Application of martensin in preparation of medicine for improving or treating myocardial remodeling and myocardial fibrosis

Abstract

The invention provides an application of martensin in preparation of a medicine for improving or treating myocardial remodeling and myocardial fibrosis. By establishing a newborn rat myocardial cell hypertrophy model and a mouse aortic arch constriction induced myocardial remodeling model, it is found that the mavibride can significantly relieve angiotensin 2 induced myocardial cell hypertrophy and myocardial cell mitochondrial damage and improve the oxidative phosphorylation function of mitochondria. An in-vitro animal experiment also proves that the macturitin can relieve TAC postoperative mouse cardiac function deterioration, reduce myocardial remodeling and myocardial fibrosis, and improve the mitochondrial structure and function damage of myocardial tissue of the myocardial remodeling mouse at the same time. The invention provides a theoretical basis for clinical application and treatment of Mactopeptide in myocardial remodeling and myocardial fibrosis.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the use of Mastolide in the preparation of drugs for improving or treating myocardial remodeling and myocardial fibrosis. Background Art

[0002] Myocardial remodeling is a process in which the heart is stimulated by various factors, resulting in thickening of the ventricular wall and decreased myocardial compliance, which is specifically manifested as cardiomyocyte hypertrophy, myocardial interstitial fibrosis, and cardiac systolic dysfunction. The pathogenic mechanism of myocardial remodeling is highly heterogeneous according to different etiologies. Clinically, these etiologies mainly include hypertension, myocardial infarction, metabolic heart disease, and congenital heart disease. In recent years, due to the development of science and technology and the improvement of treatment methods, clinical drugs such as β-blockers, ACEI / ARB, SGLT-2 inhibitors, and GLP-1 receptor agonists have been formed to improve pathological myocardial remodeling and myocardial fibrosis. However, the mechanism of myocardial remodeling and effective intervention measures have not been fully elucidated. In addition, since the onset of diseases such as hypertension is usually hidden, a certain degree of myocardial remodeling has occurred when it is detected. When it progresses to the late stage of severe myocardial tissue fibrosis, its treatment and intervention will be particularly difficult, the treatment effect is limited, and the prognosis is poor.

[0003] The occurrence and development of myocardial remodeling and myocardial fibrosis involve a variety of complex pathophysiological mechanisms and cell biological processes, including the regulation of neuroendocrine hormones, oxidative stress injury, inflammatory apoptosis, and changes in myocardial energy metabolism. In recent years, more and more studies have confirmed that mitochondrial dysfunction is a factor that cannot be ignored in causing myocardial remodeling. Mitochondrial dysfunction involves a variety of pathological conditions such as mtDNA mutations, impaired mitochondrial autophagy, reduced oxidative phosphorylation levels, and increased ROS levels. Therefore, intervention targets such as reducing mtDNA mutations (supplementing NAD+, nicotinamide riboside, etc.), increasing mitochondrial generation and improving energy metabolism (PCG1α, IGF-1, SIRT3), and reducing ROS damage (antioxidants) can delay the occurrence and development of myocardial remodeling, showing the clinical application prospects of developing anti-myocardial remodeling and heart failure drugs with the protection of mitochondrial structure and function as the target. In-depth exploration of targeted mitochondria and their homeostasis maintenance is of great significance for the prevention and treatment of myocardial remodeling and myocardial fibrosis.

[0004] Therefore, it is necessary to develop a drug for improving or treating myocardial remodeling and myocardial fibrosis. Summary of the invention

[0005] The purpose of the present invention is to provide the use of Mashidu peptide in the preparation of drugs for improving or treating myocardial remodeling and myocardial fibrosis. The present invention finds for the first time that Mashidu peptide can significantly improve the cardiac function of pressure overload model mice, indicating that Mashidu peptide has great application prospects in the preparation of drugs for improving or treating myocardial remodeling and myocardial fibrosis.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] In a first aspect of the present invention, there is provided a use of Masud peptide in the preparation of a drug for improving or treating myocardial remodeling and myocardial fibrosis.

[0008] Furthermore, the medicine also includes pharmaceutically acceptable excipients and carriers.

[0009] Furthermore, the auxiliary material includes at least one of a filler, a disintegrant, a binder, an excipient, a lubricant, a sweetener or a colorant.

[0010] Furthermore, the dosage form of the drug includes at least one of granules, tablets, pills, capsules, and injections.

[0011] Furthermore, the effective dose of the Mastolide peptide is 4-6 mg / kg, preferably 6 mg / kg.

[0012] Furthermore, the Mashidu peptide can significantly improve the cardiac function of pressure overload model mice.

[0013] Furthermore, in the present invention, the GLP-1R / GCGR dual agonist mastic peptide (at a dose of 1.0 μmol / L) can significantly improve the hypertrophy of isolated cardiomyocytes induced by angiotensin II (Ang II).

