Application of plant hormone mitochondrial acid 5 in preparation of medicine for treating myocardial injury caused by diabetes

Abstract

The invention provides application of plant hormone mitochondrial acid 5 in preparation of a medicine for treating myocardial injury caused by diabetes, and relates to the technical field of biological medicine. In the aspects of blood sugar and blood fat, the MA-5 reduces the blood sugar and blood fat levels of the mouse with the type 2 diabetes mellitus; in the aspect of cardiac injury involved by diabetes, MA-5 inhibits cardiac tissue fibrosis, improves mitochondrial injury, inhibits oxidative stress and reduces ROS (reactive oxygen species) generation. The MA-5 has the effects of stably reducing blood glucose and protecting diabetic cardiomyopathy. The composition is simple and clear; the taking is convenient; the price is low, and the like. Compared with the existing medicines for treating diabetes and diabetic cardiomyopathy, the chemical components of the MA-5 are simple and clear, the action mechanism of the MA-5 is easier to clarify in pharmacological research, and the quality of the medicines is easier to control in production. According to the application, a powerful experimental basis and a theoretical basis are provided for preventing and treating the type 2 diabetes mellitus and myocardial complications of the type 2 diabetes mellitus by the MA-5.

Description

Technical Field

[0001] The present application relates to the field of biomedicine technology, and in particular, to the use of the plant hormone mitochondrial acid 5 in the preparation of a drug for treating myocardial damage caused by diabetes. Background Art

[0002] Cardiomyopathy is a group of heterogeneous myocardial diseases. Myocardial lesions caused by different causes (genetic causes are more common, but there are also acquired factors) lead to myocardial mechanical and / or electrical dysfunction, often manifested as ventricular hypertrophy or dilation. Based on pathophysiology, etiology and pathogenic factors, cardiomyopathy is divided into dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy and unclassified cardiomyopathy.

[0003] At present, the clinical treatment methods for different types of cardiomyopathy are different. For dilated cardiomyopathy, the main treatment is to improve cardiac function. Commonly used drugs include diuretics (such as furosemide) which are mainly used to reduce the preload of the heart and relieve symptoms such as edema and dyspnea; angiotensin-converting enzyme inhibitors (such as enalapril) improve myocardial remodeling; beta-blockers (such as metoprolol) reduce myocardial oxygen consumption, etc. Hypertrophic cardiomyopathy mainly reduces left ventricular outflow tract obstruction and improves symptoms. Beta-blockers or non-dihydropyridine calcium channel blockers (such as verapamil) can be used; because patients with hypertrophic cardiomyopathy are prone to arrhythmias, especially ventricular arrhythmias and atrial fibrillation. Amiodarone can effectively prevent and treat arrhythmias and reduce the risk of sudden death. Restrictive cardiomyopathy is mainly treated with calcium channel blockers (such as diltiazem) ) and beta-blockers (such as atenolol) can improve myocardial diastolic function and reduce myocardial stiffness. Arrhythmogenic right ventricular cardiomyopathy can be mainly used to prevent and control ventricular arrhythmias through drugs such as sotalol and amiodarone. Beta-blockers (such as metoprolol) can reduce myocardial excitability and the occurrence of arrhythmias, and also have a certain protective effect on right ventricular function.

[0004] Diabetic cardiomyopathy (DCM) refers to a myocardial disease that occurs in diabetic patients and cannot be explained by hypertensive heart disease, coronary atherosclerotic heart disease, valvular heart disease and other heart diseases. It is a chronic complication of diabetes. The pathogenesis is relatively complex, mainly including metabolic disorders caused by hyperglycemia (such as increased glycosylation end products, activation of polyol pathways, activation of protein kinase C, etc.), increased oxidative stress, imbalance of calcium homeostasis in myocardial cells, microvascular lesions and other factors, which work together to cause damage to the structure and function of myocardial cells. For the treatment of DCM, controlling blood sugar is the key, and strict blood sugar control can delay the progression of myocardial lesions. At the same time, treatment should also be carried out for myocardial lesions, such as improving myocardial blood supply and reducing myocardial fibrosis.

