Application of Spexin active polypeptide in the preparation of drugs for preventing and treating ventricular remodeling

Abstract

The present invention relates to the use of Spexin active polypeptide in the preparation of drugs for preventing and treating ventricular remodeling, and belongs to the technical field of drugs for preventing and treating ventricular remodeling. The present invention provides a new drug use of Spexin active polypeptide, that is, the use of Spexin active polypeptide in the preparation of drugs for preventing and treating ventricular remodeling, and the amino acid sequence of the Spexin active polypeptide is NWTPQAMLYLKGAQ. Animal experimental results confirm that Spexin active polypeptide can significantly inhibit abnormal myocardial energy metabolism during ventricular remodeling, and can reduce the occurrence of structural remodeling such as myocardial hypertrophy, fibrosis and myocardial cell apoptosis, thereby improving cardiac function. The present invention applies Spexin active polypeptide to the preparation of drugs for preventing and treating ventricular remodeling, develops its new clinical use, and is expected to be developed into a new generation of safe and effective new drugs for preventing and treating heart diseases.

Description

Technical Field

[0001] The invention belongs to the technical field of drugs for preventing and treating ventricular remodeling, and in particular relates to the application of Spexin active polypeptide in the preparation of drugs for preventing and treating ventricular remodeling. Background Art

[0002] Ventricular remodeling refers to the response of the ventricle to persistent pathological stimulation (such as pressure overload, ischemia or ischemia-reperfusion), which is manifested as changes in heart mass, geometry (such as wall thickness and shape), size and function. It is a pathophysiological reaction process secondary to lesion repair and overall ventricular compensation. Ventricular remodeling is involved in the occurrence and development of cardiovascular diseases, such as heart failure, myocardial ischemia-reperfusion injury, myocardial infarction, atrial fibrillation, hypertension and metabolic diseases (such as obesity and diabetes).

[0003] Chronic heart failure has become a major global public health burden due to its high morbidity, high mortality and high medical costs. Ventricular remodeling is the pathophysiological basis of its occurrence and development, and is also a determinant of morbidity and mortality. Ventricular remodeling is characterized by hypertrophy and extracellular matrix deposition, leading to decreased myocardial compliance and diastolic dysfunction, and ultimately heart failure. Intervention of ventricular remodeling is an important means of treating heart failure.

[0004] At present, there are four main types of drugs commonly used in clinical practice to inhibit ventricular remodeling and improve the prognosis of heart failure. The first is ACEI / ARB and ARNI. The mechanism of action of ACEI / ARB is to inhibit the activation of the renin angiotensin aldosterone (RAAS) system, thereby reducing myocardial contractility, reducing myocardial oxygen consumption, and inhibiting myocardial remodeling; while ARNI has a dual blocking effect of the RAAS system and enkephalinase. The second is beta-blockers. The mechanism of action of this type of drug is to inhibit beta receptors on myocardial cells, reduce sympathetic nerve excitability, inhibit myocardial contractility, slow down heart rate, reduce myocardial oxygen consumption, and thus improve ventricular remodeling. The third is mineralocorticoid receptor antagonists, which can block the aldosterone effect, inhibit myocardial remodeling, and improve the long-term prognosis of heart failure. The fourth is sodium-glucose cotransporter 2 inhibitors, which can improve ventricular remodeling by inhibiting myocardial inflammation, oxidative stress, fibrosis, and improving energy metabolism and myocardial microcirculation. Although new treatments and drugs have improved the clinical prognosis of patients with heart failure, the number of deaths from heart failure patients is still increasing. At present, when exploring new drugs for preventing and treating ventricular remodeling, the field has been looking forward to drugs that directly act on damaged myocardial cells, improve myocardial cell structure and function, and reverse ventricular remodeling, in order to reduce mortality.

[0005] Spexin active peptide is a new type of bioactive peptide hormone. Studies have confirmed that Spexin active peptide plays a key role in regulating physiological functions such as pain, appetite, defecation, glucose and lipid metabolism, water and salt balance, and anti-inflammation. However, there are no reports on whether Spexin active peptide can be used to prevent and treat ventricular remodeling. Summary of the invention

[0006] The present invention provides a new medical application of Spexin active polypeptide, namely, application of Spexin active polypeptide in preparing medicine for preventing and treating ventricular remodeling.

