CN116808025A - Application of IBM in preparing medicament for treating cardiac hypertrophy - Google Patents
Application of IBM in preparing medicament for treating cardiac hypertrophy Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
- A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
- A61K31/404—Indoles, e.g. pindolol
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Life Sciences & Earth Sciences (AREA)
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- General Health & Medical Sciences (AREA)
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- Veterinary Medicine (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Cardiology (AREA)
- Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Heart & Thoracic Surgery (AREA)
- Epidemiology (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Dermatology (AREA)
- Hospice & Palliative Care (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses application of IBM in preparing a medicament for treating myocardial hypertrophy, and the research result of the invention shows that the treatment of IBM can obviously inhibit the ubiquitination degradation of GSNOR induced by TAC, thereby relieving myocardial hypertrophy and the reduction of cardiac contraction function of mice. IBM treatment significantly inhibited Ang II-induced ubiquitination degradation of GSNOR, thereby ameliorating cardiomyocyte hypertrophy. IBM can be used to treat cardiac hypertrophy.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to an application of IBM in preparing a medicament for treating myocardial hypertrophy.
Background
Myocardial hypertrophy is an important early-stage lesion of heart failure and is characterized by hypertrophy of cardiac myocytes, hypertrophy of heart wall, excessive increase in ventricular size, and the like. Initial hypertrophy maintains cardiac pumping function by resisting afterload increases, but under sustained pressure loads, cardiac hypertrophy undergoes decompensation, inducing heart failure, and thus life threatening. At present, the pathogenesis of myocardial hypertrophy is not completely elucidated, and the clinic is lack of effective treatment means.
Nitrosoglutathione reductase (GSNOR) belongs to the family of alcohol dehydrogenases, and is mainly involved in the metabolism of nitrosoglutathione, regulating the maintenance of nitric oxide homeostasis in the body. Studies have shown that GNSOR expression levels are significantly reduced during the course of cardiac hypertrophy, while overexpression of GSNOR reduces cardiac hypertrophy. This suggests that down-regulation of GSNOR may be an important causative factor in cardiac hypertrophy, and we have therefore explored specific reasons for down-regulation of GSNOR in cardiac hypertrophy. We found that in cardiac hypertrophy, E3 ubiquitin ligase NEDD4 promotes ubiquitination degradation of GSNOR, resulting in reduced GSNOR expression, which in turn exacerbates cardiac hypertrophy. Thus, inhibitors of NEDD4 activity may inhibit GSNOR degradation, alleviating cardiac hypertrophy.
Currently known NEDD4 inhibitors are mainly Indole-3-methanol (I3C), but I3C is not a selective inhibitor of NEDD4, and it also inhibits serine protease, E3 ubiquitin enzyme WWP1, lacking specificity. The compound IBM is NEDD4 activity inhibitor obtained by mass spectrum screening and structure optimization, has no inhibition effect on other ubiquitin enzymes with HECT structural domain or catalytic activity cysteine site, and has higher selectivity; meanwhile, the inhibition efficiency is greatly improved. But IBM has not yet been reported on its role and mechanism in cardiovascular disease. Development or screening of drugs for treating cardiovascular diseases is a technical problem to be solved in the art.
Disclosure of Invention
The invention aims to provide an application of novel NEDD4 activity inhibitor IBM in preparing medicaments for treating myocardial hypertrophy, and researches show that the IBM can obviously relieve myocardial hypertrophy.
The aim of the invention can be achieved by the following technical scheme:
the use of a compound IBM having the structural formula (I) in at least one of the following (1) to (3),
(1) Preparing a medicament for preventing and/or treating myocardial hypertrophy;
(2) Preparing a medicament having an improved systolic function;
(3) Preparing an agent having a function of improving myocardial cell hypertrophy;
preferably: the medicine or reagent is a medicine composition or reagent composition prepared from a compound IBM with a structural formula shown as (I) serving as a main active ingredient and a pharmaceutically acceptable carrier or auxiliary material.
Preferably: the medicament or the reagent is in the form of injection.
