Application of AVEN as target spot in preparation of medicine for treating adriamycin cardiomyopathy

Abstract

The invention relates to the technical field of biological medicines, and discloses application of AVEN as a target spot in preparation of a medicine for treating adriamycin cardiomyopathy. Wherein the AVEN is taken as the target spot, the AVEN is overexpressed by targeting cardiac myocardial cells, and further, the overexpressed AVEN by targeting cardiac myocardial cells comprises the step of improving the expression of the AVEN gene in the cardiac myocardial cells by adopting a substance capable of activating or up-regulating the expression of the AVEN in a targeting manner. It is found for the first time that specific overexpression of AVEN in myocardial cells can inhibit ferroptosis of myocardial cells, relieve myocardial fibrosis and myocardial atrophy, significantly improve adriamycin-induced cardiac dysfunction, and improve life quality and long-term prognosis of patients.

Description

Technical Field

[0001] The present application relates to the field of biomedicine technology, and more specifically, to the use of AVEN as a target in the preparation of drugs for treating doxorubicin cardiomyopathy. Background Art

[0002] Doxorubicin (Dox) is an anthracycline anti-tumor drug used to treat solid tumors and hematological malignancies. Since the 1970s, doxorubicin has been one of the most commonly used chemotherapy drugs in clinical practice. However, long-term use of doxorubicin can cause dose-dependent and cumulative cardiotoxicity, leading to the occurrence of doxorubicin-induced cardiomyopathy (DIC). Doxorubicin-induced cardiotoxicity is closely related to increased mortality and poor prognosis in cancer survivors. At present, there is no very effective clinical prevention and treatment method for heart failure caused by doxorubicin cardiotoxicity. Exploring the mechanism of doxorubicin cardiotoxicity and finding effective defense targets are of great theoretical and clinical significance for reducing the cardiotoxicity caused by doxorubicin and similar anti-tumor drugs and improving the survival rate of cancer patients.

[0003] Regarding doxorubicin cardiomyopathy, common gross morphological changes include acute left ventricular dysfunction, delayed cardiomyopathy, and eventually heart failure, which may lead to the decline of the overall structure and function of the heart. At the cellular level, the damage caused by doxorubicin is mainly through its toxic effects on mitochondria, causing the destruction of the mitochondrial oxidative respiratory chain of cardiomyocytes, leading to mitochondrial iron overload, promoting the opening of the mitochondrial permeability transition pore, and increasing oxidative stress, thereby causing cell death, especially ferroptosis. The death and functional impairment of cardiomyocytes further aggravate the structural disorder of myocardial tissue. Therefore, it is very important to find drug targets for the prevention and treatment of new doxorubicin cardiomyopathy.

[0004] Apoptosis and caspase activation inhibitor (AVEN) is an apoptosis inhibitor protein that mainly exerts its anti-apoptotic effect by inhibiting the activation of caspases in the apoptotic pathway. Studies have shown that AVEN can inhibit the activation of caspase-9 and prevent the initiation of the apoptotic cascade by interacting with caspase-9 and other apoptosis-related proteins (such as Apaf-1 and Bcl-2 family proteins); or it can protect cells from apoptosis or cancer by binding to targeted mRNA with RNA-binding proteins. However, there have been no reports on the use of AVEN in the preparation of drugs for the treatment of doxorubicin cardiomyopathy, which needs to be studied. Summary of the invention

[0005] The purpose of the present invention is to improve heart failure caused by the chemotherapy drug doxorubicin by targeted overexpression of AVEN, and to study the application of AVEN as a target in the preparation of drugs for treating doxorubicin cardiomyopathy. The present invention specifically overexpresses AVEN in cardiomyocytes and finds that overexpression of AVEN can inhibit myocardial cell ferroptosis, reduce myocardial fibrosis and myocardial atrophy, and significantly improve doxorubicin-induced cardiac dysfunction.

[0006] In order to achieve the above-mentioned invention object, the present application adopts the following technical solution:

[0007] In a first aspect, the present application provides the use of AVEN as a target in the preparation of a drug for treating doxorubicin-induced cardiomyopathy.

