CN117462654A - Application of semaglutide in the preparation of drugs for preventing or treating myocardial remodeling - Google Patents

Abstract

The invention provides an application of semaglutin in preparing a medicament for preventing or treating myocardial remodeling, which can improve cardiac dysfunction of mice with myocardial remodeling induced by pressure load, and relieve myocardial cell hypertrophy and matrix deposition of the mice, wherein the main manifestations are reduced myocardial cell cross-sectional area, fibrosis and myocardial hypertrophy and reduced fibrosis marker transcription level; the structure and the dysfunction of myocardial mitochondria of a myocardial reconstruction mouse can be improved, and the expression of mitochondrial function related genes is up-regulated at the transcriptional sequencing level and the transcription of mitochondrial structure function related molecular markers and protein level recovery are realized; further experiments indicate that semaglutin exerts an anti-myocardial remodeling effect by improving cardiac energy metabolism. The result of the invention shows that the semaglutin has potential to be a medicament for preparing myocardial remodeling prevention and treatment, and has better clinical application prospect.

Description

Application of semaglutin in preparing medicine for preventing or treating myocardial remodeling

Technical Field

The invention relates to the technical field of biological medicines, in particular to application of semaglutin in preparation of a medicament for preventing or treating myocardial remodeling.

Background

Heart failure can be caused by the end-stage of the development of a variety of causative cardiovascular diseases, and heart failure is considered to be one of the most frequent diseases. Myocardial remodeling is an important stage in the development and progression of heart failure. Myocardial remodeling is primarily manifested by pathological cardiomyocyte hypertrophy and interstitial fibrosis, leading to systolic and diastolic dysfunction. Further development of myocardial remodeling can lead to structural and functional changes in the left ventricle that are poorly adapted, ultimately leading to heart failure. Myocardial energy metabolism is closely related to the occurrence and development of myocardial remodeling. The flexibility of healthy heart energy metabolism ensures that heart cells are able to switch metabolic substrates in response to changes in substrate concentration and oxygen supply, enabling the heart to maintain adequate ATP production. Cardiac energy flows primarily through mitochondrial oxidative phosphorylation and glycolysis (approximately 5%). In healthy hearts, 60-90% of the energy comes from fatty acid oxidation. But heart failure patients lose metabolic flexibility and undergo myocardial metabolic remodeling. The relative lack of oxygen results in a heart that tends to ingest and utilize glucose as an energy substrate. However, due to defects in mitochondrial structure and function, an increase in glucose uptake primarily induces an increase in glycolysis rather than an increase in glucose oxidation. Clinical studies have shown that heart energy metabolism in heart failure patients goes from fatty acid oxidation to glycolysis, rather than glucose oxidation, resulting in reduced ATP productivity and shortage of heart energy supply and further increasing the progression of heart failure. Thus, improving mitochondrial defects and myocardial energy insufficiency is an important target for improving myocardial remodeling. No drug has been found clinically that can prevent and treat myocardial remodeling based on improving myocardial energy metabolism.

Therefore, there is a need to develop a drug for preventing and treating myocardial remodeling.

Disclosure of Invention

The invention aims to provide application of semaglutin in preparing medicines for preventing or treating myocardial remodeling, and finds application of semaglutin as medicines for preventing and treating myocardial remodeling, including relieving myocardial cell hypertrophy, fibrosis and cardiac function deterioration caused by pressure load.

In order to achieve the above purpose, the invention adopts the following technical scheme:

the invention provides an application of semaglutin in preparing a medicament for preventing or treating myocardial remodeling.

Further, the medicine for preventing or treating myocardial remodeling further comprises pharmaceutically acceptable auxiliary materials or carriers.

Further, the auxiliary materials comprise at least one of a filler, a disintegrating agent, a binder, an excipient, a diluent, a lubricant, a sweetener or a colorant.

Further, the dosage form of the medicament for preventing or treating myocardial remodeling comprises at least one of granules, tablets, pills, capsules and injections.

Further, the means for preventing or treating myocardial remodeling comprising: improving cardiac dysfunction of mice with myocardial remodeling under pressure load, relieving myocardial cell hypertrophy and fibrosis deposition of mice with myocardial remodeling, recovering structure and dysfunction of myocardial mitochondria of mice with myocardial remodeling, and improving cardiac energy metabolism to play an anti-myocardial remodeling role.

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

the application of the semaglutin in preparing the medicine for preventing or treating myocardial remodeling provided by the invention confirms that the semaglutin can relieve myocardial remodeling by researching the influence of the semaglutin on a myocardial remodeling model mouse induced by aortic arch constriction, and firstly reverses and improves heart functions such as left ventricular ejection fraction and the like; secondly, pathologically stained cardiac hypertrophy and reduced fibrosis levels and reduced levels of markers thereof; finally, the method also finds that the mitochondrial morphology, dysfunction and glycolipid metabolic disturbance in the reconstructed heart tissue have obvious improvement effects, and provides more possibility for targeted prevention and treatment of myocardial reconstruction targets.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is the effect of different doses of semaglutin on mouse body weight and liver function in example 1, and final experimental concentrations were determined. FIG. 1A is a modeling drug delivery flow of an experiment; FIG. 1B is the effect of different doses on mouse body weight during the course of the experiment; fig. 1C is the effect of different doses on liver function in mice.

