Diagnostic and pharmaceutical application of SMOC2

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to diagnosis and pharmaceutical application of SMOC2. Compared with normal people, the expression level of the SMOC2 gene in the heart of a clinical heart failure patient is increased, and the SMOC2 as a marker has clinical value in the diagnosis of cardiac hypertrophy and heart failure; in addition, by inhibiting the expression of the SMOC2 gene, the cardiac hypertrophy progress of the pressure overload model mouse can be delayed, and then the occurrence of heart failure is prevented. Therefore, a reagent for detecting the expression level of the SMOC2 gene can be used for preparing a reagent for detecting cardiac hypertrophy or heart failure, and an SMOC2 gene expression inhibitor can be used for preparing a medicine for preventing, relieving and / or treating cardiac hypertrophy.

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceutical technology, and particularly to the diagnostic and pharmaceutical uses of SMOC2. Background Art

[0002] Heart failure (HF) is the end-stage lesion of cardiovascular diseases caused by various etiologies and is also the leading cause of death in patients. Clinically, it is mainly manifested as the decline of left ventricular pump function, insufficient perfusion of peripheral tissues, and reduced exercise tolerance. In particular, the increased cardiac pressure afterload caused by aortic coarctation and hypertension is an important inducer of cardiac structural remodeling changes such as myocardial hypertrophy and myocardial fibrosis, and ultimately evolves into chronic heart failure. Over the years, scholars from various countries have made unremitting explorations in the fields of treating myocardial remodeling and heart failure, and have achieved some progress. However, the overall curative effect is still not satisfactory and there has been no fundamental improvement. The treatment means are still limited and the prognosis of patients is still poor. The cardiac dysfunction caused by systemic diseases such as hypertension and diabetes and myocardial tissue lesions such as dilated cardiomyopathy, hypertrophic cardiomyopathy, and mitochondrial cardiomyopathy are all manifested as varying degrees of ventricular wall thickening and hypertrophy of myocardial fibroblasts. At present, the drug treatment strategies for myocardial hypertrophy have poor effects. Therefore, studying the occurrence and development mechanisms of myocardial hypertrophy and heart failure, discovering specific factors and signal transduction pathways involved in myocardial hypertrophy lesions, and exploring drugs targeting these new therapeutic targets have important theoretical and practical significance.

[0003] SMOC2 (Secreted Modular Calcium-binding protein 2) is a secreted extracellular matrix protein and belongs to the SMOC (Secreted Modular Calcium-binding) protein family. SMOC2 plays a role in the extracellular matrix and is involved in various biological processes. SMOC2 plays a role in the extracellular matrix and affects cell behavior and tissue structure. It may regulate cell proliferation, migration, and differentiation by interacting with cell surface receptors or other extracellular matrix molecules. Previous studies have found that in fibrotic diseases, SMOC2 is involved in regulating the deposition of extracellular matrix and tissue sclerosis. Therefore, as a multifunctional extracellular matrix protein, the role of SMOC2 in various biological processes and its importance in diseases are being gradually revealed. However, the role of SMOC2 in myocardial hypertrophy has not been reported yet. Summary of the Invention

[0004] The present invention discovers that, compared with normal people, the expression level of the SMOC2 gene in the heart of patients with heart failure is increased. Statistics show that SMOC2 has clinical value as a biomarker in the diagnosis of myocardial hypertrophy or heart failure; in addition, inhibiting the expression of the SMOC2 gene can delay the progression of myocardial hypertrophy in pressure overload model mice, thereby preventing the occurrence of heart failure. Based on this, the present invention provides the diagnostic and pharmaceutical uses of SMOC2.

[0005] Specifically, in the first aspect of the present invention, there is provided the use of an inhibitor of SMOC2 gene expression in the preparation of a drug for preventing, alleviating, and / or treating myocardial hypertrophy, wherein the inhibitor of SMOC2 gene expression is a substance that is screened or prepared using the SMOC2 gene as a target and has an inhibitory effect on the expression of the SMOC2 gene.

[0006] In combination with the first aspect of the present invention, in some embodiments, the inhibitor of SMOC2 gene expression is selected from at least one of the following:

[0007] 1) Nucleic acid molecules, including at least one of siRNA, shRNA, antisense oligonucleotides, and their chemically modified analogs;

[0008] 2) Gene editing systems, CRISPR / Cas9 or TALEN targeting the SMOC2 gene;

[0009] 3) Nucleic acid constructs, plasmid or viral vectors containing at least one of siRNA, shRNA, antisense oligonucleotides, and their chemically modified analogs;

[0010] 4) Proteinaceous inhibitors, including at least one of antibodies, polypeptides, and fusion proteins designed based on the SMOC2 structure;

[0011] 5) Small molecule compounds, including at least one of natural products, synthetic compounds, and proteolysis-targeting chimeras.

[0012] In combination with the first aspect of the present invention, in some embodiments, the small molecule compound is the compound shown in Formula I (hereinafter referred to as SMOC2I-1) or a pharmaceutically acceptable salt thereof:

[0013]

[0014] In combination with the first aspect of the present invention, in some embodiments, the myocardial hypertrophy is pathological myocardial hypertrophy, and the pathological myocardial hypertrophy is caused by one of hypertension, aortic stenosis, and mitral insufficiency.

[0015] In combination with the first aspect of the present invention, in some embodiments, the drug is selected from:

[0016] (i) An SMOC2 gene expression inhibitor and other drugs for treating myocardial hypertrophy, each independently packaged; or,

[0017] (ii) A compound preparation formulated from an SMOC2 gene expression inhibitor and other drugs for treating myocardial hypertrophy.

