → 100% ethanol for 5 min → 95% ethanol for 5 min → 90% ethanol → 80% ethanol for 5 min → Tap water for 5 min → Distilled water for 5 min → Hematoxylin for 40 s → Tap water for 5 min → Distilled water for 5 min → Eosin staining solution for 45 s → 80% ethanol for 5 min → 90% ethanol → 95% ethanol for 5 min → 100% ethanol for 5 min → 100% ethanol for 5 min → Xylene for 5 min → Xylene for 5 min

[0166] After staining, seal the slides with neutral resin. Take pictures after air drying.

[0167] (6) Meanwhile, perform frozen sections on the heart tissues of mice in each group, wash three times with PBS, incubate with 5 μg / ml wheat germ agglutinin (WGA) staining solution in the dark at room temperature for 15 min, and wash three times with PBS. Seal the slides with an anti-fluorescence quenching agent and take pictures after air drying.

[0168] 2) Result analysis:

[0169] As can be seen from Table 19, compared with the control group, the EF and FS of mice in the isoproterenol group decreased significantly, indicating that the cardiac function of mice was significantly affected (P < 0.0001); compared with the isoproterenol group, the EF of mice in the isoproterenol + SD49-7L group and the isoproterenol + SD49-7H group increased significantly (P < 0.001; P < 0.01), and the FS of mice in the isoproterenol + SD49-7L group and the isoproterenol + SD49-7H group also increased significantly (P < 0.0001; P < 0.0001). These data indicate that SD49-7 has a significant improvement effect on the decrease in cardiac function induced by isoproterenol in mice.

[0170] Table 19. Effects of SD49-7 on cardiac function in ISO-induced myocardial hypertrophy in mice

[0171]

[0172] Note: **P < 0.01, ****P < 0.0001, compared with the control group; ##P < 0.01, #P < 0.0001, compared with the ISO group

[0173] From Figure 4 it can be seen that the appearance of mice in each group was lively, with shiny hair, smooth skin, quick movement, and no difference in mental state, indicating that isoproterenol and SD49-7 had no obvious effect on the state of mice.

[0174] From Figure 5It can be seen that, compared with the control group, the heart weight of the mice in the isoproterenol group increased significantly (P<0.001). Compared with the isoproterenol group, the heart weight of the mice in the isoproterenol + SD49-7H group decreased significantly (P<0.05), indicating that after induction with isoproterenol, the hearts of the mice had a tendency to hypertrophy, and high-dose SD49-7 could significantly inhibit isoproterenol-induced cardiac hypertrophy in mice.

[0175] From Figure 6 the HE staining results, it can be seen that, compared with the control group, the relative cross-sectional area of the hearts of the mice in the isoproterenol group increased significantly (P<0.05), indicating that the hearts of the mice became larger and the myocardium became thicker after induction with isoproterenol. The relative cross-sectional areas of the hearts of the mice in the isoproterenol + SD49-7L and isoproterenol + SD49-7H groups decreased significantly (P<0.05; P<0.01), indicating that SD49-7 could significantly inhibit isoproterenol-induced cardiac hypertrophy in mice.

[0176] From Figure 7 the WGA staining results, it can be seen that, compared with the control group, the relative cross-sectional area of the myocardial cells of the mice in the isoproterenol group increased significantly (P<0.0001). The relative cross-sectional areas of the myocardial cells of the mice in the isoproterenol + SD49-7L and isoproterenol + SD49-7H groups decreased significantly (P<0.0001; P<0.0001), indicating that SD49-7 could significantly inhibit isoproterenol-induced myocardial hypertrophy in mice.

[0177] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than a limitation on the protection scope of the present invention. Any simple modification or equivalent replacement made by those of ordinary skill in the art to the technical solution of the present invention shall not depart from the essence and scope of the technical solution of the present invention.

Claims

1. Use of SD49-7 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing and treating myocardial hypertrophy, wherein SD49-7 has the following chemical structure:

2. The application according to claim 1, characterized in that, Including for inhibiting cardiomyocyte hypertrophy.

3. The application according to claim 1, characterized in that The medicament is any pharmaceutically acceptable drug.

4. The application according to claim 1, characterized in that, The dosage form of the medicament is selected from oral dosage forms or dosage forms for parenteral administration.

5. The application according to claim 1, characterized in that, The dosage form of the medicament contains a therapeutically effective amount of SD49-7 or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers.

6. The application according to claim 5, characterized in that, The dosage form of the medicament is an oral dosage form.

7. The application according to claim 6, characterized in that, The oral dosage form is selected from tablets, capsules, granules, pills, powders, oral liquids.

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