Application of Eraffibulaol in preparation of medicine for treating myocardial remodeling of diabetic cardiomyopathy

Abstract

The invention discloses application of Eraffibulaol in preparation of a medicine for treating myocardial remodeling of diabetic cardiomyopathy, and belongs to the technical field of medicines. By researching the influence of the Eraffibularox on diabetic cardiomyopathy-induced myocardial remodeling model mice, it is confirmed that the Eraffibularox can relieve myocardial remodeling, and heart functions such as left ventricular ejection fraction and the like are reversed and improved at first; secondly, the pathological staining cardiac hypertrophy and fibrosis level is reduced, and the marker level is reduced; finally, it is found that the traditional Chinese medicine composition has an obvious improvement effect on glucose and lipid metabolism disorder in reconstructed heart tissue. The invention provides a novel application of Eraffibulanol in preparation of a medicine for treating myocardial remodeling of diabetic cardiomyopathy, and provides more treatment strategies for clinical treatment of diabetic cardiomyopathy.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of erafibrino in the preparation of a therapeutic drug for myocardial remodeling in diabetic cardiomyopathy. Background Technology

[0002] Diabetes mellitus is a chronic metabolic non-communicable disease with a rapidly rising global incidence. Diabetic cardiomyopathy is a specific microvascular complication of the myocardium caused by diabetes, and it is a leading cause of heart failure and death in diabetic patients. Metabolic disorders play a crucial role in the development and progression of diabetic cardiomyopathy. Diabetic cardiomyopathy causes metabolic shifts in cardiomyocytes, increased fatty acid metabolism, and the accumulation of incompletely oxidized fatty acids, leading to myocardial lipotoxicity and ultimately cardiomyocyte death and myocardial remodeling. Myocardial remodeling is a fundamental mechanism leading to the occurrence and development of heart failure. Myocardial remodeling is mainly manifested as pathological myocardial cell hypertrophy and intercellular fibrosis, thereby triggering systolic and diastolic dysfunction. Despite decades of research into the pathogenesis and clinical characteristics of diabetic cardiomyopathy, effective methods for preventing and treating this disease remain limited. Current clinical treatments primarily focus on controlling blood sugar and improving blood lipids, and cannot effectively reverse myocardial remodeling in diabetic cardiomyopathy.

[0003] Elafibranor (trade name Iqirvo in some regions) is a novel peroxisome proliferation-activating receptor (PPAR) agonist that primarily works by activating PPAR-α and PPAR-δ receptors. It is currently used to treat adult patients with primary biliary cholangitis (PBC) who have an inadequate response to ursodeoxycholic acid (UDCA) and is in phase 3 clinical trials. Clinical studies have demonstrated that erafibranor has a high safety profile and is well-tolerated, but it has not yet been put into clinical use. To date, there are no reports on the effects of erafibranor on myocardial remodeling in diabetic cardiomyopathy. Summary of the Invention

[0004] This invention has discovered that erafibrano has a therapeutic effect on myocardial remodeling in diabetic cardiomyopathy. The purpose of this invention is to provide a new application of erafibrano in the preparation of drugs for the treatment of myocardial remodeling in diabetic cardiomyopathy, and to expand the clinical indications of erafibrano.

[0005] This invention uses male C57 / BL6 mice as experimental subjects to establish a mouse model of type 2 diabetic cardiomyopathy by feeding them a high-fat diet (containing 60% fat) combined with a low dose of streptozotocin (30 mg / kg / day, intraperitoneal injection for 7 days). At 16 weeks of high-fat feeding, the systolic and diastolic functions of the mice's hearts showed a significant decline, and an 8-week treatment with gavage administration was initiated. Simultaneously, spontaneously induced type 2 diabetic mice (db / db mice) were used as experimental subjects, fed a normal diet, and treated with gavage administration for 8 weeks at 16 weeks of age. These two animal models were used to verify whether erafibrinolate could improve cardiac dysfunction and myocardial remodeling induced by diabetic cardiomyopathy. The results confirmed that erafibrinolate can significantly reverse myocardial hypertrophy, fibrosis, and cardiac dysfunction caused by diabetic cardiomyopathy. Specifically, the following results were observed: (1) Compared to the diabetic cardiomyopathy model group, the cardiac function of mice in the model + drug group was significantly improved, such as the left ventricular fractional shortening (LVFS), left ventricular ejection fraction (LVEF), the ratio of early diastolic blood flow velocity to atrial systolic blood flow velocity (E / A), and the ratio of early diastolic blood flow velocity at the mitral valve orifice to early diastolic motion velocity of the mitral valve annulus (E / E'); (2) Pathological eosin staining (HE) and wheat germ agglutinin (WGA) showed a decrease in the cross-sectional area of ​​cardiomyocytes and a significant decrease in the transcriptional levels of myocardial hypertrophy markers (ANP and BNP); (3) Masson staining showed a reduction in myocardial fibrosis deposition and a significant downregulation of the transcriptional levels of fibrosis markers (Col1 and Col3); (4) Transmission electron microscopy and Oil Red O staining showed a reduction in lipid deposition in the left ventricle. In summary, erafibrino can alleviate myocardial remodeling induced by diabetic cardiomyopathy and can be used to prepare drugs for the treatment of myocardial remodeling in diabetic cardiomyopathy.