[0018] In a second aspect of the present invention, there is provided a drug for preventing, alleviating, and / or treating myocardial hypertrophy, wherein the drug uses the compound represented by Formula I or a pharmaceutically acceptable salt thereof as the sole active ingredient or the main active ingredient for preventing, alleviating, and / or treating myocardial hypertrophy:

[0019]

[0020] In combination with the second aspect of the present invention, in some embodiments, the drug further comprises a pharmaceutically acceptable carrier.

[0021] In combination with the second aspect of the present invention, in some embodiments, the drug is used for preventing, alleviating, and / or treating myocardial hypertrophy or heart failure in patients diagnosed with one of hypertension, aortic stenosis, and mitral insufficiency.

[0022] In a third aspect of the present invention, there is provided the use of a reagent for detecting the expression level of the SMOC2 gene in the preparation of a reagent for examining myocardial hypertrophy or heart failure, wherein the examination reagent is used for patients diagnosed with one of hypertension, aortic stenosis, and mitral insufficiency to evaluate their risk of suffering from myocardial hypertrophy or heart failure.

[0023] In a fourth aspect of the present invention, there is provided an auxiliary diagnostic kit, the auxiliary kit comprising a reagent for detecting the expression level of the SMOC2 gene and being used for auxiliary diagnosis of the differential diagnosis of myocardial hypertrophy or heart failure.

[0024] In a fifth aspect of the present invention, there is provided an auxiliary diagnostic device, the auxiliary diagnostic device comprising the following two modules:

[0025] A detection module that detects the expression level of the SMOC2 gene in a blood sample of a patient; and

[0026] An evaluation module that evaluates the risk of the patient suffering from myocardial hypertrophy or heart failure based on the detection result of the detection module;

[0027] wherein the patient is a patient diagnosed with one of hypertension, aortic stenosis, and mitral insufficiency.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] 1. The present invention for the first time discovers that the expression level of the SMOC2 gene is elevated in the hearts of patients with myocardial hypertrophy or heart failure, and statistically confirms that SMOC2 has clinical value as a biomarker in the diagnosis of myocardial hypertrophy or heart failure. Therefore, an auxiliary diagnostic kit can be prepared using SMOC2 as a biomarker, and in combination with other clinical diagnostic means, it can be confirmed whether a patient has myocardial hypertrophy or heart failure.

[0030] 2. By inhibiting the expression of the SMOC2 gene, the present invention discovers that the progression of myocardial hypertrophy in pressure overload model mice is delayed, and confirms that inhibitors of SMOC2 gene expression can be used for the prevention, alleviation, and / or treatment of myocardial hypertrophy, thereby delaying or avoiding the occurrence of heart failure. Based on the above mechanism, it is expected to develop a variety of innovative drugs targeting the SMOC2 molecule in the art, opening up a new path for the clinical treatment of myocardial hypertrophy, myocardial remodeling, and heart failure. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0032] Figure 1 : Protein expression of SMOC2 in the hearts of normal people and clinical heart failure patients; wherein, A: SDS-PAGE-immunoblotting test (Western blot, WB) result diagram; B: Statistical histogram; C: ROC curve.

[0033] Figure 2 : Protein expression of SMOC2 in the hearts of C57BL / 6 mice after sham operation and transverse aortic constriction (TAC); wherein, A: WB result diagram; B: Statistical histogram; TAC 2w represents the 15th day after the TAC operation, and TAC 4w represents the 29th day after the TAC operation.

[0034] Figure 3 : Protein expression of SMOC2 after grouping treatment of neonatal rat cardiac fibroblasts in vitro; wherein, A: Comparison diagram of WB results of the first group and the second group, PBS represents the first group, and Ang represents the second group; B: Statistical histogram, PBS represents the first group, and Ang represents the second group; C: Grouping treatment flow chart; D: Comparison diagram of WB results of the second group and the third group, DMSO represents the second group, and SMOC2I-1 represents the third group.

[0035] Figure 4 : Treatment flow chart of pressure overload-induced myocardial hypertrophy mice.

[0036] Figure 5 : A: Protein expression and statistical graphs of Collagen I and α-SMA in heart tissues of mice treated with DMSO and SMOC2I-1 after sham operation and TAC surgery, with GAPDH as the internal reference; B: Protein quantification results in Figure A (p values are shown in the figure). The results suggest that after using SMOC2I-1, the expression levels of Collagen1 and α-SMA, which reflect fibrosis, are downregulated compared to mice treated with DMSO. In the figure, Sham represents the sham operation group, TAC 4w represents the myocardial hypertrophy model group, the sampling time is the 29th day after surgery, DMSO represents mice injected with DMSO intraperitoneally, and SMOC2I-1 represents mice injected with SMOC2I-1 intraperitoneally.

[0037] Figure 6 : A: Protein expression and statistical graphs of Vimentin and Ki67 in heart tissues of mice treated with DMSO and SMOC2I-1 after sham operation and TAC surgery, with GAPDH as the internal reference; B: Protein quantification results in Figure A (p values are shown in the figure). The results suggest that after using SMOC2I-1, the expression levels of Vimentin and Ki67, which reflect cell proliferation, are downregulated compared to the DMSO group. In the figure, Sham represents the sham operation group, TAC 4w represents the myocardial hypertrophy model group, the sampling time is the 29th day after surgery, DMSO represents mice injected with DMSO intraperitoneally, and SMOC2I-1 represents mice injected with SMOC2I-1 intraperitoneally.

