Application of medicine for inhibiting increase of SOD2 acetylation level in preparation of medicine for preventing or treating heart failure with reduced ejection fraction

Abstract

The invention relates to the technical field of biological medicine, in particular to application of a medicine for inhibiting SOD2 acetylation level increase in preparation of a medicine for preventing or treating ejection fraction reduction type heart failure. Compared with a control group, the myocardial cell SOD2 acetylation of the ejection fraction reduced heart failure is increased, the activity of the SOD2 in the myocardial cell in the ejection fraction reduced heart failure is reduced, the SOD2 acetylation is increased, and occurrence and development of the heart failure can be aggravated by promoting the increase of active oxygen; the phenomenon of SOD2 acetylation increase is also found in the heart tissue of a heart failure mouse model with reduced ejection fraction; therefore, inhibition of SOD2 acetylation can be used for inhibiting ROS rise of myocardial cells so as to relieve occurrence and development of heart failure.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the use of a drug for inhibiting the increase of SOD2 acetylation level in the preparation of a drug for preventing or treating heart failure due to reduced ejection fraction. Background Art

[0002] Heart failure with reduced ejection fraction (HFrEF) is a heart disease characterized by a left ventricular ejection fraction (LVEF) below 40%, resulting in a weakened heart pumping function. Although progress has been made in the treatment of HFrEF in recent years, from the "golden triangle" regimen to the "new quadruple" regimen, there are still problems to be solved. The main challenges include: most patients have clinical conditions such as hypotension and low heart rate, poor drug tolerance, and poor treatment effects; target dose titration is difficult during medication, and some patients cannot add other drugs; traditional drug treatment regimens have a long titration time and cannot combine all mortality-reducing drugs in the early stage. In addition, the guideline-directed medical therapy (GDMT) compliance rate of HFrEF patients is not ideal, and drug treatment faces many challenges. Therefore, in-depth exploration of the pathogenesis of HFrEF and finding more effective and personalized treatment options are important tasks in current research and clinical practice.

[0003] Superoxide dismutase 2 (SOD2), also known as manganese superoxide dismutase (MnSOD), is an enzyme that plays a key role in the cellular antioxidant process. SOD2 is mainly distributed on the inner membrane of mitochondria, and its function is to convert superoxide anions into oxygen and hydrogen peroxide, thereby preventing oxidative damage caused by free radicals. In the context of heart failure, the role of SOD2 is particularly important. Heart failure is closely related to mitochondrial dysfunction, and mitochondria are the main site of reactive oxygen species (ROS) production in cells. SOD2 helps maintain mitochondrial function and intracellular redox balance by neutralizing ROS, reducing oxidative stress damage to the myocardium. Therefore, studying the relationship between SOD2 and heart failure is of great significance for understanding the pathophysiological mechanism of heart failure and developing new treatment strategies.

[0004] On this basis, the present invention provides the use of a drug for inhibiting the increase of SOD2 acetylation level in the preparation of a drug for preventing or treating heart failure with reduced ejection fraction. Summary of the invention

[0005] The purpose of the present invention is to provide an application of a drug for inhibiting the increase of SOD2 acetylation level in the preparation of a drug for preventing or treating heart failure with reduced ejection fraction.

[0006] Furthermore, the drug for inhibiting the increase of SOD2 acetylation level includes any one of tablets, capsules, pills, granules, oral liquids and solutions.

[0007] Furthermore, the drug for heart failure with reduced ejection fraction includes any one of tablets, capsules, pills, granules, oral liquids and solutions.

[0008] Furthermore, the drug contains a pharmaceutically acceptable carrier.

[0009] Furthermore, the pharmaceutically acceptable carrier is selected from at least one of pharmaceutically acceptable solvents, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrities, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids, release retardants, polymer skeleton materials and film-forming materials.

