Application of cortistatin in preparation of medicine for treating doxorubicin-induced cardiomyopathy

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of cortistatin in preparation of a medicine for treating doxorubicin-induced cardiomyopathy. Experiments and exploration find that in a doxorubicin-induced cardiomyopathy mouse model, intraperitoneal injection of cortistatin can effectively inhibit the occurrence of heart failure, so that the death rate and the occurrence rate of doxorubicin cardiomyopathy are reduced, meanwhile, cortistatin can also effectively relieve the activation of NLRP3 inflammasome and the occurrence of pyroptosis in cardiac muscle tissues of a mouse, and the application of cortistatin to the treatment of cardiomyopathy is realized. In-vitro experiments show that the human myocardial cells (AC-16) stimulated by adriamycin can inhibit the activation of NLRP3 inflammasome and the pyroptosis process under the intervention of cortistatin. The invention develops a new application field of cortistatin, provides a new prevention and treatment method for adriamycin-induced cardiomyopathy, and provides a new way for development of a novel medicine for adriamycin-induced cardiomyopathy.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to the application of cortistatin in the preparation of a drug for treating doxorubicin-induced cardiomyopathy. Background Art

[0002] As one of the classic anthracycline anti-tumor chemotherapy drugs, doxorubicin is widely used in the treatment of hematological malignancies and solid tumors, including acute leukemia, lymphoma, breast cancer, and liver cancer, etc. The chemotherapy regimen based on doxorubicin is often a classic first-line treatment. Although the anti-tumor drug efficacy of doxorubicin is definite, the toxic and side effects such as myelosuppression and cardiotoxicity caused by it are also problems that cannot be ignored clinically, and also limit the use of doxorubicin. Therefore, finding an effective drug to alleviate doxorubicin cardiomyopathy is particularly important for the prevention and treatment of hematological malignancies and solid tumors.

[0003] As the newly discovered and recognized cell death mode, pyroptosis is considered to be the root cause of the occurrence and development of most cardiomyopathies. Studies have found that doxorubicin can directly activate NLRP3 inflammasome-mediated pyroptosis of cardiomyocytes, and knocking out the genes related to NLRP3 inflammasome components (NLRP3 and Caspase-1) can effectively improve myocardial injury and cardiac dysfunction in doxorubicin cardiomyopathy mice. These findings strongly prove that NLRP3 inflammasome-mediated pyroptosis is a key factor inducing the occurrence and development of doxorubicin cardiomyopathy, and the development of drugs targeting this has feasibility, and targeted inhibition of NLRP3 inflammasome activation has become a new research direction for the prevention and treatment of doxorubicin cardiomyopathy.

[0004] Cortistatin (CST), a neuropeptide belonging to the somatostatin family, has been established in the treatment of depression, sepsis, Crohn's disease, and rheumatoid arthritis. The modes of action include immunomodulation, anti-angiogenesis, anti-inflammatory, and anti-proliferation, etc. Studies have shown that cortistatin reduces the damage of inflammatory factors and inflammatory cells to the myocardium by reducing the secretion of TNF-α and IL-6 by macrophages, and alleviates heart failure symptoms. Other studies have shown that cortistatin inhibits proliferation and migration in smooth muscle cells by specifically binding to its receptor, and reduces the formation of neointima. However, whether cortistatin can inhibit the occurrence of doxorubicin-induced cardiomyopathy and its mechanism have not been reported in any relevant studies so far. Summary of the Invention

[0005] The purpose of the present invention is to overcome the deficiencies in the prior art and provide the application of cortistatin in the preparation of a drug for treating doxorubicin-induced cardiomyopathy, so as to effectively inhibit the occurrence of doxorubicin cardiomyopathy.

[0006] To achieve the above purpose, the technical solutions adopted by the present invention include:

[0007] In a first aspect, the present invention provides the use of somatostatin in the preparation of a medicament for preventing or treating anthracycline-induced cardiomyopathy.

[0008] Preferably, the anthracycline includes doxorubicin.

[0009] Preferably, the somatostatin targets and inhibits the activation of the NLRP3 inflammasome in cardiomyocytes in doxorubicin-induced cardiomyopathy.

[0010] Preferably, the somatostatin inhibits the occurrence of pyroptosis in cardiomyocytes in doxorubicin-induced cardiomyopathy.

[0011] Through experimental exploration, it is found in the present invention that at the animal level, in a mouse model of doxorubicin-induced cardiomyopathy, after intraperitoneal injection of somatostatin, cardiac function can be improved, myocardial cell damage can be inhibited, the mortality rate and the incidence of heart failure can be reduced, and at the same time, the over-activation of the NLRP3 inflammasome and cell pyroptosis in cardiomyocytes of mice with doxorubicin-induced cardiomyopathy can be significantly inhibited; at the cellular level, after treatment with somatostatin, the activation of the NLRP3 inflammasome and cell pyroptosis in human cardiomyocytes (AC-16) induced by doxorubicin can be inhibited; the above results indicate that somatostatin can play a cardioprotective role by targeting and inhibiting the activation of the NLRP3 inflammasome and cell pyroptosis, providing a new treatment strategy for doxorubicin-induced cardiomyopathy.