[0014] In a second aspect of the present invention, a drug for improving or treating myocardial remodeling and myocardial fibrosis is provided, wherein the active ingredient of the drug includes Mastolide.

[0015] In the second aspect of the present invention, there is provided the use of Mashidu peptide in the preparation of a drug for improving or treating mitochondrial damage and metabolic disorders.

[0016] Furthermore, the Mashidu peptide can effectively improve cell mitochondrial damage and energy metabolism disorder in the myocardial remodeling model.

[0017] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0018] (1) The present invention is the first to apply the GLP-1R / GCGR dual agonist Mastolide to the preparation of products for the treatment of myocardial remodeling and myocardial fibrosis. The GLP-1R / GCGR dual agonist Mastolide can significantly improve the cardiac function of pressure overload model mice, providing a theoretical basis for the clinical treatment of myocardial remodeling and myocardial fibrosis;

[0019] (2) The present invention constructs an in vivo myocardial remodeling and myocardial fibrosis mouse model through pressure overload, and uses a series of phenotypic experiments to confirm the positive therapeutic effects of GLP-1R / GCGR dual agonist Mashidu peptide on myocardial remodeling and myocardial fibrosis. The experimental data is sufficient and the theoretical basis is reliable. The experimental techniques involved in the content of the present invention are all mature technologies in this field, the data are highly representative, and the results are highly credible, which fully demonstrates the good benefits of Mashidu peptide in myocardial remodeling, which is convenient for active promotion and use;

[0020] (3) The present invention provides the use of GLP-1R / GCGR dual agonist Mashidu peptide in the treatment of mitochondrial damage and metabolic disorders. Mashidu peptide can improve myocardial cell mitochondrial damage after TAC surgery, improve myocardial cell oxidative phosphorylation, inhibit myocardial cell hypertrophy and myocardial fibrosis, and promote the repair process after myocardial injury. At the same time, Mashidu peptide actively improves myocardial cell energy metabolism and has clinical application value in actively treating myocardial mitochondrial dysfunction after myocardial injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings in the following description are some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative work.

[0022] Figure 1 Schematic diagram of the molecular structure of Mashidu peptide.

[0023] Figure 2 The ultrasonic detection results in Example 2, A. Representative M-mode echocardiograms of mice in the sham operation group and TAC group after the mice were given solvent or Masdu peptide; BG. Analysis of cardiac function indexes of mice in the sham operation group and TAC group after the mice were given solvent or Masdu peptide: left ventricular ejection fraction, left ventricular short-axis shortening rate, left ventricular end-systolic diameter, left ventricular end-diastolic diameter, E / A ratio and heart rate.

[0024] Figure 3 The results in Example 1, A. Gross images of mouse hearts taken from sham-operated and TAC-group mice after administration of solvent or Mashidu peptide; B. Statistical analysis of body weights of mice in sham-operated and TAC-group mice on days 0, 7, 21 and 28 after administration of solvent or Mashidu peptide; C. HE staining and WGA staining of mouse hearts taken from sham-operated and TAC-group mice after administration of solvent or Mashidu peptide; D. Cross-sectional area analysis of mouse hearts taken from sham-operated and TAC-postoperative mice after administration of solvent or Mashidu peptide; E. Gross images of heart weight / tibia length and lung weight / tibia length ratios of mice in sham-operated and TAC-postoperative mice after administration of solvent or Mashidu peptide.

[0025] Figure 4 The results in Example 3 are as follows: A. PSR staining images of the hearts of mice in the sham operation group and the TAC group after administration of solvent or Mashidu peptide, with red showing perivascular and interstitial fibrosis; B. Quantitative analysis of the myocardial fibrosis levels of mice in each group after PSR staining; C. Protein expression levels and quantitative analysis results of myocardial remodeling indicators (BNP, β-MHC) and myocardial fibrosis indicators (α-SMA, COL-1α1) of mice in the sham operation group and the TAC group after administration of solvent or Mashidu peptide, with GAPDH as an internal reference.

[0026] Figure 5 The results of the two-dimensional speckle tracking analysis in Example 2 are as follows: A. Two-dimensional speckle tracking imaging (Vevo Strain) was used to analyze the radial and longitudinal strain of the left ventricle during the cardiac cycle of each group of mice (parasternal long axis view) to evaluate the myocardial strain of the hearts of each group of mice. BD. Analysis of the global longitudinal strain rate (GLS), long-axis radial strain rate, and maximum relative delay rate of radial strain during left ventricular systole of each group of mice.