[0006] This invention provides the use of erafibrinolate in the preparation of a therapeutic agent for cardiomyopathy. Further, the cardiomyopathy is diabetic cardiomyopathy. In this application, erafibrinolate is the sole active ingredient.

[0007] The structural formula for Erafibrano is shown below.

[0008]

[0009] This invention also provides the use of compositions containing erafibrinolubrano in the preparation of medicaments for the treatment of cardiomyopathy. Further, the cardiomyopathy is diabetic cardiomyopathy. In this application, the composition may also contain other active ingredients with therapeutic effects on cardiomyopathy.

[0010] This invention also provides the use of erafibrino in the preparation of anti-myocardial remodeling drugs. Further, the myocardial remodeling is myocardial remodeling induced by diabetic cardiomyopathy. In this application, erafibrino is the sole active ingredient.

[0011] This invention also provides the use of a composition containing erafibrinolate in the preparation of an anti-myocardial remodeling drug. Further, the myocardial remodeling is myocardial remodeling induced by diabetic cardiomyopathy. In this application, the composition may also contain other active ingredients with anti-myocardial remodeling effects.

[0012] Furthermore, the aforementioned cardiomyopathy treatment drug or anti-myocardial remodeling drug, in addition to containing the active ingredient erafibrino, may also contain a pharmaceutically acceptable carrier or diluent. The dosage forms of the aforementioned cardiomyopathy treatment drug or anti-myocardial remodeling drug include powders, tablets, granules, or injections.

[0013] Furthermore, the aforementioned cardiomyopathy treatment drugs or anti-myocardial remodeling drugs are drugs that reduce cardiomyocyte hypertrophy, fibrosis and / or cardiac function deterioration caused by diabetic cardiomyopathy.

[0014] Advantages and beneficial effects of this invention: This invention, through studying the effects of erafibrano on a mouse model of myocardial remodeling induced by diabetic cardiomyopathy, confirms that erafibrano can alleviate myocardial remodeling, firstly reversing and improving cardiac functions such as left ventricular ejection fraction; secondly, decreasing the levels of myocardial hypertrophy and fibrosis and their markers in pathological staining; and finally, significantly improving glucose and lipid metabolism disorders in remodeled cardiac tissue, providing more treatment strategies for the clinical treatment of diabetic cardiomyopathy. Attached Figure Description

[0015] Figure 1 This describes the effect of 20 mg / kg / d of erafibrino administered by gavage in Example 1 on liver and kidney function in mice. Figure 1 A represents the effect of erafibrano on serum alanine aminotransferase (ALT) levels in mice; Figure 1 B represents the effect of erafibrano on serum aspartate aminotransferase (AST) levels in mice; Figure 1 C represents the effect of erafibrano on serum creatinine (Cr) levels in mice.

[0016] Figure 2 The effects of 20 mg / kg / d of erafibrano administered by gavage in Example 2 on body weight, blood glucose, and blood lipids in diabetic cardiomyopathy mice induced by high-fat diet combined with low-dose streptozotocin. Figure 2 A represents the change in the mouse's body weight over age; Figure 2 B represents the change in blood glucose levels in mice over age; Figure 2 C represents the oral glucose tolerance test in mice fed a standard diet or HFD / STZ for 24 weeks. Figure 2 D represents the effect of erafibrino treatment on serum triglyceride (TG) levels in mice; Figure 2 E represents the effect of erafibrino treatment on the serum total cholesterol (TC) level in mice.