[0038] Figure 7 : Results of pathological examinations of myocardial hypertrophy and fibrosis in mice; among them, A: Comparative graphs of the heart sizes of mice; B-C: After using SMOC2I-1, the heart HW / BW and HW / TL decreased compared to the control group (p values are shown in the figure); D-E: WGA staining graphs and statistical graphs of mice treated with DMSO and SMOC2I-1 after sham operation and TAC surgery; F-G: Masson staining and statistical graphs of the interstitium and perivascular area of mice treated with DMSO and SMOC2I-1 after sham operation and TAC surgery. In the figure, TAC 4w represents the myocardial hypertrophy model group, sham operation represents the sham operation group, the sampling time is the 29th day after surgery; DMSO represents mice injected with DMSO intraperitoneally, and SMOC2I-1 represents mice injected with SMOC2I-1 intraperitoneally.

[0039] Figure 8: Results of echocardiogram in detecting cardiac function of mice after TAC surgery; among them, A: Mice treated with DMSO and SMOC2I-1 in sham operation and TAC surgery, M-mode echocardiogram, ventricular wall motion echocardiogram and statistical charts of cardiac function detection results, including ejection fraction, global longitudinal strain (GLS); B: Quantitative results of Figure A (p values are shown in the figure). The results suggest that after using SMOC2I-1, the left ventricular ejection fraction and GLS are up-regulated compared with the control group injected with DMSO intraperitoneally, indicating improved cardiac function; in the figure, the control group represents the sham operation group, TAC 4 weeks represents the myocardial hypertrophy model group, and the sampling time is the 29th day after surgery. DMSO represents mice injected with DMSO intraperitoneally, and SMOC2I-1 represents mice injected with SMOC2I-1 intraperitoneally.

[0040] Figure 9 : Effects of SMOC2I-1 on mitochondrial structure and function; among them, A: Transmission electron micrographs of left ventricular tissues of mice in each group, and the enlarged part mainly shows the morphology of mitochondria; B-C: Statistical quantitative charts showing the number of mitochondria and the proportion of mitochondria with damaged cristae under the electron microscope from left to right. In the figure, Sham represents the sham operation group, TAC 4w represents the myocardial hypertrophy model group, and the sampling time is the 29th day after surgery. DMSO represents mice injected with DMSO intraperitoneally, and SMOC2I-1 represents mice injected with SMOC2I-1 intraperitoneally.

[0041] Figure 10 : Synthetic route of SMOC2I-1. <s Specific implementation manners

[0042] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0043] Experimental animals and feeding:

[0044] Experimental animals: C57BL / 6J mice aged 8-10 weeks and weighing 24.5±1.0 g (purchased from Beijing Huafukang Biotechnology Co., Ltd.) were selected as experimental subjects.

[0045] Feeding environment: All experimental mice were fed in the Specific Pathogen Free (SPF) - level experimental animal center of the Cardiovascular Disease Research Institute of Wuhan University. Feeding conditions: Room temperature was between 22 and 24 °C, humidity was between 50% and 70%, and the lighting time with alternating light and darkness was 12 hours. They had free access to water and food.

[0046] Synthesis of SMOC2I-1:

[0047] I. Reaction Route Analysis

[0048] The synthesis route of SMOC2I-1 is as Figure 10 shown. This reaction is a three-component coupling reaction, and the key steps include: nucleophilic substitution of -NH2 of aminopyrimidine for sulfamide bromide; condensation of the intermediate with nitroaromatic heterocycle; and conjugate recombination of the sulfur / nitrogen heterocyclic system.

[0049] II. Specific Operation Process

[0050] 1. Raw Material Pretreatment: Accurately weigh bromosulfamide benzene ring (containing Cl, Br, -SO2NH-) + nitrothiazole benzene ring + aminopyrimidine (molar ratio 1:1:1), and pre-dry all raw materials (vacuum drying at 60 °C for 4 h).

[0051] 2. Construction of Reaction System: Add the following into a 250 mL three-necked flask:

[0052] - Bromosulfamide (5.0 g, 12.5 mmol);

[0053] - Nitrothiazole compound (4.2 g, 12.5 mmol);

[0054] - Aminopyrimidine (2.8 g, 12.5 mmol);

[0055] - 100 mL of anhydrous DMF;

[0056] - K2CO3 (5.2 g, 37.5 mmol);

[0057] Replace with nitrogen three times, and heat to 110 °C under magnetic stirring.

[0058] 3. Process Monitoring: TLC monitoring (developing agent: petroleum ether / triethylamine = 3:1, volume ratio) → Rf of raw material spot ≈ 0.6 → Rf of product spot ≈ 0.3 → Typical reaction time: 18 - 24 h. Rf = solute migration distance / solvent migration distance.

[0059] 4. Post-treatment Optimization → Cool down to 50 °C and quench with water (200 mL of ice water) → Extract with ethyl acetate (3 × 100 mL) → Combine the organic phases → Wash with saturated brine → Dry over anhydrous Na2SO4 → Column chromatography parameters: silica gel 200 - 300 mesh + gradient elution: petroleum ether / triethylamine 5:1 → 3:1 + Yield is about 65% - 72%.

[0060] 5. Key Control Points

[0061] 1) Moisture Control: Keep <50 ppm throughout the process (monitored by Karl Fischer method).

[0062] 2) Temperature sensitivity: The reaction stagnates below 100 °C, and the amount of by-products increases above 130 °C.

[0063] 3) Insufficient nitrogen protection will lead to amino oxidation.

[0064] III. Key points for structural confirmation

[0065] 1. 1 Characteristic peaks of 1H-NMR

[0066] δ 7.8 - 8.2 (m, aromatic ring H);

[0067] δ 6.5 (s, pyrimidine NH);

[0068] δ 4.2 (q, SO2NH - J = 8 Hz);

[0069] 2. Mass spectrometry verification

[0070] ESI-MS: [M + H]+ calculated value 589.2, measured value 589.3.