[0027] Figure 6 The results in Example 4 are as follows: A. High-resolution transmission electron microscopy analysis of the size, number, arrangement and ultrastructure of mitochondria in the myocardial tissue of each group of mice in the sham operation group and TAC group after administration of solvent or Mashidu peptide, with mitochondria and magnified areas highlighted in the figure; B. Quantitative analysis of mitochondrial density in the myocardial tissue of each group of mice in the sham operation group and TAC group after administration of solvent or Mashidu peptide; C. ATP levels in the myocardial tissue of each group of mice in the sham operation group and TAC group after administration of solvent or Mashidu peptide were detected by the kit.

[0028] Figure 7 The results of molecular biological detection in Example 5, AB. Quantitative analysis results of mRNA levels of myocardial hypertrophy indicators (Nppa, Nppb) of primary cardiomyocytes in the control group and AngII treatment group after administration of solvent or Masdu peptide, GAPDH as internal reference; CD. Quantitative analysis results of mRNA levels of myocardial fibrosis indicators (COL-1α1, CTGF) of primary cardiomyocytes in the control group and AngII treatment group after administration of solvent or Masdu peptide, GAPDH as internal reference; E. Protein expression levels and quantitative analysis results of myocardial hypertrophy indicators (BNP, β-MHC) and myocardial fibrosis indicators (α-SMA, COL-1α1) of primary cardiomyocytes in the control group and AngII treatment group after administration of solvent or Masdu peptide, GAPDH as internal reference.

[0029] Figure 8The results of cell immunofluorescence detection in Example 5, A. Representative images of α-actinin immunofluorescence staining of each group of cells and α-SMA immunofluorescence staining of each group of myocardial fibroblasts in the control group and AngII-treated group after administration of solvent or Masdu peptide; B. Analysis results of myocardial cell cross-sectional area of ​​α-actinin immunofluorescence staining of primary cardiomyocytes in the control group and AngII-treated group after administration of solvent or Masdu peptide; C. α-SMA fluorescence quantitative analysis of myocardial fibroblasts in the control group and AngII-treated group after administration of solvent or Masdu peptide.

[0030] Fig. 9 The results in Example 6, AB. The primary cardiomyocytes of the control group and the AngII treatment group were given solvent or Masdu peptide, and the JC-1 staining of each group of cells showed the changes in the mitochondrial membrane potential of the cardiomyocytes and fluorescence quantitative analysis; C. The kit detected the ATP levels of each group of cells in the control group and the AngII treatment group after the solvent or Masdu peptide was given. DETAILED DESCRIPTION

[0031] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0032] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0033] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or obtained by existing methods.

[0034] To achieve the purpose of the present invention, the overall concept of the present invention is as follows:

[0035] The Mashidu peptide of the present invention is a GLP-1R / GCGR dual agonist, and its chemical formula is as follows Figure 1 As shown, it is commercially available.

[0036] The inventors of the present application found in an in vivo model that Mashidu peptide can significantly reduce angiotensin 2-induced cardiomyocyte hypertrophy and cardiomyocyte mitochondrial damage, and improve mitochondrial oxidative phosphorylation function. In an in vitro model, it was also confirmed that Mashidu peptide can alleviate the deterioration of cardiac function in mice after TAC surgery, reduce myocardial remodeling and myocardial fibrosis, and improve the mitochondrial structure and functional damage of myocardial tissue in mice with myocardial remodeling.

[0037] The in vivo model: The present invention uses C57BL / 6J mice aged 8-10 weeks and weighing 23.5g-27.5g as experimental subjects, and induces myocardial hypertrophy and myocardial remodeling animal models through transverse aortic constriction (TAC). Compared with the placebo group, the cardiac function of mice was significantly improved after the mice were given the drug Masdu peptide after TAC surgery. At the same time, Masdu peptide significantly reduced the heart weight / body weight, lung weight / body weight, myocardial cell cross-sectional area and myocardial fibrosis level of mice. In addition, electronic images of transmission electron microscopy showed that Masdu peptide can reduce mitochondrial damage in the heart of TAC mice. The above results show that the application of GLP-1R / GCGR dual agonist Masdu peptide can alleviate the disease progression of pathological myocardial remodeling and myocardial fibrosis.

[0038] The in vitro model: The present invention provides an in vitro myocardial cell hypertrophy model by extracting isolated cardiomyocytes from neonatal rats and administering angiotensin II (AngII). The experimental results show that compared with the control group, i.e., the phosphate buffered saline (PBS) treatment group, Mashidu peptide can significantly reduce myocardial cell hypertrophy in vitro. In addition, the myocardial cell hypertrophy index BNP was significantly reduced. At the same time, the mitochondrial oxidative respiration rate (OCR) curve results showed that compared with the control group, the oxidative phosphorylation level of myocardial cells after Mashidu peptide treatment was significantly increased, and the ATP generation level was significantly increased.