[0017] Figure 3 The effects of 20 mg / kg / d of erafibrano administered by gavage in Example 3 on cardiac function, myocardial hypertrophy, myocardial fibrosis, and myocardial lipid deposition in diabetic cardiomyopathy mice induced by high-fat diet combined with low-dose streptozotocin. Figure 3 A is an M-mode ultrasound image of diabetic cardiomyopathy mice after treatment with erafibrino; Figure 3 B represents the left ventricular ejection fraction (LVEF) and LVFS shortening fraction (LVFS) of mice in the control group and the ellafibrano treatment group at specific time points. Figure 3 C shows representative images of hematoxylin-eosin (H&E) staining and wheat germ agglutinin (WGA) staining; Figure 3 D represents the quantification of the cross-sectional area of ​​cardiomyocytes based on WGA staining; Figure 3 E represents the in vivo transcriptional level of ANP, a marker of myocardial hypertrophy; Figure 3 F represents the in vivo transcriptional level of BNP, a marker of myocardial hypertrophy; Figure 3 G is a representative cardiac marson staining image of the cardiac interstitium and perivascular area; Figure 3 H represents the quantitative analysis of left ventricular collagen volume; Figure 3 I represents the in vivo transcriptional level of Col1, a marker of myocardial fibrosis; Figure 3 J represents the in vivo transcriptional level of Col3, a marker of myocardial fibrosis; Figure 3 K represents the accumulation of lipid droplets in the heart in a transmission electron microscope image; LDs label the lipid droplets; Figure 3 L represents the quantitative measurement of the number of lipid droplets in myocardial tissue; Figure 3 M represents a representative image and quantitative analysis of Oil Red staining in cardiac tissue.

[0018] Figure 4 The effects of 20 mg / kg / d of erafibrano administered by gavage in Example 4 on body weight, blood glucose, and blood lipids in spontaneously diabetic mice (db / db mice) with induced diabetic cardiomyopathy. Figure 4 A represents the effect of erafibrino treatment on the body weight of db / db or db / m mice; Figure 4 B represents the effect of erafibrino treatment on random blood glucose levels in db / db or db / m mice; Figure 4 C represents the effect of erafibrino treatment on oral glucose tolerance in db / db or db / m mice; Figure 4 D represents the effect of erafibrino treatment on serum triglyceride (TG) levels in db / db or db / m mice; Figure 4 E represents the effect of erafibrino treatment on serum total cholesterol (TC) levels in db / db or db / m mice.

[0019] Figure 5The effects of 20 mg / kg / d of erafibrano administered by gavage in Example 5 on cardiac function, myocardial hypertrophy, myocardial fibrosis, and myocardial lipid deposition in spontaneously diabetic mice (db / db mice) induced diabetic cardiomyopathy. Figure 5 A shows representative M-mode echocardiograms of four groups of mice at 24 weeks of age; Figure 5 B represents the effect of erafibrino treatment on left ventricular ejection fraction (LVEF) in db / db or db / m mice; Figure 5 C represents the effect of erafibrino treatment on the reduced left ventricular fraction (LVFS) in db / db or db / m mice; Figure 5 D represents the effect of erafibrino treatment on the E / A peak ratio in db / db or db / m mice; Figure 5 E is a representative image of hematoxylin-eosin staining (H&E) and wheat germ lectin (WGA) staining; Figure 5 F represents the quantification of the cross-sectional area of ​​cardiomyocytes based on WGA staining; Figure 5 G represents the in vivo transcriptional level of ANP, a marker of myocardial hypertrophy; Figure 5 H represents the in vivo transcriptional level of BNP, a marker of myocardial hypertrophy; Figure 5 Image I shows a representative Sirius red (PSR) staining image of the cardiac interstitium and perivascular space; Figure 5 J represents the quantitative analysis of left ventricular collagen volume; Figure 5 K represents the in vivo transcriptional level of Col1, a marker of myocardial fibrosis. Figure 5 L represents the in vivo transcriptional level of Col3, a marker of myocardial fibrosis; Figure 5 M represents a representative image and quantitative analysis of Oil Red staining in cardiac tissue. Detailed Implementation

[0020] The following embodiments are used to further illustrate the present invention, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0021] The experimental animals and drugs used in the following examples: Laboratory animal housing: Eight-week-old male C57 / BL6 mice with an initial weight of 23-25g were used as experimental subjects in this invention. They were purchased from the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences, and housed at the Laboratory Animal Center of the Institute of Cardiovascular Diseases, Wuhan University. All animals were housed in a specific pathogen-free (SPF) facility.