FIG. 2 is the effect of semaglutin on mouse cardiac function, myocardial fibrosis and myocardial hypertrophy induced by eight weeks of TAC in example 2. FIG. 2A is a B-mode and M-mode ultrasound map after TAC post-surgery given semaglutin for a week of treatment; FIG. 2B is a graph showing statistics of left ventricular ejection fraction and short axis shortening rate; FIG. 2C is a graph of the hemodynamic parameters of the mouse heart; FIG. 2D is a statistical plot of the HW/BW ratio, the cardiac/Tibial length (Tibial, TL) ratio, and the pulmonary weight/body weight (Lung weight, LW) ratio of the mice;

FIG. 2E is a graph showing the statistics of mouse LVEDd and LVEDs; FIGS. 2F-G are in vivo transcript levels of markers ANP and BNP and markers ColI and ColIII for fibrosis; FIGS. 2H-I are quantitative results of wheat germ lectin (WGA) staining and sirius red-picric acid (PSR) staining for cell area and fibrosis area.

FIG. 3 is a reverse effect of semaglutin on TAC four week induction in reconstituted mice for cardiac function, myocardial fibrosis and myocardial hypertrophy in example 3. FIG. 3A is a modeling flow for an experimental design; FIG. 3B is a B-mode and M-mode ultrasound map of different groups after TAC surgery; FIGS. 3C-D are graphs of statistical comparisons of left ventricular ejection fraction and short axis shortening results for four weeks post-surgery and four weeks post-administration; FIGS. 3E-G are hematoxylin-eosin (HE) staining and WGA staining results and PSR staining results and cardiomyocyte area measurement and fibrosis area measurement results thereof; FIG. 3H is a statistical plot of HW/BW and HW/TL, and FIG. 3I is the in vivo transcript levels of the cardiac hypertrophy markers ANP and BNP and the fibrosis markers ColI and ColIII.

Fig. 4 is the effect of semaglutin on mitochondrial structure and function in mice with myocardial remodeling in example 4. FIG. 4A shows the results of gene expression related to mitochondrial function in the transcriptome sequencing of mouse left ventricular heart tissue; FIGS. 4B-C are immunoblots of mouse left ventricular heart tissue mitochondrial structural proteins DRP1, OPA1, mfn2, tom20 and functionally related proteins COX IV, SDHB, NDUFV2, ATP5A1 and quantitative statistical results thereof, including statistical analysis of in vivo transcription levels of these proteins; fig. 4D is a transmission electron microscope photograph of mitochondria of heart tissue of the left ventricle of a representative mouse.

FIG. 5 shows the results of metabolite detection in left ventricular heart tissue of the mice with myocardial remodeling in example 5. FIG. 5A is a statistical analysis of the mass spectrometry metabolic analysis of the left ventricular heart tissue of the mouse with respect to the products related to the glycolytic pathway and the tricarboxylic acid cycle substrate content; FIG. 5B is lipid levels in heart tissue of mice analyzed by mass spectrometry; FIG. 5C is the transcriptional levels of mouse left ventricular cardiac tissue glycolysis, and the tricarboxylic acid cycle key enzymes HK2, IDH2, and PDH.

Detailed Description

The advantages and various effects of the present invention will be more clearly apparent from the following detailed description and examples. It will be understood by those skilled in the art that these specific embodiments and examples are intended to illustrate the invention, not to limit the invention.

Throughout the specification, unless specifically indicated otherwise, the terms used herein should be understood as meaning as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification will control.

Unless specifically indicated otherwise, the various raw materials, reagents, instruments, equipment, etc., used in the present invention are commercially available or may be obtained by existing methods.

The technical scheme of the embodiment of the application aims to solve the technical problems, and the overall thought is as follows:

the molecular weight of the semaglutin is 956.114, and the molecular formula is C ₄₂ H ₆₉ N ₉ O ₁₄ S, the structural formula is as follows:

semaglutin is a novel Glucagon-like peptide-1receptor agonist (glucon-like peptide-1receptor agonist,GLP-1 RA) and is widely used clinically as a hypoglycemic agent for treating Type2diabetes (Type 2diabetes mellitus, T2 DM). Recently, the U.S. food and drug administration approved semaglutin as the first oral GLP1-RA drug to treat T2 DM. Importantly, semaglutin has remarkable weight-losing effect. At present, the traditional Chinese medicine composition is mainly used as a weight-losing therapeutic medicine clinically. The presently well-defined pharmacological actions of semaglutin include: (1) the semaglutin can reduce blood sugar, and is clinically used as blood sugar reducing treatment for patients with T2 DM; (2) the semaglutin has an improving effect on metabolism and is used for treating obesity syndromes and the like; (3) the semaglutin has obvious weight-losing effect and is clinically used as a weight-losing therapeutic drug; (4) semex Lu Tai has therapeutic effect on non-alcoholic fatty liver. The above shows that semaglutinin plays an important role in different diseases through its influence on glycolipid metabolism and body weight, but no related report on whether semaglutinin has an anti-myocardial remodeling effect has been found so far.