[0005] There is no effective cure for DCM, and patients need to take medication all the time to relieve symptoms. At present, traditional drugs used clinically for DCM include blood sugar control drugs, including biguanides (such as metformin), sulfonylureas (such as glibenclamide), glinides (such as repaglinide), α-glucosidase inhibitors (such as acarbose), thiazolidinediones (such as pioglitazone), DPP-4 inhibitors (such as sitagliptin) and SGLT-2 inhibitors (such as dapagliflozin). These drugs lower blood sugar through different mechanisms and reduce the damage of high blood sugar to myocardial cells. However, they often have side effects of varying degrees, such as headache, fatigue, diarrhea, hypoglycemia, weight gain, and jaundice may occur in some patients. There are also drugs for the treatment of myocardial lesions, such as drugs to improve myocardial remodeling, including angiotensin converting enzyme inhibitors (ACEI), angiotensin II receptor antagonists (ARB) and β-blockers. Drugs that improve myocardial metabolism include trimetazidine and L-carnitine. When heart failure occurs, anti-heart failure drugs are used. The disadvantages of this type of drug are that it can cause dry cough, angioedema, aggravate bronchospasm, electrolyte imbalance, etc. Therefore, the research on innovative DCM drugs that are highly effective, low-toxic and easy to use is of great significance. Summary of the invention

[0006] In order to make up for the shortcomings of the prior art, the purpose of the present invention is to provide an application of the plant hormone mitochondrial acid 5 (MA-5) for the protective effect on the heart of type 2 diabetic mice. MA-5 has the effect of preventing and treating heart damage in type 2 diabetes and can be used to prepare drugs for the treatment of diabetes and diabetic cardiomyopathy.

[0007] In order to solve the above technical problems, the technical solution adopted in this application is:

[0008] The present application provides the use of plant hormone mitochondrial acid 5 in the preparation of a drug for treating myocardial damage caused by diabetes. The drug is an oral dosage form and further includes medically acceptable excipients.

[0009] Furthermore, the plant hormone mitochondrial acid 5 of the present application improves and treats diabetic myocardial damage by improving mitochondrial damage and inhibiting oxidative stress.

[0010] Compared with the prior art, the embodiments of the present application have at least the following advantages or beneficial effects:

[0011] After research, the present application discovered for the first time that MA-5 has the effect of preventing and treating diabetes and diabetic cardiomyopathy: in terms of blood sugar and blood lipids, MA-5 reduces the blood sugar and blood lipid levels of type 2 diabetic mice; in terms of heart damage caused by diabetes, MA-5 inhibits cardiac tissue fibrosis, MA-5 improves mitochondrial damage, inhibits the occurrence of oxidative stress, and reduces the generation of ROS. MA-5 has a stable blood sugar-lowering effect and has a protective effect on diabetic cardiomyopathy. It has the advantages of definite efficacy, simple and clear composition, convenient administration and low price. Compared with existing drugs for the treatment of diabetes and diabetic cardiomyopathy, MA-5 has a simple and clear chemical composition, which makes it easier to clarify its mechanism of action in pharmacological research and easier to control the quality of drugs in production. The present application provides a strong experimental basis and theoretical basis for the use of MA-5 in the prevention and treatment of type 2 diabetes and its myocardial complications, and broadens its application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present application and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0013] Figure 1 This is a graph showing the effect of MA-5 on blood glucose and blood lipid levels in diabetic mice;

[0014] Figure 2 This is a diagram showing the effect of MA-5 on myocardial structure and myocardial fibrosis in diabetic mice;

[0015] Figure 3 This is a diagram showing the effect of MA-5 on myocardial mitochondrial damage in diabetic mice;

[0016] Figure 4 The imaging diagram and DHE fluorescence density measurement results of the MA-5 in the present application example on the oxidative stress level in the myocardial tissue of diabetic mice;

[0017] Figure 5 This is a diagram showing the effect of MA-5 in the present application on mitochondrial fission and fusion-related proteins in the myocardial tissue of diabetic mice;

[0018] Figure 6 This is a diagram showing the effect of MA-5 of the present application example on pyroptosis-related proteins in the myocardial tissue of diabetic mice. DETAILED DESCRIPTION

[0019] In order to make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the technical scheme in the embodiments of the present application will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0020] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present application will be described in detail below with reference to specific embodiments.

[0021] This application uses a diabetic model mouse to give different concentrations of MA-5 intervention to observe whether it has a protective effect on the myocardium and explore the specific mechanism, providing a theoretical basis and experimental support for the development of MA-5 to treat the disease. The specific research steps are:

[0022] Step 1: Synthesize MA-5 by chemical method.

