Application of reagent for knocking out Sam68 gene or inhibiting Sam68 gene expression in preparation of medicine for preventing and / or treating pathological cardiac hypertrophy

Abstract

The invention relates to the technical field of biology, and particularly discloses application of a reagent for knocking out a Sam68 gene or inhibiting Sam68 gene expression in preparation of a medicine for preventing and / or treating pathological cardiac hypertrophy. According to the application, the function and effect of the Sam68 gene in pathological cardiac hypertrophy are identified through experiments, further, the application of a reagent for knocking out the Sam68 gene or inhibiting the expression of the Sam68 gene in preparation of a medicine for preventing and / or treating pathological cardiac hypertrophy is provided, and a reagent for effectively protecting the cardiac function and inhibiting the expression of the Sam68 gene is provided. And a brand new means for restraining or relieving cardiac hypertrophy and heart failure is provided.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to the use of an agent for knocking out the Sam68 gene or inhibiting the expression of the Sam68 gene in the preparation of a drug for preventing and / or treating pathological myocardial hypertrophy. Background Art

[0002] At present, the incidence and mortality of cardiovascular diseases are increasing year by year. The "China Cardiovascular Health and Disease Report 2022" pointed out that the number of people with cardiovascular diseases in my country is as high as 330 million, of which 8.9 million are suffering from heart failure (hereinafter referred to as "heart failure"), and the 5-year mortality rate of heart failure patients exceeds 50%. Therefore, finding effective methods to prevent and treat heart failure is one of the major problems that need to be solved in clinical practice and basic medicine.

[0003] Pathological myocardial hypertrophy is a precursor of heart failure. It is the heart's response to pathological stimuli such as volume overload and pressure overload. It begins with the enlargement of myocardial cells and the centripetal growth of the ventricles, followed by the elongation of myocardial cells, thinning of ventricular walls, dilation of ventricular cavities, and decreased cardiac contractile function. Pathological myocardial hypertrophy is in the compensatory stage at the early stage, and its clinical manifestations are not obvious and easy to be ignored. However, as the disease slowly develops into severe heart failure and fatal arrhythmias, it often causes irreparable damage. Therefore, pathological myocardial hypertrophy is a strong risk factor for cardiovascular disease. At present, due to the complexity of the etiology of pathological myocardial hypertrophy, the specific pathogenic mechanism of its development has not been clearly studied, so there is a lack of effective treatments and drugs for myocardial hypertrophy in clinical practice. Drugs such as beta-blockers, calcium ion blockers and angiotensin-converting enzyme inhibitors that can only relieve patients' clinical symptoms, as well as surgery to remove the hypertrophic ventricular septal muscle, do not have ideal prognostic effects on patients. Therefore, the exploration and development of new drugs based on the pathological mechanism of myocardial hypertrophy is an important means to effectively prevent and intervene in pathological myocardial hypertrophy, curb the progression of heart failure, and improve patients' quality of life. Summary of the invention

[0004] In view of the above-mentioned deficiencies in the prior art, the purpose of the present application is to provide a reagent for knocking out the Sam68 gene or inhibiting the expression of the Sam68 gene for use in the preparation of a drug for preventing and / or treating pathological myocardial hypertrophy, aiming to solve the problem that the prior art lacks effective means for preventing and intervening in pathological myocardial hypertrophy.

[0005] The technical solution of this application is as follows:

[0006] In a first aspect of the present application, a reagent for knocking out the Sam68 gene or inhibiting the expression of the Sam68 gene is provided for use in the preparation of a drug for preventing and / or treating pathological myocardial hypertrophy.

[0007] Optionally, the reagent is a gene editing system.

[0008] Optionally, the agent is a reverse peptidomimetic.

[0009] Optionally, the reverse analog peptide includes at least one of YB-0158, ICG-001, CWP232228, and CWP232291.

[0010] Optionally, the agent knocks out the Sam68 gene, reduces or inhibits the transcription of the Sam68 gene, and / or reduces or inhibits the translation of the mRNA product of Sam68.

[0011] In a second aspect of the present application, a drug for preventing and treating pathological myocardial hypertrophy is provided, wherein the drug comprises an agent for knocking out the Sam68 gene or inhibiting the expression of the Sam68 gene.

[0012] Optionally, the reagent is a gene editing system; or the reagent is a reverse analog peptide.

[0013] The third aspect of the present application provides a method for screening drugs for preventing and / or treating pathological myocardial hypertrophy, which comprises screening Sam68 inhibitors.

[0014] Optionally, the Sam68 inhibitor can inhibit or reduce the expression of the Sam68 gene, or inhibit or block the activity of the Sam68 protein.

[0015] Optionally, the step of screening Sam68 inhibitors includes: detecting whether the test agent can inhibit Sam68 gene expression or Sam68 protein activity; and selecting the test agent that can inhibit Sam68 gene expression or Sam68 protein activity as a candidate drug.