[0039] The present application will be described in detail below with reference to embodiments and experimental data.

[0040] Example 1: Construction of mouse myocardial remodeling model

[0041] Transverse aortic constriction (TAC) was used to establish a mouse myocardial remodeling and myocardial fibrosis model. The specific process is as follows:

[0042] The experimental subjects were wild-type C57 BL / 6J mice aged 8-10 weeks and weighing 23.5-27.5 g. All experimental mice were housed in the Specific Pathogen Free (SPF) experimental animal center of the Institute of Cardiovascular Diseases of Wuhan University. The breeding conditions were as follows: room temperature between 22-24°C, humidity between 50-70%, 12-hour light and dark alternating lighting, and sufficient water and food.

[0043] (1) Preoperative preparation

[0044] ① Prepare experimental mice, 3% sodium pentobarbital, ear tags, surgical instruments, medical gauze tape, etc. Randomly take out mice of the required age from the IVC cages, weigh the mice, group the mice weighing between 23.5-27.5g, and mark them with ear tags.

[0045] ② Anesthesia: Use 3% sodium pentobarbital solution with a concentration of 40 mg / kg to anesthetize the mice by intraperitoneal injection.

[0046] ③ Skin preparation: After the mouse is completely anesthetized, use a small animal shaver to shave the hair on the mouse's chest area, use alcohol for local disinfection, and wait for surgery.

[0047] (2) TAC

[0048] The mice were intubated non-invasively and connected to a ventilator. The limbs were fixed on a heating pad in a supine position. A transverse incision was made along the midpoint of the upper limbs of the mice. The incision was placed so that the second rib could be clearly seen. The subcutaneous fascia was separated in sequence by clamping the incision. The thymus was bluntly separated to expose the aortic arch and its branches. A small opening was made in the soft tissue between the aortic arch, the brachiocephalic trunk and the left common carotid artery. The surgical suture was passed under the aortic arch. The 27-gauge needle with a sharp tip was placed on the suture parallel to the aortic arch. The aortic arch and the needle were firmly ligated together, and then the needle was immediately withdrawn, which could cause 70% stenosis of the aortic arch. In the Sham group, only the thread was hung without ligation. The chest cavity gas was extracted and the chest cavity was closed. The incision was sutured layer by layer. The mice were left on the heating pad and waited for anesthesia to wake up. Then they were placed in a clean IVC cage and returned to the breeding room.

[0049] (3) Postoperative observation and grouping

[0050] After surgery, mice were free to eat and drink. The basic vital signs and activity status of mice were observed every day in the first week after surgery and every week thereafter. Mice in the sham operation group and TAC group were randomly selected and intraperitoneally injected with Mastolide once a week for 4 consecutive weeks. The solvent was normal saline with a concentration of 6 mg / kg. The control group was only given an equal dose of solvent. After the above treatment, the mice were divided into the following four groups: simple sham operation group (sham group), sham operation group + Mastolide group (sham+MAZ group), surgery group (TAC group) and surgery group + Mastolide group (TAC+MAZ group), with 12 mice in each group. The weight changes of mice were detected during the administration period. After 4 weeks of administration, gross specimens were taken and the heart weight, lung weight and tibia length of the mice were recorded.

[0051] (4) General materials:

[0052] ① Preparations before heart sampling: Instrument preparation: high temperature and high pressure sterilized ophthalmic scissors, ophthalmic straight forceps, ophthalmic curved forceps, scalpels and other surgical instruments; Instrument preparation: small animal weighing balance, precision dynamic analytical balance, -80℃ low temperature refrigerator; Consumables preparation: surgical pads, sterile gauze, cryopreservation tubes, 10% formalin, 10% potassium chloride, liquid nitrogen, utensils, pathology preservation tubes, rulers, markers, and sampling record sheets.

[0053] ② Heart pathological sampling: After the mice were killed by cervical dislocation, they were placed in a supine position on a surgical pad, their limbs were fixed, and the skin of the mouse xiphoid process was lifted with ophthalmic straight forceps. The chest cavity was opened and the heart and large blood vessels were fully exposed after being cut open quickly with ophthalmic scissors. The root of the large blood vessels was clamped with ophthalmic curved forceps, and the heart was quickly removed and placed in a 10% potassium chloride solution. After the heart was fully ejected and there was no residual blood, the heart was gently squeezed with sterile gauze and trimmed, and the weight of the mouse heart was weighed and recorded. The heart was then fixed in a 10% formalin solution. The mouse heart should be fully immersed in the fixative. After 24 hours at room temperature on a shaking table, subsequent treatments such as dehydration, embedding, and slicing were performed. After processing the heart sample, the left and right lungs were removed, and the residual blood on the surface was cleaned with sterile gauze, and then weighed and recorded; at the same time, both ends of the tibia on one side were exposed, and the length of the mouse femur was measured with a ruler and recorded in the sampling registration form.

