Use of SIAH2 inhibitors for treating heart failure or improving myocardial cell function

Abstract

The invention discloses application of an SIAH2 inhibitor to treatment of heart failure or improvement of myocardial cell functions, relates to the technical field of medical treatment, and is used for researching the treatment effect and the treatment mechanism of SIAH2 on heart failure. According to the application of the SIAH2 gene or protein inhibitor in preparing the medicine for treating the heart failure, the SIAH2 inhibitor is used for intervening a TAC-induced mouse heart failure model, and it is found that the SIAH2 inhibitor can improve myocardial energy metabolism and protect the heart function; the invention provides a new target and strategy for clinically diagnosing and treating heart failure or improving myocardial cell functions.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medical technology, in particular to the application of SIAH2 inhibitor for treating heart failure or improving myocardial cell function. BACKGROUND

[0002] Heart is a high-energy-demanding organ, and must continuously produce ATP to maintain the contractile function of the heart. The ATP stored in the heart can only sustain the beating of the heart for 2-10 seconds.

[0003] Mitochondrial oxidative metabolism is the main source of cardiac energy. The energy supply of a healthy heart comes from a variety of energy substrates, about 40%-60% from fatty acid oxidation, and 20%-40% from glucose metabolism. In the failing heart, there is a change in energy metabolism. Compared with a healthy heart, the ATP content in the heart at the end stage of heart failure is reduced by about 30%, thereby leading to insufficient energy supply, and ultimately leading to myocardial contractile dysfunction. This change in energy metabolism can be due to impaired mitochondrial oxidative metabolism, changes in cardiac energy substrate preference, and reduced cardiac efficiency. Metabolic remodeling plays an important role in regulating cardiac energy substrate utilization, ion and redox homeostasis, and maintaining ATP content, and is crucial for maintaining cardiac contractile function. During cardiac metabolic remodeling, the accumulation of some metabolic products further aggravates metabolic disorders. However, the signaling pathways involved in cardiac metabolic remodeling during the development of heart failure are very complex, and the regulatory mechanisms are not clear. Current research is not sufficient.

[0004] Ubiquitin-Proteasome System (UPS) is a key protein degradation pathway in cells, responsible for regulating various cellular processes, including cell cycle, signal transduction, gene expression, stress response, and metabolic balance. In recent years, E3 ubiquitin ligase has gradually increased in the study of cardiovascular diseases. By specifically recognizing substrate proteins and promoting their ubiquitination degradation or stabilization, it regulates the processes of signal transduction, apoptosis, and inflammatory response in the cardiovascular system. E3 ubiquitin ligase plays an important role in cardiovascular diseases. It is very important to further explore its specific mechanisms in the occurrence and development of cardiovascular diseases, as well as its potential clinical application value as a therapeutic target.

[0005] In our study, we jointly analyzed the transcriptomics of myocardial cells with β receptor overactivation (ISO stimulation) with the transcriptomics of mouse heart failure and the transcriptomics of human heart failure, and found that the ubiquitination-related signaling pathways were significantly enriched in the commonly changed genes. Further analysis of the differential genes in the ubiquitination pathway found that SIAH2 was the E3 ubiquitin ligase with the most obvious changes.

[0006] SIAH2 (Seven in Absentia Homolog2) is an E3 ubiquitin ligase belonging to the SIAH family (Siah proteins), and its role in various cellular processes has been gradually revealed. It mainly participates in cell cycle, transcription regulation, metabolic regulation, and cellular stress response processes by regulating protein ubiquitination. The mechanism of action of SIAH2 and its function in different diseases are being paid more and more attention, especially in cardiovascular diseases, cancer, neurodegenerative diseases and metabolic diseases, SIAH2 as a key regulatory factor is becoming more and more clear. For example, in the process of fat formation, SIAH2 mediates the ubiquitination and degradation of ZFP521, promoting the formation of fat in fat precursor cells; SIAH2 can also regulate DNA damage repair by promoting CtIP ubiquitination; SIAH2 promotes cancer cell hypoxia adaptation by degrading PHD2 and PHD3, enhancing the stability of HIF-1α, thereby enhancing the invasion and metastasis ability of cancer cells. Therefore, based on the above related research, SIAH2 has been confirmed to be closely related to cell stress, inflammatory response and apoptosis in various disease states, but the role of SIAH2 in heart failure is still unclear. SUMMARY

[0007] In view of the above analysis, the present application aims to provide an application of a SIAH2 inhibitor for treating heart failure or improving myocardial cell function, in order to study the therapeutic effect and mechanism of SIAH2 on heart failure.