The inventor finds the application of the semaglutin as the myocardial remodeling prevention and treatment drug through experiments:

in the application process of the semaglutin as the myocardial remodeling prevention and treatment medicine, the safe dosage range of the semaglutin should be determined firstly, and the equivalent dosages of 4 mug/kg/day, 12 mug/kg/day and 60 mug/kg/day are used for carrying out the preliminary experiment on the intraperitoneal injection of the mice by referring to the research dosages and effective bioconversion in other diseases in the past, and because the semaglutin is mainly clinically used as the weight-losing medicine at present, the dosage which is most obvious for weight loss and heart function improvement of the mice is selected, namely, the equivalent dosage of 60 mug/kg/day is the experimental dosage for subsequently verifying the semaglutin as the myocardial remodeling prevention and treatment medicine.

In the specific embodiment, a male C57BL/6N mouse is mainly used as an experimental object, and an aortic arch constriction (Transverse aortic constriction, TAC) is utilized to construct a myocardial reconstruction model; two schemes of four weeks of beginning administration after operation and eight weeks of beginning administration after operation are adopted; the aim was to verify whether semaglutin has an effect on both improving myocardial remodeling and reversing myocardial remodeling. Ultrasonic and hemodynamic detection are respectively carried out before eight weeks of material collection after operation, and heart tissues are collected for respectively carrying out histological and molecular biological detection, so as to verify the application of the semaglutin as a medicament for preventing and treating myocardial remodeling. The results prove that the semaglutin can obviously prevent and reverse myocardial hypertrophy, fibrosis and heart function deterioration caused by pressure load, and is specifically expressed as follows: (1) Compared to the surgery group, the mice in the surgery+drug group had a reduced Heart Weight (HW)/Body Weight (BW) ratio. The heart functions are obviously improved, such as the left ventricular fractional shortening (Left ventricular fractional shortening, LVFS), the left ventricular ejection fraction (Left ventricular ejection fraction, LVEF), the left ventricular end-diastole inner diameter (Left ventricular end-diastolic diameter, LVEDd) and the left ventricular end-systole inner diameter (Left ventricular end-systolic diameter, LVEDs) are improved; (2) Histological staining showed reduced cardiomyocyte cross-sectional area and reduced levels of myocardial fibrosis; (3) The transcript level of markers of cardiac hypertrophy and fibrosis (ANP, BNP, col i and Col iii) was significantly reduced; (4) From the mechanism aspect, compared with an operation group, the mitochondrial morphology and the function in heart tissues of the operation and drug group are obviously improved, and after the treatment of the semaglutinin is given, the glycolipid metabolism of the heart tissues is obviously improved, and the glycolysis product enters tricarboxylic acid circulation to obviously increase so that the myocardial ATP supply is restored. In conclusion, the semaglutin can relieve myocardial remodeling induced by pressure load and can be applied as a myocardial remodeling prevention and treatment drug.

In conclusion, the result of the invention shows that the compound semaglutin has potential to prepare the medicine for preventing or treating myocardial remodeling, and has better clinical application prospect.

The invention provides an application of semaglutin in preparing a medicament for preventing or treating myocardial remodeling, wherein the application refers to the addition of pharmaceutically acceptable auxiliary materials and carriers to the semaglutin, the auxiliary materials comprise at least one of filling agents, disintegrating agents, adhesives, excipients, diluents, lubricants, sweeteners or colorants, and different auxiliary materials are selected according to the requirements of pharmaceutical dosage forms. The preparation is granule, tablet, pill, capsule, injection or dispersing agent.

It can be understood that the further structural optimization is performed by taking the semaglutin as a lead compound, and the preparation of the medicament for preventing or treating myocardial remodeling also belongs to the scope of the invention.

The application of the semaglutin in the present application in preparing a medicament for preventing or treating myocardial remodeling will be described in detail below with reference to examples and experimental data.

Feeding experimental animals: the invention adopts 8-10 week old male C57BL/6N mice with initial weight of 23-28g as experimental objects, which are purchased from the medical laboratory animal institute of Chinese medical science sciences and fed to the laboratory animal center of the cardiovascular disease institute of the university of Wuhan. All animals were housed in specific pathogen free (SPF grade) facilities.

Drug sources: the medicine used in the invention is purchased from MedChemexpress, the catalog number is HY-114118, and the purity is about 99.84%.