[0010] Beneficial effects:

[0011] The present invention found that the acetylation of SOD2 in myocardial cells of heart failure with reduced ejection fraction increased. The present invention found that compared with the control group, the acetylation of SOD2 in myocardial cells of heart failure with reduced ejection fraction increased. The activity of SOD2 in myocardial cells is reduced and the acetylation of SOD2 is increased in heart failure with reduced ejection fraction, which can aggravate the occurrence and development of heart failure by promoting the increase of reactive oxygen species; and the phenomenon of increased SOD2 acetylation was also found in the heart tissue of the mouse model of heart failure with reduced ejection fraction. Therefore, inhibiting SOD2 acetylation can be used to inhibit the increase of ROS in myocardial cells to alleviate the occurrence and development of heart failure. Therefore, the present invention provides the use of a drug that inhibits the increase of SOD2 acetylation level in the preparation of a drug for preventing or treating heart failure with reduced ejection fraction. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is a diagram showing reactive oxygen species in H9c2 cells induced by ISO;

[0013] Figure 2 This is a statistical diagram of the fluorescence intensity of reactive oxygen species in H9c2 cells induced by ISO;

[0014] Figure 3 This is a diagram showing reactive oxygen species induced by PE in H9c2 cells;

[0015] Figure 4 This is a statistical diagram of the fluorescence intensity of reactive oxygen species in H9c2 cells induced by PE;

[0016] Figure 5 This is a diagram showing the inhibition of SOD activity in H9c2 cells by ISO;

[0017] Figure 6 This is a diagram showing the inhibition of SOD activity in H9c2 cells by PE;

[0018] Figure 7 This is a diagram showing the inhibition of SOD2 activity in H9c2 cells by ISO;

[0019] Figure 8 This is a diagram showing the inhibition of SOD2 activity in H9c2 cells by PE;

[0020] Fig. 9 This is a statistical diagram of SOD activity in heart tissue of TAC model mice;

[0021] Fig.10 The diagram shows the deterioration of cardiac function and reactive oxygen species in TAC model mice. Fig.10 A is a schematic diagram of ultrasound in TAC model mice. Fig.10 B is the statistical diagram of ejection fraction of TAC model mice. Fig.10 C is the statistical chart of shortening scores in TAC model mice. Fig.10 D is a schematic diagram of the thickness of the left ventricular posterior wall during systole in TAC model mice. Fig.10 E is a schematic diagram of the left ventricular end-systolic diameter of TAC model mice. Fig.10 F is a schematic diagram of the maximum movement distance of TAC model mice. Fig.10 G is a schematic diagram of WGA and MASSON staining of TAC model mice. Fig.10 H is the statistical diagram of WGA staining of TAC model mice. Fig.10 I is the MASSON staining statistical diagram of TAC model mice, Fig.10 J is a statistical graph of reactive oxygen content in the heart tissue of TAC model mice;

[0022] Fig.11 This is a diagram showing the acetylation of SOD2 induced by ISO in H9c2 cells;

[0023] Fig.12 This is the statistical diagram of SOD2 acetylation induced by ISO in H9c2 cells;

[0024] Fig.13 This is a diagram showing PE-induced SOD2 acetylation in H9c2 cells;

[0025] Fig.14 This is the statistical diagram of SOD2 acetylation induced by PE in H9c2 cells;

[0026] Fig.15 This is a diagram showing SOD2 acetylation in heart tissue of TAC model mice;

[0027] Fig.16 This is a statistical chart of SOD2 acetylation in heart tissue of TAC model mice. DETAILED DESCRIPTION

[0028] The technical solution of the present invention is described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] The embodiments of the present invention will be described in detail below in conjunction with examples. The following examples take the best examples as illustrations, but those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. The specific conditions not specified in the examples are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased commercially.

[0030] The cell line in the present invention is from rat H9c2 purchased from Procell, using complete culture medium (DMEM medium + 10% serum + 1% penicillin-streptomycin), placed at 37°C, 5% CO 2 Culture in a cell culture incubator.

