Application of Rev-erbalpha as therapeutic target in preparation of medicine for treating pressure load-induced early myocardial injury
The adenovirus interference of Rev-erbα as a therapeutic target reduces its expression, which solves the shortcomings in the development of Rev-erbα target drugs in the prior art, significantly inhibits cardiac remodeling and fibrosis, and provides a diagnosis of myocardial injury. and new treatments have improved cardiac function.
Patent Information
- Application Number
- CN202411263166.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-10
- Publication Date
- 2024-12-13
AI Technical Summary
The prior art has not yet effectively solved the development of stress overload damage drugs for Rev-erbα as a target, limiting the self-repair ability of cardiomyocytes after damage, leading to cardiac function decline and heart failure.
Rev-erbα is used as a therapeutic target to prepare drugs for early myocardial injury induced by reducing the expression of Rev-erbα by reducing the adenovirus vector interference, and use Rev-erbα as a diagnostic marker to detect its expression level to assist in diagnosis. and treatment.
It significantly inhibited the remodeling and fibrosis process of mouse hearts, improved cardiac contraction function, provided biological indicators for early diagnosis and treatment of myocardial injury, and alleviated early myocardial injury induced by stress load.
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Figure CN119113116A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomedicine technology, and in particular to the application of Rev-erb alpha as a therapeutic target in the preparation of a drug for treating early myocardial injury induced by pressure load. Background Art
[0002] Given the limited proliferation capacity of mature cardiomyocytes, the death of cardiac cells is considered the root cause of heart attacks. In damaged myocardial tissue, researchers have identified a variety of cell death modes, including apoptosis, pyroptosis, programmed cell death (necrosis), and autophagy. These death modes limit the ability of myocardial cells to repair themselves after damage, thereby causing a decline in cardiac function and may gradually evolve into heart failure.
[0003] As an indispensable member of the nuclear receptor (NRs) superfamily, Rev-erbα (Nr1d1) plays a key role as a ligand-regulated transcriptional repressor, which has a profound impact on the physiological activities of organisms. Rev-erbα performs important functions in multiple key organ systems, such as the liver, skeletal muscle, and kidney, and participates in regulating various physiological processes such as glucose and lipid metabolism, immune response, and injury repair. However, the development of pressure overload injury drugs targeting Rev-erbα has not yet made a breakthrough. Summary of the invention
[0004] In order to solve the above problems, the present invention provides the use of Rev-erbα as a therapeutic target in the preparation of a drug for treating early myocardial injury induced by pressure overload. The present invention finds that Rev-erbα can be used as a new target for the development of drugs for treating early myocardial injury induced by pressure overload, and this discovery is of great significance for the early identification, accurate diagnosis and effective treatment of myocardial injury in heart failure.
[0005] In order to achieve the above object, the present invention provides the following technical solutions:
[0006] The present invention provides the use of Rev-erbα as a therapeutic target in the preparation of a drug for treating early myocardial injury induced by pressure load. The active ingredients of the drug include: an agent for reducing the expression amount of Rev-erbα.
[0007] Preferably, the agent for reducing the expression level of Rev-erbα includes: an agent that interferes with the expression of the Rev-erbα encoding gene.
[0008] Preferably, the agent interfering with the expression of the Rev-erbα encoding gene comprises an adenovirus vector interfering with the expression of the Rev-erbα encoding gene.
[0009] Preferably, the target sequence of the adenoviral vector that interferes with the expression of the Rev-erbα encoding gene is as shown in SEQ ID NO.1.
[0010] The present invention provides the use of Rev-erbα as a diagnostic marker in the preparation of a kit for diagnosing or assisting in diagnosing early myocardial injury induced by pressure load.
[0011] Preferably, the kit comprises a reagent for detecting Rev-erbα.
[0012] Beneficial effects:
[0013] The present invention provides the use of Rev-erbα as a therapeutic target in the preparation of a drug for treating early myocardial injury induced by pressure load, wherein the active ingredient of the drug includes: an agent that reduces the expression of Rev-erbα. The present invention finds that adenovirus interference targeting Rev-erbα can significantly inhibit the remodeling of the mouse heart and the fibrosis process of the cardiac interstitium, which plays an important role in improving the contractile function of the heart and reducing early myocardial injury induced by pressure load. In addition, the present invention also finds that after early myocardial injury induced by pressure load, the expression level of Rev-erbα protein in mouse heart tissue is significantly upregulated. By detecting the expression level of Rev-erbα, the functional state of myocardial cells can be more accurately understood, thereby providing a powerful biological indicator for the diagnosis and treatment of early myocardial injury induced by pressure load. BRIEF DESCRIPTION OF THE DRAWINGS
[0014] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.