Preparing the medicine: the semaglutin is dissolved in normal saline to prepare a storage solution of 300ug/ml, the storage solution is conveniently packaged and stored in a refrigerator at the temperature of minus 20 ℃, and the experimental drug is administered by intraperitoneal injection once every three days with the diluted concentration of the normal saline being 12 mug/kg, 36 mug/kg and 180 mug/kg.

The administration mode is as follows: the mice of the surgical or sham control were given by intraperitoneal injection every three days at a dose of 12 μg/kg,36 μg/kg and 180 μg/kg body weight, with the drug being administered as it is, starting four or three days after surgery, once every three days, and continuing for 4 or 8 weeks.

Example 1 influence of different doses of semaglutin on body weight and liver function in mice

(1) Establishing a mouse myocardial reconstruction model and semaglutin treatment

Male C57BL/6N with age of 8-10 weeks are randomly divided into 4 groups, and each group ensures at least 12 mice: (1) sham surgery group (Sham); (2) semaglutin group (Sema); (3) aortic arch constriction (TAC); (4) aortic arch constriction + semaglutin different dose groups (TAC + Sema).

Wherein, only the hanging wire is not ligated after the breast is opened by the artificial operation group, the operation group specifically operates as follows: the mice were anesthetized with 3% pentobarbital sodium, a percutaneous backward noninvasive cannula was prepared, fixed in the right lateral recumbent position, hearts were exposed layer by layer along 2/3 intercostals, thoracic aorta was freed, 7-0 surgical sutures were passed under the aorta, and a despinned 27-gauge needle was placed parallel to the aorta on the surgical sutures, after firm ligation of the aorta and needle, the needle was rapidly withdrawn to achieve a stenosis of 70% of the aorta, the chest was closed after surgery, and the mice were observed until awakening.

Starting intraperitoneal injection administration after 3 days or four weeks after operation, dissolving split-charging semaglutinin in normal saline to make the final concentration of semaglutinin solution be 12 μg/kg,36 μg/kg and 180 μg/kg; the semaglutin mice are 180 mug/kg; mice in the surgery + semaglutin group were dosed at 12 μg/kg,36 μg/kg and 180 μg/kg body weight, once every three days for 4 or eight weeks.

(2) The body weight of the mice was recorded weekly at the beginning of the experiment until the eighth week of sampling and statistical analysis was performed

The results of fig. 1B show: the different doses of semaglutin had a significant down-regulation effect on the body weight of mice compared to the control group, wherein the high dose group (180 μg/kg) was most pronounced to reduce the body weight of mice compared to the control group (P < 0.05).

Collecting blood from orbital vein of mice, placing the collected orbital vein blood on ice, centrifuging at 12000rpm at 4deg.C for five minutes to separate serum, collecting supernatant from the centrifuge tube, and placing into a new centrifuge tube, and adopting full-automatic biochemical analyzer2400, siemens, usa) to determine serum alanine aminotransferase and aspartate aminotransferase concentrations.

The results of fig. 1C represent: compared with the control group, the serum alanine aminotransferase and the aspartate aminotransferase of the mice are not significantly affected by the different doses of the semaglutin, which indicates that the liver function of the mice is not affected by the different doses of the semaglutin.

Example 2 influence of semaglutin on reconstruction of mouse cardiac Functions, myocardial fibrosis and myocardial hypertrophy under eight weeks of TAC Induction

(1) Establishing a mouse myocardial reconstruction model and semaglutin treatment

The content is the same as in example 1.

(2) Echocardiography assessment of mouse cardiac function

After weighing and recording the mice, the mice are smeared with depilatory cream on the chest of the mice to carry out depilatory, so that the skin of the precordial region of the mice is exposed for subsequent ultrasonic detection. Next, the precordial region was smeared with an ultrasound couplant, and after anesthesia with 1.5-2.5% isoflurane, the mice were left lying in position and examined for evaluation of LVEF, LVFS, LVEDd and LVEDs and hemodynamic parameters at the level of the short axis of the left ventricular papillary muscle. And leave the B-mode and M-mode ultrasonography video of the mice.

FIG. 2A is a representative B-mode and M-mode ultrasound plot of mice showing that TAC post-surgery mice had deteriorated cardiac function and left ventricular hypertrophy as compared to sham-surgery controls, whereas cardiac function and left ventricular hypertrophy were significantly improved following semaglutin treatment; fig. 2B-C, E results show that semaglutin treatment can improve LVEF, LVFS, LVEDd and LVEDs and changes in hemodynamic parameters (P < 0.001) in reconstituted mice simultaneously compared to the surgical group.

