Use of alpha-ketoglutarate salts in the preparation of a medicament for improving diabetic cardiomyopathy

Abstract

The application belongs to the technical field of biological medicine and treatment of cardiovascular and cerebrovascular diseases, and particularly relates to application of alpha-ketoglutarate in preparation of a medicine for improving diabetic cardiomyopathy, and the alpha-ketoglutarate can reduce hyperglycemia and hyperlipidemia, improve insulin resistance and cardiac dysfunction caused by hyperglycemia and insulin resistance, for example, significant reduction of fasting blood glucose insulin and blood lipids, and significant improvement of glucose tolerance and insulin resistance; meanwhile, the alpha-ketoglutarate can effectively improve cardiac systolic dysfunction caused by diabetic cardiomyopathy, and reduce the degree of myocardial tissue pathological damage and fibrosis. The alpha-ketoglutarate is used for the treatment of diabetic cardiomyopathy for the first time, and important basis is provided for using the alpha-ketoglutarate to treat diabetic cardiomyopathy in clinic.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine and treatment of cardiovascular and cerebrovascular diseases, and particularly relates to application of alpha-ketoglutarate in preparation of a medicine for improving diabetic cardiomyopathy. BACKGROUND

[0002] Diabetic cardiomyopathy (DCM) is a primary myocardial injury independent of hypertension and coronary artery disease, mainly characterized by left ventricular hypertrophy and impaired diastolic function in the early stage, and characterized by cardiac fibrosis and systolic dysfunction in the late stage, and finally inducing heart failure, arrhythmia and cardiogenic shock, and even sudden death in severe patients. The pathophysiological processes involved in the pathogenesis of DCM include oxidative stress, endoplasmic reticulum stress, mitochondrial dysfunction, inflammatory response, autophagy, apoptosis and microvascular dysfunction. Hyperglycemia, insulin resistance and metabolic disorders can eventually lead to impaired cardiac function through various mechanisms. According to the latest version of the heart failure treatment guidelines jointly formulated by the American College of Cardiology Foundation (ACCF) and the American Heart Association (AHA), the prognosis of patients with diabetic cardiomyopathy is poor and there is currently no better treatment drug.

[0003] Alpha-ketoglutaric acid (alpha-KG) is an important part of the tricarboxylic acid cycle, which is not only an important metabolic intermediate in the tricarboxylic acid cycle, but also a biosynthetic precursor of various amino acids, vitamins and organic acids such as L-glutamic acid, L-glutamine, L-proline and L-arginine in the body. Animal experiments have also shown its safety for oral administration. Previous studies have reported that exogenous addition of alpha-KG can significantly improve the exercise capacity of normal mice, activate protein synthesis-related signaling pathways, and increase muscle protein synthesis. Alpha-KG can reduce the level of systemic inflammatory cytokines, improve the coat and hair condition, gait and kyphosis of female mice, and help maintain the muscle mass, gait and grip strength of male mice, and prolong the lifespan of both male and female mice. Therefore, alpha-KG is considered an important active metabolic intermediate, which plays an important role in stem cell development, anti-aging, anticancer and energy metabolism regulation. Research and drug development around alpha-KG are currently the forefront of research at home and abroad. A series of studies have shown that alpha-KG has a wide prospect as a drug target in clinical drug development. However, its application in improving and treating diabetic cardiomyopathy has rarely been reported. SUMMARY

[0004] The purpose of the present application is to overcome the shortcomings of the prior art and provide the application of alpha-ketoglutarate in the preparation of a medicine for improving diabetic cardiomyopathy.

[0005] In order to achieve the above-mentioned purpose, the application provides application of alpha-ketoglutarate in preparation of a medicine for improving diabetic cardiomyopathy, wherein the alpha-ketoglutarate can reduce hyperglycemia and hyperlipidemia, improve insulin resistance and cardiac dysfunction caused by hyperglycemia and insulin resistance, for example, significant reduction of fasting blood glucose insulin and blood lipids, significant improvement of glucose tolerance and insulin resistance; meanwhile, the alpha-ketoglutarate can effectively improve cardiac systolic dysfunction caused by diabetic cardiomyopathy, and reduce the degree of myocardial tissue pathological damage and fibrosis.