[0008] The purpose of the present application is mainly realized by the following technical solutions:

[0009] The present application provides an application of a SIAH2 gene or protein inhibitor in the preparation of a drug for heart failure.

[0010] Further, in the application, the inhibitor includes a specific small molecule inhibitor, and the chemical structure of the specific small molecule inhibitor is as shown in the following formula, the molecular formula is C 28 H 44 N4O8, and the molecular weight is 564.67, and the CAS number is 666843-10-3.

[0011]

[0012] Further, in the application, the inhibitor also includes a SIAH2 gene knockout reagent.

[0013] Further, in the application, the SIAH2 gene knockout reagent is siRNA, the sense strand nucleotide sequence of the siRNA is as shown in SEQ ID NO: 1, and the antisense strand nucleotide sequence of the siRNA is as shown in SEQ ID NO: 2.

[0014] The second aspect of the present application also provides an inhibitor of SIAH2 gene or protein for use in inhibiting the expression level of a heart failure marker mRNA in heart tissue of heart failure.

[0015] Further, in the use, the heart failure marker mRNA comprises ANP, BNP, beta-MHC, col1a1 and / or col3a1.

[0016] The third aspect of the present application also provides an inhibitor of SIAH2 gene or protein for use in inhibiting the decrease of expression amount of a mitochondrial function marker in heart tissue of heart failure.

[0017] Further, in the use, the mitochondrial function marker is Nudfa11, SDHB, Uqcrb, Cox6a2 and / or ATP5e.

[0018] Compared with the prior art, the present application can achieve at least one of the following beneficial effects:

[0019] The use of the SIAH2 inhibitor provided by the present application for treating heart failure or improving myocardial cell function, using the SIAH2 inhibitor to intervene in a TAC-induced mouse heart failure model, finding that the SIAH2 inhibitor can improve myocardial energy metabolism and protect cardiac function, and the present application provides a new target and strategy for clinically diagnosing and treating heart failure or improving myocardial cell function.

[0020] Other features and advantages of the present application will be set forth in the following description, and in part will become apparent to those skilled in the art from the description, or can be learned by practice of the present application. The objects and other advantages of the present application will be realized and attained by the structure particularly pointed out in the written description and claims hereof. BRIEF DESCRIPTION OF DRAWINGS

[0021] The accompanying drawings are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description serve to explain the principles of the application. In the drawings:

[0022] Figure 1 Figure 1 is a graph showing the experimental results of detecting the expression level of SIAH2 protein in a TAC-induced mouse heart failure model and sham operation in the present embodiment by Western blotting;

[0023] Figure 2 Figure 2 is a graph showing the experimental results of detecting the expression level of SIAH2 protein in heart tissue of heart failure patients and the control group of non-cardiac disease patients in the present embodiment by Western blotting;

[0024] Figure 3Quantitative analysis of qRT-PCR expression of heart failure markers ANP, BNP, β-MHC, col1a1 and col3a1 in TAC-WT model mice and TAC-KO model mice in this embodiment;

[0025] Figure 4 Quantitative analysis of qRT-PCR expression of mitochondrial function markers Nudfa11, SDHB, Uqcrb, Cox6a2 and ATP5e in TAC-WT model mice and TAC-KO model mice in this embodiment;

[0026] Figure 5 Transmission electron microscope contrast diagram of myocardial mitochondria in TAC-WT model mice and TAC-KO model mice in this embodiment;

[0027] Figure 6 Seahorse monitoring of the effect of shSIAH2 and ISO treatment on mitochondrial respiratory function of cells in this embodiment;

[0028] Figure 7 Seahorse monitoring of the effect of SIAH2 expression regulation on ISO-stimulated mitochondrial function of neonatal mouse myocardial primary cells in this embodiment;

[0029] Figure 8 After treating the TAC-induced wild mouse heart failure model with specific small molecule inhibitors, the heart failure-related gene RT-PCR expression quantitative analysis was detected. DETAILED DESCRIPTION

[0030] The present application is further described in the following examples with reference to the accompanying drawings. The following provides specific materials used in the embodiments of the present application and their sources. However, it should be understood that these are merely exemplary and are not intended to limit the present application. Materials identical or similar to the following reagents and instruments in terms of type, model, quality, properties or functions can be used to implement the present application. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents and the like used in the following examples can be obtained from commercial channels unless otherwise specified.