[0022] Drug source: The drug erafibrino used in this invention was purchased from Selleck, catalog number S3720, with a purity >99.90%.

[0023] Drug preparation: Dissolve erafilbrano in 0.5% CMC-Na solution to prepare a concentration of 3 mg / mL. Prepare fresh before use.

[0024] Administration method: Mice in the diabetic cardiomyopathy group or control group were administered the drug once daily by gavage at a dose of 20 mg / kg / d body weight. The drug was prepared and used immediately. The drug was administered to mice at 24 weeks of age, i.e., 16 weeks after being fed a high-fat diet or a normal diet.

[0025] Example 1: Effects of Erafibrano on Liver and Kidney Function in Mice (1) Establishment of a mouse model of diabetic cardiomyopathy and treatment with erafibrano Eight-week-old C57BL / 6 mice were fed either a high-fat diet (HFD, 60.3% fat, ResearchDiets, D12492) or a standardized diet (ND, 10% fat) for 24 weeks. Mice on the high-fat diet for 8 weeks received intraperitoneal injections of streptozotocin (STZ, 30 mg / kg / day, citrate buffer, pH 4.5) for 7 consecutive days to induce insulin resistance, while mice in the ND group received an equivalent dose of citrate buffer. Blood glucose levels were measured one week after the last injection. Mice with a random blood glucose level >16.7 mmol / L were considered to have successfully induced diabetes and were selected for subsequent experiments. The diabetic mice were then continued to be fed a high-fat diet, and cardiac function was assessed by echocardiography to establish a diabetic cardiomyopathy model. To assess the nutritional status of the mice, diet type (ND or HFD), body weight, food intake, blood lipid levels, and blood glucose levels were recorded in detail. Significant cardiac dysfunction appeared in mice after 16 weeks of high-fat feeding.

[0026] To investigate the mechanism of action of erafibrinolate, mice treated with ND or HFD / STZ were randomly divided into four groups: ND+CMC-Na (ND-CON), ND+erafibrinolate (ND-ELA), HFD / STZ+CMC-Na (HFD / STZ-CON), and HFD / STZ+erafibrinolate (HFD / STZ-ELA). Mice in the ND-ELA and HFD / STZ-ELA groups received erafibrinolate (20 mg / kg / day, dissolved in 0.5% sodium carboxymethyl cellulose solution (CMC-Na), administered by gavage, Selleck product S3720) for 8 weeks. Mice in the ND-CON and HFD / STZ-CON groups received the same amount of 0.5% CMC-Na (administered orally) for 8 weeks concurrently. At the end of treatment, mouse body weight, blood glucose levels, and echocardiography were measured, and tissue and serum samples were collected.

[0027] (2) Blood was collected from the orbital vein during mouse sampling. The collected orbital vein blood was placed on ice and centrifuged at 12,000 rpm for five minutes at 4°C to separate the serum. The supernatant of the centrifuge tube was collected into a new centrifuge tube, and the serum alanine aminotransferase and aspartate aminotransferase concentrations were determined using an automated biochemical analyzer (ADVIA®2400 Siemens Ltd, Tarrytown, NY, USA).

[0028] Figure 1 The AC results showed that, compared with the control group, the erafibrino treatment group had no significant effect on the serum alanine aminotransferase, aspartate aminotransferase and creatinine levels in mice, indicating that erafibrino treatment had no effect on the liver and kidney function of mice compared with the control group.

[0029] Example 2: Effects of erafibrano on body weight, blood glucose, and blood lipids in mice with high-fat diet combined with low-dose streptozotocin-induced diabetic cardiomyopathy. (1) Establishment of a mouse model of diabetic cardiomyopathy and treatment with erafibrano The content is the same as in Example 1.

[0030] (2) Monitoring of mouse weight: The weight of mice was measured every 4 weeks.

[0031] Figure 2 The results showed that, compared to the model group, erafibrino treatment had no significant effect on the body weight of mice.