The compound shown in the structural formula (I) is a novel NEDD4 activity inhibitor, and is named as: IBM. IBM is renamed according to the main groups indole, butenoic acid, methyl ester in the structure of this compound (I: indole, indole; B: butenoic acid, butenoic acid; M: methyl, methyl ester), the references for the preparation process of which are: JAm Chem soc.2015oct 7;137 (39) 12442-5, IBM is in accordance with the Compound3 architecture in the reference.
The synthetic route of IBM is shown below:
the beneficial effects of the invention are that
The research results of the invention show that IBM can relieve the increase of the surface area of the myocardial cells induced by Ang II, inhibit the increase of atrial natriuretic peptide (atrial natriuretic peptide, anp), brain natriuretic peptide (brain natriureticpeptide, bnp) and beta-myosin heavy chain (beta-myosin heavy chain, beta-mhc) which are markers of myocardial cell center muscle hypertrophy induced by Ang II, and indicate that the invention can be used for improving myocardial cell hypertrophy; IBM can improve EF, FS values of aortic arch stenosis (transverse aortic constriction, TAC) mouse hearts, suggesting that the present invention may be used to improve the systolic function under cardiac hypertrophy; IBM can reduce the ratio of heart weight to body weight of mice, reduce the expression level of cardiac hypertrophy markers Anp, bnp, beta-mhc in heart tissues, reduce the cross-sectional area of cardiac myocytes of TAC mice, and suggest that the invention can be used for treating cardiac hypertrophy.
Drawings
FIG. 1 is a schematic diagram of TAC modeling and IBM dosing regimen.
FIG. 2 is an illustration of IBM alleviating TAC-induced myocardial hypertrophy and GSNOR ubiquitination degradation;
a, echocardiography detects mouse EF, FS, IVS and LVPW value changes (n=10);
b, first behavioural mouse heart photograph, second behavioural HE staining (scale 1mm, n=10), third behavioural WGA staining (scale 100 μm, n=10). On the right are quantitative analysis of heart to body weight ratio (HW/BW) and cardiomyocyte cross-sectional area (n=10);
c, qRT-PCR detects the levels of myocardial hypertrophy markers Anp, bnp and beta-mhc (n=10) of the heart tissue of the mouse, the expression level of RNA is uniform according to the expression level of 18s, and the statistical figures are relative values compared with sham groups;
d, western-blot detects GSNOR ubiquitination levels (n=10) in heart tissue. * P <0.001.
FIG. 3 is an IBM alleviation of Ang II-induced cardiomyocyte hypertrophy and GSNOR ubiquitination degradation;
a, observing the surface area of myocardial cells by alpha-actinin immunofluorescence staining and carrying out statistical analysis (the scale is 100 μm, n=3), wherein the surface area statistical graphs are all relative values compared with CON groups;
b, qRT-PCR detects the levels of Anp, bnp, β -mhc (n=3) in mouse cardiomyocytes, the RNA expression levels are uniform according to the expression level of 18s, and the statistical figures are relative values compared with the CON group;
c, western blot detects GSNOR ubiquitination levels (n=3) in cardiomyocytes. * P <0.001.
Detailed Description
The following examples will provide those skilled in the art with a more complete understanding of the invention, but are not intended to limit the invention in any way.
Example 1: therapeutic effect of IBM on TAC-induced cardiac hypertrophy mice
1. TAC-induced mouse myocardial hypertrophy model:
male C57BL/6J mice (purchased from Experimental animal center of Nanjing medical university) of 25-28 g in weight at 8 weeks of age were selected and randomly classified as: sham surgery (sham) group, TAC group, sham+ibm group, tac+ibm group, 10 per group. The mice are anesthetized and fixed, chest is opened through a sternum handle, and the mice are connected with a rodent breathing machine through an trachea cannula to carry out artificial ventilation. Opening the chest between the 2 nd rib and the 3 rd rib on the left side of the chest, and separating the aortic arch; threading after the innominate artery, winding the aortic arch, tying two dead knots above the aortic arch, restoring thymus and heart to original positions, carefully closing the chest cavity, and suturing the skin; sham group mice repeat the above procedure except that only the threading is not narrowed.