[0031] The present application provides a use of a SIAH2 gene or protein inhibitor in the preparation of a heart failure drug.

[0032] Further, the inhibitor includes a specific small molecule inhibitor, and the chemical structural formula of the specific small molecule inhibitor is shown in the following formula, the molecular formula is C 28 H 44 N4O8, and the molecular weight is 564.67, and the CAS number is 666843-10-3;

[0033]

[0034] Further, the inhibitor also includes a SIAH2 gene knockout reagent.

[0035] Further, the SIAH2 gene knockout reagent is siRNA, the sense strand nucleotide sequence of the siRNA is 5'-CUGAUAAAGAGUUAUGCCATT-3'(SEQ ID NO: 1), and the antisense strand nucleotide sequence of the siRNA is 5'-UGGCAUAACUCUUUAUCAGTT-3'(SEQ ID NO: 2).

[0036] The second application of the application also provides application of an inhibitor of a SIAH2 gene or protein in inhibiting expression of a heart failure marker mRNA in heart tissue of heart failure.

[0037] Further, the heart failure marker mRNA includes ANP, BNP, β-MHC, col1a1 and / or col3a1.

[0038] The third aspect of the application also provides application of an inhibitor of a SIAH2 gene or protein in inhibiting decrease of expression of a mitochondrial function marker in heart tissue of heart failure.

[0039] Further, the mitochondrial function marker is Nudfa11, SDHB, Uqcrb, Cox6a2 and / or ATP5e.

[0040] Embodiment

[0041] 1. Construction of a mouse heart failure model induced by transverse aortic constriction (TAC) of a mouse aortic arch:

[0042] The TAC-induced mouse heart failure model includes a TAC-induced wild-type mouse heart failure model (TAC-WT model) and a TAC-induced SIAH2 knockout mouse heart failure model (TAC-KO model); the TAC-WT model uses 8-week-old, 20-25 g adult male C57BL / 6 mice, and the TAC-KO model uses SIAH2 knockout mice (SIAH2 knockout mice KO, from Seye (Suzhou) Biotechnology Co., Ltd.).

[0043] The specific construction process of the mouse heart failure model induced by transverse aortic constriction of a mouse aortic arch is as follows:

[0044] Mice were fixed in supine position after anesthesia, the hair on the front chest was shaved and the skin was routinely disinfected. The skin was cut from the upper edge of the suprasternal fossa, and was cut to the second intercostal space along the midline of the sternum to form a longitudinal incision of about 1 cm. The fascia was bluntly separated at the suprasternal fossa, the trachea was exposed, and the thymus tissue was separated to both sides with a small curved forceps to enlarge the operation field. A 5# surgical suture was inserted into the brachiocephalic trunk using a self-made syringe needle (the front end was cut off and bent at a right angle), and the knot was tied in advance for subsequent ligation. Then a 4mm 27G needle was placed under the threading site and the ligation operation was completed. After ligation, the 27G needle was slowly removed, and the muscle layer and skin were sutured. After the operation, the mice were placed in a warm environment to wake up, and the model was completed.

[0045] The mice in the sham operation group were treated according to the same operation procedure, but only threading was performed without vascular ligation.

[0046] 2. Collection of clinical heart failure heart samples:

[0047] After passing the ethics review and informed consent, 4 cases of left ventricular myocardial tissue of patients diagnosed as heart failure (HFrEF) were collected in Tongji Hospital of Huazhong University of Science and Technology (located in Wuhan). At the same time, 3 cases of left ventricular myocardial tissue of non-heart disease patients were collected as a control group of non-heart failure.

[0048] 3. Western blot analysis of clinical heart failure heart samples, TAC-induced mouse heart failure model and sham operation group:

[0049] Extraction of total protein of myocardial tissue: Take the myocardial tissue stored in liquid nitrogen, put it into a mortar and grind it with liquid nitrogen, take two-thirds (the other third is used to extract RNA) and add tissue lysis buffer (20 mmol / L Tris-HCl pH 7.4, 150 mmol / L NaCl, 2.5 mmol / L EDTA, 50 mmol / L NaF, 0.1 mmol / L Na4P2O7, 1 mmol / L Na3VO4, 1% Triton X-100, 10% glycerol, 0.1% SDS, 1% deoxycholic acid, 1 mmol / L PMSF, 1 μg / mL aprotinin) and mix well, then stand on ice for 15 minutes. Add 800 μL lysis buffer for about 50 mg of myocardial tissue. Collect the homogenate, ultrasonic breakage (45%, open 5s, close 5s, 4 cycles), centrifuge at 12000 rpm for 15 minutes at 4°C, transfer part of the supernatant to a new EP tube, quantify the protein and store at -80°C, add one quarter of the volume of 5X loading buffer, mix well, boil at 100°C for 5 minutes, freeze, and save for subsequent detection of related proteins by Western blot.