[0031] Source of animals in the present invention: The experimental animals are mainly from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., strain: C57BL / 6JGpt, genotype: (C57BL / 6JGpt) wt / wt, male mice 8 weeks old, production license number: SCXK (Su) 2023-0009. The use of C57BL / 6J mouse handling procedures complies with the requirements of the Animal Ethics Committee of the First Affiliated Hospital of the University of Science and Technology of China. There are 6 mice per cage, and water and standard laboratory diets are provided free of charge. The animal room maintains a constant temperature of 25±1°C, humidity of 55±5%, and a 12-hour light / 12-hour dark cycle.

[0032] The sources of drugs in the present invention are as follows: (1) isoproterenol hydrochloride (ISO): purchased from Taoshu Biological, the cell stimulation concentration is 10umol / L, and the time is 24h; (2) phenylephrine (PE): purchased from Taoshu Biological, the cell stimulation concentration is 100umol / L, and the time is 24h.

[0033] Example

[0034] 1. Detection of cell ROS content

[0035] The fluorescent probe DCFH-DA was used to measure the level of cellular reactive oxygen species (ROS). DCFH-DA (2',7'-dichlorofluorescein diacetate) can penetrate the mitochondrial membrane and is hydrolyzed by esterase to form non-fluorescent DCFH, which is then oxidized by ROS to produce fluorescent DCF. The probe was diluted with serum-free culture medium at a ratio of 1:1000 to a final concentration of 5 μM. The cell culture medium was removed, and an appropriate volume of diluted DCFH-DA was added. The cells were incubated in a cell culture incubator at 37°C for 30 minutes, and the cells were washed three times with PBS. DAPI staining solution was then added to stain the nuclei, and the cells were incubated in a cell culture incubator at 37°C for 20 minutes. The cells were washed three times with PBS, and finally observed with a confocal microscope. The excitation wavelength of the DCFH-DA probe is 488 nm, and the emission wavelength is 525 nm; the excitation wavelength of DAPI is 350 nm, and the emission wavelength is 460 nm.

[0036] When the drug source is isoproterenol hydrochloride (ISO), the test results are as follows Figure 1 and Figure 2 As shown, the results of the measurement of ISO-induced reactive oxygen species in cardiomyocytes showed that the reactive oxygen species level in H9c2 cells stimulated by ISO for 24 hours was significantly increased compared with the control group.

[0037] When the drug source is phenylephrine (PE), the test results are as follows Figure 3 and Figure 4 As shown, the results of the measurement of reactive oxygen species content in cardiomyocytes induced by PE showed that compared with the control group, the reactive oxygen species level in H9c2 cells stimulated by PE for 24 hours was significantly increased.

[0038] 2. SOD activity detection (cell tissue)

[0039] Using CheKine TM Superoxide dismutase (SOD) activity detection kit (Abbkine, catalog number: KTB1030). The activity of SOD is negatively correlated with the amount of formazan produced. Cell sample: 5×10 6 cells, wash the cells with pre-cooled PBS, centrifuge at 800g for 2 minutes, and discard the supernatant. Add 1mL of pre-cooled 1×LysisBuffer to suspend the cells, incubate on ice for 10 minutes, centrifuge at 4℃, 12,000g for 5 minutes, and take the supernatant for detection. Animal tissue samples: Weigh 0.1g of tissue and add 1mL of pre-cooled 1×LysisBuffer to homogenize the sample. After homogenization, centrifuge the sample at 4℃, 12,000g for 5 minutes, and take the supernatant for detection. Mix the sample and detection reagent thoroughly in a 96-well plate, incubate at 37℃ for 30 minutes, measure the absorbance value A of each well at 450nm, adjust to zero with deionized water, and finally calculate the result from the measured value. (Bradford method is used for protein quantification)

[0040] When the drug source is isoproterenol hydrochloride (ISO), the test results are as follows Figure 5 As shown, the results of the PE-induced cardiomyocyte SOD activity assay showed that compared with the control group, the cell SOD activity of H9c2 cells was significantly reduced after PE stimulation for 24 hours.