[0023] Step 2, select SPF-grade C57BL / 6J male mice weighing 16-18g, and after one week of adaptive feeding with ordinary feed, use a high-sugar and high-fat diet combined with streptozotocin to simulate the myocardial injury model of type 2 diabetic mice; the mice are randomly divided into a normal control group and a diabetic group; the normal control group is fed with ordinary feed, and the diabetic group is fed with a high-sugar and high-fat feed, and the feeding lasts for 16 weeks.

[0024] Step 3: The diabetic mice were fasted for 10-12 h and given STZ at a dose of 100 mg kg on the first and fifth days. -1 ·d -1 The mice were injected with tail vein, and the normal control group was given an equal volume of citric acid-sodium citrate buffer. Blood was collected from the tail vein of the mice at 3d and 7d after STZ injection for random blood glucose measurement. The model was considered to be successfully established when the random blood glucose was ≥16.7mmol / L twice, and the mice that did not form the model were screened out.

[0025] Step 4: Oral gavage was started and the mice were randomly divided into six groups: normal control group (Control group, n = 15), diabetic model group (DM group, n = 15), MA-5 low-dose group (DM + 25 mg kg -1 MA-5 group, n = 15), MA-5 medium dose group (DM + 50 mg kg -1 MA-5 group, n = 15), MA-5 high-dose group (DM + 100 mg kg -1 MA-5 group, n = 15) and MA-5 high-dose normal control group (Control + 100 mg kg -1The normal group and the MA-5 high-dose control group continued to be fed with ordinary feed for 8 weeks, but the MA-5 high-dose normal control group was given MA-5 by gavage at the same time, and the mice in other groups were still fed with high-sugar and high-fat feed and treated with different concentrations of MA-5 by gavage at the same time for 8 weeks.

[0026] Step 5, observe and record general conditions such as baby weight.

[0027] Step 6: At the end of the experiment, the mice are processed to obtain blood and heart tissue.

[0028] Step 7, calculate organ coefficients such as tibia coefficient (HeartWeight / Tibia Length, HW / TL) and heart coefficient (HeartWeight / Body Weight, HW / BW).

[0029] Step 8, detecting blood glucose and blood lipid levels of mice.

[0030] Step 9: H&E staining was used to observe the pathological changes of mouse myocardial tissue; Sirius red staining was used to observe the level of myocardial fibrosis in mice; and transmission electron microscopy was used to observe the ultrastructural changes of mouse myocardial mitochondria.

[0031] Step 10, detecting the oxidative stress level of the mouse myocardial tissue.

[0032] Step 11, detecting the expression levels of mitochondrial fission and fusion-related proteins in mouse myocardial tissue.

[0033] Step 12, detecting the expression level of pyroptosis-related proteins in mouse myocardial tissue.

[0034] Step 13, statistical analysis.

[0035] The features and performance of the present application are further described in detail below in conjunction with the embodiments.

[0036] Example

[0037] 1. Chemical method for preparing mitochondrial acid 5

[0038] Aluminum chloride (2.7 g, 20 mmol) was slowly added to a CH2Cl2 (40 mL) solution of maleic anhydride (0.98 g, 10 mmol) and 1,3-difluorobenzene (1.14 g, 10 mmol) and stirred at room temperature for 4 h. 1N HCl (100 mL) was poured into the reaction mixture and the product was extracted with ethyl acetate (100 mL). Then, the organic layer was washed with saturated NH4Cl solution and brine and dried over Na2SO4. Using benzene as the starting material, (E)-4-(2,4-difluorophenyl)-4-oxobut-2-enoic acid (yield 75%) was obtained by recrystallization. Equimolar acrylic acid (2.12 g, 10 mmol) and indole (1.16 g, 10 mmol) were refluxed in benzene (25 mL) for 8 h, and the solvent was evaporated after the reaction. The residue was subjected to silica gel column chromatography to obtain 4-(2,4-difluorophenyl)-2-(1H-indiol-3-yl)-4-oxobutyric acid, namely, mitochondrial acid 5 (MA-5) in a yield of 54%.