[0054] ③ Cardiac molecular biology sampling: The mice were quickly killed by cervical dislocation, and the mouse weight was weighed and the mouse ear tag number was recorded. The mouse was then placed on a surgical pad in a supine position, and the mouse chest was quickly opened with ophthalmic straight forceps and ophthalmic scissors to separate and expose the mouse heart and the roots of large blood vessels. The roots of the heart and large blood vessels were clamped with ophthalmic curved forceps, the heart was lifted, and the heart was quickly removed with ophthalmic scissors and placed on sterile gauze. After cleaning the residual blood in the heart, the weight of the mouse heart was accurately weighed and recorded. The atrium and right ventricle were removed with a scalpel, and the left ventricular tissue was retained. The left ventricular tissue was then immediately placed in a cryotube marked with key information such as group, number, and sampling date in advance, and then transferred to a -80°C low-temperature refrigerator with liquid nitrogen for subsequent molecular biology analysis.

[0055] Figure 3 The results of gross specimens of mice in the sham operation group and mice after aortic arch constriction modeling with solvent and Mashidu peptide. Compared with the sham operation group, the heart volume of mice increased significantly 4 weeks after TAC, and the heart-to-tibia length ratio and lung-to-tibia length ratio increased. Compared with the TAC group, the heart volume of mice in the TAC group decreased 4 weeks after intraperitoneal injection of Mashidu peptide, and the heart-to-tibia length ratio and lung-to-tibia length ratio decreased.

[0056] Example 2: Ultrasound detection of mouse heart

[0057] (1) Preparation

[0058] ① Instrument and reagent preparation: Turn on the Vevo 3100 small animal high-resolution high-frequency ultrasound imaging system, set the parameters related to mouse cardiac ultrasound, prepare the anesthetic isoflurane, small animal shaver, depilatory cream, coupling agent, sterile gauze and 75% ethanol disinfectant;

[0059] ② Mouse skin preparation: Weigh the mouse, record the weight and the corresponding ear tag, then grab the mouse, shave the hair on the chest, left armpit and other ultrasound detection sites, and evenly apply small animal hair removal cream to remove excess hair;

[0060] ③Isoflurane anesthesia: Fix the skin-prepared mouse on the anesthesia mask and adjust the isoflurane concentration in the anesthesia mask to 2.5%. After the mouse is fully anesthetized, adjust the concentration to 1.5% to maintain the mouse's anesthesia state until the subsequent cardiac ultrasound operation.

[0061] (2) Cardiac ultrasound

[0062] Place the anesthetized mouse on the ultrasound platform, fix the mouse's limbs with tape, evenly apply an appropriate amount of ultrasound coupling agent on the mouse's precordial skin and ultrasound probe, aim the ultrasound probe at the strongest point of the mouse's heart beat, and detect it parallel to the long axis of the mouse's heart. First determine the long axis section of the left ventricle by ultrasound, then adjust the probe and continue to detect the short axis section of the mouse's heart. After the heart rate of each mouse stabilizes, switch to M-mode ultrasound mode, record and measure video and image data under multiple cardiac cycles. Analyze cardiac function indicators such as left ventricular ejection fraction (LVEF), short axis shortening rate (LVFS), E / A, left ventricular end-systolic internal diameter (LVIDs), left ventricular end-diastolic internal diameter (LVIDd) and heart rate.

[0063] (3) 2D Speckle Tracking Analysis

[0064] Vevo strain software of Vevo 3100 was used to perform two-dimensional speckle tracking on the dynamic images of the long-axis section of the heart obtained by ultrasound, and the trajectory of myocardial motion was traced. At least 6 independent cardiac cycles were analyzed for each heart. Myocardial strain indicators such as global longitudinal strain rate (GLS), long-axis radial strain rate, and maximum relative delay rate of radial strain during left ventricular systole were obtained through analysis.

[0065] Figure 2 and Figure 5The results of cardiac function test of mice in the sham operation group and mice after aortic arch constriction modeling were established by solvent and Mastolide. Compared with the sham operation group, the mice showed weakened cardiac contractile function and weakened local motion of the ventricular wall 4 weeks after TAC, mainly manifested as reduced left ventricular ejection fraction and global longitudinal strain. Compared with the TAC group mice, the cardiac function of the TAC group mice improved 4 weeks after intraperitoneal injection of Mastolide, and the left ventricular ejection fraction and global longitudinal strain increased, indicating that Mastolide can significantly improve the postoperative cardiac dysfunction of TAC mice.