[0041] When the drug source is phenylephrine (PE), the test results are as follows Figure 6 As shown, the results of the PE-induced cardiomyocyte SOD activity assay showed that compared with the control group, the cell SOD activity of H9c2 cells was significantly reduced after PE stimulation for 24 hours.

[0042] The SOD activity in the heart tissue of TAC model mice was measured. Figure 7 As shown in Figure 3, compared with the sham operation group, the SOD activity in the heart tissue of the TAC group was significantly decreased.

[0043] 3. SOD2 activity detection

[0044] Since SOD2 is mainly present in the mitochondria of cells, the mitochondria of cells and tissues are extracted first. The mitochondrial extraction method is as follows: weigh about 0.1g of tissue or collect 5 million cells, add 1mL Extraction Buffer and 10μLReagentⅡ, homogenize with an ice bath, centrifuge at 600g, 4℃ for 5min, collect the supernatant into a new centrifuge tube, and discard the precipitate. Centrifuge the supernatant again, 11,000g, centrifuge at 4℃ for 10min, and the precipitate is the extracted mitochondria. Discard the supernatant, add 200μLReagentⅠ to the precipitate and resuspend it, and place it on ice for testing. Then use the SOD activity kit to thoroughly mix the sample and detection reagent in a 96-well plate, incubate at 37℃ for 30min, measure the absorbance value A of each well at 450nm, adjust to zero with deionized water, and finally calculate the result from the measured value. (Bradford method is used for protein quantification)

[0045] When the drug source is isoproterenol hydrochloride (ISO), the test results are as follows Figure 8 As shown, the results of the ISO-induced cardiomyocyte SOD2 activity assay showed that compared with the control group, the cell SOD2 activity was significantly reduced after ISO stimulation of H9c2 cells for 24 hours.

[0046] When the drug source is phenylephrine (PE), the test results are as follows Fig. 9 As shown, the results of the PE-induced cardiomyocyte SOD2 activity assay showed that compared with the control group, the cell SOD2 activity was significantly reduced after PE stimulation of H9c2 cells for 24 hours.

[0047] 4. Mouse heart ultrasound detection

[0048] Mouse TAC model (aortic arch stenosis model): 8-10 week old male C57BL / 6J mice were adaptively fed for one week and randomly divided into control group and model group. After intraperitoneal injection of 0.08% sodium pentobarbital for anesthesia, the chest was depilated and the abdomen was fixed with the head facing the operator. The operation was performed under a strong light. A 10 mm longitudinal incision was made at the line connecting the mouse axilla to separate the cortex and expose the ribs. Then a transverse incision was made above the second rib of the mouse, the ribs were cut, the two white thymus were bluntly separated, the left and right common carotid arteries were exposed, and the aortic arch was blindly punctured and ligated at the bilateral common carotid artery branches, followed by a 27-gauge needle and 4-0 silk thread. After ligation, the needle was withdrawn to form a narrowing area to control the aortic arch stenosis rate (60±5%). After the operation, the chest wall layer was closed and sutured with sutures. The surgical operation of the sham group mice was consistent with that of the TAC operation group except that the aortic arch was not ligated. Four weeks after the operation, Doppler ultrasound was used to detect the cardiac function of the mice, including ejection fraction (EF), fractional shortening (FS), reduction of left ventricular posterior systolic wall thickness (LVPWs), and left ventricular end-systolic internal diameter (LVIDs).

[0049] The results of mouse cardiac ultrasound examination showed that the cardiac contractile function of mice was significantly deteriorated after TAC surgery ( Fig.10 A). Compared with the sham operation group, the ejection fraction (EF) and fractional shortening (FS) of mice in the TAC group were significantly decreased ( Fig.10 BC), left ventricular posterior systolic wall thickness (LVPWs) decreased ( Fig.10 D) Increased left ventricular end-systolic diameter (LVIDs) Fig.10 E).