[0071] Example 1: Detection of the expression of SMOC2 in the hearts of normal people and clinical heart failure patients

[0072] Normal human hearts (individuals donated due to non-cardiac death) and clinical heart failure patient hearts (recipients replaced during heart transplantation surgery or heart biopsy tissues) were selected. Proteins were extracted from the hearts for SDS-PAGE-immunoblotting experiments, and SMOC2 protein was detected in combination. The level of SMOC2 (Abcam, ab198715) was measured, and GAPDH (CST, #2118) was used as an internal reference. The detection results are as Figure 1 shown. Compared with normal human hearts, the level of SMOC2 in the heart tissues of clinical heart failure patients was significantly upregulated.

[0073] The method for detecting SMOC2 expression by SDS-PAGE-immunoblotting is as follows:

[0074] 1) Prepare a 12% SDS polyacrylamide gel according to the instructions on the gel preparation kit;

[0075] 2) Electrophoresis: Dilute the 5× electrophoresis buffer to 1× electrophoresis buffer with double-distilled water. Use a constant voltage of 75 V for stacking gel electrophoresis for 20 min. After all samples reach the same horizontal line, change to a constant voltage of 10 5 V for electrophoresis until the electrophoresis is completed;

[0076] 3) Transfer membrane: Place the cut polyvinylidene fluoride membrane (PVDF membrane) of appropriate size in a bowl containing methanol and soak for half a minute for activation. Remove the gel after electrophoresis and place it in the transfer buffer. Then assemble it in the order of sponge pad - 3 layers of filter paper - gel - PVDF membrane - 3 layers of filter paper. Install it in the transfer tank with the gel corresponding to the negative electrode and the PVDF membrane corresponding to the positive electrode. Place the transfer tank in an ice box and press an ice pack on it to maintain low-temperature transfer. Adjust to constant current transfer at 200 mA for 1.5 h;

[0077] 4) Blocking: After the transfer is completed, take out the PVDF membrane and place it in 5% BSA-TBST and block it on a shaker at room temperature for 1 h;

[0078] 5) Prepare the corresponding primary antibody dilution (1:1000) with 3% BSA-TBST according to the instructions. Cut the PVDF membrane according to different molecular weights, add the corresponding primary antibody and incubate overnight on a shaker at 4°C;

[0079] 6) After the incubation with the primary antibody is completed, wash the membrane 3 times with TBST, 5 min each time. Add the secondary antibody (1:2000) diluted with TBST and the HRP-labeled internal reference antibody and incubate for 1 h. After the incubation with the secondary antibody is completed, wash the membrane 3 times with TBST, 5 min each time;

[0080] 7) Prepare the developing solution for developing in the dark. Use the Bio-Rad Laboratories chemiluminescence imaging system and perform quantitative analysis using Quantity One software after the development is completed.

[0081] As Figure 1 A, Figure 1 B shown, compared with normal people, the level of SMOC2 in the heart tissue of clinical heart failure patients is up-regulated, indicating that SMOC2 is a potential biomarker for heart failure.

[0082] In addition, the present invention collected the sera of clinical heart failure patients and used the SMOC2 ELISA kit (antibodies-online, ABIN4884589) to measure the content of SMOC2 in the sera of clinical heart failure patients, and performed ROC statistical analysis to evaluate the clinical value of SMOC2 as a biomarker in the diagnosis of heart failure.

[0083] The experimental steps for measuring the content of SMOC2 in the sera of clinical heart failure patients using the SMOC2 ELISA kit are as follows:

[0084] 1) Prepare the reagents, samples and standards according to the instructions.

[0085] 2) Add 100 μL of the standard or sample to each well and incubate at 37°C for 2 hours.

[0086] 3) Take out the liquid from each well without washing.

[0087] 4) Add 100 μL of biotin antibody (1×) to each well and incubate at 37 °C for 1 hour.

[0088] 5) Aspirate and wash 3 times.

[0089] 6) Add 100 μL of HRP-avidin (1×) to each well and incubate at 37 °C for 1 hour.

[0090] 7) Aspirate and wash 5 times.

[0091] 8) Add 90 μL of TMB substrate to each well and incubate at 37 °C for 15 - 30 minutes. Protect from light.

[0092] 9) Add 50 μL of stop solution to each well and read the absorbance at 450 nm within 5 minutes.

[0093] Figure 1 The area under the curve (AUC value) of the ROC curve in C is equal to 0.74***0751, indicating that SMOC2 can effectively distinguish clinical heart failure patients from healthy individuals and has moderate disease diagnostic value. As the 1 - specificity increases (i.e., the false positive rate increases), the sensitivity usually also increases, indicating that while the model improves the true positive recognition rate, it also brings more false positives.

[0094] Example 2: Detection of SMOC2 expression in angiotensin II - stimulated cardiac fibroblasts

[0095] The main purpose of stimulating cardiac fibroblasts with angiotensin II in vitro is to simulate the pathogenesis of cardiac fibrosis under pathological conditions (hypertension, cardiac pressure overload) and study the specific role of SMOC2 in myocardial remodeling (such as myocardial hypertrophy, fibrosis). The specific steps are as follows:

[0096] 1. Primary cardiac fibroblast culture

[0097] 1) Take 10 Sprague - Dawley neonatal rats (hereinafter referred to as neonatal rats) aged 1 - 3 days after birth. Disinfect the area below the neck with 75% alcohol. Use ophthalmic scissors and forceps to remove the heart and place it in a glass petri dish containing 10 mL of DMEM / F12 solution.