[0032] (3) Monitoring of blood glucose and oral glucose tolerance in mice: Mice were fasted for 12 hours prior to the oral glucose tolerance test. During the glucose loading administration, mice were administered 1.0 g / kg body weight of glucose by gavage, and blood glucose levels were measured via tail vein at 0 minutes before gavage and at 30, 60, 90, and 120 minutes after gavage (China Sannuo Health).

[0033] Figure 2 The results from BC showed that, compared with the carrier (CMC-Na) treatment, erafibrano treatment reduced random blood glucose levels and improved glucose tolerance in diabetic cardiomyopathy mice.

[0034] (4) Measurement of blood lipid levels in mice: Blood was collected from the orbital vein in mice. The collected orbital venous blood was placed on ice and centrifuged at 12,000 rpm for five minutes at 4°C to separate the serum. The supernatant was collected into a new centrifuge tube, and the serum total cholesterol and triglyceride concentrations were determined using a fully automated biochemical analyzer (ADVIA®2400 Siemens Ltd, Tarrytown, NY, USA).

[0035] Figure 2 The results of DE showed that, compared with the model group, erafibrino treatment significantly reduced the levels of total cholesterol and triglycerides in the serum of diabetic cardiomyopathy mice.

[0036] Example 3: Effects of erafibrano on cardiac function, myocardial hypertrophy, myocardial fibrosis, and lipid deposition in diabetic cardiomyopathy mice induced by high-fat diet combined with low-dose streptozotocin. (1) Establishment of a mouse model of diabetic cardiomyopathy and treatment with erafibrano The content is the same as in Example 1.

[0037] (2) Echocardiographic assessment of cardiac function in mice After weighing and recording the mice, hair removal cream was applied to the mice's chests to remove hair, exposing the precordial skin for subsequent ultrasound examination. Next, ultrasound coupling gel was applied to the precordial area, and after anesthetizing with 1.5-2.5% isoflurane, the mice were placed in the left lateral decubitus position. Left ventricular ejection fraction, fractional shortening, end-diastolic diameter, end-systolic diameter, and hemodynamic parameters were assessed at the level of the left ventricular papillary muscle short-axis view. B-mode and M-mode ultrasound images and videos were recorded.

[0038] Figure 3 The results of AB showed that, compared with the model group, erafibrino treatment significantly improved cardiac dysfunction in diabetic cardiomyopathy mice, manifested by increasing left ventricular ejection fraction and shortening the ejection fraction.

[0039] (3) Take left ventricular heart tissue from mice and detect the degree of myocardial hypertrophy and fibrosis at the histological level. At the end of the experiment, mice whose weight had been recorded and who had undergone ultrasound were euthanized by dislocation. The heart was dissected by cutting along the left ribcage with surgical scissors. Heart tissue for pathological sections was quickly placed in a 10% KCl solution to stop beating in diastole. Blood was squeezed out of the heart chambers, and the tissue was trimmed and weighed (mg). The tissue was then soaked in formalin. Heart tissue for meristems was directly squeezed out of the heart chambers, trimmed, and weighed (mg). Lung tissue was removed and weighed (mg). One tibia was exposed and its length (cm) was measured. The weighed mouse hearts were fixed in formalin for 24–48 hours, then trimmed, placed in an embedding frame for dehydration, clearing, and embedding to prepare pathological sections for subsequent experiments.

[0040] Hematoxylin-eosin (H&E) staining for cardiomyocyte hypertrophy: Pathological sections were baked at 55°C for at least 30 minutes. The sections were then dewaxed by immersing them in xylene twice for 5 minutes each time, hydrated with a gradient of 100%–70% ethanol, and then soaked in pure water for 10 minutes. Next, the cell nuclei were stained with hematoxylin. After differentiation and blueing treatment, the cytoplasm was stained with eosin. Finally, the sections were dehydrated, cleared, and mounted. The morphology and structure of the cardiac tissue were observed under a microscope.