[0050] 5. Detection of the maximum movement distance of mice

[0051] Before the experiment, the mice ran at a speed of 5m / min for 10min on a treadmill with a slope of 5°. The experiment began 3 days after training. During the experiment, the mice first ran at a speed of 5m / min for 4min, then increased the speed to 14m / min. After 2min of exercise, the speed was increased to 16m / min, and so on. Every 2min, the speed was increased by 2m / min. The maximum movement distance of the mice was recorded until the mice were exhausted. The electric shock current was set to 0.4mA, and the exhaustion range was set to 18 electric shocks / 10s.

[0052] The results of the maximum movement distance test of mice showed that the maximum movement distance of mice in the TAC group was significantly shortened compared with that in the sham operation group ( Fig.10 F);

[0053] 6. Specimen collection and WGA staining and MASSON staining

[0054] After the mice were modeled, they were anesthetized with isoflurane and then killed by dislocating the neck. The mouse hearts were perfused with KCI to cause cardiac arrest. Then the heart tissue was removed, and part of the ventricle was immersed in 4% paraformaldehyde for fixation, and the rest was stored at -80°C for later use. The heart tissue of the mouse fixed in 4% paraformaldehyde was taken, embedded in paraffin, sliced, stained with WGA (wheat germ agglutinin) and MASSON (trichrome), stained with nuclei, and sealed, and images were collected under a microscope.

[0055] Based on histological analysis of the cross-sectional area of ​​cardiomyocytes, we found that cardiomyocytes of TAC-operated mice showed hypertrophic lesions compared with those of the sham-operated group ( Fig.10 G, Fig.10 H); and Masson staining results showed that the relative collagen fiber content in the heart tissue of TAC model mice was significantly higher than that in the sham operation group, indicating that significant fibrosis had occurred in their heart tissue ( Fig.10 G, Fig.10 I).

[0056] 7. Detection of tissue ROS content

[0057] Using CheKine TM Mitochondrial ROS production rate fluorescence assay kit (Abbkine, catalog number: KTB1911), the rate of increase of DCF fluorescence intensity is proportional to the ROS production rate. Extract fresh heart tissue or cell mitochondria, mix with the detection reagent in a 96-well plate, and incubate at 37°C in the dark for 15 minutes. After incubation, use a multifunctional microplate reader to measure the fluorescence value within 10 minutes, with an excitation wavelength of 488nm and an emission wavelength of 525nm. The instrument temperature is maintained at 37°C, and the change in fluorescence value within 10 minutes is recorded. The sampling data points, i.e., the change of fluorescence intensity over time, are subjected to linear regression fitting processing, and the regression coefficient, i.e., the slope of the straight line (k), is calculated. The actual mitochondrial ROS production rate is equal to the slope of the linear regression line of the data point of the sample fluorescence intensity change over time (s) minus the slope of the linear regression line of the data point of the background fluorescence intensity change over time (k), k = (RFU10min-RFU0min) / 600. (Bradford method is used for protein quantification)

[0058] The results of ROS determination in mouse heart tissue showed that the reactive oxygen species in heart tissue of TAC group increased significantly ( Fig.10 J).

[0059] 8. Western Blot of cell samples

[0060] When extracting total cell protein, first prepare a mixed lysis solution of IP lysis solution and PMSF with a mixed volume ratio of 100:1; for H9C2 cells cultured on a six-well plate, discard the culture medium, wash with pre-cooled PBS, dry, add 150 μL of mixed lysis solution to each well, scrape the cells at the bottom of the well plate with a cell scraper, and transfer the mixed lysis solution with cell fragments to a centrifuge tube; shake quickly in an ice bath for 30 minutes, centrifuge at 12000rpm for 15 minutes in a 4℃ centrifuge, take the supernatant, and then add 5× protein loading buffer at a ratio of lysis solution: loading buffer = 4:1, mix well by pipetting, and heat in a metal bath at 100℃ for 10 minutes. Then, use the rapid gel preparation kit to prepare polyacrylamide gel, prepare electrophoresis solution and pre-cooled transfer solution, then load the sample, electrophoresis (200V, 35min), transfer to PVDF membrane (300mA, 90min or 200mA, 75min), block with rapid protein-free blocking solution for 20min, wash with 1×PBST, and incubate the primary antibody (1:1000 dilution) in a 4℃ refrigerator overnight. Take out the membrane the next day (10min / time), incubate the secondary antibody corresponding to the primary antibody (1:10000 dilution) at room temperature for 1h, wash three times with 1×PBST after incubation, and finally place the membrane in the developer, add the chemiluminescent solution prepared in 1:1, expose and observe the protein expression.