[0006] Further, the alpha-ketoglutarate is alpha-ketoglutarate sodium salt, alpha-ketoglutarate calcium salt or alpha-ketoglutarate magnesium salt.

[0007] Further, the medicine dosage form is granules, capsules, tablets, powders, oral liquids, suspensions or emulsions.

[0008] The medicine is a medicine or a pharmaceutical composition containing alpha-ketoglutarate, and the administration route of the medicine or the pharmaceutical composition is oral administration.

[0009] Compared with the prior art, the application first uses alpha-ketoglutarate in the treatment of diabetic cardiomyopathy, the alpha-ketoglutarate can improve myocardial hypertrophy, myocardial fibrosis and cardiac systolic function of mice with diabetic cardiomyopathy, and reduce blood glucose of the mice, improve blood lipids and reduce insulin resistance, thereby providing an important basis for using alpha-ketoglutarate to treat diabetic cardiomyopathy in clinic. BRIEF DESCRIPTION OF DRAWINGS

[0010] Figure 1 Fig. 8 is a schematic diagram of the results of OGTT and ITT of the DCM model group mice involved in the application in the eighth week, wherein A is OGTT, B is the area under the OGTT curve, C is ITT, and D is the area under the ITT curve.

[0011] Figure 2 Fig. 9 is a schematic diagram of the experimental results of the effect of alpha-KG calcium salt on cardiac function of DCM mice involved in the application, wherein A is the result of cardiac M-Mode ultrasound in the 24th week, B is LVIDs in the 24th week, C is LVIDd in the 24th week, D is LVEF in the 24th week, and E is LVFS in the 24th week.

[0012] Figure 3 Fig. 10 is a schematic diagram of the experimental results of the effect of alpha-KG calcium salt on body weight and cardiac organ coefficient of DCM mice involved in the application, wherein A is body weight, and B is the ratio of cardiac weight to body weight.

[0013] Figure 4Figures of the experimental results of the influence of the α-KG calcium salt involved in the present application on the blood biochemical indicators, blood glucose, insulin, etc. of the DCM mice, wherein A is an oral glucose tolerance test (OGTT), B is a fasting insulin level (FINS), C is an insulin resistance index (HOMA-IR), D is total cholesterol (TC), and E is triglyceride (TG).

[0014] Figure 5 Figures of the experimental results of the influence of the α-KG calcium salt involved in the present application on the heart tissue pathology of the DCM mice, wherein A is HE staining, and B is Masson staining. DETAILED DESCRIPTION

[0015] The present application will be further described in detail below by means of specific examples and in conjunction with the accompanying drawings.

[0016] Example 1

[0017] This example relates to the influence of α-ketoglutarate on high-fat diet-induced diabetic cardiomyopathy mice. To verify the improvement of α-ketoglutarate on diabetic cardiomyopathy, a diabetic cardiomyopathy mouse model was constructed by using a high-fat diet, and the changes in heart function and tissue structure of the mice after α-ketoglutarate intervention were observed. The specific experimental steps are as follows:

[0018] 1. Establishment and grouping of diabetic cardiomyopathy animal models

[0019] In this example, 50 SPF 8-week-old male C57BL / 6 mice (20±2 g) purchased from Beijing Weitong Lihua were used for testing. All animal experiments were performed in accordance with the Guide for the Animal Experimentation Ethics Review Committee of Qingdao University (Approval No.: 2022-472), and every effort was made to minimize animal suffering. The mice were allowed to eat and drink freely at a temperature of (24±2) °C and a humidity of (50±5) %. Ten mice were randomly selected as a normal control group (NC group) and fed with a 10% fat energy ratio of ordinary feed (D12450J); the remaining 40 mice were fed with a 60% fat energy ratio of high-fat feed (D12492). After 8 weeks, OGTT and ITT were performed on the mice in the two groups, and the experimental procedures and results are as follows:

[0020] (1) At 8 weeks, OGTT was performed on the mice in the normal control group and the model group. After the mice were fasted for 12 h overnight, each mouse was given a 50% glucose solution (1 g / kg body weight) by gavage. At 0 min, 30 min, 60 min, 90 min, and 120 min, respectively, the blood glucose concentration in the tail vein of the mice was measured using a Roche blood glucose meter, and the area under the OGTT curve was calculated according to the following formula. Area under the curve (AUC) (mmol·h-1 ·L -1 ) = 0h glucose value / 2 + 0.5h glucose value + 1h glucose value + 1.5h glucose value + 2h glucose value / 2, the results are shown in Figure 1 As shown in Figure 1 A-1B, compared with the NC group, the area under the OGTT curve of the model group increased significantly, and the difference between the two groups was statistically significant (P<0.05), indicating that the model mice had impaired glucose tolerance, and the blood glucose was maintained at a high level.

[0021] (2) ITT test was performed on normal control group and model group mice. After the mice were fasted for 8h overnight, the initial blood glucose at 0min was measured as the initial blood glucose, and insulin (0.5U / kg) was injected intraperitoneally immediately. The blood glucose value of the tail vein was measured at 30min, 60min, 90min and 120min after injection respectively, and the area under the ITT curve was calculated according to the following formula. Area under the curve (AUC) (mmol·h -1 ·L -1 ) = 0h glucose value / 2 + 0.5h glucose value + 1h glucose value + 1.5h glucose value + 2h glucose value / 2, the results are shown in Figure 1 As shown in Figure 1 C-1D, after 60min of intraperitoneal injection of insulin, the blood glucose of the model mice was significantly different from that of the NC mice (P<0.05), and the area under the ITT curve increased significantly, and the difference between the two groups was statistically significant (P<0.05), indicating that the model group had abnormal insulin secretion and glucose metabolism.

[0022] Further, 30 mice in the model group were randomly selected and divided into: diabetic cardiomyopathy mouse group (DCM model group), α-ketoglutaric acid group (α-KG group) and positive control metformin group (MET group), 10 mice in each group; the DCM model group and the MET group continue to feed high-fat diet (D12492), and the α-KG group is fed with high-fat feed with 2% w / w α-ketoglutarate calcium salt (α-KG calcium salt) and 60% fat energy ratio; the feeding amount of each group of mice is 5g / d. At the same time, the MET group of mice is given metformin (150mg / kg·bw) by gavage every day, and the NC group, the DCM model group and the α-KG group of mice are given the same volume of 0.5% hydroxymethyl cellulose sodium buffer by gavage every day. After 24 weeks of treatment, the heart function of the mice was evaluated by echocardiography, and then the animals were sacrificed, and blood and heart tissue were taken for further study.

[0023] 2, Effect of α-KG calcium salt on heart function of DCM mice

[0024] At week 24, the body weight of each group of mice was measured, and the mice were fasted for 12 h without water restriction, intraperitoneally injected with 50 mg / kg sodium pentobarbital for anesthesia, and subjected to color Doppler ultrasound examination of the heart. After anesthesia, the mice were fixed in a supine position, the chest and abdomen were exposed and shaved, and after cleaning, an appropriate amount of ultrasonic coupling agent was applied. The color Doppler program was started, and the left ventricular anterior wall diastolic thickness (LVAWd), left ventricular anterior wall systolic thickness (LVAWs), left ventricular posterior wall diastolic thickness (LVPWd), left ventricular posterior wall systolic thickness (LVPWs), left ventricular systolic diameter (LVIDs), and left ventricular diastolic diameter (LVIDd) were measured. The left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were calculated, and the results are shown in Figure 2

[0025] Figure 2 A-2C shows the results of M-Mode ultrasound of the hearts of mice in each group at week 24. From Figure 2 As can be seen from the A-2C graph, the LVIDs and LVIDd of the DCM model group mice were significantly higher than those of the NC group (P<0.01), indicating that the left ventricular cavity of the DCM model group mice was enlarged and the ventricular wall was thinned, accompanied by impaired cardiac systolic function; the LVIDs and LVIDd of the α-KG group mice were significantly lower than those of the DCM model group mice (P<0.05).