[0039] The reaction equation is as follows:

[0040]

[0041] 1 H NMR (400MHz, DMSO): δ12.23(s,1H),11.01(s,1H),7.99(td,J=8.7,6.8Hz,1H),7.64(d, J=7.9Hz,1H),7.43(ddd,J=11.7,9.3,2.4Hz,1H),7.37(d,J=8.1Hz,1H),7.27(t,J=2.5H z,1H),7.24(dd,J=8.4,2.4Hz,1H),7.05(dtd,J=15.9,7.1,1.0Hz,2H),4.34(dd,J=10.5 ,3.9Hz,1H),3.90(ddd,J=18.4,10.6,2.8Hz,1H),3.41-3.31(m,4H),3.29-3.16(m,1H).

[0042] 13 C NMR (101MHz, DMSO): δ195.50,175.01,169.10,161.22,136.74,132.94,126.57,123 .59,122.44,121.65,119.22,118.90,112.96,112.80,12.04,105.57,45.89,38.18.

[0043] 2. Make animal models

[0044] After 1 week of adaptive feeding, SPF C57BL / 6J male mice (16-18g) were randomly divided into a normal group and a diabetic group. The mice in the normal group were fed with ordinary feed, and the mice in the diabetic group were fed with a high-sugar and high-fat feed. After 6 weeks, all mice received intraperitoneal injections of an equal volume of citric acid-sodium citrate buffer (pH 4.5, normal group) or 100 mg kg -1 ·d -1 STZ (dissolved in citric acid-sodium citrate buffer, pH 4.5, diabetic group).

[0045] At 3d and 7d after STZ injection, blood was collected from the tail vein of mice for random blood glucose measurement. The model was considered to be successfully established when the random blood glucose was ≥16.7mmol / L twice, and the mice that failed to establish the model were screened out.

[0046] The subjects were randomly divided into six groups: normal control group (Control group, n = 15), diabetic model group (DM group, n = 15), MA-5 low-dose group (DM + 25 mg kg -1 MA-5 group, n = 15), MA-5 medium dose group (DM + 50 mg kg -1 MA-5 group, n = 15), MA-5 high-dose group (DM + 100 mg kg -1 MA-5 group, n = 15) and MA-5 high-dose normal control group (Control + 100 mg kg -1 The normal group and the MA-5 high-dose control group continued to be fed with ordinary feed for 8 weeks, but the MA-5 high-dose normal control group was given MA-5 by gavage at the same time, and the mice in other groups were still fed with high-sugar and high-fat feed and treated with different concentrations of MA-5 by gavage at the same time for 8 weeks.

[0047] During the entire experiment, the overall condition of each group of mice was observed every morning and evening, including general condition, mental and hair condition, urination, defecation, and changes in diet and drinking.

[0048] 3. Calculate organ weight coefficient

[0049] After continuous administration for 8 weeks, 1% sodium pentobarbital (50 mg / kg) was intraperitoneally injected to anesthetize the mice. When collecting samples, the heart weight (HW) of the mice was weighed, the tibia length (TL) and body weight (BW) were measured, and the tibia coefficient (HW / TL) and heart coefficient (HW / BW) were calculated. The results are shown in Table 1.

[0050] Table 1

[0051]

[0052]

[0053] As can be seen from Table 1, compared with the normal group, the HW of the DM group mice increased. After treatment with MA-5, the myocardial weight decreased, and it was significantly reduced in the MA-5 high-dose group (P<0.05). In the experiment, we also measured the ratio of myocardial organ coefficient (HW / BW). The HW / BW of the DM group mice was significantly higher than that of the Control group (P<0.05), indicating that the DCM group mice had myocardial hypertrophy. After treatment with MA-5, it was significantly reduced in the MA-5 low-dose group. In addition, the tibia length no longer changes after the mice mature and can be used as a quantitative comparison standard, so the tibia coefficient (HW / TL) can measure the degree of cardiac hypertrophy. As can be seen from the table, compared with the normal group, the HW / TL of the DM group was higher, and compared with the DM group, it was slightly reduced in the MA-5 low-dose group and had a significant difference (P<0.05). HW, HW / BW and HW / TL showed significant differences from the MA-5 low dose and decreased in a concentration gradient-dependent manner. This indicates that MA-5 has different degrees of protective effects on myocardial hypertrophy caused by diabetes.

[0054] 4. Determination of blood glucose and blood lipids in mouse serum

[0055] (1) Blood glucose measurement

[0056] Blood glucose was tested using the Anwen+ blood glucose meter (model: Anwen+) purchased from Sino Biosensor Co., Ltd. The tail of the mouse was wiped with a medical sterile cotton ball, and the tail tip vein was punctured with a disposable blood collection needle. The blood quickly filled the blood glucose test strip, and the blood glucose meter reading was read and recorded.