(3) Taking left ventricular heart tissue of a mouse, and detecting myocardial hypertrophy and fibrosis degree of the mouse at a histological level

After 8 weeks of operation, the mice with weight recorded and ultrasound done are dislocation killed, the small scissors are used for cutting along the left rib of the sternum, the heart of the mice is separated, the heart tissue for pathological sections is taken out and then is rapidly placed in 10% KCl solution, so that the heart tissue stops jumping in diastole, blood in the heart cavity is squeezed out, and the heart tissue is weighed (unit: mg) after trimming and is placed in formalin for soaking; the heart tissue used for the meristem can be directly squeezed out of the heart cavity blood and then trimmed for weighing (unit: mg); lung tissue was removed and weighed (unit: mg); one side of the tibia was exposed and its length (unit: cm) was measured. The weighed mouse heart is fixed in formalin for 24-48 hours, then trimmed and placed in an embedding frame for dehydration, transparency and embedding, and then pathological sections are prepared for subsequent experiments.

WGA staining to detect cardiomyocyte cross-sectional area: baking the pathological section at 65 ℃ for at least 30min, taking out the section, sequentially putting the section into xylene twice for 5min each for dewaxing, hydrating with 100% -70% gradient alcohol, and then putting into pure water for soaking for 10min; trypsin method antigen repair slice (50 ul/heart is placed in a wet box for incubation at 37 ℃ for 15 min), PBS rinsing, dropwise adding prepared WGA-AF488 working solution into heart tissue for incubation for 2h, and thoroughly rinsing with PBS; finally, DAPI sealing sheets are used and are placed under a fluorescence microscope for photographing. WGA staining photograph: at least 20 cells with clear boundaries on each plot and nuclei in the center of the cells were required, and after photographing, the myocardial cell cross-sectional area was measured using Image-Pro Plus 6.0 Image analysis software.

PSR staining detects fibrosis levels: baking the sections, dewaxing and hydrating steps were the same as above WGA staining; then placing the cleaned slice into 0.2% phosphomolybdic acid for about 2min, then dripping 0.1% picric acid-sirius red dye liquor on the tissue, placing the slice into a wet dyeing box for dyeing, throwing away the dye liquor after 1.5 h, placing the slice into 0.01N hydrochloric acid for about 1 second each time, then sequentially placing the slice into 70% -100% gradient alcohol for dehydration, finally placing the slice into dimethylbenzene until the slice is transparent, sealing the slice with neutral resin while the dimethylbenzene is not dried, and drying the slice in a fume hood to take a picture.

From the data measured at the time of drawing, the mice HW/BW and HW/TL were calculated for each group, and the results in FIG. 2D represent: compared with the sham operation group, the semaglutinin group has no statistical significance with the difference, while HW and LW of the TAC operation group are increased, and the result of using semaglutinin after operation can be reversed; the results of FIGS. 2H-I show: compared to sham surgery group, TAC surgery group central myocyte cross-sectional area increased, myocardial fiber increased and arrangement disordered, interstitial overt edema (P < 0.001); semaglutin treatment can reduce myocardial cell cross-sectional area, alleviate interstitial edema and myocardial fiber deposition (P < 0.001).

(4) RT-PCR evaluation of mice on the transcriptional level of markers of cardiac hypertrophy and fibrosis

After TAC operation for 8 weeks, the mice are sacrificed to obtain materials after echocardiography is completed, the hearts squeeze out blood and left ventricular heart tissues are trimmed and placed in a freezing tube; a certain amount of left ventricular heart tissue was cut and ground to extract total RNA, and in order to detect mRNA expression, total RNA was extracted from each sample heart tissue using TRIzol reagent, cDNA synthesis was performed by purchasing a transcribed first strand cDNA synthesis kit (Roche, switzerland) and then real-time fluorescent quantitative PCR was performed using 480SYBR green dye. Gene expression was determined by reverse transcription polymerase chain reaction Luo Shiguang cycler 1 detection system. Comparing the expression of the target genes of each group according to the variation of the CT period value among groups compared with the betA-Actin level, adopting 2 ^-ΔΔCt Methods and normalized statistical analysis. The transcript levels of the hypertrophic markers ANP and BNP and the fibrosis markers ColI and ColIII in the heart tissue of the mice were examined in the same manner as in example 2. The primer sequences were as follows:

TABLE 1

The results of FIGS. 2F-G show: compared to sham surgery, the transcript levels of ANP, BNP, col i and Col iii were both elevated in TAC surgery, the differences were statistically significant (P < 0.01); whereas semaglutin treatment inhibited myocardial hypertrophy and fibrosis marker transcript levels in reconstituted mice (P < 0.01) compared to TAC surgery group.

Sustained pressure load stimulation can cause disturbances in nerve and body fluid regulation, leading to myocardial remodeling, which refers to changes in cardiomyocyte structure and function, leading to cardiac hypertrophy, left ventricular enlargement, and systolic/diastolic dysfunction, which in turn progress to heart failure; myocardial remodeling due to pressure load is often mainly caused by myocardial hypertrophy, and at this time, the number of myocardial cells is not increased, and myocardial fibers are mainly increased, so that cavitation is also increased in mitochondria as a supply energy substance, and thus myocardial ATP supply is also lost. Semaglutin is effective in inhibiting the occurrence of myocardial remodeling when sustained pressure loading causes pathological changes in the heart.