[0066] Example 3: Molecular biology and pathological detection of myocardial remodeling and myocardial fibrosis model mice

[0067] (1) Molecular biology detection (protein extraction and Western-blotting detection): Take the myocardial tissue sample to be tested and add the lysis solution to grind it thoroughly in a grinder, ultrasonically lyse it, centrifuge it, and use the protein BCA quantitative kit to quantify it. Heat it in a dry hot pot to denature the protein and then collect it in a -80℃ low-temperature refrigerator for frozen storage. Prepare SDS-PAGE gel, take the above extracted protein for spotting, electrophoresis, transfer to the membrane, block it, and incubate it with the primary antibody overnight. Incubate it with the corresponding species secondary antibody the next day, and finally scan the membrane for development. Figure 4 As shown, compared with the mice in the TAC group, the protein expression levels of myocardial remodeling indicators (BNP, β-MHC) and myocardial fibrosis indicators (α-SMA, COL-1α1) in the myocardial tissue of the mice in the TAC group were significantly reduced 4 weeks after intraperitoneal injection of Mastolide.

[0068] (2) Pathological examination

[0069] ①HE staining: Place the paraffin sections of the heart in a 55℃ oven for more than 30 minutes; soak the baked sections in xylene for 5 minutes, repeat 3 times; place the dewaxed heart sections in anhydrous ethanol for 3 minutes, repeat 2 times; 95% ethanol for 3 minutes, repeat 2 times; soak in 70% ethanol and PBS for 3 minutes; soak in hematoxylin dye tank for 5 minutes, then rinse with running water for 5 minutes; differentiate in 1% hydrochloric acid alcohol for 3 seconds, then rinse with running water for 5 minutes; soak in Scott solution for 3 minutes, then rinse with running water for 5 minutes; soak in 0.5% eosin dye tank for 3 minutes, then wash away the floating color with pure water; soak in 70% ethanol for 1 minute; soak in 95% ethanol for 1 minute; soak in anhydrous ethanol for 3 minutes, repeat 3 times; soak in xylene for 3 minutes, repeat 3 times; seal with neutral resin and use pathological sections to scan and analyze the cross-sectional area of ​​each group of cardiomyocytes.

[0070] ②PSR staining: bake, dewax and hydrate the paraffin sections of the heart according to the above steps, then soak in running water for 10 minutes, repeat twice; soak in pure water for 3 minutes; soak in 0.2% phosphomolybdic acid for 3 minutes; discard the residual phosphomolybdic acid on the surface of the heart, add picric acid-sirius red dye to cover the heart while it is still wet, and stain at room temperature for 1.5 hours; discard the picric acid-sirius red dye on the surface of the heart, soak the sections in 0.01N hydrochloric acid for 3 seconds; soak in 70% ethanol for 1 minute; soak in 95% ethanol for 1 minute; soak in anhydrous ethanol for 3 minutes, repeat 3 times; soak in xylene for 3 minutes, repeat 3 times; after sealing with neutral resin, scan the pathological sections and statistically analyze the level of myocardial cell fibrosis.

[0071] ③WGA staining: bake, dewax and hydrate the paraffin sections of the heart according to the above steps, then place the sections in 1x sodium citrate repair solution, repair under high pressure for 5 minutes, cool in running water, and then rinse in PBS 3 times, 5 minutes each time; during this process, prepare the WGA-AF488 working solution with a concentration of 10μg / ml and dilute it with PBS 1:50; take out the sections washed in PBS, wipe the liquid around the heart tissue with gauze, but be careful not to damage the heart tissue, circle the heart with an immunohistochemistry pen, add WGA-AF488 working solution to cover the heart, and incubate in a wet box at 37℃ for 2h; rinse with PBS for 3 minutes, repeat three times; add DAPI on the heart tissue and cover the slide with a coverslip; collect fluorescent images of heart tissue in different areas using a fluorescence microscope and statistically analyze the cross-sectional area of ​​myocardial cells.

[0072] The results are as follows Figure 4 As shown, HE, PSR and WGA staining showed that intraperitoneal injection of Mastolide for 4 weeks could improve cardiac hypertrophy and myocardial fibrosis in mice in the TAC group.

[0073] Example 4: Detection of mitochondrial structure and function of cardiomyocytes in each group of mice

[0074] At the end point of modeling in each group of mice, the size, number, morphology and damage of mitochondrial cristae in the mouse myocardial tissue were evaluated by transmission electron microscopy. Consistent with the method of sampling for mouse heart molecular biology, the mice were quickly killed by cervical dislocation, the chest cavity was opened, the heart and large blood vessels were fully exposed, and the heart was quickly removed. While the heart was still beating, a scalpel was used to quickly take about 1mm3 of mouse myocardial tissue from the new apex, and the sample was immediately placed in a 4°C precooled electron microscope fixative for preservation. The samples were then sent to the Electron Microscopy Center of Renmin Hospital of Wuhan University for subsequent processing such as embedding and slicing, and were collected by high-resolution transmission electron microscopy. At the same time, a kit was used to detect the ATP level in the myocardial tissue of each group of mice.

