Application of sarcosine in preparation of medicine for treating ischemic heart disease

Abstract

The invention relates to the technical field of biological medicines, and particularly discloses application of sarcosine in preparation of a medicine for treating ischemic heart disease. According to the technical scheme provided by the invention, the expression of HIF1-alpha protein can be remarkably increased through sarcosine, so that angiogenesis is enhanced, the heart function of the ischemic heart disease is improved, ventricular remodeling is relieved, and the ischemic heart disease can be effectively prevented and / or treated.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to the use of sarcosine in the preparation of a drug for treating ischemic heart disease. BACKGROUND

[0002] Ischemic heart disease is the most common fatal cardiovascular disease, and the incidence and mortality are increasing year by year. About 180 million people died of cardiovascular disease in 2019, accounting for 32% of global deaths, and it has been established as the leading cause of death from all diseases.

[0003] Ischemic heart disease is a heart disease caused by coronary artery stenosis or obstruction leading to insufficient blood supply to the myocardium. Large area of myocardial ischemia infarction will cause serious complications such as ventricular arrhythmia and heart rupture, leading to patient death. Only about 10% of patients can survive from large area of myocardial infarction, but due to the lack of endogenous regeneration ability of myocardial cells in adult mammals, the infarct area is often repaired by scar formation and ventricular remodeling, so these patients often have severe heart failure symptoms in the late stage. With the progress of medicine, vascular recanalization and drug therapy have reduced the mortality rate of patients after myocardial ischemia, but restoring normal cardiac function and maintaining normal cardiac structure after myocardial ischemia death is still a worldwide problem. Therefore, exploring the pathophysiological mechanism of myocardial repair of ischemic heart disease and finding effective intervention targets and treatment strategies are of great significance for improving cardiac function after myocardial ischemia, reducing cardiac death, and improving the survival time and quality of life of myocardial infarction patients.

[0004] Studies have found that angiogenesis after cardiac ischemia is one of the important endogenous mechanisms for maintaining myocardial viability during cardiac ischemia, and is considered one of the predictors of long-term survival of patients. Promoting angiogenesis can improve the prognosis of myocardial ischemia by increasing blood supply to the peripheral area of cardiac infarction.

[0005] Sarcosine is an amino acid catalyzed by glycine or dimethyl glycine. Studies have shown that the level of sarcosine in circulating blood decreases with age, and exogenous supplementation of sarcosine can activate autophagy and enhance the level of autophagy in the body, so sarcosine can be used as a biomarker of aging. In addition, exogenous supplementation of sarcosine can rapidly activate the AMPAR-mTOR signaling pathway and GluR1 phosphorylation to play an antidepressant role, but its role and specific mechanism in ischemic heart disease have not been reported. It is of great significance to clarify the therapeutic effect of sarcosine in ischemic heart disease. SUMMARY

[0006] The technical problem to be solved by the present application is to provide the use of sarcosine in the preparation of a drug for treating ischemic heart disease in view of the deficiencies of the prior art.

[0007] To solve the above technical problems, the present application discloses the following technical solutions: In the first aspect, the present application discloses the use of sarcosine in the preparation of a drug for preventing and / or treating ischemic heart disease.

[0008] In some embodiments, the sarcosine is used as the only effective ingredient in the preparation of the drug for preventing and / or treating ischemic heart disease.

[0009] In some embodiments, the prevention and / or treatment of ischemic heart disease includes reducing ventricular remodeling.

[0010] In some embodiments, the prevention and / or treatment of ischemic heart disease includes improving heart function.

[0011] In some embodiments, the prevention and / or treatment of ischemic heart disease includes reducing myocardial necrosis.

[0012] In some embodiments, the prevention and / or treatment of ischemic heart disease includes increasing the expression of HIF1-a protein.

[0013] In the second aspect, the present application discloses a drug for preventing and / or treating ischemic heart disease.