[0012] Preferably, the effective concentration of the somatostatin is 80 - 120 nM.

[0013] Somatostatin shows potential therapeutic effects in doxorubicin-induced cardiomyopathy by targeting and inhibiting the activation of the NLRP3 inflammasome and cell pyroptosis. And based on comprehensive consideration of multiple factors such as the biological activity, therapeutic effect, and toxicity level of somatostatin, the effective concentration of somatostatin in the range of 80 - 120 nM is the optimal dosage range.

[0014] Preferably, the effective concentration of the somatostatin is 100 nM.

[0015] In a second aspect, the present invention provides a medicament for preventing or treating anthracycline-induced cardiomyopathy, and the medicament includes somatostatin.

[0016] Preferably, the medicament is used in at least one of the following aspects:

[0017] a. Inhibiting the activation of the NLRP3 inflammasome in cardiomyocytes;

[0018] b. Inhibiting the occurrence of pyroptosis in cardiomyocytes;

[0019] c. Inhibiting myocardial injury, cardiac dysfunction, or ventricular abnormal dilation.

[0020] Preferably, the drug further comprises a pharmaceutically acceptable carrier or excipient.

[0021] Preferably, the dosage form of the drug includes an injection or an oral preparation.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] Through experimental exploration, the present invention first discovers that in a mouse model of doxorubicin-induced cardiomyopathy, intraperitoneal injection of cortistatin can effectively inhibit the occurrence of heart failure, thereby reducing the mortality rate and the incidence of doxorubicin cardiomyopathy. At the same time, cortistatin can also effectively reduce the activation of NLRP3 inflammasome and the occurrence of pyroptosis in the myocardial tissue of mice. Moreover, in vitro experiments show that human cardiomyocytes (AC-16) stimulated by doxorubicin can also inhibit the activation of NLRP3 inflammasome and the process of pyroptosis under the intervention of cortistatin. These results fully confirm that cortistatin can regulate the NLRP3 inflammasome and the pyroptosis pathway, thereby playing a therapeutic role in doxorubicin-induced cardiomyopathy, providing new ideas and strategies for the clinical treatment of doxorubicin cardiomyopathy. Description of the Drawings

[0024] Figure 1 It is a graph showing the curative effect after intraperitoneal injection of cortisone to the mice in the doxorubicin-induced cardiomyopathy model in Example 1. Among them, Figure 1 A is the echocardiogram of the mouse heart; Figure 1 B is to detect the levels of LDH and EF values of the mice, n = 5, **** P < 0.001; Figure 1 C is to detect the survival rate of the mice, n = 8 - 10; *P < 0.05, # P < 0.001;

[0025] Figure 2 It is a schematic diagram for detecting the activation of NLRP3 inflammasome and myocardial pyroptosis in the myocardial tissue of the mice in Example 1;

[0026] Figure 3 It is a schematic diagram for detecting the activation of NLRP3 inflammasome and immunostaining of pyroptosis by treating with doxorubicin or doxorubicin + cortistatin at the cellular level in Example 2;

[0027] Figure 4 It is a schematic diagram for detecting the expression levels of NLRP3 inflammasome-related component proteins (NLRP3, caspase-1 / cleaved caspase-1, IL-1β / cleaved IL-1β) and the pyroptosis marker protein (GSDMD / GSDMD-NT) in cardiomyocytes by treating with doxorubicin or doxorubicin + cortistatin at the cellular level in Example 2 by WB. Detailed Embodiments

[0028] To make the objectives, technical solutions and effects of the present invention clearer and more definite, the present invention will be further described in detail below with reference to the embodiments and the accompanying drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0029] Unless otherwise specified, the reagents used in the embodiments are all conventional reagents in the art and can be obtained through commercial channels. The experimental operations not specifically described in the embodiments are all conventional operations in the art or can be understood or known by those skilled in the art based on the existing technology or common general knowledge they have mastered.

[0030] In the embodiments of the present invention, both cortistatin and doxorubicin were purchased from MCE.cn.

[0031] Example 1. Exploration of the therapeutic effect of cortistatin on doxorubicin-induced cardiomyopathy at the animal level

[0032] To explore the therapeutic effect of cortistatin on doxorubicin-induced cardiomyopathy, in this study, SPF-grade male C57BL / 6J mice aged 8 - 12 weeks (purchased from the Animal Experiment Center of Sun Yat-sen University) were selected and fed with a regular diet. The mice were randomly divided into 3 groups: a control group, a doxorubicin-induced cardiomyopathy model group, and a group of doxorubicin-induced cardiomyopathy treated with cortistatin by gavage. Doxorubicin was administered by single intraperitoneal injection at a dose of 15 mg / kg for 5 days to induce the occurrence of doxorubicin cardiomyopathy, that is, the model group; the cortistatin administration group was continuously intraperitoneally injected at a dose of 175 mg / kg 3 days before the administration of doxorubicin; the control group was given an equal amount of normal saline. After 5 days, blood was taken from the orbital cavity of the mice, and LDH was detected by ELISA experiment to evaluate the level of myocardial injury. The left ventricular ejection fraction (EF) of the cardiac function of each group of mice was detected using a Vevo2100 imaging system. In addition, the mouse hearts were isolated, embedded with tissue embedding agent OCT, and after frozen section, triple immunofluorescence staining with Cleaved caspase-1, TUNEL, and α-actinin (a cardiomyocyte marker) was used to detect whether inflammasome activation and pyroptosis occurred in cardiomyocytes in the myocardial tissue, and the scale bar was 25 μm.