[0007] The technical solution of the present invention:

[0008] Application of Spexin active polypeptide in the preparation of drugs for preventing and treating ventricular remodeling.

[0009] Furthermore, the amino acid sequence of the Spexin active polypeptide is NWTPQAMLYLKGAQ.

[0010] Furthermore, the drug for preventing and treating ventricular remodeling has Spexin active polypeptide as the only active ingredient or one of the active ingredients.

[0011] Furthermore, the content of Spexin active polypeptide in the drug for preventing and treating ventricular remodeling is 0.1wt% to 99wt%.

[0012] Furthermore, the drug for preventing and treating ventricular remodeling also includes pharmaceutically acceptable excipients and / or carriers.

[0013] Furthermore, the drug for preventing and treating ventricular remodeling is used for preventing and treating ventricular remodeling in mammals.

[0014] Furthermore, the mammal is a human or a mouse.

[0015] Furthermore, the drug for preventing and treating ventricular remodeling is a parenteral dosage form, specifically an injectable dosage form.

[0016] Furthermore, the pathophysiological changes of ventricular remodeling include myocardial hypertrophy, myocardial fibrosis, myocardial cell apoptosis, and abnormal myocardial cell energy metabolism and mitochondrial function.

[0017] Furthermore, the diseases associated with ventricular remodeling include coronary heart disease, hypertension, arrhythmia, valvular disease, myocarditis, cardiomyopathy, diabetes, metabolic syndrome, sleep apnea syndrome, tumor-related heart disease, or a combination thereof.

[0018] Beneficial effects of the present invention:

[0019] The present invention provides a new drug use of Spexin active polypeptide for preventing and treating ventricular remodeling. Animal experimental results confirm that Spexin active polypeptide can, on the one hand, significantly inhibit abnormal myocardial energy metabolism during ventricular remodeling, and on the other hand, reduce the occurrence of structural remodeling such as myocardial hypertrophy, fibrosis and myocardial cell apoptosis, thereby ultimately improving cardiac function.

[0020] The present invention applies Spexin active polypeptide to the preparation of drugs for preventing and treating ventricular remodeling, and develops new clinical uses thereof. Since Spexin active polypeptide has significant function in preventing and treating ventricular remodeling, and is safe and non-toxic, it is expected to be developed into a new generation of safe and effective drugs for preventing and treating heart diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a comparison chart of HW / BW of each group of mice in Example 1;

[0022] Figure 2 This is a comparison chart of HW / TL of each group of mice in Example 1;

[0023] Figure 3 Schematic diagram of cardiac ultrasound of each group of mice in Example 2

[0024] Figure 4 This is a comparison chart of LVEF of mice in each group in Example 2;

[0025] Figure 5 This is a comparison chart of LVFS of each group of mice in Example 2;

[0026] Figure 6 This is a comparison chart of LVmass of each group of mice in Example 2;

[0027] Figure 7 These are HE staining photos of the hearts of mice in each group in Example 2;

[0028] Figure 8 HE staining photos of ventricular muscle tissue of each group of mice in Example 2;

[0029] Fig. 9 These are Masson staining photos of ventricular muscle tissue of each group of mice in Example 3;

[0030] Fig.10 This is a comparison chart of the collagen volume fraction of the heart tissue of each group of mice in Example 3;

[0031] Fig.11 These are photos of TUNEL staining of ventricular muscle tissue of each group of mice in Example 4;

[0032] Fig.12 This is a comparison chart of the percentage of apoptotic cells in the ventricular muscle tissue of each group of mice in Example 4;

[0033] Fig.13 These are transmission electron microscopic photographs of the heart tissues of the mice in each group in Example 5;

[0034] Fig.14 This is a comparison of the number of mitochondria in the ventricular muscles of mice in each group in Example 5;

[0035] Fig.15 This is a comparison of the length / width ratio of mitochondria in the ventricular muscles of each group of mice in Example 5;

[0036] Fig.16 Comparison of the expression of key proteins of mitochondrial function in ventricular muscle tissue of mice in each group in Example 5, Figure A and Figure B are UCP2 and TFAM, Figure C and Figure D are mitochondrial respiratory chain complexes CⅠ, CⅡ, CⅢ and CⅤ;