[0014] In some embodiments, the effective ingredient of the drug includes sarcosine; in some embodiments, the effective ingredient of the drug only contains sarcosine, i.e., sarcosine is the only effective ingredient of the drug; in some embodiments, the effective ingredient of the drug includes other effective ingredients in addition to sarcosine, such as other effective ingredients that can be used for preventing and / or treating ischemic heart disease.

[0015] In some embodiments, the prevention and / or treatment of ischemic heart disease includes reducing ventricular remodeling.

[0016] In some embodiments, the prevention and / or treatment of ischemic heart disease includes improving heart function.

[0017] In some embodiments, the prevention and / or treatment of ischemic heart disease includes reducing myocardial necrosis.

[0018] In some embodiments, the prevention and / or treatment of ischemic heart disease includes increasing the expression of HIF1-a protein.

[0019] The present application has the following beneficial effects:

[0020] The technical solution provided by the present application can significantly increase the expression of HIF1-α protein through sarcosine, thereby enhancing angiogenesis to improve the heart function of ischemic heart disease and reduce ventricular remodeling, so that ischemic heart disease can be effectively prevented and / or treated. BRIEF DESCRIPTION OF DRAWINGS

[0021] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0022] Figure 1 The vertical and horizontal axes of echocardiograms of C57 / BL6 mice at 28 days after treatment, representing different treatment groups.

[0023] Figure 2 Statistical plot of left ventricular ejection fraction (LVEF) and left ventricular shortening fraction (FS) data in mice (n = 17).

[0024] Figure 3 The images show representative HE and Masson staining images of mouse hearts, and a statistical graph of left ventricular thickness in mice from different treatment groups (n = 5) (**P<0.01).

[0025] Figure 4 The expression and statistical graph of HIF1-α in the myocardial tissue of mice in different treatment groups (n = 2) (**P<0.01). Detailed Implementation

[0026] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the descriptions in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as detailed in the claims.

[0027] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; the reagents and materials described are commercially available unless otherwise specified.

[0028] Example 1:

[0029] A mouse model of ischemic heart disease is ligation of the left anterior descending artery. This example provides a model of ischemic heart disease and investigates it by injecting creatine solution, as detailed below.

[0030] 1. Mouse feeding and drug preparation All mice were housed in strictly regulated laboratory animal facilities, with no more than 5 mice per cage, and provided with lighting (a 12-hour light and 12-hour dark cycle), suitable temperature (24 ± 2 °C), humidity (50 ± 10%), and sufficient food and water.

[0031] The creatine solution is prepared fresh daily (concentration of 50 mg / mL, solvent is physiological saline).

[0032] 2. Mouse grouping and administration To clarify the role of creatine in treating ischemic heart disease by promoting angiogenesis, mice were divided into four groups: the Sham group (surgical procedures were the same as the MI group except for ligation of the left anterior descending artery), the Sarcosine group (surgical procedures were the same as the MI group except for ligation of the left anterior descending artery, with intraperitoneal injection of creatine starting on the day of surgery), the MI group (permanent ligation of the left anterior descending artery), and the MI+Sarcosine group (permanent ligation of the left anterior descending artery, with intraperitoneal injection of creatine starting on the day of surgery). Details are as follows: MI group: 9-week-old male C57BL / 6 mice were anesthetized by intraperitoneal injection of 1.25% tribromoethanol (500ml / kg) before surgery. Tracheal intubation and ventilation were performed before thoracotomy. During surgery, the left anterior descending branch was permanently ligated through a left thoracic incision, and specific toes were removed for marking. After the experiment, the chest was closed layer by layer, and the mice were placed in a warm blanket to await recovery.

[0033] MI+Sarcosine group: Permanent ligation of the anterior descending branch was performed, and intraperitoneal injection of creatine was started on the same day after the operation, with daily intraperitoneal injections (500 mg / kg).

[0034] Sham group: Except for coronary artery ligation, the rest of the surgical procedure was the same as that of MI group.

[0035] Sarcosine group: Except for ligation of the left anterior descending artery, the other surgical procedures were the same as those in the MI group. Sarcosine was injected intraperitoneally on the day of surgery and administered intraperitoneally at regular intervals every day (500mg / kg).