[0041] Wheat germ lectin (WGA) staining for cardiomyocyte cross-sectional area detection: Pathological sections were baked at 55℃ for at least 30 min. After removal, the sections were dewaxed by immersing them in xylene twice for 5 min each time, hydrated with a gradient of 100%–70% ethanol, and then soaked in pure water for 10 min. Antigen retrieval sections were prepared using trypsin method (50 μL / heart tissue, incubated in a humidified chamber at 37℃ for 15 min), rinsed with PBS, and then incubated with prepared WGA-AF488 working solution for 2 h. The tissue was then thoroughly rinsed with PBS. Finally, the sections were mounted with DAPI and photographed under a fluorescence microscope. WGA staining and photography: Each image should contain at least 20 cells with clear boundaries and centrally located nuclei. After photography, the cross-sectional area of ​​cardiomyocytes was measured using Image-Pro Plus 6.0 image analysis software.

[0042] Masson staining: The pathological sections were baked at 55°C for at least 30 minutes. The sections were then dewaxed by immersing them in xylene twice for 5 minutes each time, hydrated with a gradient of 100% to 70% ethanol, and then soaked in pure water for 10 minutes. The nuclei were stained with Weigert iron hematoxylin, differentiated by hydrochloric acid-ethanol, and then returned to blue by running water. The myocardial cytoplasm was then stained with Ponceau S and acid fuchsin, differentiated by phosphomolybdic acid, and then stained with aniline blue. Finally, the sections were rapidly rinsed with 1% glacial acetic acid, dehydrated by a gradient of ethanol, cleared with xylene, and mounted with neutral resin. The contrast between the red myocardial fibers and the blue collagen fibers was observed under a microscope.

[0043] Figure 3 The results from CD indicated that, compared with vector-treated diabetic cardiomyopathy mice, erafibrano treatment significantly improved cardiomyocyte hypertrophy in diabetic cardiomyopathy mice. Figure 3 The results from GH showed that, compared with vector-treated diabetic cardiomyopathy mice, erafibrano treatment significantly reduced the degree of left ventricular myocardial fibrosis in diabetic cardiomyopathy mice.

[0044] (4) RT-PCR was used to assess the transcriptional levels of markers of myocardial hypertrophy and fibrosis in mice in vivo. At the end of the experiment, mice were euthanized after echocardiography and tissue samples were collected. Blood was squeezed from the heart, and left ventricular heart tissue was trimmed and placed in cryovials. A certain amount of left ventricular heart tissue was extracted and ground to extract total RNA. To detect mRNA expression, total RNA was extracted from each heart tissue sample using TRIzol reagent. First-strand cDNA synthesis was performed using a purchased Roche (Basel, Switzerland) cDNA synthesis kit, followed by real-time quantitative PCR using 480 SYBR green dye. Gene expression was measured using a Roche reverse transcription polymerase chain reaction (RT-PCR) system. The expression of target genes in each group was compared with β-actin levels based on changes in CT cycle values, using the 2-ΔΔCT method and normalized statistical analysis. The transcription levels of hypertrophy markers ANP and BNP, and fibrosis markers Col1 and Col3 in mouse heart tissue were detected. Primer sequences are as follows (SEQ ID NO. 1-10):

[0045] Figure 3 The results of EF showed that, compared with the vector-treated diabetic cardiomyopathy mice, erafibrino treatment significantly reduced the levels of ANP and BNP, markers of myocardial hypertrophy. Figure 3 The results from IJ showed that, compared with vector-treated diabetic cardiomyopathy mice, erafibrano treatment significantly reduced the levels of Col1 and Col3, markers of myocardial fibrosis.

[0046] (5) Take left ventricular heart tissue from mice and observe the mitochondrial structure under an electron microscope. Mice were anesthetized and euthanized at the end of the experiment, and their hearts were quickly removed and placed in electron microscopy fixative for subsequent dissection. Left ventricular heart tissue was cut into small pieces approximately 2-3 mm in size and immediately fixed in electron microscopy solution (2.5% glutaraldehyde) for 24 hours. It was then stained with toluidine blue, followed by staining with 4% methanol-uranyl acetate and lead renoate. The ultrastructure of mouse myocardial mitochondria was observed using a transmission electron microscope (TM-3000; Hitachi, Tokyo, Japan).

[0047] Figure 3 KL's results showed that, compared with vector-treated diabetic cardiomyopathy mice, erafibrano treatment significantly reduced lipid droplet accumulation in cardiac tissue.