[0015] Figure 1 The results of Rev-erbα protein level detection in TAC group or sham operation;
[0016] Figure 2 The results of HE staining of the hearts of mice in different groups; the scale bar is 500 μm;
[0017] Figure 3 The results of Masson trichrome staining of mice in different groups; the scale bar is 100 μm;
[0018] Figure 4 Results of TRITC-conjugated wheat germ agglutinin staining and cross-sectional area of cardiomyocytes in different groups of mice; scale bar is 50 μm;
[0019] Figure 5 The left ventricular fractional shortening of the different groups of mice by echocardiography;
[0020] Figure 6The results of lactate dehydrogenase activity test of mice in different groups;
[0021] Figure 7 The results are for the protein level of Rev-erbα in cardiomyocytes induced by AngⅡ;
[0022] Figure 8 Results of cTNT staining of H9C2 cells in different groups; scale bar is 50 μm;
[0023] Fig. 9 Results of lactate dehydrogenase activity detection of H9C2 cells in different groups;
[0024] Fig.10 The results of mitochondrial membrane potential detection of H9C2 cells in different groups; the scale bar is 100 μm;
[0025] Among them, * means P<0.05. DETAILED DESCRIPTION
[0026] The present invention provides the use of Rev-erbα as a therapeutic target in the preparation of a drug for treating early myocardial injury induced by pressure load, wherein the active ingredient of the drug includes: an agent that reduces the expression of Rev-erbα. In the present invention, the agent that reduces the expression of Rev-erbα preferably includes: an agent that interferes with the expression of the Rev-erbα encoding gene; the agent that interferes with the expression of the Rev-erbα encoding gene preferably includes an adenovirus vector that interferes with the expression of the Rev-erbα encoding gene; the target sequence of the adenovirus vector that interferes with the expression of the Rev-erbα encoding gene is preferably as shown in SEQ ID NO.1.
[0027] The present invention provides the use of Rev-erbα as a diagnostic marker in the preparation of a kit for diagnosing or assisting in the diagnosis of early myocardial injury induced by pressure load. In the present invention, the kit comprises a reagent for detecting Rev-erbα.
[0028] The present invention uses Rev-erbα as a target to construct a Rev-erbα interfering adenovirus, and uses experimental methods at the animal and cell levels to discover the influence of Rev-erbα on early myocardial injury induced by pressure load and its potential therapeutic effect. The present invention provides a new drug target for the treatment of early myocardial injury induced by pressure load, and provides a new idea and method for the clinical treatment of early myocardial injury induced by pressure load.
[0029] The advantages of the solution provided by the present invention are as follows:
[0030] 1. Rev-erbα is a protein that is widely present and highly expressed in cardiac tissue and cardiomyocytes. Its content in these cells is relatively high, which makes it more convenient in the detection and research process. Since cardiomyocytes are the basic structural and functional units of the heart, the specific expression of Rev-erbα in this tissue makes it an important marker for studying cardiac physiological functions and pathological changes. By detecting the expression level of Rev-erbα, the present invention can more accurately understand the functional state of cardiomyocytes, thereby providing a powerful biological indicator for the diagnosis and treatment of early myocardial damage induced by pressure load;
[0031] 2. The expression level of Rev-erbα increased significantly under the conditions of aortic coarctation (TAC) surgery and angiotensin II (AngⅡ) induction. This change provides favorable conditions for early detection and diagnosis, allowing medical workers to carry out targeted treatment for the specific target of Rev-erbα in a timely manner;
[0032] 3. Adenovirus interference targeting Rev-erbα can significantly inhibit cardiac remodeling and cardiac interstitial fibrosis in mice, which plays an important role in improving cardiac contractile function and reducing myocardial damage;
[0033] 4. For the treatment of heart failure, both clinical thoracotomy and conservative drug treatment have certain effects. Compared with intravenous administration, in situ cardiac injection can effectively avoid the first-pass effect of the virus after entering the blood circulation system and passing through the liver. In this way, the virus will be enriched in the heart, thereby producing a good expression effect in the local area of injection, and the virus is not easy to spread to the surrounding area. In addition, the virus dose required for in situ cardiac injection is relatively small, which can not only reduce the patient's pain, but also save medical economic costs.
[0034] To further illustrate the present invention, the application of Rev-erbα provided by the present invention as a therapeutic target in the preparation of a drug for treating early myocardial injury induced by pressure overload is described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.