[0008] Furthermore, the AVEN as a target refers to overexpression of AVEN by targeting cardiac cardiomyocytes.

[0009] Furthermore, the method of overexpressing AVEN by targeting cardiac cardiomyocytes includes increasing the expression of AVEN gene in cardiomyocytes by using a substance that can target and activate or upregulate the expression of AVEN.

[0010] In a second aspect, the present application provides the use of a substance that targets activation or upregulation of AVEN expression in the preparation of a drug for treating doxorubicin-induced cardiomyopathy.

[0011] In a third aspect, the present application provides a drug for treating doxorubicin-induced cardiomyopathy, comprising a substance that targets activation or upregulation of AVEN expression as the sole active ingredient.

[0012] In a fourth aspect, the present application provides a drug for treating doxorubicin-induced cardiomyopathy, including a substance that targets and activates or upregulates AVEN expression and other active ingredients for treating doxorubicin-induced cardiomyopathy.

[0013] Furthermore, the dosage form of the drug includes tablets, powders, granules, capsules, oral liquids, injections or sustained-release preparations.

[0014] In a fifth aspect, the present application provides the use of AVEN in the preparation of a doxorubicin cardiomyopathy prognosis diagnostic kit.

[0015] The present invention discovers for the first time that overexpression of AVEN in cardiomyocytes specifically can inhibit cardiomyocyte ferroptosis, reduce myocardial fibrosis and myocardial atrophy, significantly improve doxorubicin-induced cardiac dysfunction, and improve the quality of life and long-term prognosis of patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 : The modeling process of the mouse model in the examples of the present application;

[0017] Figure 2: Specific overexpression of AVEN in cardiomyocytes can improve the deterioration of cardiac function caused by DOX. A: Flow chart of the establishment of doxorubicin cardiomyopathy model; BC: AVEN mRNA and protein levels are upregulated in the heart tissue of adenovirus-overexpressed mice; D: The protein levels of ferroptosis markers PTGS2 and GPX4 in mice treated with different treatment groups; E: M-mode ultrasound results showed that specific overexpression of AVEN in cardiomyocytes can improve the ejection fraction (EF%) and shortening fraction (FS%) of doxorubicin mice; FI: The size, HE staining, picrosirius red staining and WGA staining of the hearts of mice treated with different treatment groups. Specific overexpression of AVEN in cardiomyocytes can improve cardiac fiber damage and disorder, reduce cardiac fibrosis, atrophy and cell apoptosis; ns: No statistical difference. DETAILED DESCRIPTION

[0018] The technical scheme and effects of the present application are further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the invention, rather than to limit the invention.

[0019] Long-term treatment with doxorubicin will cause progressive and irreversible cardiotoxicity in cancer patients, often leading to heart failure. Pathological processes such as mitochondrial iron overload and endogenous cell apoptosis caused by doxorubicin seriously affect the contraction and relaxation function of the heart, accelerating the progression of heart failure. Therefore, the present invention focuses on targeted overexpression of AVEN, an apoptosis and metabolism-related protein, to block DOX-induced myocardial cell ferroptosis and apoptosis, and improve cardiac function.

[0020] As an important apoptosis inhibitor protein, AVEN plays an important role in inhibiting apoptosis pathways and regulating cell metabolic processes. This study started from the starting point of doxorubicin-induced heart failure and found that overexpression of AVEN in cardiomyocytes reduced myocardial fibrosis and myocardial atrophy and significantly improved abnormal cardiac function. This study involved the construction of a doxorubicin model, the method was authentic and reliable, and preliminary research results have confirmed that overexpression of AVEN can block cardiac remodeling and improve cardiac contractile function.