As shown in FIG. 1, all animals were injected intraperitoneally with IBM (10 mg/kg/d) or their control solvents (saline containing 10% Solutol (g/100 ml, 10g Solutol per 100ml control solvent) and 5% (v/v) DMSO) at once daily frequency; after one week, TAC modeling is carried out, and sham group carries out false operation; after molding for 4 weeks, an ultrasonic cardiography examination is performed, and heart tissues are taken for subsequent myocardial hypertrophy index examination.
2. Echocardiography:
4 weeks after TAC molding, echocardiography images were acquired by cardiac M-mode ultrasound techniques using a small animal ultrasound imaging system Visual Sonics Vevo 2100 to assess the thickening and contractile function of the mouse's heart, including left ventricular posterior wall thickness (left ventricular posterior wall, LVPW), ventricular septum thickness (interventricular septum, IVS), left ventricular posterior wall ejection fraction (ejection fraction, EF), short axis foreshortening rate (fraction shortening, FS).
3. Mice heart weighing:
after euthanasia of the mice, hearts were taken, weighed, and the ratio of Heart Weight (HW) to Body Weight (BW) was calculated.
4. Heart tissue paraffin sections HE staining, WGA staining:
heart tissue was collected, paraffin sections were prepared, after dewaxing and rehydration, hematoxylin stained nuclei for 3min, after washing with clear water, eosin stained for 3min, after washing, air dried, and blocked, and observed using an Olympus BX63 microscope.
After baking heart paraffin sections in an oven at 55 ℃ for 3h, membrane rupture, washing, wheat germ lectin (Wheat Germ Agglutinin, WGA) staining at 37 ℃ for 1h, dapi staining and sealing, observations were made using an Olympus BX63 microscope.
5. RT-qPCR detects heart tissue Anp, bnp, beta-mhc levels:
after extracting RNA of heart tissue and synthesizing cDNA by reverse transcription, the expression of myocardial hypertrophy markers Anp, bnp and beta-mhc is detected by adopting a real-time fluorescence quantitative PCR (RT-qPCR) technology. Wherein the reverse transcription system (20. Mu.L) is: super Mix (5×) 4. Mu.L, total RNA 1000ng, RNase Free dH 2 O makes up the volume to 20 mu L at 50 ℃,15 min-85 ℃ and 5 s-12 ℃. The RT-qPCR system (10. Mu.L) was: SYBR GREEN (2×) 5. Mu.L, primer F0.2. Mu.L, primer R0.2. Mu. L, cDNA 1. Mu.L, RNase Free dH 2 O3.6. Mu.L, provided that: pre-denaturation and enzyme activation, 95 ℃ for 10min; denaturation, 95 ℃,10s, annealing and stretching, 60 ℃,60s,40 cycles; dissolution profile, 95 ℃,15 s-60 ℃,60 s-95 ℃,15s. The primer sequences used for RT-qPCR are shown in Table 1.
TABLE 1 primer sequences for RT-qPCR of mouse heart tissue
| Gene | Forward(5'to 3') | Reverse(5'to 3') | Species of species |
| Anp | ATTGACAGGATTGGAGCCCAGAGT | TGACACACCACAAGGGCTTAGGAT | A mouse |
| Bnp | CTCAAGCTGCTTTGGGCACAAGAT | AGCCAGGAGGTCTTCCTACAACAA | A mouse |
| β-mhc | ACTGTCAACACTAAGAGGGTCA | TTGGATGATTTGATCTTCCAGGG | A mouse |
| 18s | AGTCCCTGCCCTTTGTACACA | CGATCCGAGGGCCTCACTA | A mouse |
6. Detection of GSNOR ubiquitination levels in cardiac tissue:
20mg of heart tissue was weighed, sheared, washed 1 time with ice PBS, 1mL of ubiquitinated lysate (50 mM Tris-HCl, pH7.5, 150mM NaCl,1mM EDTA,1Mm EGTA,1% (v/v) Triton X-100,1mM PMSF,20mM NEM, protease inhibitor added prior to use) was added, and homogenized with a homogenizer until no solids were present. The homogenate was spun at 4℃for 30min. The cell suspension was centrifuged at 12,000rcf,4℃for 10min. Protein concentration was measured, 50. Mu.g of protein was retained as input according to the measured protein concentration, 500. Mu.g of protein was added to protein G beads and GSNOR antibody, and the mixture was spun overnight at 4 ℃. Washing the beads for 5 times, adding 2×loading buffer equal to the volume of the beads, shaking, centrifuging, and boiling the protein at 100deg.C for 5min.