[0035] Example 1 Detection of Rev-erbα expression in early myocardial injury induced by pressure overload
[0036] Mice were housed under pathogen-free conditions by maintaining the temperature of the housing room at 21–27 °C, humidity at 50%–60%, and providing a 12-h light and 12-h dark cycle.
[0037] Construction of a pressure load-induced early myocardial injury mouse model (referred to as TAC): 8-week-old male C57BL / 6 mice were anesthetized with 2% isoflurane, fixed in a supine position, and intubated orally and connected to a ventilator. The skin was cut along the left edge of the sternum and the chest cavity was opened along the left side of the sternum to expose the aortic arch. A 6-0 silk thread was passed through the aortic arch in advance, and a 26G pad needle placed parallel to the blood vessel was ligated together with the aortic arch, and then the pad needle was removed. The sham operation group (referred to as Sham) was operated in the same way as the TAC operation group except that the silk thread was not tied. Four weeks after the TAC operation, the mice were euthanized by the administration of pentobarbital, the heart tissue was removed by thoracotomy, the blood was washed with saline, and the protein in the heart tissue was extracted after lysis with RIPA lysis buffer. Western blotting was performed to detect the expression of Rev-erbα.
[0038] Results Figure 1 The results showed that after early myocardial injury induced by pressure load, the expression level of Rev-erbα protein in mouse heart tissue was significantly upregulated.
[0039] Example 2 Construction of Rev-erbα interfering adenovirus to detect the role of Rev-erbα in early myocardial injury induced by pressure overload
[0040] Genema was commissioned to construct an adenovirus that interferes with Rev-erbα (gene ID in NCBI: 217166), and the target sequence was: 5'-GCACCAGCAACATTACCAAGC-3' (SEQ ID NO.1). Eight-week-old male C57BL / 6 mice were anesthetized with 2% isoflurane and infected with adenovirus that interferes with Rev-erbα. The infection dose was 1×10 8 PFU / mL (the infection group was recorded as Ad-Rev-erbα); a control group (WT) was set up at the same time: a negative control adenovirus (provided by Genema) was injected at a volume of 1×10 8 PFU / mL. A 0.5 cm skin incision was made along the line connecting the xiphoid process and the axilla, the pectoralis major and pectoralis minor muscles were bluntly separated, and the third rib was opened to expose the heart. The virus was injected into the myocardium through a syringe filled with adenovirus. After the injection, the skin was sutured with 5-0 silk suture. The infected mice and the control mice underwent TAC surgery and sham surgery (the method was the same as in Example 1), respectively, and then echocardiography and histological staining were performed, including: HE staining to assess the size of the heart, Masson trichrome staining to analyze the fibrosis area, TRITC-coupled wheat germ agglutinin (WGA) staining to analyze the cross-sectional area of myocardial cells, analysis of the shortening fraction (FS) of the left ventricle by echocardiography, and detection of lactate dehydrogenase (LDH) activity. During the experiment, the mice were randomly grouped according to the experimental design. The results are shown in Figure 2 to Figure 6 .
[0041] The results showed that Rev-erbα knockout mice showed enlarged heart chambers as assessed by hematoxylin-eosin staining. Figure 2 Masson trichrome staining showed that Rev-erbα interference inhibited cardiac interstitial fibrosis ( Figure 3 Attenuated cardiac remodeling and improved cardiac function were also observed in hearts with disruption of Rev-erbα ( Figure 4 , Figure 5 ). Lactate dehydrogenase (LDH) activity assay showed that interference with Rev-erbα inhibited myocardial necrosis ( Figure 6 ).
[0042] Example 3
[0043] Detection of Rev-erbα expression in AngⅡ-induced heart failure model at the cellular level
[0044] The embryonic cardiomyocyte-derived H9C2 cell line was cultured in an incubator containing 5% CO2 at 37°C in Dulbecco's modified Eagle's medium (DMEM). H9C2 cells were induced with 1 μM angiotensin II (Ang II) for 12 hours. Proteins in the cells were extracted and immunoblotted to detect the expression of Rev-erbα. The results are shown in Figure 7 .
[0045] The results showed that Rev-erbα was significantly upregulated in AngⅡ-treated cardiomyocytes, indicating that Rev-erbα was involved in AngⅡ-induced heart failure.