[0057] (2) Determination of blood lipids

[0058] After continuous administration for 8 weeks, all mice were anesthetized, and blood was collected from the vein. The blood was placed at 4°C overnight, and centrifuged at 3000 rpm for 10 minutes at 4°C the next day. The supernatant was taken for blood lipid testing. The low-density lipoprotein (LDL) kit and high-density lipoprotein (HDL) kit purchased from Nanjing Jiancheng Bioengineering Research Institute Co., Ltd. were used for testing. The specific steps were carried out according to the kit instructions.

[0059] According to the instructions, we need to prepare blank wells, calibration wells and sample wells. The amount required for each well is μL, which requires distilled water, standard, sample to be tested and R1 liquid in the kit. The blank well only needs distilled water and R1 liquid in a ratio of 2.5:180, the calibration well needs standard and R1 in a ratio of 2.5:180, and the sample well needs to add the sample to be tested in a ratio of 2.5:180. Stir the mixture in the 96-well plate evenly, put it in a 37℃ constant temperature mixer and incubate it for 5 minutes, and then use a wavelength of 546nm to measure the absorbance of each well as A1. Then add R2 liquid, 60 (μL) to each well. Stir the mixture in the 96-well plate evenly, put it in a 37℃ constant temperature mixer and incubate it for 5 minutes, and then use a wavelength of 546nm to measure the absorbance of each well as A2. Calculate according to the following formula:

[0060]

[0061] The results are as follows Figure 1 As shown. Figure 1 It can be seen that the DM group mice showed a continuous increase in blood sugar. After intervention with different concentrations of MA-5, the blood sugar of the mice showed a downward trend, especially in the MA-5 low-dose group and the MA-5 high-dose group, which showed a significant decrease (P<0.05) ( Figure 1 A).

[0062] The serum LDL value of the DM group mice was significantly higher than that of the normal group. After intervention with MA-5, the serum LDL value decreased in a concentration-dependent manner at low doses ( Figure 1 B). As shown in the figure, the HDL value of the DM group was significantly lower than that of the normal group. After administration, the HDL value of the MA-5 low-dose group increased in a concentration-dependent manner (P<0.05) ( Figure 1 C).

[0063] 5. Heart disease pathology

[0064] After 8 weeks of continuous administration, all mice were anesthetized, the mouse hearts were isolated and placed in 4% paraformaldehyde fixative, and after fixation, they were embedded in paraffin, and the heart tissues were stained with H&E and picrosirius red, respectively. The specific steps are as follows:

[0065] (1) Paraffin section

[0066] a. Move the myocardial tissue into melted paraffin for immersion and embedding. Finally, use a microtome to cut slices with a thickness of about 4 μm.

[0067] b. The subsequent time in 50% ethanol, 70% ethanol, 85% ethanol and 95% ethanol was 90 min, and the final time in anhydrous ethanol I and anhydrous ethanol II was 60 min.

[0068] (2) HE staining

[0069] a. Bake in a 60℃ constant temperature drying oven for 30 minutes, and the tissue on the slide will become transparent. Then proceed to the HE operation steps.

[0070] b. Prepare reagents: xylene I, II (15 min each), 50% xylene (5 min), anhydrous ethanol I, II (15 min each), 95%, 90%, 80%, 70% ethanol (5 min each), distilled water (1-3 min), hematoxylin (5-10 min), 1% hydrochloric acid alcohol (5-20 s), distilled water (same as above), 1% dilute ammonia water (30 s), distilled water (same as above), eosin (5 min), 80% ethanol (5 min), 95% ethanol I (5 min), ethanol II (5 min), 100% anhydrous ethanol (5 min), xylene I and II (3-10 min each).

[0071] c. After completing the above operations, observe the microstructural changes of myocardial tissue under a light microscope.

[0072] (3) Sirius red staining

[0073] a. After dewaxing, wash the paraffin sections three times with tap water and then three times with ultrapure water.

[0074] b. Staining: Place the sections in the prepared Sirius staining solution for 8 minutes.

[0075] c. Dehydration of sections: The sections were placed in anhydrous ethanol I, anhydrous ethanol II, and anhydrous ethanol III for 3s, 5s, and 30s respectively.