[0037] Fig.17 These are comparison charts of the key protein expressions of energy metabolism in the ventricular muscle tissue of each group of mice in Example 5, with Figure A showing PGC-1α, Figure B showing GLUT4, Figure C showing CD36, and Figure D showing CPT-1. DETAILED DESCRIPTION

[0038] The technical solution of the present invention is further described below in conjunction with the embodiments, but it is not limited thereto. Any modification or equivalent replacement of the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention shall be included in the protection scope of the present invention. The process equipment or devices not specifically noted in the following embodiments are all conventional equipment or devices in the art. If not specifically specified, the raw materials used in the embodiments of the present invention can be obtained commercially; if not specifically specified, the technical means used in the embodiments of the present invention are all conventional means well known to those skilled in the art.

[0039] Example 1

[0040] This example investigates the inhibitory effect of Spexin active polypeptide on the increase of heart weight during ventricular remodeling.

[0041] The experimental animals used in the present invention are all male C57BL / 6 mice, 8-10 weeks old, purchased from Weitonglihua Experimental Animal Technology Co., Ltd.

[0042] Modeling, grouping, and drug administration methods:

[0043] Angiotensin II (AngⅡ)-induced ventricular remodeling mouse model was established: the mice were anesthetized with 1% pentobarbital intraperitoneal injection, the back hair of the mice were removed, the skin on the back of the mice was disinfected with iodine, the skin on the back of the neck of the mice was cut open, the subcutaneous tissue was bluntly separated, and a micropump pre-injected with saline or AngⅡ (2 mg / kg / d) was implanted subcutaneously in the mice, the wound was sutured, and the wound was cleaned and disinfected again.

[0044] The mice were divided into a negative control group, a ventricular remodeling positive control group and a Spexin active peptide treatment group, marked as CTR group, AngⅡ group and AngⅡ+SPX group, respectively.

[0045] The control group was implanted with a micropump containing saline subcutaneously, while the AngⅡ group and AngⅡ+SPX group were implanted with a micropump containing AngⅡ. Each group of mice was intraperitoneally injected with 0.1 ml of saline or Spexin active peptide on the day of surgery. The dosage of Spexin active peptide was 70 μg / kg per day. The model and treatment lasted for 4 weeks.

[0046] After the administration, the hearts of mice in each group were sampled to calculate the heart / body weight ratio (HW / BW), heart / tibia length ratio (HW / TL), Figure 1 It is the HW / BW comparison diagram of each group of mice; Figure 2 The HW / TL comparison diagram of each group of mice; Figure 1 and Figure 2 As shown, compared with the CTR group, the HW / BW and HW / TL in the AngⅡ group were significantly increased; compared with the AngⅡ group, the HW / BW and HW / TL in the AngⅡ+SPX group were significantly decreased, which indicates that Spexin active polypeptide can significantly reduce the increase in heart weight.

[0047] Example 2

[0048] This example investigates the inhibitory effect of Spexin active polypeptide on myocardial hypertrophy and decreased cardiac contractile function during ventricular remodeling.

[0049] In order to observe the inhibitory effect of Spexin active polypeptide on myocardial hypertrophy, the cardiac structure and function of each group of mice in Example 1 were first detected by small animal ultrasound. Figure 3 Schematic diagram of cardiac ultrasound of mice in each group; Figure 4 The left ventricular ejection fraction (LVEF) comparison chart of mice in each group; Figure 5 The left ventricular fractional shortening (LVFS) comparison chart of mice in each group; Figure 6 The figure is a comparison of the left ventricular mass (LVmass) of mice in each group.

[0050] like Figures 3 to 6 As shown, 4 weeks after subcutaneous implantation of AngⅡ micropump, the cardiac contractile function (LVEF, LVFS) of mice was significantly lower than that of the control group, and the left ventricular weight (LVmass) was significantly increased; compared with the AngⅡ group, the cardiac contractile function of the AngⅡ+SPX group was significantly improved, and the heart weight was significantly reduced.

[0051] In order to further observe the therapeutic effect of Spexin in ventricular remodeling, we performed pathological tests on the ventricular muscle tissue of each group of mice. Figure 7 HE staining photos of the hearts of mice in each group. Figure 8 HE staining photos of ventricular muscle tissue of mice in each group.

