Application of NINJ1 in treatment of adriamycin-induced myocardial injury

Abstract

The invention discloses application of NINJ1 in treatment of adriamycin-induced myocardial injury, and relates to the field of biological medicine. The NINJ1 is applied to preparation of a medicine for preventing and / or treating adriamycin-induced myocardial injury, and the application comprises the step of achieving the purpose of preventing and / or treating adriamycin-induced myocardial cell injury, cardiac insufficiency, myocardial fibrosis or heart failure by inhibiting expression or activity of NINJ1. Wherein the mode of inhibiting the NINJ1 comprises the use of an NINJ1 inhibitor, the knockout of an NINJ1 gene or the silencing of the NINJ1 gene. The key effect of the NINJ1 in doxorubicin cardiotoxicity is disclosed for the first time, the NINJ1 is determined as a new therapeutic target, a new direction and theoretical basis are provided for development of drugs for preventing and treating doxorubicin cardiotoxicity, and the NINJ1 has important clinical value.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to the application of NINJ1 in the treatment of doxorubicin-induced myocardial injury. Background Technology

[0002] Doxorubicin (DOX) is a widely used anthracycline anticancer drug, but its long-term use can lead to dose-dependent cardiotoxicity, clinically manifested as decreased left ventricular ejection fraction, arrhythmias, cardiac fibrosis, and even heart failure, severely limiting its clinical application. Current research indicates that doxorubicin-induced cardiotoxicity (DIC) involves multiple mechanisms, including excessive reactive oxygen species (ROS) production, cardiomyocyte apoptosis, autophagy dysregulation, intracellular calcium homeostasis disturbances, and inflammatory responses. However, effective therapeutic targets and drugs for DIC remain very limited.

[0003] Neuroinjury-inducing protein 1 (NINJ1) is a cell membrane protein that mediates plasma membrane rupture during cell death through oligomerization. Previous studies have suggested that NINJ1 plays a role in apoptosis and inflammation, but its specific function and mechanism of action in doxorubicin-induced cardiotoxicity have not been reported, nor has its application as a therapeutic target been observed.

[0004] Therefore, this invention proposes the application of NINJ1 in the treatment of doxorubicin-induced myocardial injury. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by proposing the application of NINJ1 in the treatment of doxorubicin-induced myocardial injury.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: The application of NINJ1 in the treatment of doxorubicin-induced myocardial injury is discussed in the preparation of drugs for the prevention and / or treatment of doxorubicin-induced myocardial injury. This application includes inhibiting the expression or activity of NINJ1 to prevent and / or treat doxorubicin-induced cardiomyocyte damage, heart failure, myocardial fibrosis, or heart failure. Inhibition of NINJ1 can be achieved through the use of NINJ1 inhibitors, NINJ1 gene knockout, or NINJ1 gene silencing. This application of NINJ1 in the treatment of doxorubicin-induced myocardial injury addresses the shortcomings of traditional methods.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: The application of NINJ1 in the treatment of doxorubicin-induced myocardial injury, specifically in the preparation of drugs for the prevention and / or treatment of doxorubicin-induced myocardial injury, includes: preventing and / or treating doxorubicin-induced cardiomyocyte injury, heart failure, myocardial fibrosis, or heart failure by inhibiting the expression or activity of NINJ1; wherein, the inhibition of NINJ1 includes the use of NINJ1 inhibitors, NINJ1 gene knockout, or NINJ1 gene silencing.

[0008] Preferably, the NINJ1 inhibitor is a small molecule compound, antibody, nucleic acid aptamer, or small interfering RNA.

[0009] A pharmaceutical composition for the prevention and / or treatment of doxorubicin-induced myocardial injury, comprising a therapeutically effective amount of the NINJ1 inhibitor as described in claim 1 or 2 and a pharmaceutically acceptable carrier; the dosage form of the pharmaceutical composition is an injection, tablet, capsule, or sustained-release formulation; the route of administration is intraperitoneal injection, intravenous injection, or oral administration.