[0076] d. Slice and place in n-butanol for 10 seconds.

[0077] e. Transparency: Place the slices in xylene I and xylene II for 5 minutes respectively.

[0078] f. Seal the slides and gently cover them with a coverslip, taking care not to create any bubbles, and allow to dry at room temperature.

[0079] The microstructural changes of myocardial tissue after HE staining and picrosirius red staining are shown in Figure 2 Figure 2 (magnification = 400×) as shown:

[0080] The nuclei of myocardial cells in the normal group were uniform in size, the myofibers were uniformly stained, and the shapes were regular. Compared with the normal group, the nuclei of myocardial cells in the DM group were unevenly distributed, with different shades of color, chaotic and loose arrangement of myofibers, and uneven thickness of myofibers. Compared with the DM group, the myocardial morphology of the mice in the MA-5 low-dose group began to show more regular changes, with reduced degrees of rupture and cavitation damage, and more orderly arrangement of myofibers ( Figure 2 A).

[0081] The myocardial fibroblasts of the normal group mice were arranged in an orderly manner. The myocardial red fiber collagen deposition of the DM group mice increased significantly and was arranged in a disordered manner. Compared with the DM group, the collagen deposition of the low-dose MA-5 group mice was significantly reduced, and the myocardial fibers were arranged more regularly. The myocardial morphology and degree of fibrosis of the normal drug group mice were similar to those of the normal group ( Figure 2 B).

[0082] 6. Changes in the ultrastructure of myocardial mitochondria

[0083] (1) Dehydration treatment:

[0084] a. Take the apical tissue and immerse it in electron microscopy fixative, usually overnight; then wash it three times with 0.1mmol / L phosphate buffer, 15 minutes each time; fix it with 1% osmium acid fixative for 2 hours; then wash it with 0.1mmol / L phosphate buffer as above; then dehydrate it with 50, 70, 80, 90, 95, 100% alcohol, 15 minutes each time; finally, treat it with 100% acetone for 15 minutes.

[0085] (2) The myocardial tissue is embedded as follows:

[0086] a. The first embedding of myocardial tissue requires a ratio of acetone to embedding solution (2:1) and a time of 3 hours at room temperature.

[0087] b. Give acetone + embedding solution ratio (2:1) again and keep at room temperature overnight.

[0088] c. Finally, use embedding solution at 37℃ for 2h.

[0089] (3) The myocardial tissue is solidified according to the conditions and sequence in Table 2:

[0090] Table 2

[0091] Reaction time Reaction temperature overnight 37℃ 12h 45℃ 24h 60℃

[0092] (4) The myocardial tissue was cut into 60 nm thin slices using an ultrathin slicer, then stained with 3% uranyl acetate-lead citrate and observed using an electron microscope. Figure 3 shown.

[0093] from Figure 3 It can be seen that compared with the normal group, the mitochondrial cristae in the DM group were reduced or even disappeared. At the same time, mitochondrial swelling can also be seen. With the increase of MA-5 treatment concentration, the degree of mitochondrial swelling improved, a small amount of myofilaments were broken, and myocardial damage was significantly alleviated.

[0094] 7. Determination of oxidative stress level in myocardial tissue

[0095] ROS is a highly reactive chemical substance containing oxygen free radicals. It is one of the indicators for judging oxidative stress. Superoxide anions in cells were measured using a fluorescent probe containing dihydroethidium (DHE). Myocardial tissue was made into 10-micron frozen sections and dried for 1.5 hours. After being treated with 10μM DHE, they were placed in a light-proof chamber at 37°C for 30 minutes. The cells were then fixed with 5μg / mL DAPI for 10 minutes. Images of DAPI-stained myocardial tissue sections were captured under a fluorescence microscope, as shown in Figure 2. Figure 4 As shown, A is the image of oxidative stress level in myocardial tissue of diabetic mice, and B is the measurement result of DHE fluorescence density.

[0096] from Figure 4 It can be seen that abnormal oxidative stress levels are closely related to the occurrence of diabetic myocardial damage. DHE is the most common fluorescent probe for superoxide anion detection, and this probe is often used to detect the expression of intracellular ROS. The results of the DHE probe showed that compared with the normal group, the red fluorescence of the DM group was enhanced and the ROS content increased. After treatment with MA-5, the red fluorescence in the high-dose group dimmed, and there was a significant difference (P<0.05).