[0061] When the drug source is isoproterenol hydrochloride (ISO), the test results are as follows Fig.11 and Fig.12 As shown in the figure, the results of the measurement of changes in SOD2 acetylation levels in cardiomyocytes induced by ISO showed that compared with the control group, after ISO stimulation of H9c2 cells for 24 hours, the total protein content of cellular SOD2 had no significant change, and the acetylation level of SOD2 was significantly increased.

[0062] When the drug source is phenylephrine (PE), the test results are as follows Fig.13 and Fig.14 As shown in the figure, the results of the measurement of changes in the acetylation level of SOD2 in cardiomyocytes induced by PE showed that compared with the control group, after ISO stimulation of H9c2 cells for 24 hours, the total protein content of cellular SOD2 had no significant change, and the acetylation level of SOD2 was significantly increased.

[0063] 9. Tissue Sample Western Blot

[0064] When extracting total tissue protein, first prepare a mixed lysis solution of IP lysis solution and PMSF with a mixed volume ratio of 100:1; for mouse tissue, cut the tissue frozen in a -80℃ refrigerator and weigh it, put it into a centrifuge tube with steel balls, then add 10μL of mixed lysis solution per mg of tissue, and grind it in a tissue grinder; after scraping / grinding, shake quickly in an ice bath for 30 minutes, centrifuge at 12000rpm for 15min in a 4℃ centrifuge, take the supernatant, and then add 5× protein loading buffer at a ratio of lysis solution: loading buffer = 4:1, mix well by blowing, and heat in a metal bath at 100℃ for 10 minutes. Then, use the rapid gel preparation kit to prepare polyacrylamide gel, prepare electrophoresis solution and pre-cooled transfer solution, then load the sample, electrophoresis (200V, 35min), transfer to PVDF membrane (300mA, 90min or 200mA, 75min), block with rapid protein-free blocking solution for 20min, wash with 1×PBST, and incubate the primary antibody (1:1000 dilution) in a 4℃ refrigerator overnight. Take out the membrane the next day (10min / time), incubate the secondary antibody corresponding to the primary antibody (1:10000 dilution) at room temperature for 1h, wash three times with 1×PBST after incubation, and finally place the membrane in the developer, add the chemiluminescent solution prepared in 1:1, expose and observe the protein expression.

[0065] The results of the measurement of SOD2 acetylation levels in heart tissue of TAC mice showed that ( Fig.15 , 16 ), compared with the sham operation group, the total SOD2 protein content of mice 4 weeks after TAC surgery did not change significantly, but the SOD2 acetylation level was significantly increased.

[0066] Finally, it should be noted that the above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention. Those skilled in the art should understand that the present invention can still be modified or replaced by equivalents. All technical solutions and improvements that do not depart from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.

Claims

1. Use of drugs that inhibit the increase of SOD2 acetylation level in the preparation of drugs for preventing or treating heart failure with reduced ejection fraction.

2. The use according to claim 1, characterized in that: The drug for inhibiting the increase of SOD2 acetylation level includes any one of tablets, capsules, dripping pills, granules, oral liquids and solutions.

3. The use according to claim 1, characterized in that: The medicine for heart failure with reduced ejection fraction includes any one of tablets, capsules, pills, granules, oral liquids and solutions.

4. The use according to claim 1, characterized in that: The drug contains a pharmaceutically acceptable carrier.

5. The use according to claim 4, characterized in that: The pharmaceutically acceptable carrier is selected from at least one of pharmaceutically acceptable solvents, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrities, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids, release retardants, polymer skeleton materials and film-forming materials.

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