[0021] In the following examples, the experimental materials and methods used are listed as follows:

[0022] 1. Experiment on specific overexpression of AVEN in mouse cardiac cells

[0023] For overexpression of AVEN in mouse cardiac cells, 8-week-old C57BL / 6 male wild-type mice were injected intravenously via the tail vein with a single dose of adeno-associated serotype 9 (HBAAV2 / 9-cTNT-m-AVEN-3×flag-EGFP) viral vector or negative control (HBAAV2 / 9-cTNT-EGFP) at a concentration of 5×10 per mouse.10 The researchers isolated the viral genomes and studied them 4 weeks later.

[0024] 2. Construction of Adriamycin-induced cardiomyopathy model

[0025] To establish a chronic DIC model, 12-week-old C57BL / 6 male wild-type mice were injected intraperitoneally with DOX (5 mg / kg) once a week for 4 weeks. Cardiac function was assessed using echocardiography 4 weeks after the last DOX administration. Then, heart tissues were collected for biochemical analysis after echocardiographic evaluation. The injection method of the control group was the same, and the drug was replaced with an equal dose of normal saline. After the injection, the mice were randomly divided into 4 groups: AAV-NC / Saline, i.e., mice injected with AAV-NC+normal saline; AAV-NC / DOX, i.e., mice injected with AAV-NC+DOX; AAV-AVEN / Saline, i.e., mice specifically overexpressing AVEN in cardiomyocytes+normal saline; AAV-AVEN / DOX, i.e., mice specifically overexpressing AVEN in cardiomyocytes+DOX.

[0026] 3. Mouse Heart Ultrasound

[0027] After depilation, mice were anesthetized with isoflurane and fixed on a heating pad (1%-2%). The isoflurane flow was carefully controlled to maintain a heart rate of approximately 450 beats / min. Two-dimensional (2-D) guided M-mode echocardiography (VisualSonics Vevo 2100, Canada) equipped with a 30mHz linear transducer was used to evaluate the geometry and function of the heart. Parasternal long-axis views were obtained while heart rate was recorded. Cardiac geometry and function indices were obtained: ejection fraction (EF), fractional shortening (FS), left ventricular end-diastolic diameter (LVEDD), and left ventricular end-diastolic volume (LVESV). Each measurement was averaged over at least 5 consecutive cardiac cycles.

[0028] 4. Sirius red staining

[0029] Myocardial tissue was separated, fixed with formalin, and embedded in paraffin, with a slice thickness of 6-7 μm. Sirius red staining solution was dripped for 1 hour, rinsed slightly with running water, and the staining solution on the surface of the slice was removed. Then, the nucleus was stained with hematoxylin staining solution for 8-10 minutes, and rinsed with running water for 10 minutes. Conventional dehydration was performed to make it transparent, and then sealed with neutral gum. Under an optical microscope, collagen fibers were red, cell nuclei were blue, and other components were yellow. The collagen volume fraction (CVF) was calculated, and the calculation formula was: CVF (%) = average collagen area / total area × 100%.

[0030] 5. Histological analysis

[0031] Heart samples were first fixed in 4% paraformaldehyde at 4°C for 1-2 days and then dehydrated in ethanol. Afterwards, the samples were cleared with xylene and then embedded in paraffin. Subsequently, the samples were cut into sections with a thickness of 4 μm. To evaluate cardiac morphology, fibrosis, and cardiomyocyte area (CSA), specific staining procedures were performed on tissue sections. Hematoxylin and eosin (H&E) staining and wheat germ agglutinin (WGA) staining were used according to standard protocols. TUNEL staining of heart sections was performed using the One-Step TUNEL In Situ Apoptosis Kit. Sections were visualized using an Olympus SlideView VS200 microscope.

[0032] 6. Experimental Materials

[0033] The mice involved in the following examples were purchased from Saiye Biotechnology Co., Ltd., and the AVEN cardiomyocyte-specific overexpression adenovirus was purchased from Heyuan Biotechnology Co., Ltd. The animal experiments have been approved by the Ethics Committee of Zhongshan Hospital Affiliated to Fudan University.

[0034] Example

[0035] WT C57BL / 6 mice were randomly divided into two groups: AAV-NC and AAV-AVEN, and were injected with a single tail vein injection of adeno-associated virus type 9 (HBAAV2 / 9-cTNT-m-AVEN-2×flag-EGFP) viral vector or negative control (HBAAV2 / 9-cTNT-EGFP).