[0026] The results of the determination of the left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) are shown in Figure 2 As can be seen from the D-2E graph, compared with the NC group, the LVEF and LVFS of the DCM model group mice were significantly reduced; the LVEF and LVFS of the α-KG group and the positive control MET group mice were significantly higher than those of the DCM model group, indicating that α-KG calcium salt and MET could effectively improve the left ventricular ejection fraction and left ventricular fractional shortening.

[0027] These results show that α-KG calcium salt can significantly improve the left ventricular systolic function of DCM mice.

[0028] 3. Effect of α-KG calcium salt on blood biochemistry, blood glucose, and insulin levels of DCM mice

[0029] Before sacrificing the mice at week 24, the mice in each group were fasted for 12 h overnight, and blood was collected from the tail vein of the mice using a Roche glucometer for an oral glucose tolerance test (OGTT), and the area under the curve was calculated by formula. Three days later, the mice were fasted and blood was collected from the eye, and the fresh sample was placed in a 4°C low-temperature centrifuge, centrifuged at 3000 rpm for 10 min, and the supernatant was taken and stored in an 80°C refrigerator for detection of total cholesterol (TC), triglycerides (TG), fasting blood glucose (FBG), and fasting insulin (FINS) in the blood, and calculation of the insulin resistance index (HOMA-IR), and the results are shown in​Figure 4 A shows the OGTT test results of the mice in each group at the 24th week.

[0030] Figure 4 A shows the OGTT test results of the mice in each group at the 24th week. Figure 4 As can be seen from A, the area under the OGTT curve of the DCM model group was significantly higher than that of the NC group (P < 0.01), indicating that the mice in the DCM model group had abnormal glucose tolerance during the entire experimental period; the area under the OGTT curve of the α-KG group was significantly lower than that of the DCM model group (P < 0.01), indicating that α-KG calcium salt can increase the sensitivity of insulin and improve the abnormal glucose tolerance phenomenon.

[0031] Figure 4 B-4C shows the fasting insulin (FINS) and insulin resistance index (HOMA-IR) test results of the mice in each group. From Figure 4 As can be seen from B-4C, compared with the DCM group, the fasting insulin (FINS) and insulin resistance index (HOMA-IR) of the mice in the α-KG group were significantly reduced, indicating that α-KG calcium salt can significantly reduce the insulin resistance index of DCM mice, and the increase in insulin resistance index is closely related to the occurrence and development of DCM, so α-KG calcium salt can be used to treat DCM.

[0032] Figure 4 D-4E shows the total cholesterol (TC) and triglyceride (TG) level test results of the mice in each group. From Figure 4 As can be seen from D-4E, compared with the NC group, the TC and TG levels of the mice in the DCM model group were significantly increased (P < 0.01); the TC and TG levels of the mice in the α-KG group were significantly lower than those in the DCM model group (P < 0.01). This indicates that α-KG calcium salt can reduce the TC and TG levels of DCM mice.

[0033] 4. Effect of α-KG calcium salt on body weight and heart organ coefficient of DCM model mice

[0034] After successful modeling at the 8th week, the body weight of the mice in each group was measured every week, and the results are shown in Figure 3 A. After 24 weeks of treatment, the mice in each group were taken blood from the eyeball, the heart was dissected and weighed, and the heart weight / body weight ratio of each mouse was calculated, and the results are shown in Figure 3 B.

[0035] From Figure 3As can be seen from A, there was no significant difference in the baseline body weight of mice in the DCM model group, the a-KG group and the MET group at the 8th week, but the body weight of mice in the three groups was significantly higher than that in the NC group (P<0.01); after the 8th week, the body weight of mice in the a-KG group and the MET group was significantly lower than that in the DCM model group (P<0.01), indicating that the a-KG calcium salt effectively reduced the body weight of the diabetic cardiomyopathy mice, that is, the a-KG calcium salt can be used to improve DCM.