[0098] 2) Wash the heart with DMEM / F12 medium and cut the heart into fragments of 1 - 2 mm 3 . Transfer them to a serum bottle with a rotor, aspirate the DMEM / F12 medium, and add trypsin digestion solution. Set the rotation speed to 120 r / min and digest for 15 min; then let it stand for a few seconds and discard the supernatant.

[0099] 3) Add trypsin digestion solution and rotate at 120 r / min for 15 min. Then let it stand for several seconds, aspirate the supernatant, terminate the digestion with DMEM / F12 medium containing 20% calf serum, and store it in a refrigerator at 4°C. Repeat this step for several cycles. When aspirating the supernatant, try to aspirate as much as possible. When the tissue pieces turn white and become significantly smaller, terminate the digestion. Collect the remaining myocardial fibroblast suspension in the serum bottle.

[0100] 4) Centrifuge the collected myocardial fibroblast suspension at 1500 rpm for 8 min, and then discard the supernatant. Add an appropriate amount of medium to the centrifuge tube, gently pipette to resuspend the cells, and then concentrate them into 1 50-mL centrifuge tube. Filter the cell suspension through a filter with a pore size of 40 μm.

[0101] 5) Inoculate the cells in a culture dish with a diameter of 100 mm, perform differential adhesion for 90 min, aspirate the non-adherent cell suspension and filter it. Add Brdu (final concentration 0.1 mM) according to the total volume of the cell suspension, mix well, and then add it to a vessel coated with 0.1% gelatin.

[0102] 6) Gently shake to disperse the cells, do not vortex. Incubate at 37°C and 5% CO2 for 48 hours, then wash once with PBS and change the medium to obtain primary myocardial fibroblasts.

[0103] 2. In vitro cell experiments

[0104] Culture the primary myocardial fibroblasts for 48 h, then change the medium, add serum-free DMEM / F12 medium, and perform starvation treatment for 12 h to synchronize the cells. Then perform grouped treatment on the cells:

[0105] Group 1: Incubate for 48 hours after adding dimethyl sulfoxide (DMSO), and then stimulate with PBS for 24 hours.

[0106] Group 2: Incubate for 48 hours after adding DMSO, and then stimulate with Ang II (1.0 μM) for 24 hours.

[0107] Group 3: Incubate for 48 hours after adding SMOC2I-1 (1.0 μM), and then stimulate with Ang II (1.0 μM) for 24 hours.

[0108] In each of the above groups, the added volumes of DMSO and SMOC2I-1 (1.0 μM) are equal, and the added volumes of PBS and Ang II (1.0 μM) are equal.

[0109] Extract proteins from the myocardial fibroblasts treated in the above three groups, and then perform SDS-PAGE-immunoblotting assay. The detection results are as Figure 3 A, Figure 3As shown in B: compared with the first group, the level of SMOC2 in the second group of AngII-stimulated cardiac fibroblasts was significantly up-regulated, that is, the expression level of SMOC2 gene in cardiac fibroblasts under pressure load was up-regulated. As Figure 3 C, Figure 3 As shown in D, the level of SMOC2 in the cells of the second group without SMOC2I-1 was higher than that in the third group, that is, the level of SMOC2 in the cardiac fibroblasts stimulated by AngII in the third group was significantly inhibited by SMOC2I-1. These results indicate that after stimulation with Ang II, the level of SMOC2 is up-regulated, while the use of SMOC2I-1 can inhibit the up-regulation of SMOC2 level.

[0110] Example 3: Expression of SMOC2 in mice with pressure overload-induced myocardial hypertrophy

[0111] The TAC surgery can significantly increase the left ventricular ejection resistance by physically narrowing the aorta (usually at the ascending aorta of mice), resulting in a long-term state of pressure overload (AfterloadIncrease) of the heart, thereby triggering compensatory cardiac hypertrophy and pathological cardiac remodeling. In the present invention, a mouse model of pressure overload-induced myocardial hypertrophy was obtained by TAC surgery for modeling.

[0112] The flow chart of this experiment is shown in Figure 4 , the date of birth of the mice was recorded as Day-4w, the date of TAC surgery was recorded as Day 0, the date of the first injection was recorded as Day 1w (i.e., the 8th day after TAC surgery), and the date of cardiac function detection and heart sampling was recorded as Day 4w (i.e., the 29th day after TAC surgery).

[0113] The following is a detailed description of the experimental procedure:

[0114] 1. The operating steps for TAC surgery for modeling are as follows:

[0115] 1.1 Preparation before surgery

[0116] 1) Anesthesia: First, weigh the mice (8-10 weeks old, body weight 24.5±1.0 g, C57BL / 6J mice), calculate the required amount of anesthetic (3% sodium pentobarbital) according to 90 mg / kg body weight, inject it intraperitoneally, and record the injection time point. The standard for successful anesthesia is that there is no obvious reaction when pinching the tail or toes and the mouse is in good condition (generally, there is no obvious reaction about 10 min after injection, the best surgical time is about 30 min after anesthesia, and the mouse's toes have a reaction about 50 min after anesthesia).

[0117] 2) Preparation of the surgical area: Remove the hair from the skin of the left chest, left side of the chest and under the left forelimb axilla of the mouse. After shaving, wipe the surgical area with a wet gauze to remove the mouse hair, so as not to affect the surgical field of view.

[0118] 3) Tracheal intubation: Fix the upper incisors of the mouse to the inclined plane of the V-shaped plate with a rubber band, and quickly insert the tracheal intubation accurately into the trachea through the glottis. Then place the mouse in the right lateral position on a heating pad (the heating pad needs to be preheated in advance), and then connect the tracheal intubation to the ventilator and fix the mouse. If the chest movement of the mouse is consistent with the ventilator frequency, it indicates successful tracheal intubation.