[0010] Preferably, the NINJ1 inhibitor is phenyl-β-D-glucopyranoside PDG, and the pharmaceutical composition is configured for administration by intraperitoneal injection to relieve doxorubicin-induced cardiomyocyte apoptosis, oxidative stress, myocardial fibrosis, or decreased ejection fraction.

[0011] Preferably, the use of a pharmaceutical composition for the prevention and / or treatment of doxorubicin-induced myocardial injury in the preparation of a medicament for improving the pathological characteristics of myocardial tissue in a doxorubicin-induced myocardial toxicity model, wherein the improvement of pathological characteristics includes: reducing myocardial cell necrosis, reducing myocardial fiber breakage and disordered arrangement, reducing inflammatory cell infiltration, and / or reducing collagen deposition and fibrosis area, the improvement being evaluated by HE staining and Sirius red staining of myocardial tissue.

[0012] Preferably, the use of a pharmaceutical composition for the prevention and / or treatment of doxorubicin-induced myocardial injury in the preparation of a medicament for inhibiting doxorubicin-induced cardiomyocyte apoptosis is achieved by reducing the proportion of TUNEL-positive cells in myocardial tissue.

[0013] Preferably: the use of a pharmaceutical composition for the prevention and / or treatment of doxorubicin-induced myocardial injury in the preparation of a medicament for alleviating doxorubicin-induced oxidative stress in myocardial tissue, wherein the use is achieved by reducing the level of reactive oxygen species (ROS) in myocardial tissue, the ROS level being detected by DHE staining.

[0014] Preferably, the method of using the pharmaceutical composition for the prevention and / or treatment of doxorubicin-induced myocardial injury includes administering a therapeutically effective amount of a NINJ1 inhibitor to an individual before, simultaneously with, or after administering doxorubicin treatment; said NINJ1 inhibitor is PDG, administered by intraperitoneal injection at a dose of 100 mg / kg once weekly, and doxorubicin treatment is initiated one week after the first administration of the inhibitor.

[0015] A method for screening candidate drugs for the prevention and / or treatment of doxorubicin-induced myocardial injury, the method comprising the following steps: (a) In the presence of doxorubicin, the candidate substance was brought into contact with the NINJ1 protein or cells expressing NINJ1; (b) To detect the effect of the candidate substance on NINJ1 expression or activity; (c) Select candidate substances that can inhibit NINJ1 expression or activity as potential therapeutic agents.

[0016] An evaluation system for assessing the therapeutic effect of NINJ1 inhibitors on doxorubicin-induced myocardial injury, the evaluation system including an in vivo animal model and multiple detection indicators; The animal model was a chronic myocardial injury model established in C57BL / 6 mice by intraperitoneal injection of doxorubicin; The detection indicators include: left ventricular ejection fraction (EF%) and left ventricular fractional shortening (FS%) by echocardiography; serum creatine kinase isoenzyme (CK-MB) and lactate dehydrogenase (LDH) levels by enzyme-linked immunosorbent assay (ELISA); and myocardial tissue pathological changes, degree of fibrosis, apoptosis and oxidative stress levels by tissue section staining, including HE staining, Sirius red staining, TUNEL staining and DHE staining.

[0017] The beneficial effects of this invention are as follows: This study reveals for the first time the crucial role of NINJ1 in doxorubicin cardiotoxicity and establishes it as a new therapeutic target, providing a new direction and theoretical basis for the development of drugs to prevent and treat doxorubicin cardiotoxicity. It has significant clinical value and scientific significance. Attached Figure Description

[0018] Figure 1 Evidence from the GSE59672 and GSE233644 databases shows that the mRNA expression level of NINJ1 in the mouse heart is upregulated after doxorubicin treatment.

[0019] Figure 2 : Display NINJ1 knockout (NINJ1 cKO Combined treatment with doxorubicin and wild-type (NINJ1) fl / flTranscriptome sequencing analysis of mouse heart tissue treated with doxorubicin, including (A) differentially expressed gene heatmap, (B) volcano plot, (C) differential gene statistics, (D) KEGG pathway enrichment analysis Top20, (E) KEGG bubble plot, and (FK) key pathway enrichment map, showed that NINJ1 knockout regulates inflammation, mitochondrial function, oxidative stress and autophagy.