[0046] Example 4 Interference with Rev-erbα at the cellular level to clarify the role of Rev-erbα in heart failure injury
[0047] The H9C2 cell line derived from embryonic cardiomyocytes was cultured in an incubator containing 5% CO2 at 37°C in an incubator containing 5% CO2. The culture medium was Dulbecco's modified Eagle medium (DMEM). The complete culture medium was discarded, and low-serum DMEM was added. The Rev-erbα interfering adenovirus (Ad-Rev-erbα) constructed in Example 3 or the negative control (NC, provided by Genema Gene) was added to infect the cardiomyocytes. The infection amount was MOI = 150. After 4 hours, the fluid was replenished. After 36 hours of the Rev-erbα adenovirus action, 1 μM AngⅡ was added to treat H9C2 cells for 12 hours, and cTNT staining was performed to indicate damaged cells. The lactate dehydrogenase (LDH) activity of cells in different groups was detected. The results are shown in Figure 8 and Fig. 9 .
[0048] The results showed that increased cell damage and enhanced LDH activity were observed in cardiomyocytes exposed to 1 μM AngⅡ, while Rev-erbα interference attenuated this phenomenon ( Figure 8 , Fig. 9 ).
[0049] The present invention then analyzes the change of mitochondrial membrane potential, and uses the analysis of mitochondrial membrane potential △Ψm to calculate the ratio of J-monomer / J-aggregate. JC-1 is a fluorescent probe for detecting mitochondrial membrane potential △Ψm, which is widely used to evaluate mitochondrial membrane potential. When the mitochondrial membrane potential is high, JC-1 will aggregate in the mitochondrial matrix to form polymers (J-aggregates), which can produce red fluorescence; and JC-1, as a monomer (J-monomer), cannot aggregate in the mitochondrial matrix when the mitochondrial membrane potential is low, and can produce green fluorescence. The decline of cell membrane potential can be easily detected by the conversion of JC-1 from red fluorescence to green fluorescence, and the conversion of JC-1 from red fluorescence to green fluorescence can also be used as a detection index for early apoptosis.
[0050] The experimental process is as follows:
[0051] 1. Preparation of JC-1 staining working solution:
[0052] The amount of JC-1 staining working solution required for each well of a 6-well plate is 1 ml, and the amount of JC-1 staining working solution required for other culture vessels is similar; for cell suspension, 0.5 ml of JC-1 staining working solution is required for every 500,000 to 1 million cells. Take an appropriate amount of JC-1 (200X) and dilute JC-1 at the ratio of adding 1 ml of JC-1 staining buffer to every 5 μl of JC-1 (200X). Use a pipette to blow repeatedly and mix well to obtain the JC-1 staining working solution.
[0053] 2. For adherent cells:
[0054] a. For one well of a 6-well plate, remove the culture medium, wash the cells once with PBS or other appropriate solution if necessary according to the specific experiment, and add 1 ml of cell culture medium. The cell culture medium may contain serum and phenol red.
[0055] b. Add 1 ml of JC-1 staining working solution and mix thoroughly. Incubate in a cell culture incubator at 37°C for 20 minutes.
[0056] c. After incubation at 37°C, remove the supernatant and wash twice with JC-1 staining buffer.
[0057] d. Add 2 ml of cell culture medium, which may contain serum and phenol red.
[0058] e. Observe under fluorescence microscope.
[0059] The JC-1 test kit was purchased from Bio-Tech, catalog number: C2003S.
[0060] The present invention found that in cardiomyocytes exposed to 1 μM AngⅡ, J-aggregates decreased and J-monomers increased; and Rev-erbα interference could significantly rescue this phenomenon ( Fig.10 ). These results indicate that Rev-erbα inhibits the decrease in mitochondrial membrane potential induced by AngⅡ.
[0061] In summary, Rev-erbα inhibited AngⅡ-induced cardiomyocyte injury and decreased membrane potential.
[0062] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.
Claims
1. Use of Rev-erbα as a therapeutic target in the preparation of a drug for treating early myocardial injury induced by pressure overload, wherein the active ingredients of the drug include: An agent that reduces the expression of Rev-erbα.
2. The use according to claim 1, characterized in that: The reagents for reducing the expression of Rev-erbα include reagents that interfere with the expression of Rev-erbα encoding genes.
3. The application according to claim 2, characterized in that: The reagent interfering with the expression of Rev-erbα encoding gene includes an adenovirus vector interfering with the expression of Rev-erbα encoding gene.
4. The application according to claim 5, characterized in that: The target sequence of the adenoviral vector interfering with the expression of the Rev-erbα encoding gene is shown in SEQ ID NO.
1.
5. Application of Rev-erbα as a diagnostic marker in the preparation of a kit for diagnosing or assisting in the diagnosis of early myocardial injury induced by pressure overload.
6. The use according to claim 5, characterized in that: The kit includes a reagent for detecting Rev-erbα.