[0119] 1.2 TAC surgery

[0120] Myocardial hypertrophy model group (abbreviated as TAC group): Take the right lateral position, place the left forelimb of the mouse above the right forelimb, and fix the two forelimbs with medical tape. Pad a cotton swab under the right chest to elevate the chest wall. Disinfect the skin of the surgical area with iodine tincture and 75% (v / v) alcohol in turn. Hold the left chest skin with forceps in the left hand and cut the skin about 1 cm with scissors in the right hand. Separate the muscle and soft tissue in turn, open the chest cavity at the level of the 2nd - 3rd ribs, gently push aside the left lung with a cotton swab, free the descending branch of the aortic arch, pass a 7-0 surgical suture through the blood vessel, and place a 26G syringe needle (for mice weighing 25.0 - 27.5 g) or 27G syringe needle (for mice weighing 23.5 - 25.0 g) parallel above the blood vessel. Ligate the blood vessel and the needle together, and then withdraw the needle to achieve the corresponding degree of blood vessel constriction. After ligation, suture in turn, close the chest cavity, insert a syringe into the chest cavity through the suture and draw out 1 cc of gas to restore the negative pressure in the chest cavity. After withdrawing the syringe, quickly suture the skin incision.

[0121] Sham operation group (abbreviated as Sham group): Only pass the thread but do not ligate after freeing the descending branch of the aorta, and the remaining steps are the same as those in the myocardial hypertrophy model group.

[0122] 1.3 Postoperative care

[0123] After TAC surgery, when the mouse shows spontaneous breathing and a strong response to toe pinching, remove the tracheal intubation, and place the mouse in a breeding cage filled with autoclaved bedding, feed, and drinking water, and continue to raise and observe in the breeding room.

[0124] 2. Group intervention

[0125] Group intervention starts on the 8th day (Day 1w) after TAC surgery: Inject SMOC2I-1 or placebo (saline in this experiment) into the peritoneal cavity of the mouse, and continuously administer the drug for 2 weeks (14 days) at a dose of 10 mg / kg / d (determined by pre-experiment and basic blood drug concentration). On the 29th day (Day 4w) after TAC surgery in the mouse, use echocardiography to detect the cardiac function of the mouse (see Figure 8 A) and collect samples. The detection of echocardiography is described as follows.

[0126] The group intervention situation is as follows:

[0127] DMSO - Sham operation group: From the 8th day after sham operation, mice were intraperitoneally injected with placebo at a dose of 10 mg / kg / d for 2 consecutive weeks.

[0128] SMOC2I - 1 - Sham operation group: From the 8th day after sham operation, mice were intraperitoneally injected with SMOC2I - 1 at a dose of 10 mg / kg / d for 2 consecutive weeks.

[0129] DMSO - TAC group: From the 8th day after TAC surgery, mice were intraperitoneally injected with placebo at a dose of 10 mg / kg / d for 2 consecutive weeks.

[0130] SMOC2I - 1 - TAC group: From the 8th day after TAC surgery, mice were intraperitoneally injected with SMOC2I - 1 at a dose of ၁၀ mg / kg / d for 2 consecutive weeks.

[0131] 3. Echocardiography was used to detect the cardiac function of mice

[0132] 3.1. Preliminary preparation

[0133] 1) Preparation of anesthesia machine: First, connect the oxygen cylinder to the air inlet interface on the anesthesia machine, then unscrew the sealing cap of the medicine adding port on the anesthesia machine, quickly add isoflurane to the safe concentration and then tighten the sealing cap. Unscrew the main valve on the oxygen cylinder and adjust the knob of the flow control valve so that the outlet pressure is maintained at 0.2 - 0.3 mPa.

[0134] 2) Preparation of mice to be measured: After the mice to be measured were quickly anesthetized with isoflurane, the hair on the left precordial area was shaved, and the head of the treated mice was inserted into the anesthesia catheter sleeve, and the mice were maintained in a stable anesthetic state with 1.5% - 2.0% isoflurane.

[0135] 3.2 Cardiac function detection

[0136] The mice were placed in the left lateral position or supine position, and ultrasonic coupling agent was evenly applied to the shaved area. A high - frequency ultrasonic diagnostic instrument with a frequency of 15 MHz was used to select the standard short - axis section of the left ventricular papillary muscle to measure the left ventricular end - diastolic diameter, left ventricular end - systolic diameter, left ventricular ejection fraction, and fractional shortening.

[0137] 3.3 Proteins were extracted from the heart for SDS - PAGE - immunoblotting assay, and detected by combining with an antibody (Abcam, ab198715) that specifically recognizes SMOC2 protein to determine the level of SMOC2, with GAPDH as the internal reference. The results are as Figure 2 shown. The expression of SMOC2 was significantly up - regulated on the 15th day and 29th day after TAC surgery, indicating that SMOC2 is a potential biomarker for myocardial hypertrophy.

[0138] The results of cardiac function detection of mice in each group after grouped intervention are as Figure 8As shown. Compared with the mice in the sham operation group, the mice in the myocardial hypertrophy model group showed weakened cardiac function on the 29th day, with decreased ejection fraction and fractional shortening, which are indicators reflecting cardiac function. In contrast, compared with the mice injected with DMSO, the cardiac insufficiency of the mice given SMOC2I-1 injection was alleviated ( Figure 8 ), indicating that inhibiting the expression of SMOC2I-1 gene can relieve the symptoms of heart failure.