7. Western-blot detection of GSNOR ubiquitination level:
12% of the separation gel and 3% of the concentrate gel were prepared as shown in Table 2, and the loading per well was about 30. Mu.g. And (3) carrying out 110V constant-pressure electrophoresis for about 90min until bromophenol blue completely disappears. After electrophoresis, the concentrated gel was cut off, and the protein band was transferred to a PVDF membrane by wet transfer (SDS-gel at negative electrode and PVDF membrane at positive electrode), followed by 0.3A constant-current electrophoresis for 90min. After the transfer, the PVDF membrane was removed and immersed in TBST containing 5% skimmed milk powder for 1h. After blocking, the membrane was shaken with the antibody overnight at 4 ℃. After washing the membrane, horseradish peroxidase-labeled secondary antibody was added and incubated for 2h at room temperature. After washing the membrane, ECL developed and the results were observed.
TABLE 2 preparation of SDS-PAGE
| Composition of the components | Release adhesive (12%) | Concentrated glue (3%) |
| Deionized water | 1.6mL | 1.8mL |
| 30% (v/v) AA mother liquor | 2.0mL | 0.3mL |
| 1.5M Tris-HCl(pH8.8) | 1.3mL | |
| 1M Tris-HCl(pH6.8) | 0.75mL | |
| 10％(g/100ml)SDS | 50μL | 30μL |
| 10% (g/100 ml) Ammonium Persulfate (APS) | 50μL | 50μL |
| TEMED | 5μL | 5μL |
| Total volume of | 5mL | About 3mL |
8. Statistical methods:
two-way ANOVA (two-way ANOVA) was used between the different groups in the experiment, and it was considered that P <0.05 had statistical significance; statistical plots were drawn using prism8.0 statistical software.
9. Analysis of results:
echocardiography showed that IBM reduced IVS and LVPW in the TAC mice and increased hearts EF and FS in the TAC mice compared to the TAC group, indicating that IBM improved left ventricular hypertrophy and systole function in the TAC mice (as shown in fig. 2A). IBM significantly reduced the ratio of heart weight to body weight (HW/BW) of mice compared to the TAC group, and heart tissue section HE staining and WGA staining showed that IBM was able to significantly reduce heart volume and cardiomyocyte cross-sectional area of TAC mice (as shown in fig. 2B). At the same time, IBM reduced the expression level of cardiac markers of hypertrophy (Anp, bnp, β -mhc) in heart tissue (as shown in fig. 2C). The above results indicate that IBM significantly improves myocardial hypertrophy and contractile function in TAC mice. The ubiquitination results showed that IBM significantly reduced the TAC-induced increase in GSNOR ubiquitination levels in myocardial tissue (as shown in fig. 2D).
In conclusion, IBM treatment significantly inhibited TAC-induced ubiquitination degradation of GSNOR, thereby alleviating myocardial hypertrophy and decreased systolic function in mice.
Example 2: protection of Ang II-induced cardiomyocyte hypertrophy by IBM
1. Ang II induced cardiomyocyte hypertrophy model:
SD rat rats (purchased from Experimental animal center of Nanjing medical university) born for 1-3 days were isolated from primary cardiomyocytes and divided into Control (CON) group, ang II group, CON+IBM group, ang II+IBM group. The cells were cultured in DMEM medium containing 10% (v/v) fetal bovine serum. Before the experiment, the cells were treated with serum-free medium for 12 hours, then replaced with DMEM medium containing 10% fetal bovine serum, and incubated with 2.5 μm IBM or equivalent control solvent (DMSO) for 4 hours, then incubated with 1 μm Ang II or equivalent control solvent (PBS) for 24 hours, and then subjected to detection such as RT-qPCR, immunofluorescent staining, etc.