Example 3 reversal of mouse cardiac function, myocardial fibrosis and myocardial hypertrophy by semaglutin after four weeks of TAC induction

(1) Establishing a mouse myocardial reconstruction model and semaglutin treatment

Injection of semaglutin into the abdominal cavity was started four weeks after TAC surgery, the remainder being the same as in example 2.

(2) Echocardiography assessment of mouse cardiac function

Echocardiographic evaluations were performed four and eight weeks after surgery, as in example 2.

The results of fig. 3B show: compared to sham operated control group, TAC operated group mice had deteriorated cardiac function and ventricular hypertrophy, and semaglutin treatment reversed this change; the results of fig. 3C-D show: compared with the TAC operation group, the left ventricle ejection fraction and the short axis shortening rate of the mice with myocardial reconstruction can be effectively reversed by the treatment of the semaglutin after the TAC is treated for four weeks; this suggests that semaglutin has a significant reversal of cardiac function in mice that have undergone myocardial remodeling (P < 0.05).

(3) Taking a mouse left ventricular heart, and detecting the reversion degree of the semaglutin to the mouse myocardial hypertrophy and fibrosis at a histological level

Example 2 was prepared by taking materials and pathological sections.

HE staining detects gross changes in myocardium: baking slices, dewaxing and hydrating steps are the same as before; then hematoxylin dye liquor, 1% hydrochloric acid alcohol differentiation, scott liquor bluing, eosin dye liquor and distilled water rinsing; the sealing plate and the observation under a microscope are the same as before. HE staining and photographing: the requirements are the same as before.

WGA staining to detect cardiomyocyte cross-sectional area: the content is the same as in example 2.

PSR staining detects fibrosis levels: the content is the same as in example 2.

The results of fig. 3E-G show: the heart generally has an increased cross-sectional area after TAC compared to sham surgery, and administration of semaglutin treatment after reconstitution can also reverse this hypertrophy; meanwhile, compared with the TAC group, the semaglutin has a reverse effect on the increase of the myocardial cross section and the fibrosis deposition of a mice with myocardial reconstruction after the treatment; the results in fig. 3H show that: the semaglutin treatment also reduced the HW/BW ratio compared to the TAC group.

(4) RT-PCR evaluation of mice on the transcriptional level of markers of cardiac hypertrophy and fibrosis

The procedure is as in example 2.

The results of fig. 3I show: compared to sham surgery, the transcript levels of ANP, BNP, col i and Col iii were both elevated in TAC surgery, the differences were statistically significant (P < 0.01); whereas, compared to TAC surgery group, semaglutin treatment can reverse the myocardial hypertrophy and fibrosis marker transcript levels (P < 0.01) in reconstituted mice.

In view of the fact that early stages of myocardial remodeling are not easily found, most clinical hospitalization patients are already present in early stages of myocardial remodeling, it is important to reverse existing myocardial remodeling and prevent the existing myocardial remodeling from further developing into heart failure. Therefore, we further evaluate the reversal effect of semaglutin on existing myocardial remodeling, and found that semaglutin can reverse existing myocardial remodeling, preventing further development.

Example 4 influence of semaglutin on mitochondrial Structure and function in mice with myocardial remodeling

(1) Establishing a mouse myocardial reconstruction model and intervention of semaglutin

The procedure is as in example 2.

(2) Results of expression of mitochondrial function-related genes by transcriptome sequencing of left ventricular heart tissue of mice

Firstly, total high-purity RNA in heart tissue of a mouse is separated, the RNA is converted into an original sequencing sequence (sequential Reads) through Base recognition (Base Calling), quality control treatment is carried out by using software fastq, then hisat2 software is used for comparison to a reference genome, the number of featureCount soft statistical genes after the reference genes are effectively compared is obtained, data analysis and drawing are carried out by using resources and software such as GSEA, metascope, cytoscope and R programs after basic data are obtained, and the inter-group differential expression genes are corrected by adopting Limma relaxation t detection (P < 0.1).

(3) Taking heart tissue of left chamber of mouse, observing mitochondrial structure under electron microscope

After successful molding, mice were anesthetized and euthanized 8 weeks after TAC treatment, and their hearts were rapidly removed and placed in a liquid nitrogen freezer for subsequent cutting. Left ventricular heart tissue was cut into small pieces of about 2-3mm, immediately fixed in an electron microscope solution (2.5% glutaraldehyde) for 24 hours, then stained with toluidine blue, and stained with 4% uranyl methoxide and lead ranolacitrate, and the ultrastructure of mouse cardiac mitochondria was observed with a transmission electron microscope (TM-3000; hitachi, japan).