[0036] 3. Mouse ultrasound Baseline echocardiographic data were collected from mice three days prior to modeling, and data were collected on days 7, 14, and 28 after modeling. Mice were induced to anesthetize using gas anesthesia (1.5% isoflurane and 1 L / min O2). After anesthesia, cardiac function was assessed using a Vevo 770 imaging system (VisualSonics, Toronto, Canada) with a 30 MHz transducer. The long axis of the mice was measured in B-mode to observe ventricular enlargement, and the short axis view recorded in M-mode was used to measure parameters such as LVEF (%), LVFS (%), D:s, D:d, LVPW:s, and LVPW:d.

[0037] 4. Obtaining mouse heart tissue and performing HE and Masson staining To further explore whether creatine can alleviate ventricular remodeling in ischemic heart disease, we performed paraffin embedding, sectioning, and HE and MASSON staining on heart tissues from different groups of mice.

[0038] (1) After anesthetizing mice with an overdose of 1.25% tribromoethanol solution, intact heart tissue was obtained by dissection and immediately immersed in 4% PFA for fixation for 4-24 hours. After fixation, the tissue was dehydrated so that it could be embedded in water-insoluble paraffin. The tissue was gradually dehydrated by immersing it in a dehydrating agent of increasing concentration, such as ethanol. Then, it was incubated in xylene to replace the dehydrating agent and to make the tissue transparent. The paraffin was heated to 60°C to embed the tissue, and then allowed to harden overnight. The tissue was then sectioned using a microtome.

[0039] (2) HE staining: Paraffin sections were dewaxed to water, rinsed with ddH2O, and then immersed in hematoxylin staining solution for 3 minutes to stain the nuclei. The sections were then rinsed in running water for 1 minute. Next, the sections were immersed in 1% hydrochloric acid-ethanol solution for 8 seconds to induce differentiation, followed by immersion in ammonia water for 8 seconds for inversion blue staining, and then rinsed in running water for 1 minute. The cytoplasm was then stained with 1% eosin solution for 1 minute, followed by rinsing in running water for 1 minute. The sections were dehydrated with anhydrous ethanol, cleared with xylene, and mounted with neutral resin.

[0040] (3) Masson staining: Paraffin-embedded tissue blocks fixed in paraformaldehyde were sectioned at a thickness of 5 μm using a microtome for Masson's three-color histochemical analysis. Differences in the multicolor staining scheme revealed extracellular matrix components: the collagen matrix appeared sky blue, cytoplasmic elements showed eosinophilic granulocyte affinity, and nuclear chromatin showed basophilic characteristics.

[0041] 5. Tissue protein homogenate lysis and Western blotting To clarify whether creatine enhances angiogenesis and thus reduces myocardial necrosis and left ventricular remodeling by upregulating HIF1-a expression, we validated this at both the mRNA and protein levels. After 285 days of echocardiography, the heart was dissected, and cardiac tissue proteins were extracted and subjected to Western blotting experiments.

[0042] (1) Dissect the mouse aortic tissue using clean instruments, place the tissue in centrifuge tubes and immerse them in liquid nitrogen for quick freezing, then place them on ice and homogenize immediately. Add 300 μL of lysis buffer to every 5 mg of tissue, homogenize with an electric homogenizer, and shake continuously at 4°C for 2 hours.

[0043] (2) Centrifuge at 11,200 rpm (12,000 × g) at room temperature for 15 min. Gently remove the centrifuge tube from the centrifuge and place it on ice. Aspirate the supernatant and transfer it to a new tube that has been pre-cooled on ice. Discard the precipitate.

[0044] (3) Take a small amount of lysis buffer for protein quantitative analysis, and add 1 / 4 volume of 5× loading buffer to the remaining lysis buffer and boil at 100°C for 5 minutes for reduction and denaturation.

[0045] (4) Gel running: Load equal amounts of protein and molecular weight markers into the wells of an SDS-PAGE gel. The total protein loading amount of aortic tissue homogenate is 20-30 μg, and run the gel at 100 V for 1-2 hours.