[0048] (6) Take left ventricular heart tissue from mice and use Oil Red O staining to assess lipid accumulation in the mouse heart. Mice were anesthetized and euthanized at the end of the experiment, and their hearts were quickly removed and flash-frozen in liquid nitrogen. First, the heart tissue blocks were embedded in OCT and then cut into 8-12 μm thick frozen sections using a -20℃ cryostat, which were then attached to glass slides. The sections were then fixed with 4% paraformaldehyde for 10 minutes, rinsed with PBS, air-dried, and stained with Oil Red O working solution in the dark for 15-30 minutes. Next, the sections were rapidly differentiated with 60% isopropanol until the background was colorless, and then the nuclei were counterstained with hematoxylin for 1-2 minutes. Finally, the sections were blued with running water, mounted with glycerol gelatin, and the staining results—intracellular lipid droplets appearing red and cell nuclei blue—were observed under a microscope.

[0049] Figure 3 The results from M showed that, compared with vector-treated diabetic cardiomyopathy mice, erafibrano treatment significantly reduced lipid accumulation in cardiac tissue.

[0050] Example 4: Effects of erafibrano on body weight, blood glucose, and blood lipids in spontaneously diabetic mice (db / db mice) induced diabetic cardiomyopathy. (1) Establishment of a mouse model of diabetic cardiomyopathy and treatment with erafibrano The db / db mouse is an autosomal recessive spontaneous type 2 diabetes mellitus (T2DM) model derived from the C57BL / 6J inbred strain, characterized by the deletion of the leptin receptor gene, serving as a classic model for studying myocardial complications in T2DM. Male db / db and db / m mice were randomly assigned to four groups: db / m + CMC-Na (db / m-CON), db / m + erafibrano (db / m-ELA), db / db + CMC-Na (db / db-CON), and db / db + erafibrano (db / db-ELA). Mice were fed a standardized diet (fat comprising 10% of total calories). Mice in the db / m-ELA and db / db-ELA groups received erafibrano (20 mg / kg / day, dissolved in 0.5% sodium carboxymethyl cellulose solution, administered intragastrically, Selleck, S3720) for 8 weeks at 16 weeks of age, while the control group received an equal volume of 0.5% CMC-Na solution (administered intragastrically). Eight weeks after treatment, mice were monitored for body weight, blood glucose, and cardiac function, and then sacrificed for further analysis.

[0051] (2) Monitoring of mouse weight: The weight of mice was monitored at the end of the experiment.

[0052] Figure 4 The results from A indicated that, compared with vector-treated db / db mice, erafibrano treatment had no significant effect on the body weight of the mice.

[0053] (3) Monitoring of blood glucose and oral glucose tolerance in mice: Mice were fasted for 12 hours prior to the oral glucose tolerance test. During the glucose loading administration, mice were administered 1.0 g / kg body weight of glucose by gavage, and blood glucose levels were measured via tail vein at 0 minutes before gavage and at 30, 60, 90, and 120 minutes after gavage (China Sannuo Health).

[0054] Figure 4 The results from BC showed that, compared with vector-treated db / db mice, erafibrano treatment significantly reduced blood glucose and glucose tolerance in db / db mice.

[0055] (4) Measurement of blood lipid levels in mice: Blood was collected from the orbital vein in mice. The collected orbital venous blood was placed on ice and centrifuged at 12,000 rpm for five minutes at 4°C to separate the serum. The supernatant was collected into a new centrifuge tube, and the serum total cholesterol and triglyceride concentrations were determined using a fully automated biochemical analyzer (ADVIA®2400 Siemens Ltd, Tarrytown, NY, USA).

[0056] Figure 4 The results of DE showed that, compared with vector-treated db / db mice, erafibrano treatment significantly reduced the levels of total cholesterol and triglycerides in the serum of diabetic cardiomyopathy mice.

[0057] Example 5: Effects of erafibrano on cardiac function, myocardial hypertrophy, myocardial fibrosis, and lipid deposition in spontaneously diabetic mice (db / db mice) induced diabetic cardiomyopathy. (1) Establishment of a mouse model of diabetic cardiomyopathy and treatment with erafibrano The content is the same as in Example 4.

[0058] (2) Echocardiographic assessment of cardiac function in mice The content is the same as in Example 3.

[0059] Figure 5 The results of AD showed that, compared with the model group, erafibrino treatment significantly improved cardiac systolic and diastolic dysfunction in diabetic cardiomyopathy mice, as evidenced by a significant increase in left ventricular ejection fraction, shortening fraction, and the ratio of E-wave to A-wave.