(4) Western Blot detection of mitochondrial structure and function related proteins in myocardial remodeling

The mouse heart was obtained as in example 2, and the mouse heart was ground and lysed to extract total protein and protein quantification was performed. After spotting, electrophoresis, transfer and sealing, the corresponding primary antibody was applied overnight at 4 ℃. Rinsing for 3 times in TBST for 5 min/time, then incubating the secondary antibodies of the corresponding species for 1h at room temperature, and rinsing for 3 times in TBST to obtain the membrane scanning analysis. Wherein the primary antibody used comprises: DRP1, OPA1, mfn2, tom20, COX IV, SDHB, NDUFV2, ATP5A1 and VDAC. Specific primary antibody information is as follows:

TABLE 2

(1) RT-PCR detection of mouse cardiac mitochondrial function and structure related marker transcript levels

mRNA levels of mitochondrial structural and functional correlations, such as DRP1, in mouse heart tissue were detected and performed as in example 2. The specific sequence is as follows.

TABLE 3 Table 3

The results of fig. 4A show: compared with the control group, the expression of the mitochondrial function related gene of the heart of the mice in the TAC group is reduced, and the mouse is recovered after the treatment of the semaglutin; the results of FIGS. 4B-C show: further validation of expression levels of mitochondrial fission protein (Drp 1), fusion protein (Opa 1 and Mfn 1/2), apoptosis (Tom 20) and mitochondrial respiration (COX IV, SDHB, NDUFV2 and ATP5 A1) markers by western blot and PCR, the results indicate that TAC induction resulted in increased mitochondrial fission apoptosis, reduced fusion with respiratory dysfunction (P < 0.01), whereas semaglutinin treatment improved this imbalance and mitochondrial respiratory dysfunction (P < 0.05) compared to sham control; the results of fig. 4D show: transmission electron microscopy showed that compared to the sham-operated control group, TAC post-operation mice had a swelling of the matrix, cavitation and a reduction in mitochondrial ridges in the cardiac tissue mitochondria, whereas semeglucoside treatment restored the disorder of mitochondrial structure. These results indicate that semaglutin treatment can ameliorate mitochondrial respiratory impairment and mitochondrial structural disruption in TAC-induced pathological myocardial remodeling.

Mitochondria are closely related to myocardial energy metabolism, and myocardial remodeling can lead to mitochondrial structure and dysfunction, and can further lead to myocardial energy supply deficiency, thereby promoting heart failure. Therefore, it is important to improve mitochondrial structure and dysfunction, reverse myocardial remodeling, and prevent heart failure. Mitochondrial structure and dysfunction are mainly manifested by swelling of the mitochondrial matrix, cavitation and reduction of mitochondrial ridges. The semaglutin can effectively restore the structure and function of the reconstructed cardiac muscle mitochondria, thereby playing a role in heart protection.

Example 6 influence of semaglutin on myocardial energy metabolism in mice with myocardial remodeling

(1) Establishing a mouse myocardial reconstruction model and intervention of semaglutin

The content is the same as in example 2.

(2) Mouse left ventricular heart tissue material

The content is the same as in example 2.

(3) Non-targeted metabonomics analysis of mouse left ventricular heart tissue based on HPLC-Zeno TOF-MS/MS

The heart tissue of the mice was isolated and homogenized in ultrafiltration water. Subsequently 4 volumes of acetonitrile-methanol solution (50% v/v) were added to the tissue homogenate, followed by sonication for 10 minutes and refrigeration for 1 hour to precipitate the protein. Centrifuging to obtain supernatant, and purifying with high purity nitrogenAnd (5) blow-drying. The supernatant was reconstituted in water using 50% v/v acetonitrile and centrifuged again. The supernatant was then collected and transferred to vials and quality control samples were made. The metabolite was dissolved by liquid chromatography using mobile phase a, followed by ExionLC ^TM Serial UHPLC separations. Mobile phase a is an aqueous solution containing 0.1% formic acid; mobile phase B was acetonitrile. Setting a liquid phase gradient: 0-2.0 min, 1% b;2.0-12.0 minutes, 1% -99% B;12.0-19.0 min, 99% B;19.0-19.1 minutes, 99% -1% B;19.1-21.9 minutes, 1% B, flow rate was maintained at 300nL/min. The metabolites were isolated by UHPLC system, then injected into ESI ion source for ionization, then SCIEX-ZenoTOF 7600 system (AB SCIEX, USA). TOF-MS experimental setup: spray voltage, 5.5kV; unblustering potential, 60V; collision energy, 10V; the scan range was set to 60-1000Da with an accumulation time of 0.15s. TOF-MSMS setup: the declustering potential, 60V; resonance energy, 35±15V. The secondary mass spectrum scanning range is between 25 and 1000 Da. The data acquisition mode was performed using a data independent acquisition (IDA) procedure with the zeno threshold set at 2000000cps. Metabolites were identified based on self-constructed libraries and MetDNA (http:// www.metdna.zhulab.cn). Identification settings of the self-built library: parent ion m/z and fragment ion spectra fault tolerance 5ppm; the retention time varies by less than 5% relative to the purified standard metabolite. The relative quantification of the metabolites is determined by the peak area of the parent ion, which is normalized by the total protein concentration. Metabolite data were statistically analyzed using MetaboAnalyst 5.0 (https:// www.metaboanalyst.ca). Data graphs were generated by Rstudio and GraphPad Prism 9.4.1.