[0033] Figure 1 (A - C) The results showed that in the mice with doxorubicin-induced cardiomyopathy model alone, obvious dilation of the left ventricle occurred, obvious damage to the myocardial tissue and a decrease in the ejection fraction were observed, and the mortality rate was significantly increased compared with the control group. However, in the mice with doxorubicin-induced cardiomyopathy, after pretreatment with cortistatin (175 mg / kg / day) for 3 days, the dilation of the left ventricle, myocardial injury, cardiac function, and mouse mortality were all inhibited, indicating that cortistatin can inhibit the occurrence of doxorubicin-induced cardiomyopathy.

[0034] Moreover, it is known that NLRP3 inflammasome activation and cardiomyocyte pyroptosis are widely involved in the occurrence of doxorubicin-induced cardiomyopathy. Therefore, in this study, immunofluorescence staining was used to detect whether cortistatin could reduce NLRP3 inflammasome activation and cardiomyocyte pyroptosis. The results showed that the fluorescence intensities of active caspase-1 and TUNEL in the myocardial tissues of doxorubicin-induced cardiomyopathy mice increased significantly, and the myocardial structure was significantly damaged. However, in the cortistatin-treated group, the fluorescence intensities of active caspase-1 and TUNEL in the myocardial tissues decreased significantly, and the myocardial structure damage was inhibited( Figure 2 ), indicating that cortistatin can improve myocardial tissue integrity by inhibiting NLRP3 inflammasome activation and cardiomyocyte pyroptosis.

[0035] Example 2. Exploration of the therapeutic effect of cortistatin on doxorubicin-induced cardiomyopathy at the cellular level

[0036] In this study, the therapeutic effect of cortistatin on doxorubicin-induced cardiomyopathy at the cellular level was explored using human myocardial tissue (AC-16). The human myocardial tissue was starved in serum-free medium for 24 hours, and then treated with doxorubicin (0.1 μM) and cortistatin (100 nM) for 24 hours. Immunofluorescence staining was used to detect the expression intensities of NLRP3 and active caspase-1 (scale bar: 5 μm), as well as the expression intensities and co-localization of TUNLE and active capase-1b (scale bar: 50 μm); Western blotting (WB) was used to detect the changes in the expression levels of NLRP3 inflammasome-related component proteins (NLRP3, caspase-1 / cleaved caspase-1, IL-1β / cleaved IL-1β) and cardiomyocyte pyroptosis marker proteins (GSDMD / GSDMD-NT) in cardiomyocytes.

[0037] Immunofluorescence staining detection found that after doxorubicin treatment in human cardiomyocytes, the expression intensities of NLRP3 and active caspase-1, and TUNEL and active caspase-1 increased significantly, and the co-localization increased; on this basis, after treatment with cortistatin, the increase in the expression intensities and co-localization of NLRP3 and active caspase-1, and TUNEL and active caspase-1 were inhibited( Figure 3 ). WB detection found that cortistatin treatment could significantly down-regulate the expression of NLRP3 inflammasome-related component proteins and cardiomyocyte pyroptosis marker proteins( Figure 4 ). The above results suggest that cortistatin can improve the occurrence and development of doxorubicin-induced cardiomyopathy by inhibiting NLRP3 inflammasome activation and cell pyroptosis.

[0038] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. Use of cortistatin in the preparation of drugs for preventing or treating anthracycline-induced cardiomyopathy.

2. The use according to claim 1, characterized in that The anthracycline drugs include doxorubicin.

3. The use according to claim 2, characterized in that The cortistatin targets and inhibits the activation of NLRP3 inflammasome in cardiomyocytes in doxorubicin-induced cardiomyopathy.

4. The use according to claim 2, characterized in that The cortistatin inhibits the occurrence of cardiomyocyte pyroptosis in adriamycin-induced cardiomyopathy.

5. The use according to claim 1, characterized in that The effective concentration of the cortistatin is 80-120 nM.

6. A drug for preventing or treating anthracycline-induced cardiomyopathy, characterized in that: Such drugs include cortistatin.

7. The drug according to claim 6, characterized in that The drug is used for at least one of the following aspects: a. Inhibit activation of NLRP3 inflammasome in cardiomyocytes; b. Inhibit cardiomyocyte pyroptosis; c. Inhibit myocardial damage, cardiac dysfunction or abnormal ventricular dilatation.

8. The drug according to claim 6, characterized in that The medicine also includes a pharmaceutically acceptable carrier or excipient.

9. The drug according to any one of claims 6 to 8, characterized in that The dosage form of the drug includes injection or oral preparation.

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