[0046] (5) Transfer: PVDF was activated with methanol for 1 minute and rinsed with transfer buffer before preparing the transfer sandwich structure. The transfer time and voltage were optimized according to the molecular weight of the target protein.

[0047] (6) Blocking: After the transfer is completed, remove the PVDF membrane and immerse it in TBST (TBS with Tween-20 added at a concentration of 0.05%) containing 5% skim milk powder for 1 h.

[0048] (7) Primary antibody incubation: After blocking, incubate the PVDF membrane in blocking buffer with a primary antibody of appropriate dilution overnight at 4°C on a shaker.

[0049] (8) Secondary antibody conjugation: Wash the membrane with TBST for 10 min × 3 times. Incubate the membrane with the conjugated secondary antibody at the recommended dilution in blocking buffer at room temperature for 1 hour. Wash the membrane again with TBST for 10 min × 3 times.

[0050] (9) ECL color development: Mix ECL color development solution A and B and add them evenly to the membrane surface. Expose the membrane to light and observe the results.

[0051] 6. Experimental Results (1) Echocardiography of C57 / BL6 mice at 28 days Figure 1 As shown, echocardiographic data revealed that creatine significantly reduced ventricular remodeling in the MI+Sarcosine group compared to the MI group. Cardiac function data of mice in different groups at 0, 7, 14, and 28 days were statistically analyzed and plotted as line graphs. The results showed that creatine administration had no significant difference in cardiac function in mice without ischemic heart disease, but significantly improved cardiac function in mice with ischemic heart disease. Figure 2 ).

[0052] (2) HE staining and Masson staining images of mouse hearts, and statistical analysis of left ventricular thickness in mice under different treatment groups, as shown below. Figure 3 As shown, the results indicate that ischemic heart disease mice injected intraperitoneally with creatine have a smaller area of ​​myocardial necrosis compared to mice with ischemic heart disease alone.

[0053] (3) The expression of HIF1-α in the myocardial tissue of mice in different treatment groups is as follows: Figure 4As shown, the results indicate that HIF1-a protein expression was significantly increased in mice with ischemic heart disease model after administration of creatine. Figure 4 ).

[0054] The above experimental results sufficiently demonstrate that creatine can treat ischemic heart disease by upregulating HIF-1α expression, thereby reducing the area of ​​myocardial necrosis and left ventricular remodeling. Increasing creatine intake can effectively reduce the incidence of heart failure following ischemic heart disease. Therefore, we believe that creatine can serve as a new and important drug for the clinical treatment of ischemic heart disease, and has potential clinical application value in the prevention and treatment of ischemic heart disease.

[0055] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. Use of sarcosine in the preparation of drugs for the prevention and / or treatment of ischemic heart disease.

2. The use according to claim 1, characterized in that, The prevention and / or treatment of ischemic heart disease includes reducing ventricular remodeling.

3. The use according to claim 1, characterized in that, The prevention and / or treatment of ischemic heart disease includes improving cardiac function.

4. The use according to claim 1, characterized in that, The prevention and / or treatment of ischemic heart disease includes reducing myocardial necrosis.

5. The use according to claim 1, characterized in that, The prevention and / or treatment of ischemic heart disease includes increasing the expression level of HIF1-a protein.

6. A drug for the prevention and / or treatment of ischemic heart disease, characterized in that, The active ingredient in the drug includes sarcosine.

7. The drug according to claim 6, characterized in that, The prevention and / or treatment of ischemic heart disease includes reducing ventricular remodeling.

8. The drug according to claim 6, characterized in that, The prevention and / or treatment of ischemic heart disease includes improving cardiac function.

9. The drug according to claim 6, characterized in that, The prevention and / or treatment of ischemic heart disease includes reducing myocardial necrosis.

10. The medicament according to claim 6, characterized in that, The prevention and / or treatment of ischemic heart disease includes increasing the expression level of HIF1-a protein.

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