[0060] (3) Take left ventricular heart tissue from mice and detect the degree of myocardial hypertrophy and fibrosis at the histological level. At the end of the experiment, mice whose weight had been recorded and who had undergone ultrasound were euthanized by dislocation. The heart was dissected by cutting along the left ribcage with surgical scissors. Heart tissue for pathological sections was quickly placed in a 10% KCl solution to stop beating in diastole. Blood was squeezed out of the heart chambers, and the tissue was trimmed and weighed (mg). The tissue was then soaked in formalin. Heart tissue for meristems was directly squeezed out of the heart chambers, trimmed, and weighed (mg). Lung tissue was removed and weighed (mg). One tibia was exposed and its length (cm) was measured. The weighed mouse hearts were fixed in formalin for 24–48 hours, then trimmed, placed in an embedding frame for dehydration, clearing, and embedding to prepare pathological sections for subsequent experiments.

[0061] Hematoxylin-eosin (H&E) staining for cardiomyocyte hypertrophy: Same as in Example 3.

[0062] Wheat germ lectin (WGA) staining to detect the cross-sectional area of ​​cardiomyocytes: Same as in Example 3.

[0063] Sirius Red (RSR) staining to detect fibrosis level: The baking, dewaxing, and hydration steps are the same as in Example 3; then, the cleaned sections are placed in 0.2% phosphomolybdic acid for about 2 minutes, and then 0.1% picric acid Sirius Red staining solution is dropped onto the tissue. The sections are placed in a moist staining box for staining. After 1.5 hours, the picric acid Sirius Red staining solution is removed, and the sections are placed in 0.01N hydrochloric acid four times, about 1 second each time. Then, the sections are placed in a series of 70% to 100% graded alcohols for dehydration, and finally in xylene until transparent. While the xylene is still wet, the sections are mounted with neutral resin and placed in a fume hood to dry before taking pictures.

[0064] Figure 5 The results of EF showed that, compared with vector-treated db / db mice, erafibrano treatment significantly improved cardiomyocyte hypertrophy in db / db mice. Figure 5 The results from IJ showed that, compared with vector-treated db / db mice, erafibrino treatment significantly improved the degree of left ventricular myocardial fibrosis in db / db mice.

[0065] (4) RT-PCR was used to assess the transcriptional levels of markers of myocardial hypertrophy and fibrosis in mice in vivo. The content is the same as in Example 3.

[0066] Figure 5 The results of GH showed that, compared with vector-treated db / db mice, erafibrano treatment significantly reduced the levels of ANP and BNP, markers of myocardial hypertrophy, in db / db mice; Figure 5KL's results showed that, compared with vector-treated db / db mice, erafibrino treatment significantly reduced the levels of Col1 and Col3, markers of myocardial fibrosis, in db / db mice.

[0067] (5) Take left ventricular heart tissue from mice and use Oil Red O staining to assess lipid accumulation in the mouse heart. The content is the same as in Example 4.

[0068] Figure 5 The results showed that, compared with vector-treated db / db mice, erafibrano treatment significantly reduced lipid accumulation in the cardiac tissue of db / db mice.

[0069] The above embodiments are only used to help illustrate the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of erafibrano in the preparation of drugs for the treatment of cardiomyopathy.

2. The use of compositions containing erafibrino in the preparation of drugs for the treatment of cardiomyopathy.

3. The application according to claim 2, characterized in that: The composition containing erafibrano also contains other active ingredients with therapeutic effects on cardiomyopathy.

4. The application according to any one of claims 1-3, characterized in that: The cardiomyopathy mentioned is diabetic cardiomyopathy.

5. Application of erafibrano in the preparation of anti-myocardial remodeling drugs.

6. The use of compositions containing erafibrano in the preparation of anti-myocardial remodeling drugs.

7. The application according to claim 6, characterized in that: The composition containing erafibrano also contains other active ingredients with anti-myocardial remodeling effects.

8. The application according to any one of claims 5-7, characterized in that: The myocardial remodeling mentioned is myocardial remodeling induced by diabetic cardiomyopathy.

9. The application according to any one of claims 1-8, characterized in that: The drug also contains a pharmaceutically acceptable carrier or diluent.

10. The application according to any one of claims 1-8, characterized in that: The dosage form of the drug includes powder, tablet, granule or injection.

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