[0050] Western blotting: After electrophoresis on a 10% SDS-PAGE gel, the membrane was transferred to a nitrocellulose membrane and blocked with 5% skim milk for 1 hour at room temperature. The membrane was then incubated overnight at 4°C with the following primary antibodies: fibronectin (ab2413, abcam, Cambridge, MA, USA), αSMA (ab32575, abcam, Cambridge, MA, USA), ColI (203002, MDBiosciences), SIAH2 (ab75105, abcam, Cambridge, MA, USA), and GAPDH (2118S, CST). The membrane was washed three times with TBST and incubated with the corresponding secondary antibody for 1 hour at room temperature. The membrane was then washed again with TBST and developed. The membrane was placed in developer (Millipore Corporation), drained, and exposed in a luminometer. Band intensity was quantified using NIH ImageJ software. GAPDH was used as a reference.

[0051] The results of Western blot experiments show that Figure 1 As shown in Figure 3, in the TAC-induced mouse heart failure model, the expression level of SIAH2 increased during the pathogenesis of heart failure compared with the sham operation group. Figure 2 As shown, SIAH2 expression levels were elevated in cardiac tissues of patients with heart failure compared with controls without heart disease.

[0052] 4. Detect the expression levels of heart failure markers in TAC-induced mouse heart failure model mice after surgery and SIAH2 knockout.

[0053] The severity of heart failure in TAC-WT model mice and TAC-KO model mice was verified by polymerase chain reaction. Figure 3 As shown, the mRNA expression levels of heart failure markers ANP, BNP, β-MHC, Col1a1, and Col3a1 in TAC-KO model mice were significantly decreased (primer sequences are shown in Table 1 ), indicating that cardiac function was restored and TAC-induced heart failure in mice was alleviated.

[0054] The mitochondrial function of the hearts of TAC-WT model mice and TAC-KO model mice was verified by polymerase chain reaction. Figure 4 As shown, the expression levels of heart failure markers Nudfa11, SDHB, Uqcrb, Cox6a2, and ATP5e (primer sequences are shown in Table 1 ) in TAC-KO model mice were significantly increased, indicating that cardiac function was restored and TAC-induced heart failure in mice was alleviated.

[0055] Transmission electron microscopy was used to verify the mitochondrial morphology of cardiomyocytes in TAC-WT and TAC-KO mice. Figure 5As shown, the TAC-KO model mouse myocardial cell mitochondria has clear inner ridge, which has protective function.

[0056] Table 1 primer sequence list

[0057]

[0058] 5. Seahorse experiment study on SIAH2 expression regulation on ISO stimulated milk mouse myocardial primary cell mitochondrial function

[0059] 5.1 Milk mouse myocardial primary cell: SPF grade milk mice of 1-3 days old were selected, and myocardial primary cells were separated by trypsin digestion method, and cultured in 10% fetal bovine serum-containing DMEM medium in a 37°C, 5% CO2 incubator.

[0060] 5.2 SIAH2 gene knockout reagent (siRNA reagent): The siRNA sequence for knocking down SIAH2 is 5'-GCAGUUCUGUUUCCCUGUATT-3'(SEQ ID NO: 1) and 5'-UACAGGGAAACAGAACUGCTT-3'(SEQ ID NO: 2), purity ≥98%.

[0061] 5.3 Other reagents and instruments: isoproterenol (ISO, purity ≥98%); Agilent Seahorse XFe24 cell energy metabolism analyzer and matching Seahorse cell culture microwell plate; 10% fetal bovine serum-containing DMEM medium.

[0062] 5.4 Specific operation

[0063] 5.4.1 SIAH2 knockdown treatment: when the milk mouse myocardial primary cells grow to 70%-80% confluence, according to the operation instruction of Lipofectamine3000 transfection reagent, the above siRNA is transfected into the myocardial cells, and the untransfected blank control group and the negative control group transfected with irrelevant sequence siRNA are set, and cultured for 24 hours after transfection.