[0139] Example 4: Pathological detection of myocardial hypertrophy and fibrosis in mice with myocardial hypertrophy model

[0140] 1. Specimen collection

[0141] 1) Preliminary work: Prepare a urine cup, add 20 mL of formaldehyde (volume fraction 10%), and label it (mouse number, group, surgical type, and specimen collection date). Place a petri dish filled with KCl solution (mass fraction 10%) at the specimen collection site. Turn on the analytical balance and zero it for standby. Then weigh and sacrifice the mice.

[0142] 2) Specimen collection: Use ophthalmic curved forceps to clamp the vascular pedicle below the auricle of the heart, cut off the heart, and quickly place it in KCl solution (mass fraction 10%). After the heart stops beating in diastole, place it on a sterilized gauze, gently squeeze the liquid in the heart cavity, blot the surface liquid, weigh and record it, and then put the heart into the corresponding urine cup. After fixation for 48 hours, it is used for pathological detection.

[0143] 3) Related measurements and calculations: Take out the hearts and lungs of the mice, trim them, blot them dry with filter paper, weigh and record. Cut open the skin at the tibia of the hind limb of the mouse, measure and record the tibia length. Calculate the ratio of heart weight to body weight (HW / BW) and the ratio of heart weight to tibia length (HW / TL).

[0144] 2. Pathological detection

[0145] 2.1 Preparation of paraffin specimen sections

[0146] The main operating procedures include: trimming the heart → treating the embedding frame → rinsing with running water → dehydration → clearing → impregnation with wax → embedding → sectioning → spreading the sections → air-drying or baking for standby.

[0147] 2.2 Wheat germ agglutinin (WGA) staining

[0148] 1) Deparaffinization of sections (for paraffin sections): Place the sections in xylene and incubate at room temperature for 5 - 10 minutes, repeat 2 times to remove paraffin.

[0149] 2) Hydration: Place the sections successively in gradient ethanol (100%, 95%, 70%, 5 minutes each time) to remove xylene, and finally place them in distilled water for 5 minutes for hydration.

[0150] 3) Hematoxylin staining: Immerse the sections in hematoxylin staining solution and stain at room temperature for 5 - 10 minutes.

[0151] 4) Differentiation (optional): Transfer the sections into the differentiation solution (such as 1% hydrochloric acid alcohol), and differentiate at room temperature for 5 - 30 seconds to remove the excess dye.

[0152] 5) Eosin staining: Transfer the sections into eosin staining solution and stain at room temperature for 1 - 3 minutes.

[0153] 6) Dehydration: Sequentially place the sections into gradient ethanol for dehydration (70%, 85%, 95%, 100%, 5 minutes each time).

[0154] 7) Clearing: Place the sections into xylene and clear at room temperature 2 times, 5 minutes each time.

[0155] 8) Mounting: Place the sections on the glass slides, drop the mounting medium (such as Canada balsam or glycerol gelatin), cover with a coverslip, and gently flatten.

[0156] 9) Microscopic observation: Observe the sections under the microscope, record and analyze the staining results.

[0157] 2.3 Masson trichrome staining

[0158] The main steps are: Bake at 55 °C for 30 min → Xylene for 2 min, 3 times → 100% alcohol for 1 min → 95% alcohol for 1 min → 70% alcohol for 1 min → Running water rinse for 10 min → Double-distilled water for 1 min → Weigert's iron hematoxylin stain for 5 min → Tap water wash for 5 min → Remove the residual liquid → Differentiate with 1% hydrochloric acid alcohol for 4 s → Tap water wash for 5 min to blue back → Stain with Ponceau acid fuchsin solution for 10 min → Rinse with distilled water for 5 min → Treat with phosphomolybdic acid aqueous solution for about 5 min → Counterstain with aniline blue solution for 5 min → Treat with 1% glacial CH3COOH for 1 min → 70% alcohol once → 90% alcohol once → 100% alcohol for 30 s, 3 times → Xylene for 2 min, 3 times → Immediately cover the coverslip for mounting while the xylene is not dry, and take microscopic photos.

[0159] 2.4 As Figure 7As shown in A, compared with the DMSO - sham group, there were no obvious differences in the gross cardiac phenotypes of the SMOC2I - 1 - sham group mice. However, the hearts of both the SMOC2I - 1 - TAC group and the DMSO - TAC group were enlarged. Compared with the DMSO - TAC group mice, the hearts of the SMOC2I - 1 - TAC group mice were significantly smaller. WGA staining also indicated that compared with the DMSO - sham group, although there were no obvious differences in the cross - sectional areas of cardiomyocytes in the SMOC2I - 1 - sham group mice, compared with the DMSO - TAC group, the cross - sectional areas of cardiomyocytes in the SMOC2I - 1 - TAC group were significantly smaller. These results together suggest that SMOC2I - 1 can alleviate myocardial hypertrophy caused by pressure overload( Figure 7 D、 Figure 7 E). In addition, among the sham - operated group mice, whether or not stimulated with SMOC2I - 1, there were no obvious differences in the heart weight / tibia length ratio (HW / TL). However, the HW / TL of the SMOC2I - 1 - TAC group mice was significantly lower than that of the DMSO - TAC group mice( Figure 7 B - C), further confirming the drug - specific inhibition of cardiac hypertrophy. In addition, WB results suggested that compared with the mice injected with DMSO, the levels of myocardial fibrosis markers collagen I and α - SMA in the mice injected with SMOC2I - 1 were both down - regulated( Figure 5 ), also verifying the previous conclusion.