2. Detecting the area size of myocardial cells by alpha-actinin staining:
after washing the cardiomyocytes with PBS, 4% (v/v) paraformaldehyde was fixed at room temperature for 20min,0.3% (v/v) Triton X-100 membrane was broken for 15min,5% (g/100 ml) BSA was blocked at room temperature for 30min, alpha-actinin antibody (1:100, sigma-Aldrich, A7811) was incubated overnight at 4℃and then secondary antibody was added, incubated at 37℃for 30min away from light, photographed under a fluorescence microscope Leica DMi8 and analyzed.
3. RT-qPCR detects cardiomyocyte Anp, bnp, beta-mhc levels:
after extracting total RNA of myocardial cells and synthesizing cDNA by reverse transcription, the expression of myocardial hypertrophy markers Anp, bnp and beta-mhc is detected by adopting a real-time fluorescence quantitative PCR (RT-qPCR) technology. The conditions are as described previously. The primer sequences used for RT-qPCR are shown in Table 3.
TABLE 3 primer sequences for myocardial cell RT-qPCR
| Gene | Forward(5'to 3') | Reverse(5'to 3') | Species of species |
| Anp | ATCTGCCCTCTTGAAAAGCA | GGATCTTTTGCGATCTGCTC | Rat (rat) |
| Bnp | ATCGGCGCAGTCAGTCGCTT | GGTGGTCCCAGAGCTGGGGAA | Rat (rat) |
| β-mhc | AGATCGAGGACCTGATGGTG | GATGCTCTTCCCAGTTGAGC | Rat (rat) |
| 18s | AGTCCCTGCCCTTTGTACACA | CGATCCGAGGGCCTCACTA | Rat (rat) |
4. Detecting GSNOR ubiquitination levels in cardiomyocytes:
cells were washed 3 times with pre-chilled PBS, scraped with 500. Mu.L of ubiquitinated lysate (50 mM Tris-HCl, pH7.5, 150mM NaCl,1mM EDTA,1Mm EGTA,1% (v/v) Triton X-100,1mM PMSF,20mM NEM, with pre-protease inhibitor added) and transferred to a 1.5mL centrifuge tube. Rotating at 4 ℃ for 30min. The cell suspension was centrifuged at 12,000rcf,4℃for 10min. Protein concentration was measured, 50. Mu.g of protein was retained as input according to the measured protein concentration, 500. Mu.g of protein was added to protein G beads and GSNOR antibody, and the mixture was spun overnight at 4 ℃. Washing the beads for 5 times, adding 2×loading buffer equal to the volume of the beads, shaking, centrifuging, and boiling the protein at 100deg.C for 5min.
5. Western-blot detection of GSNOR ubiquitination level:
as previously described.
6. Statistical methods:
two-way ANOVA (two-way ANOVA) was used between the different groups in the experiment, and it was considered that P <0.05 had statistical significance; statistical plots were drawn using prism8.0 statistical software.
7. Analysis of results:
alpha-actin staining showed that IBM treatment reduced cardiomyocyte surface area compared to Ang II group (as shown in figure 3A). qRT-PCR results showed that IBM reduced the expression levels of Anp, bnp, β -mhc in cardiomyocytes (as shown in FIG. 3B). The ubiquitination assay showed that IBM significantly reduced the rise in GSNOR ubiquitination levels in cardiomyocytes caused by Ang II (as shown in figure 3C).
In conclusion, IBM treatment significantly inhibited Ang II-induced ubiquitination degradation of GSNOR, thereby ameliorating cardiomyocyte hypertrophy.
Claims (3)
1. The use of a compound IBM having the structural formula (I) in at least one of the following (1) to (3),
(1) Preparing a medicament for preventing and/or treating myocardial hypertrophy;
(2) Preparing a medicament having an improved systolic function;
(3) Preparing an agent having a function of improving myocardial cell hypertrophy;
2. the use according to claim 1, characterized in that: the medicine or reagent is a medicine composition or reagent composition prepared from a compound IBM with a structural formula shown as (I) serving as a main active ingredient and a pharmaceutically acceptable carrier or auxiliary material.
3. Use according to claim 1 or 2, characterized in that: the medicament or the reagent is in the form of injection.
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