[0075] The results are as follows Figure 6 As shown, it was shown that Mashidu peptide could alleviate the mitochondrial structural and functional damage of cardiac myocardial cells in mice after TAC surgery.

[0076] Example 5: Effect of Mashidu peptide on in vitro AngII-stimulated cardiomyocyte hypertrophy model

[0077] (1) Extraction of primary cardiomyocytes and cardiac fibroblasts

[0078] ① Preparation: sterilize the cell clean bench with ultraviolet light 30 minutes in advance; add 200 mL of pure water to the magnetic stirring heater, and sterilize ophthalmic scissors, ophthalmic tweezers, magnetic stirring rotor, serum bottles and other consumables and reagents under high pressure one day in advance.

[0079] ② Collection of rat suckling heart: Soak the collected newborn SD rat suckling (1-3 days old) in 75% alcohol for 1-3 seconds for disinfection, take out and place on gauze; disinfect the suckling rat chest again by dipping gauze in alcohol, quickly open the suckling rat chest with ophthalmic scissors, take out the suckling rat heart, and place it in the first culture dish containing PBS buffer. Wash the residual blood of the suckling rat heart in the first culture dish, remove the atrial tissue and right ventricular tissue, and transfer the left ventricular tissue to the second culture dish, cut the heart into small pieces with ophthalmic scissors and put it into the serum bottle.

[0080] ③ Heart digestion: Add 6-8mL of 0.125% pancreatin-EDTA digestion solution to the serum bottle, transfer the serum bottle to a magnetic stirring heating instrument, and digest for 10 minutes. Discard the upper digestion solution (mainly containing red blood cells, etc.) of the first digestion, continue to add 8mL of 0.125% pancreatin-EDTA digestion solution, put it into a magnetic stirring heating instrument, and digest for 10 minutes. Use a Pasteur pipette to suck the upper digestion solution from the second digestion into a 50mL centrifuge tube with 20% FBS termination digestion solution, then continue to add 8mL of 0.125% pancreatin-EDTA digestion solution to the serum bottle, put it into a magnetic stirring heating instrument, and digest for 10 minutes; after digestion, continue to collect the upper digestion solution into the centrifuge tube, continue to add pancreatic digestion solution, and repeatedly digest the heart tissue 4-5 times until the heart tissue is completely digested, at which time the heart tissue becomes lighter and whiter visibly with the naked eye.

[0081] ④ Isolation and culture of primary cardiomyocytes: After the digested cell suspension is filtered through a 40μm cell strainer, it is centrifuged in a 4℃ precooled low-temperature centrifuge for 8 minutes at a speed of 1000rpm. After the centrifugation, the upper layer is discarded, the lower layer of cell sediment is retained, and 8mL of cell culture medium is added to suspend the cells; the filtered cell suspension is evenly inoculated in a 10cm cell culture dish, and the cells are allowed to adhere to the wall in a 37℃ cell culture incubator for 90 minutes. After the cells are attached to the wall, the attached cells at the bottom of the culture dish are myocardial fibroblasts, and those that have not yet attached are primary cardiomyocytes. The cell density is calculated according to the cell counting results, the cell suspension is diluted, and evenly added to 10cm cell culture medium, six-well plates, 24-well plates, and 96-well plates. The cells are placed in a 37℃ constant temperature cell culture incubator for continued culture. Primary cardiomyocytes can adhere to the wall and grow for 24-48 hours. BrdU can be added to the culture dish to inhibit the proliferation and growth of fibroblasts in cardiomyocytes.

[0082] (2) Cell treatment and grouping: An in vitro cardiomyocyte hypertrophy model was constructed by angiotensin II (AngII, 1 μM) treatment. The control group and AngII-treated group were given equal amounts of PBS and MAZ (1 μM), respectively. After the above treatment, primary cardiomyocytes or fibroblasts were divided into the following four groups: PBS group, AngII group, PBS+MAZ group, and AngII+MAZ group.

[0083] (3) Molecular biology detection: According to the above steps, the cell proteins of each group were extracted and Western-boltting was performed to evaluate the level of myocardial remodeling and myocardial fibrosis. The cardiomyocytes of each group were collected and added to Trizol lysis buffer for full lysis, chloroform extraction, centrifugal precipitation, ethanol rinse, and heated for dissolution. The concentration was detected and reverse transcription was performed. RT-PCR was then performed to detect the mRNA expression level of cardiomyocyte-related genes in each group.