(4) RT-PCR detection of mouse cardiac glycolysis and tricarboxylic acid cycle key rate-limiting enzyme transcription level

mRNA levels of key rate limiting enzymes for glycolysis and tricarboxylic acid cycle, such as HK2, were measured in mouse heart tissue and the procedure is followed as in example 2. The specific sequence is as follows.

TABLE 4 Table 4

The results of fig. 5A show: compared to sham surgery group, eight weeks after TAC induction, mice heart glycolysis related metabolites: glucose-6-phosphate (G-6-P), fructose-6-phosphate (F-6-P), 3-phosphoglycerate (3-PG), phosphoenolpyruvate (PEP) and pyruvic acid; pentose Phosphate Pathway (PPP) product: glucose-1-phosphate (G-1-P), glucose-6-phosphate (6-PG) and ribose-5-phosphate are all significantly elevated (P < 0.01), but tricarboxylic acid cycle substrates such as citric acid and aconitic acid (P < 0.01) demonstrate increased cardiac glycolysis and decreased tricarboxylic acid cycle ATP production, semaglutin treatment results in decreased glycolysis product (P < 0.01) and increased tricarboxylic acid cycle substrate (P < 0.01), demonstrating that semaglutin can cause glycolysis product to enter tricarboxylic acid cycle to generate more ATP to increase cardiac energy supply; the results of fig. 5B show: the TAC-induced increase in free fatty acids resulted in cardiolipin toxicity compared to control sham surgery, whereas the cardiolipin toxicity was significantly reduced following semaglutinin treatment; the results of fig. 5C represent: glycolytic key rate limiting enzymes (HK 2) were significantly up-regulated after TAC (P < 0.01), while tricarboxylic acid cycle rate limiting enzymes (IDH 2 and PDH) were significantly down-regulated after TAC (P < 0.01) and corrected after semaglutin treatment. In conclusion, the semaglutin treatment can reduce glycolysis, promote the substrate to enter TCA circulation, increase myocardial energy supply and reduce the lipotoxicity of pathological myocardial remodeling induced by TAC.

Normal hearts rely primarily on fatty acid oxidation to provide energy. Cardiac lipid oxidation and TCA are inhibited in pathologically heart restructured patients, glycolysis is enhanced but glucose oxidation is unchanged or reduced, so that insufficient myocardial energy supply further leads to HF. Therefore, myocardial remodeling and cardiac energy substrate metabolism are closely related, myocardial energy metabolism is damaged, and myocardial remodeling complement each other and promote each other. And the semaglutin can improve the myocardial energy metabolism of the pathologically reconstructed heart, thereby playing a role in protecting the pathologically reconstructed heart.

Taken together with the above examples, it was found that semaglutin can not only improve cardiac dysfunction in mice with myocardial remodeling under pressure load, but also alleviate myocardial cell hypertrophy and fibrosis deposition in mice with myocardial remodeling, which is mainly manifested by decreased myocardial cell area, decreased fibrosis deposition, decreased myocardial hypertrophy and decreased fibrosis marker transcription level; the structure and the dysfunction of myocardial mitochondria of a myocardial reconstruction mouse can be recovered, and the structure and the dysfunction are expressed as the up-regulation of mitochondrial function related genes at the transcriptional sequencing level and the transcriptional and protein level change of mitochondrial structure function related molecular markers; further experiments indicate that semaglutin exerts an anti-myocardial remodeling effect by improving cardiac energy metabolism.

Finally, it is also noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.

Claims (5)

1. The application of semaglutin in preparing medicine for preventing or treating myocardial remodeling.

2. The use according to claim 1, wherein the medicament for preventing or treating myocardial remodeling further comprises a pharmaceutically acceptable adjuvant or carrier.

3. The use according to claim 2, wherein the auxiliary material comprises at least one of a filler, a disintegrant, a binder, an excipient, a diluent, a lubricant, a sweetener or a colorant.

4. The use according to claim 1, wherein the dosage form of the medicament for preventing or treating myocardial remodeling comprises at least one of granules, tablets, pills, capsules, injections.

5. The use according to claim 1, wherein the means for preventing or treating myocardial remodeling comprises: improving cardiac dysfunction of mice with myocardial remodeling induced by pressure load, relieving hypertrophy and fibrosis of myocardial cells of mice, improving structure and dysfunction of myocardial mitochondria of mice with myocardial remodeling, and improving heart energy metabolism to play an anti-myocardial remodeling role.