[0097] 8. Western blot analysis

[0098] (1) Protein extraction

[0099] Use an electronic analytical balance to weigh about 40 mg of myocardial tissue and add it to the freshly prepared tissue lysis buffer (i.e., RIPA: Cocktail: PMSF: Na3VO4 = 100: 1: 1: 1) in a ratio of volume: weight = 10: 1. Grind it completely on ice and let it stand for 0.5 h. Aspirate it into a 1.5 mL centrifuge tube and centrifuge it at 12000 rpm and 4°C for 15 min. Aspirate the supernatant for later use.

[0100] (2) Protein concentration determination

[0101] The BCA protein concentration determination kit is used to determine the sample concentration. For the extraction of total protein from tissues, the supernatant sample and standard protein extracted above are dissolved in a low temperature environment, and the protein standard is diluted to 1 mg / mL with ultrapure water. Add to a 96-well plate according to the concentration ratio (0, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1 mg / mL), and make up to 20 μL with ultrapure water. In the 96-well plate, leave one secondary well for each standard protein. Take 2 μL of the sample supernatant, add 18 μL of double distilled water, and make up to 20 μL (that is, each sample concentration is diluted 10 times for detection, and the final calculation of protein concentration needs to be multiplied by the dilution multiple). Each sample well also retains a secondary well. Calculate the number of wells required, and each well needs to add 200 μL of the configured BCA working solution (A solution: B solution = 50:1). After the above operations are completed, the 96-well plate needs to be placed in a 37°C constant temperature metal bath and heated for 30 minutes. The absorbance value of each well at a wavelength of 562nm is measured using an enzyme reader. The standard curve is calculated using the standard protein sample and the OD value of the sample to be tested is substituted into the standard curve to calculate the sample concentration. Take an appropriate amount of supernatant, mix it with the lysate and 5× protein loading buffer, and heat it in a constant temperature metal bath at 97°C for denaturation for 5-10 minutes. After cooling, store it in a -20°C refrigerator for later use.

[0102] (3) Preparation of separation gel and concentration gel

[0103] a. Preparation of SDS-PAGE gel This application is divided into three ratios, namely 10%, 12% and 15%.

[0104] b. The reagents are ultrapure water, 30% acrylamide, Tris-HCL (pH 8.8), Tris-HCL (pH 6.8), 10% SDS, 10% ammonium persulfate, 10% ammonium persulfate and TEMED.

[0105] c. The molecular weight used was 10% and 12% glue. The ratio of the above reagents for 10% glue was: 4 (mL): 3.3 (mL): 2.5 (mL): 0.5 (mL): 0.1 (mL): 0.1 (mL): 5 (μL), and the total volume was 10mL; the ratio of the above reagents for 12% glue was: 3.3 (mL): 4 (mL): 2.5 (mL): 0.5 (mL): 0.1 (mL): 0.1 (mL): 5 (μL).

[0106] d. Mix and prepare the glue, ready for use

[0107] (4) Electrophoresis

[0108] In the experiment, the amount of protein loaded on the myocardial tissue sample was about 50 μg, and the volume of the rainbow marker added to both sides of the loading well was about 1.5 μL. The sample should be added accurately to avoid sample overflow. After the sample is loaded, check whether there is any abnormality in the electrophoresis instrument connection equipment, and adjust the electrophoresis instrument voltage to 60V to start electrophoresis. It is necessary to observe whether electrophoresis bubbles are generated at the bottom and whether the internal liquid between the two plates changes. After the protein sample is electrophoresed to the contact surface of the separation gel and the rainbow marker is separated, adjust the electrophoresis instrument voltage to 80V. Stop the electrophoresis when the electrophoresis reaches the required height.

[0109] (5) Transfer

[0110] Take a PVDF membrane that is close to the size of the gel, remove the glass plate and cut the gel into appropriate sizes, immerse it in the transfer solution, remove the transfer clip, soak the PVDF membrane with methanol solution and activate it. Rinse the sampler before collecting the sample to ensure that there is no residue left to avoid affecting the subsequent test results. Wash the methanol solution on the surface of the PVDF film with the transfer solution, move it to the gel surface, and fix one end to drive out the bubbles between the gel and the membrane so that they are tightly combined. Lock the transfer clip and put it into the transfer tank. Set the transfer conditions: 300mA, 120min. After the transfer is completed, take it out, mark it, and wash it with 1×TBST.