[0020] Figure 3 The following images show the therapeutic effect of the NINJ1 inhibitor PDG in an doxorubicin-induced cardiomyopathy model. (A) Schematic diagram of the in vivo experimental timeline; (B) Effect of PDG on mouse body weight changes; (C) Effect of PDG on the heart weight / tibia length ratio; (D) Effect of PDG on ejection fraction in mice; (E) HE staining results of myocardial tissue; (F) Sirius red staining results of myocardial tissue; (G) TUNEL staining results of myocardial tissue; (H) DHE staining results of myocardial tissue. Detailed Implementation

[0021] The technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0022] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," "linking," and "setting" should be interpreted broadly. For example, they can refer to a fixed connection or setting, a detachable connection or setting, or an integral connection or setting. Those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances.

[0023] Example 1: Confirmation of NINJ1 as a key target for doxorubicin cardiotoxicity Analysis of data from the Gene Expression Database (GEO) (GSE59672 and GSE233644) revealed that on day 5 following a single intraperitoneal injection of 15 mg / kg doxorubicin into C57BL / 6J mice, the mRNA expression level of NINJ1 in their cardiac tissue was significantly higher than that in the saline control group. This result indicates that NINJ1 is involved in the pathogenic mechanism of doxorubicin-induced cardiomyopathy.

[0024] Example 2: Investigation into the protective effect and mechanism of NINJ1 gene knockout against doxorubicin-induced myocardial injury Constructing cardiomyocyte-specific NINJ1 knockout mice (NINJ1 cKO ) and wild-type mice (NINJ1) fl / fl As a control, both groups of mice were treated with doxorubicin, and then heart tissue was harvested for RNA transcriptome sequencing (n=3). The results showed that, compared with NINJ1... fl / fl Compared to the DOX group, NINJ1 cKO+ In the DOX group, 69 genes were significantly upregulated and 44 genes were significantly downregulated. KEGG enrichment analysis showed that differentially expressed genes were significantly enriched in multiple protective signaling pathways, including: the tumor necrosis factor negative regulatory signaling pathway (indicating suppression of the inflammatory response), suppression of the MAPK signaling pathway, enrichment of mitochondrial complex I assembly and oxidative phosphorylation (indicating improved mitochondrial function), enrichment of the Nrf2 signaling pathway (indicating relief of oxidative stress), and activation of autophagy. These results confirm at the molecular level that knockout of NINJ1 can alleviate doxorubicin-induced myocardial injury through multiple pathways.

[0025] Example 3: Validation of the therapeutic effect of the NINJ1 inhibitor PDG Wild-type C57 mice were randomly divided into groups. Mice in the treatment group received intraperitoneal injections of the NINJ1 inhibitor PDG at a dose of 100 mg / kg once weekly for five consecutive weeks. One week after the first PDG injection, mice were given intraperitoneal injections of doxorubicin at a dose of 4 mg / kg once weekly for four consecutive weeks. The control group received the appropriate solvent. One week after the last doxorubicin injection, various indicators were measured. The results are as follows: Weight and cardiac morphology: PDG treatment alleviated doxorubicin-induced weight loss and decreased heart weight / tibia length ratio, indicating improved myocardial atrophy.

[0026] Cardiac function: Echocardiography showed that PDG treatment significantly alleviated the doxorubicin-induced decrease in ejection fraction in mice.

[0027] Histopathology: HE staining showed that PDG improved cardiomyocyte necrosis, fiber breakage and inflammatory infiltration; Sirius red staining showed that PDG alleviated myocardial fibrosis.

[0028] Apoptosis and oxidative stress: Tunel staining showed that PDG inhibited cardiomyocyte apoptosis; DHE staining showed that PDG alleviated the level of oxidative stress in myocardial tissue.

[0029] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. The application of NINJ1 in the treatment of doxorubicin-induced myocardial injury, characterized in that, Its application is in the preparation of drugs for the prevention and / or treatment of doxorubicin-induced myocardial injury, the application including: preventing and / or treating doxorubicin-induced cardiomyocyte injury, heart failure, myocardial fibrosis or heart failure by inhibiting the expression or activity of NINJ1; wherein, the inhibition of NINJ1 includes the use of NINJ1 inhibitors, NINJ1 gene knockout or NINJ1 gene silencing.