[0160] As is well known, in tissue injury models, if interstitial cells (such as fibroblasts) positive for intermediate filament protein (Vimentin) also show high Ki67 expression, it indicates that these cells are in an active proliferation state and may be involved in tissue repair or the pro - fibrosis process. However, in the mice injected with SMOC2I - 1, vimentin was down - regulated along with ki67( Figure 6 ), and these results together proved that fibrosis was alleviated after SMOC2I - 1 treatment, reducing myocardial remodeling. As Figure 7 F、 Figure 7 The Masson staining results in G showed that the collagen content in the left ventricular tissue of the mice after TAC surgery increased significantly, while intraperitoneal injection of SMOC2I - 1 could significantly reduce the rising rate of the fibrosis level in the left ventricular tissue, thus delaying the occurrence of heart failure.

[0161] Example 5: Effects of SMOC2I - 1 on mitochondrial structure and function

[0162] Analysis of mitochondrial morphology in mouse myocardial tissue:

[0163] 1) Preliminary work: Prepare EP tubes, add 100 μL of tissue electron microscopy fixative (G1102, Servicebio), and label them (mouse number, group, surgical type, and sampling date). Place a petri dish containing 10 mL of tissue electron microscopy fixative at the sampling site. Weigh and sacrifice the mice.

[0164] 2) Sampling: Use an ophthalmic curved forceps to clamp the vascular pedicle below the auricle, cut off the heart, quickly place it on a sterilized gauze, gently squeeze the liquid in the heart cavity, dry the surface liquid, then put the heart into the tissue electron microscopy fixative, and use an ophthalmic scissors to cut a tissue block about 1.0 mm 3 in size from the apex of the heart, avoiding squeezing the apex tissue, and place it in a labeled EP tube. Prepare a transmission electron microscopy tissue sample within 48 hours.

[0165] 3) Related measurements and calculations: Observe the mitochondrial morphology under a transmission electron microscope, take pictures of the mitochondrial morphology of myocardial tissues in each group, and use Image J software to count the number of mitochondria, the proportion of mitochondria in the tissue, and the proportion of mitochondria with damaged cristae.

[0166] 4) The results of mitochondrial morphological analysis showed that: compared with the sham-operation group of mice, the number of mitochondria in the apex tissue of mice after 4 weeks of TAC surgery decreased, the proportion of mitochondria with damaged cristae increased, while the number of mitochondria in mice intervened with SMOC2I-1 increased, and the proportion of mitochondria with damaged cristae decreased, suggesting that the application of SMOC2I-1 can significantly reduce the mitochondrial damage in the myocardial tissue of mice with pressure overload ( Figure 9 ).

[0167] The above research results indicate that SMOC2I-1 can significantly reduce the mitochondrial damage in the myocardial tissue of mice with pressure overload, inhibit the fibrosis level in the heart tissue under pressure overload, delay the occurrence of compensatory cardiac hypertrophy and pathological cardiac remodeling, and is expected to be used for preventing, alleviating or treating pathological myocardial hypertrophy caused by hypertension heart disease, aortic stenosis, mitral insufficiency, etc., and delaying the occurrence of chronic heart failure.

[0168] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. Use of an inhibitor of SMOC2 gene expression in the preparation of a drug for preventing, alleviating and / or treating myocardial hypertrophy, characterized in that: The SMOC2 gene expression inhibitor is a substance prepared or screened using the SMOC2 gene as a target and having an inhibitory effect on SMOC2 gene expression.

2. The use according to claim 1, wherein: The SMOC2 gene expression inhibitor is selected from at least one of the following: 1) Nucleic acid molecules, including at least one of siRNA, shRNA, antisense oligonucleotides, and their chemically modified analogs; 2) Gene editing systems, CRISPR / Cas9 or TALEN targeting the SMOC2 gene; 3) Nucleic acid constructs, plasmids or viral vectors containing at least one of siRNA, shRNA, antisense oligonucleotides, and their chemically modified analogs; 4) Proteinaceous inhibitors, including at least one of antibodies, polypeptides, or fusion proteins designed based on the SMOC2 structure; 5) Small molecule compounds, including at least one of natural products, synthetic compounds, and proteolysis-targeting chimeras.

3. The use according to claim 2, characterized in that: The small molecule compound is the compound of formula I or a pharmaceutically acceptable salt thereof:

4. The use according to claim 1, characterized in that: The myocardial hypertrophy is pathological myocardial hypertrophy.

5. A drug for preventing, alleviating and / or treating myocardial hypertrophy, characterized in that, The drug uses the compound of formula I or a pharmaceutically acceptable salt thereof as the sole active ingredient or the main active ingredient for preventing, alleviating, and / or treating myocardial hypertrophy:

6. The drug according to claim 5, characterized in that: The drug further includes a pharmaceutically acceptable carrier.

7. The drug according to claim 5, characterized in that: The drug is used for preventing, alleviating, and / or treating myocardial hypertrophy or heart failure in patients diagnosed with one of hypertension, aortic stenosis, and mitral insufficiency.

8. Use of a reagent for detecting the expression level of the SMOC2 gene in the preparation of a reagent for examining myocardial hypertrophy or heart failure, characterized in that: The test reagent is used for patients diagnosed with one of hypertension, aortic stenosis, and mitral insufficiency to evaluate their risk of developing myocardial hypertrophy or heart failure.

9. An auxiliary diagnostic kit, characterized in that: The auxiliary kit includes reagents for detecting the expression level of the SMOC2 gene and is used for assisting in the differential diagnosis of myocardial hypertrophy or heart failure.

10. An auxiliary diagnosis device, characterized in that, The auxiliary diagnostic device includes the following two modules: A detection module that detects the expression level of the SMOC2 gene in a patient's blood sample; and an evaluation module that evaluates the risk of the patient developing myocardial hypertrophy or heart failure based on the detection result of the detection module; wherein the patient is a patient diagnosed with one of hypertension, aortic stenosis, and mitral insufficiency.

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