[0052] like Figure 7 As shown in the figure, HE staining showed that the heart volume of mice in the AngⅡ group increased and the ventricular myocardium was hypertrophic, and the AngⅡ+SPX group was significantly improved compared with the AngⅡ group. Figure 8 As shown, the ventricular myocytes of the control group mice were arranged regularly and tightly, while the myofilaments of the ventricular myocytes of the AngⅡ group mice were arranged loosely, and myocardial cell swelling, myofiber rupture and inflammatory cell infiltration were observed. The ventricular myocytes of the AngⅡ+SPX group were arranged more regularly, and the above phenomena were significantly improved.

[0053] The results confirmed that Spexin active peptide treatment can significantly inhibit cardiomyocyte hypertrophy, reduce myocardial hypertrophy, and improve cardiac contractile function.

[0054] Example 3

[0055] This example investigates the inhibitory effect of Spexin active polypeptide on ventricular myofibrosis during ventricular remodeling.

[0056] In order to observe the inhibitory effect of Spexin active polypeptide on ventricular myofibrosis, the heart tissues of the mice in each group in Example 1 were subjected to Masson staining and the myocardial collagen volume fraction (CVF) was analyzed in combination with Masson staining. Fig. 9 Masson staining photos of ventricular muscle tissue of mice in each group; Fig.10 The figure is a comparison of the collagen volume fraction of heart tissue in each group of mice.

[0057] like Fig. 9 and Fig.10 As shown, the myocardium of mice in the control group was tightly arranged, and no obvious collagen deposition was observed; a large amount of blue collagen deposition was observed in the ventricular myocardium sections of mice in the AngⅡ group, and CVF increased significantly; collagen deposition in the AngⅡ+SPX group was significantly improved, and a small amount of collagen deposition was observed between the muscle fibers. The results showed that Spexin active polypeptide can significantly reduce ventricular myocardial fibrosis.

[0058] Example 4

[0059] This example investigates the inhibitory effect of Spexin active polypeptide on myocardial cell apoptosis during ventricular remodeling.

[0060] In order to observe the inhibitory effect of Spexin active polypeptide on cardiomyocyte apoptosis, the heart tissues of the mice in each group in Example 1 were subjected to TUNEL staining. Fig.11 These are photos of TUNEL staining of ventricular muscle tissues of mice in each group; Fig.12 The figure shows the comparison of the percentage of apoptotic cells in the ventricular muscle tissue of each group of mice.

[0061] like Fig.11 and Fig.12 As shown, the myocardial cell nuclei of the control group mice were arranged regularly, with most of the normal cell nuclei stained blue and less stained brown; in the ventricular muscle sections of the AngⅡ group mice, a large number of apoptotic cells showed brown signals and were arranged irregularly; the apoptosis level of myocardial cells in the AngⅡ+SPX group was significantly improved, and the brown signals in the cells were significantly reduced.

[0062] The results confirmed that Spexin active peptide can significantly inhibit cardiomyocyte apoptosis.

[0063] Example 5

[0064] This example investigates the inhibitory effect of Spexin active polypeptide on myocardial energy metabolism and mitochondrial dysfunction during ventricular remodeling.

[0065] (1) Transmission electron microscopy was used to observe the ultrastructure of ventricular muscle tissue in each group of mice. Fig.13 Transmission electron microscopic photographs of heart tissues of mice in each group.

[0066] like Fig.13 As shown, in the normal group, the nuclei of the ventricular myocytes of mice were normal, the myofilaments were tightly arranged, and the mitochondria were mostly rod-shaped or club-shaped; in the AngⅡ group, the myofilaments were loosely arranged, the number of mitochondria increased, the mitochondria were obviously swollen, and some mitochondria changed from long rods to nearly round shapes; the AngⅡ+SPX group significantly improved the above ultrastructural abnormalities.

[0067] (2) Further analysis of electron microscope images using image pro plus software, Fig.14 The comparison of the number of mitochondria in the ventricular muscles of mice in each group. Fig.15 The figure shows the comparison of the length / width ratio of mitochondria in the ventricular muscles of each group of mice.