[0036] From Figure 3 As can be seen from B, the heart weight / body weight ratio of the DCM model group was significantly higher than that of the NC group (P<0.05); while the heart weight / body weight ratio of mice in the a-KG group and the MET group was significantly lower than that in the DCM model group (P<0.01), indicating that the a-KG calcium salt treatment can reduce the heart weight / body weight ratio of the diabetic cardiomyopathy mice, and the heart weight / body weight ratio is related to the pathological myocardial hypertrophy of DCM, so the a-KG calcium salt can be used to improve the pathological myocardial hypertrophy of DCM mice.

[0037] 5. Detection of the effect of a-KG calcium salt on the heart tissue pathology of DCM mice

[0038] At the 24th week, the mice in each group were sacrificed, the heart was dissected, the tissue at the same part of the heart apex was taken, the ventricular muscle was quickly separated and placed in 10% neutral formalin buffer for 24h fixation, and after conventional treatment, embedding, sectioning, the thickness was 2μm, then hematoxylin-eosin (HE) staining and Masson staining were performed, conventional sectioning was performed, and the section was scanned by a digital scanner. Semi-quantitative analysis was performed by using Image-Pro plus 6.0 image analysis software, and the results are shown in Figure 5 .

[0039] Figure 5 A-5B shows the heart tissue pathology results of mice in the NC group, the DCM model group, the a-KG group and the MET group [Figure A: HE staining (x400); Figure B: Masson staining (x400)]. The myocardial fibers of the normal control group mice were arranged in order, the cell structure was clear, the cell nucleus size was uniform, and the collagen fibers between the myocardium were few; the myocardial fibers of the DCM model group mice were arranged in disorder, the myocardial cells were hypertrophic and degenerative, and the collagen fibers around the blood vessels were obviously increased; compared with the DCM model group, the myocardial cell hypertrophy and collagen arrangement disorder of mice in the a-KG group and the MET group were improved, and the collagen fibers between the myocardium were obviously reduced. Therefore, it is proved that the a-KG calcium salt can obviously improve the myocardial cell hypertrophy and myocardial fibrosis of DCM mice.

Claims

1. Use of alpha-ketoglutarate salt for the preparation of a medicament for improving diabetic cardiomyopathy, characterized in that, The diabetic cardiomyopathy is a heart dysfunction caused by diabetes induced by a high-fat diet.

2. The use of α-ketoglutarate salt according to claim 1 for the preparation of a medicament for ameliorating diabetic cardiomyopathy, characterized in that, The alpha-ketoglutarate salt can effectively improve the heart dysfunction caused by hyperglycemia and insulin resistance, increase the left ventricular ejection fraction and systolic and diastolic functions of the heart, and slow down the myocardial hypertrophy and fibrosis; the alpha-ketoglutarate salt can also reduce hyperglycemia and hyperlipidemia, and improve insulin resistance.

3. The use of α-ketoglutarate salt according to claim 1 for the preparation of a medicament for ameliorating diabetic cardiomyopathy, characterized in that, The alpha-ketoglutarate salt is alpha-ketoglutarate sodium salt, alpha-ketoglutarate calcium salt or alpha-ketoglutarate magnesium salt.

4. The use of α-ketoglutarate salt according to claim 1 for the preparation of a medicament for ameliorating diabetic cardiomyopathy, characterized in that, The dosage form of the medicine is granules, capsules, tablets, powders, oral liquids, suspensions or emulsions.

5. The use of α-ketoglutarate salt according to claim 1 for the preparation of a medicament for ameliorating diabetic cardiomyopathy, characterized in that, The medicine is a medicine or a pharmaceutical composition containing the alpha-ketoglutarate salt, and the administration route of the medicine or the pharmaceutical composition is oral administration.

Images