[0064] 5.4.2 Cell planting and stimulation: the transfected myocardial cells and control group cells were inoculated in the Seahorse cell culture microwell plate at a density of 5×10 4 cells / well, after 24 hours of culture, isoproterenol (ISO) was added to each well to make the final concentration 10 μmol / L, and continued to culture for 48 hours.

[0065] 5.4.3 Seahorse experiment detection: the cell culture microplate was taken out and replaced with Seahorse XF base medium (containing 25 mmol / L glucose, 1 mmol / L pyruvic acid, 2 mmol / L glutamine), and equilibrated at 37°C without CO2 for 1 hour. Then the microplate was placed in the Agilent Seahorse XFe24 instrument, and the oxygen consumption rate of the cells was detected according to the instrument operation manual. The detection indexes included basal respiration oxygen consumption, idle respiration oxygen consumption, ATP generation oxygen consumption and maximum respiration oxygen consumption.

[0066] 5.5 Results

[0067] As shown in Figure 6 , knocking down SIAH2 can enhance the mitochondrial function of milk mouse myocardial primary cells under ISO stimulation (maximal respiration, ATP generation, basal respiration oxygen consumption all increase), indicating that SIAH2 gene knockout reagent (SIAH2 knockdown) may affect the energy metabolism response of myocardial cells to ISO by regulating mitochondrial respiration.

[0068] Figure 7 The results show that overexpression of SIAH2 impairs mitochondrial function in myocardial cells, suggesting that mitochondrial function is significantly impaired. Seahorse experiments show that overexpression of SIAH2 leads to a decrease in mitochondrial maximal respiration oxygen consumption, idle respiration oxygen consumption, ATP generation oxygen consumption and basal respiration oxygen consumption in myocardial cells.

[0069] 6. Polymerase chain reaction verification of the effect of specific small molecule inhibitors (Mizagliflozin) on TAC-induced heart failure.

[0070] According to the above method, aortic constriction (TAC) was performed to construct a TAC-induced wild mouse heart failure model caused by pressure overload. On the 3rd day after the operation, the experimental mice were randomly divided into a treatment group and a control group.

[0071] The treatment group was given specific small molecule inhibitors (Mizagliflozin) by gavage, 20 mg / kg / day; the control group was given DMSO solvent, 20 mg / kg / day. Continuous administration for 2 weeks until the 4th week after the operation. Subsequently, the expression levels of heart failure-related marker genes and genes related to collagen synthesis and extracellular matrix deposition were detected by RT-qPCR, as shown in Figure 8 , it is clear that specific small molecule inhibitors (Mizagliflozin) can reduce the mRNA levels of heart failure markers ANP, BNP, β-MHC, Col1a1 and Col3a1, and inhibit the progression of pressure overload-induced heart failure.

[0072] The above merely provides the preferred but not limiting embodiments of the present application, and any modification or substitution within the technical scope of the present application should be covered within the protection scope of the present application.

Claims

1. Use of inhibitors of SIAH2 gene or protein in the preparation of drugs for heart failure.

2. The use according to claim 1, characterized in that The inhibitor includes a specific small molecule inhibitor, and the chemical structure of the specific small molecule inhibitor is shown below. The molecular formula is C 28 H 44 N4O8, molecular weight is 564.67, CAS number is 666843-10-3; 3. The use according to claim 1, characterized in that The inhibitors also include SIAH2 gene knockout agents.

4. The use according to claim 3, characterized in that The SIAH2 gene knockout reagent is siRNA, the nucleotide sequence of the sense strand of the siRNA is such as SEQ ID NO: 1, and the nucleotide sequence of the antisense strand of the siRNA is such as SEQ ID NO:

2.

5. Use of an inhibitor of SIAH2 gene or protein in suppressing the expression level of heart failure marker mRNA in heart tissue of patients with heart failure.

6. The use according to claim 5, characterized in that The heart failure marker mRNAs include ANP, BNP, β-MHC, col1a1 and / or col3a1.

7. Use of an inhibitor of the SIAH2 gene or protein in inhibiting the decreased expression of mitochondrial function markers in cardiac tissue of heart failure.

8. The use according to claim 7, characterized in that The mitochondrial function markers are Nudfa11, SDHB, Uqcrb, Cox6a2 and / or ATP5e.

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