[0068] like Fig.14 and 15 As shown, compared with the control group, the number of mitochondria per unit area in the cardiomyocytes of the AngⅡ group increased and the mitochondrial length / width ratio decreased. Spexin active peptide treatment could reduce the number of mitochondria and increase the length / width ratio.

[0069] (3) Western blot was used to detect the expression of mitochondrial function-related proteins UCP2, TFAM and mitochondrial respiratory chain complex proteins. Fig.16This is a comparison chart of the expression of key proteins of mitochondrial function in the ventricular muscle tissue of each group of mice in Example 5.

[0070] like Fig.16 As shown, compared with the normal group, the expression of UCP2 in the myocardial tissue of mice in the AngⅡ group was significantly increased, and the expression of TFAM and mitochondrial respiratory chain complexes CⅡ, CⅢ and CⅤ was significantly decreased, indicating abnormal mitochondrial respiratory function and ATP synthesis disorder; the AngⅡ+SPX group could significantly reduce the expression of UCP2 and up-regulate the expression of TFAM, CⅡ, CⅢ and CⅤ proteins.

[0071] The results confirmed that Spexin active peptide can significantly reduce the abnormalities of mitochondrial structure and function in cardiomyocytes.

[0072] (4) Western blot was used to detect the expression of PGC-1α, GLUT4, CD36, and CPT-1, key proteins that regulate mitochondrial biogenesis and lipid metabolism. Fig.17 This is a comparison chart of the expression of key proteins in energy metabolism in the ventricular muscle tissue of each group of mice in Example 5.

[0073] like Fig.17 As shown, compared with the normal group, the expression of PGC-1α, GLUT4, CD36 and CPT-1 in the myocardial tissue of mice in the AngⅡ group was significantly downregulated, and Spexin active polypeptide treatment significantly upregulated the expression levels of the above proteins.

[0074] The results confirmed that Spexin active peptide has a protective effect on abnormal myocardial energy metabolism in mice.

[0075] The above results confirm that Spexin active peptide can significantly reduce structural remodeling such as ventricular hypertrophy, fibrosis, and myocardial cell apoptosis on the one hand, and improve metabolic remodeling such as abnormal myocardial energy metabolism and mitochondrial dysfunction on the other hand, and is expected to become a safe and effective new drug for the prevention and treatment of heart disease.

Claims

1. Use of Spexin active polypeptide in the preparation of a drug for preventing and treating ventricular remodeling, characterized in that: The amino acid sequence of the Spexin active polypeptide is NWTPQAMLYLKGAQ, and the ventricular remodeling is ventricular structural remodeling caused by hypertension.

2. The use of the Spexin active polypeptide according to claim 1 in the preparation of a drug for preventing and treating ventricular remodeling, characterized in that: The drug for preventing and treating ventricular remodeling has Spexin active polypeptide as the only active ingredient or one of the active ingredients.

3. The use of the Spexin active polypeptide according to claim 2 in the preparation of a drug for preventing and treating ventricular remodeling, characterized in that: The content of Spexin active polypeptide in the drug for preventing and treating ventricular remodeling is 0.1wt%-99wt%.

4. The use of the Spexin active polypeptide according to claim 3 in the preparation of a drug for preventing and treating ventricular remodeling, characterized in that: The drug for preventing and treating ventricular remodeling further comprises pharmaceutically acceptable adjuvants and / or carriers.

5. The use of the Spexin active polypeptide according to claim 4 in the preparation of a drug for preventing and treating ventricular remodeling, characterized in that: The drug for preventing and treating ventricular remodeling is used for preventing and treating ventricular remodeling in mammals.

6. The use of the Spexin active polypeptide according to claim 5 in the preparation of a drug for preventing and treating ventricular remodeling, characterized in that: The mammal is a human or a mouse.

7. The use of the Spexin active polypeptide according to claim 6 in the preparation of a drug for preventing and treating ventricular remodeling, characterized in that: The drug for preventing and treating ventricular remodeling is a parenteral dosage form, specifically an injectable dosage form.

8. The use of the Spexin active polypeptide according to claim 7 in the preparation of a drug for preventing and treating ventricular remodeling, characterized in that: The pathophysiological changes of ventricular remodeling include myocardial hypertrophy, myocardial fibrosis, and myocardial cell apoptosis.

Images