[0111] (6) Closed

[0112] Place the PVDF membrane washed with TBST in a 5% skim milk blocking solution, place it on a shaker at room temperature for 1-2 hours, and then wash it with 1×TBST. Then place the activated membrane on the adhesive layer. You can also adjust the blocking time and washing times according to the test results.

[0113] (7) Antibody incubation

[0114] After the blocking is completed, wash with 1×TBST solution for 10 minutes, wash 3 times. Cut the PVDF membrane as required, use the primary antibody diluent at 1:1000 to prepare the primary antibody solution, and place the cut film in the primary antibody solution and incubate it in a shaker at 4°C overnight. After the enzymatic hydrolysis is complete, take the supernatant and add a certain amount of physiological saline to wash until neutral. On the second day, remove the incubated film and rinse it with 1×TBST for 10 minutes, rinse 3 times. Then dry the soaked membrane to obtain a polyamide / chitosan composite hydrogel with good biocompatibility. Then put the PVDF membrane into 1×TBST diluted antibody HRP-labeled goat anti-rabbit IgG (1:10000) or HRP-labeled rabbit anti-mouse IgG (1:10000) and incubate it in a shaker at room temperature for 1h, then wash the PVDF membrane in 11×TBST 3 times, 5-10 minutes each time.

[0115] (8) Development

[0116] Use equal volumes of Meilunbio Felixmin ECL luminescent liquid A and ECL luminescent liquid B to prepare supersensitive luminescent liquid, soak the PVDF membrane evenly in the luminescent liquid, expose it using a gel imaging system, and analyze the grayscale value using Image Lab Software.

[0117] Figure 5 The effect of MA-5 on mitochondrial fission and fusion-related proteins in the myocardial tissue of diabetic mice was shown. The results showed that in the life cycle of mitochondria, the biogenesis of new mitochondria depends on mitochondrial fission. Mitochondria can also remove dysfunctional mitochondria through fission. When mitochondria are damaged, peripheral fission occurs. Drp1 is an essential protein for regulating mitochondrial fission. The important mechanism for regulating activity is phosphorylation. Among them, phosphorylation of Drp1 S637 site promotes increased mitochondrial fission. Mitochondrial MFN1, MFN2 and OPA1 are fusion proteins. These proteins decreased in the DM group compared with the Control group. After treatment with MA-5, P-Drp1S637 / Drp1 decreased in the MA-5 high-dose group. There was a certain improvement, with significant differences (P<0.05); MFN1 and OPA1 increased in the MA-5 low-dose group; it can be seen in the MA-5 medium-dose group that MFN2 increased to a certain extent, with significant differences (P<0.05).

[0118] Figure 6 The results show the effect of MA-5 on pyroptosis-related proteins in the myocardial tissue of diabetic mice. It can be seen that cell pyroptosis is another way of programmed cell death. When cells undergo pyroptosis, they often swell and enlarge until the cell membrane ruptures, and a large amount of cell contents are released, causing a severe inflammatory response. In order to determine the role of inflammatory changes in the myocardium of diabetic mice, the expression of pyroptosis-related proteins such as IL-1β, IL-18, NLRP3, and Caspase-1 was determined by Western blot. Compared with the DM group and the Control group, the expression levels of IL-1β, Caspase-1, IL-18, and NLRP3 proteins in the myocardial tissue of mice were significantly increased (P<0.05); compared with the DM group, the expression of Caspase-1, IL-1β, NLRP3, and IL-18 proteins of MA-5 at different doses was significantly decreased (P<0.05).

[0119] The embodiments described above are part of the embodiments of the present application, rather than all of the embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the present application for protection, but merely represents the selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work belong to the scope of protection of the present application.

Claims

1. Use of the plant hormone mitochondrial acid 5 in the preparation of a drug for treating myocardial damage caused by diabetes.

2. The use of the plant hormone mitochondrial acid 5 according to claim 1 in the preparation of a drug for treating myocardial damage caused by diabetes, characterized in that: The medicine is in oral dosage form and also includes medically acceptable auxiliary materials.

3. The use of the plant hormone mitochondrial acid 5 according to claim 1 in the preparation of a drug for treating myocardial damage caused by diabetes, characterized in that: The plant hormone mitochondrial acid 5 improves mitochondrial damage and inhibits oxidative stress.

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