[0084] The results are as follows Figure 7 As shown, Mastolide can reduce the expression levels of AngII-induced myocardial remodeling and myocardial fibrosis genes in in vitro cardiomyocytes.

[0085] (4) Cell immunofluorescence detection: The culture medium of the 24-well plate of treated cardiomyocytes or fibroblasts is completely discarded, PBS is slowly dripped along the side wall of the well with a dropper, and the plate is placed on a shaker for 3 minutes. After rinsing three times, 4% paraformaldehyde solution is used for fixation. After rinsing three times with PBS, 0.2% Triton X-100 is added, and the plate is gently shaken for 10 minutes for full permeabilization. The permeabilization solution is discarded, and after rinsing three times with PBS, 200 μL of pre-prepared 10% sheep serum is dripped into each well for blocking for 1 hour. A primary antibody solution containing rabbit anti-mouse α-actinin or α-SMA monoclonal antibody is prepared in advance with PBS at a ratio of 1:100. 30 μL of primary antibody is dripped onto each slide to fully cover the slide, and the slide is placed in a black humidified box at 4°C overnight. The next day, take out the wet box and rewarm it in a 37℃ incubator for 30 minutes, discard the primary antibody, transfer the slide to a 24-well plate, slowly add PBS to the wells and rinse on a shaker for 3 minutes, repeat the rinse 5 times. Pick up the slide again and put it on the slide, add 30μL of the corresponding species secondary antibody solution to the slide, and place the slide in a black wet box at 37℃ for 1 hour. After DAPI sealing, observe with a fluorescence microscope or confocal camera.

[0086] The results are as follows Figure 8 As shown, it was shown that Mastolide could significantly improve AngII-induced cardiomyocyte hypertrophy and myocardial fibrosis in vitro.

[0087] Example 6: Effects of Mashidu peptide on AngII-stimulated cardiomyocyte mitochondria in vitro

[0088] Use the JC-1 detection kit to prepare 2mL JC-1 staining buffer (5X) as the JC-1 staining working solution; JC-1 staining buffer (1X): 2mL JC-1 staining buffer (5X) is mixed evenly with 8Ml pure water; positive control setting: CCCP (10mM) marked in the kit is added to the culture medium at a ratio of 1:1000 and diluted to 10μM. Take out the six-well plate, discard the culture medium, replace the new culture medium and add 1mL JC-1 staining working solution to each well, mix well and incubate in the cell culture incubator at 37℃ for half an hour, and the positive control group is treated with CCCP. Precool the staining buffer (1X) on ice in advance, discard the culture medium after incubation, and wash twice with JC-1 staining buffer, and replace the new culture medium after washing. Most cells usually lose their mitochondrial membrane potential completely after half an hour of CCCP treatment. After JC-1 staining, it should show green fluorescence. Cells with normal membrane potential show red fluorescence when JC-1 staining. Mitochondrial membrane potential analysis is performed according to the fluorescence intensity of different colors. In addition, the ATP detection kit was used to detect the ATP level of cardiomyocytes in each group.

[0089] Test results such as Fig. 9As shown, it was shown that Mashidu peptide could improve AngII-induced abnormal mitochondrial membrane potential and cardiomyocyte ATP level.

[0090] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0091] Although preferred embodiments of the present invention have been described, additional changes and modifications may be made to these embodiments by those skilled in the art once the basic inventive concepts are known. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention. Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Use of Mashidu peptide in the preparation of drugs for improving or treating myocardial remodeling and myocardial fibrosis.

2. The use according to claim 1, characterized in that: The medicine also includes pharmaceutically acceptable excipients and carriers.

3. The use according to claim 1, characterized in that: The auxiliary material includes at least one of a filler, a disintegrant, a binder, a lubricant, a sweetener or a colorant.

4. The use according to claim 1, characterized in that: The dosage form of the drug includes at least one of granules, tablets, pills, capsules and injections.

5. The use according to claim 1, characterized in that: The effective dose of the Mastu peptide is 4-6 mg / kg.

6. The use according to claim 1, characterized in that: The Mashidu peptide can significantly improve the cardiac function of pressure overload model mice and significantly improve the hypertrophy of isolated cardiomyocytes caused by angiotensin II.

7. A drug for improving or treating myocardial remodeling and myocardial fibrosis, characterized in that: The active ingredient of the medicine includes Mastu peptide.

8. The drug for improving or treating myocardial remodeling and myocardial fibrosis according to claim 7, characterized in that: The drug also includes pharmaceutically acceptable excipients.

9. Use of Mashidu peptide in the preparation of drugs for improving or treating mitochondrial damage and metabolic disorders.

10. The use according to claim 9, characterized in that: The Mashidu peptide improves cell mitochondrial damage and energy metabolism disorder in the myocardial remodeling model.

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