[0036] Four weeks after adenovirus injection, the mice were randomly divided into two groups: the DOX group was injected with doxorubicin to establish the model, and the saline group was injected with an equal amount of normal saline. The injection method and frequency were consistent with those of the experimental group.

[0037] Four weeks after the end of administration, cardiac ultrasound was performed on the mice to detect cardiac function. qPCR and Western blot were used to detect the expression levels of ferroptosis markers GPX4 and PTGS2 in mice in different treatment groups. HE staining, picrosirius red staining, and WGA staining were used to detect the degree of myocardial fibrosis and disorder and the level of myocardial cell atrophy. TUNEL staining was used to detect the level of myocardial cell apoptosis.

[0038] The process of establishing doxorubicin (DOX)-induced heart failure model is as follows Figure 2 As shown in A. Figure 2BC detected the mRNA and protein levels of AVEN in the myocardial tissue of different groups of mice, confirming that the adenovirus was effective. PTGS2 and GPX4 levels are important indicators for evaluating the degree of cell ferroptosis. We used qPCR and Western blot to detect the expression of PTGS2 and GPX4 in mouse heart tissue. The results showed that compared with the control group, AVEN specific overexpression significantly increased the expression of GPX4, but significantly reduced the expression of PTGS2, indicating a reduction in ferroptosis ( Figure 2 D). The cardiac function of mice in different intervention groups was evaluated based on the left ventricular ejection fraction (EF%) and the fractional shortening (FS%) of the left ventricle. Figure 2 As shown in E, the results showed that compared with the control group sham, the myocardial EF and FS values ​​of mice in the DOX group were significantly reduced. After the injection of adenovirus overexpressing AVEN in myocardial cells, the EF and FS values ​​after DOX were significantly increased. We used HE staining and picrosirius red staining to determine the degree of myocardial fiber damage and fibrosis, and found that after the injection of adenovirus overexpressing AVEN in myocardial cells, myocardial fiber damage and disorder were alleviated, and the overall fibrosis level was reduced. Figure 2 F, G. Cardiac atrophy is an important feature of doxorubicin-induced cardiomyopathy. In our study, WGA staining was used to evaluate the level of myocardial atrophy in different treatment groups. The results showed that AVEN overexpression significantly reduced DOX-induced cardiomyocyte atrophy compared with the control group ( Figure 2 H). In this study, apoptotic cells were marked by TUNEL staining. Figure 2 The results showed that AVEN overexpression in cardiomyocytes significantly reduced DOX-induced myocardial apoptosis in mice.

[0039] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed. However, as long as it is within the scope of the claims of the present application, it shall be protected by the patent law.

Claims

1. Application of AVEN as a target in the preparation of drugs for the treatment of doxorubicin-induced cardiomyopathy.

2. The use according to claim 1, characterized in that: The AVEN as a target refers to overexpression of AVEN in cardiac cardiomyocytes.

3. The use according to claim 2, characterized in that: The method of overexpressing AVEN by targeting cardiac myocardial cells includes using a substance capable of targeted activation or upregulation of AVEN expression to increase the expression of AVEN gene in myocardial cells.

4. Application of substances that target activation or upregulation of AVEN expression in the preparation of drugs for the treatment of doxorubicin-induced cardiomyopathy.

5. A drug for treating adriamycin cardiomyopathy, characterized in that: Includes a substance that targets and activates or upregulates AVEN expression as the sole active ingredient.

6. A drug for treating adriamycin cardiomyopathy, characterized in that: Including substances that target activation or upregulation of AVEN expression and other active ingredients for treating doxorubicin-induced cardiomyopathy.

7. The drug according to claim 5 or 6, characterized in that The dosage form of the drug includes tablets, powders, granules, capsules, oral liquids, injections or sustained-release preparations.

8. Application of AVEN in the preparation of doxorubicin cardiomyopathy prognosis diagnostic kit.

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