2. The application of NINJ1 according to claim 1 in the treatment of doxorubicin-induced myocardial injury, characterized in that, The NINJ1 inhibitor is a small molecule compound, antibody, nucleic acid aptamer, or small interfering RNA.

3. A pharmaceutical composition for the prevention and / or treatment of doxorubicin-induced myocardial injury, characterized in that, The pharmaceutical composition comprises a therapeutically effective amount of the NINJ1 inhibitor as described in claim 1 or 2 and a pharmaceutically acceptable carrier; the dosage form of the pharmaceutical composition is an injection, tablet, capsule or sustained-release formulation; the route of administration is intraperitoneal injection, intravenous injection or oral administration.

4. The pharmaceutical composition according to claim 3 for the prevention and / or treatment of doxorubicin-induced myocardial injury, characterized in that, The NINJ1 inhibitor is phenyl-β-D-glucopyranoside PDG, and the pharmaceutical composition is formulated for administration by intraperitoneal injection to relieve doxorubicin-induced cardiomyocyte apoptosis, oxidative stress, myocardial fibrosis, or decreased ejection fraction.

5. The use of the pharmaceutical composition according to claim 4 for the prevention and / or treatment of doxorubicin-induced myocardial injury in the preparation of a medicament for improving the myocardial histopathological characteristics in a doxorubicin cardiotoxicity model, characterized in that, Improvements in pathological features include: reduced cardiomyocyte necrosis, reduced myocardial fiber breakage and disorder, reduced inflammatory cell infiltration, and / or reduced collagen deposition and fibrosis area. These improvements are evaluated by HE staining and Sirius red staining of myocardial tissue.

6. The use of the pharmaceutical composition according to claim 4 for the prevention and / or treatment of doxorubicin-induced myocardial injury in the preparation of a medicament for inhibiting doxorubicin-induced cardiomyocyte apoptosis, characterized in that, The application is achieved by reducing the proportion of TUNEL-positive cells in myocardial tissue.

7. The use of the pharmaceutical composition according to claim 4 for the prevention and / or treatment of doxorubicin-induced myocardial injury in the preparation of a medicament for alleviating doxorubicin-induced oxidative stress in myocardial tissue, characterized in that, The application achieves its effect by reducing the level of reactive oxygen species (ROS) in myocardial tissue, and the ROS level is detected by DHE staining.

8. A method of using a pharmaceutical composition according to any one of claims 3-7 for the prevention and / or treatment of doxorubicin-induced myocardial injury, characterized in that, This includes administering a therapeutically effective amount of a NINJ1 inhibitor to an individual before, simultaneously with, or after doxorubicin treatment; the NINJ1 inhibitor being PDG, administered via intraperitoneal injection at a dose of 100 mg / kg once weekly, with doxorubicin treatment initiated one week after the first administration of the inhibitor.

9. A method for screening candidate drugs for the prevention and / or treatment of doxorubicin-induced myocardial injury, characterized in that, The method includes the following steps: (a) In the presence of doxorubicin, the candidate substance was brought into contact with the NINJ1 protein or cells expressing NINJ1; (b) To detect the effect of the candidate substance on NINJ1 expression or activity; (c) Select candidate substances that can inhibit NINJ1 expression or activity as potential therapeutic agents.

10. An evaluation system for assessing the therapeutic effect of NINJ1 inhibitors on doxorubicin-induced myocardial injury, characterized in that, The evaluation system includes an in vivo animal model and multiple detection indicators; The animal model was a chronic myocardial injury model established in C57BL / 6 mice by intraperitoneal injection of doxorubicin; The detection indicators include: left ventricular ejection fraction and left ventricular fractional shortening by echocardiography; serum creatine kinase isoenzyme and lactate dehydrogenase levels by enzyme-linked immunosorbent assay; and myocardial tissue pathological changes, degree of fibrosis, apoptosis and oxidative stress levels by tissue section staining, including HE staining, Sirius red staining, TUNEL staining and DHE staining.

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