[0016] Beneficial effects of the present application: The present application experimentally identifies the function and role of Sam68 in pathological myocardial hypertrophy, and further proposes the use of an agent for knocking out the Sam68 gene or inhibiting the expression of the Sam68 gene in the preparation of a drug for preventing and / or treating pathological myocardial hypertrophy, so as to provide a new means of effectively protecting heart function, curbing or alleviating myocardial hypertrophy and heart failure. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for describing the embodiments are briefly introduced below.

[0018] Figure 1 Sam68 transcription level analysis diagram of 2 cardiac RNA sequencing result data sets provided in Example 1 of the present application:

[0019] A is the transcriptome analysis of Sam68 in the GSE116250 dataset; B is the transcriptome analysis of Sam68 in the GSE160997 dataset;

[0020] Figure 2 This is an analysis chart of the Sam68 protein expression in the heart of the myocardial hypertrophy mouse model constructed in Example 2 of the present application:

[0021] A is a protein immunoblotting analysis; B is an immunofluorescence co-staining analysis;

[0022] Figure 3 This is an analysis diagram of the effects of the heart / body weight ratio and the heart / tibia length ratio on the myocardial hypertrophy mouse model provided in Example 3 of the present application;

[0023] Figure 4 HE staining image of mouse heart tissue provided in Example 3 of the present application;

[0024] Figure 5 The wheat germ agglutinin staining and Sirius red staining images of the heart tissue of mice provided in Example 3 of the present application are as follows:

[0025] A is wheat germ agglutinin staining; B is cell area analysis; C is Sirius red staining; D is collagen ratio analysis;

[0026] Figure 6 This is a gene expression analysis diagram of the myocardial hypertrophy mouse model provided in Example 3 of the present application:

[0027] A is the transcription level analysis of ANP and BNP; B is the protein immunoblot analysis of ANP, BNP and Sam68;

[0028] Figure 7 This is an analysis diagram of cardiac function ultrasound detection of a myocardial hypertrophy mouse model provided in Example 4 of the present application:

[0029] A is the thickness of the posterior wall at the end of diastole; B is the thickness of the posterior wall at the end of systole; C is the ejection fraction; D is the fractional shortening;

[0030] Figure 8 This is an analysis chart of the expression of ANP, BNP and Sam68 in the heart of the myocardial hypertrophy mouse model provided in Example 5 of the present application:

[0031] A is the mRNA transcription level of ANP and BNP; B is the expression analysis of ANP, BNP and Sam68;

[0032] Fig. 9 This is an analysis diagram of the effects of the heart / body weight ratio and the heart / tibia length ratio on the myocardial hypertrophy mouse model provided in Example 5 of the present application:

[0033] A is the ratio of heart to body weight; B is the ratio of heart to tibia length;

[0034] Fig.10 This is a cardiac function ultrasound detection analysis diagram of the myocardial hypertrophy mouse model provided in Example 5 of the present application:

[0035] A is the left ventricular internal diameter at the end of systole; B is the left ventricular internal diameter at the end of diastole; C is the ejection fraction; D is the fractional shortening;

[0036] Fig.11 This is an analysis chart of the effects of YB0518 on the degree of myocardial hypertrophy, degree of fibrosis and cardiac function in a myocardial hypertrophy mouse model provided in Example 6 of the present application:

[0037] A is the YB0518 drug treatment flow chart; B is the measurement of systolic and diastolic ventricular septum thickness in the myocardial hypertrophy mouse model; C is the heart / body weight ratio and heart / tibia length ratio in the myocardial hypertrophy mouse model; D is the analysis of ANP, BNP and Sam68 expression levels in the heart of the myocardial hypertrophy mouse model. DETAILED DESCRIPTION

[0038] In order to make the purpose, technical solutions and advantages of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below in combination with the drawings and embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments of the present application, all other embodiments obtained by ordinary technicians in this field without making creative work belong to the protection scope of the present application. In the absence of conflict, the following embodiments and features in the embodiments can be combined with each other.

[0039] It should be noted that if there are descriptions involving "first", "second", etc. in the implementation of this application, the descriptions of "first", "second", etc. are only used for descriptive purposes and cannot be understood as indicating or suggesting their relative importance and implicitly indicating the number of technical features indicated. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In addition, the technical solutions between the various embodiments can be combined with each other, but they must be based on the fact that ordinary technicians in the field can implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be deemed that such combination of technical solutions does not exist and is not within the scope of protection required by this application.

[0040] An embodiment of the present application provides a use of an agent for knocking out the Sam68 gene or inhibiting the expression of the Sam68 gene in the preparation of a drug for preventing and / or treating pathological myocardial hypertrophy.

[0041] Among them, the term "treatment" refers to a method implemented in order to obtain a beneficial or desired clinical result. For the purposes of the present invention, a beneficial or desired clinical result includes, but is not limited to, alleviating symptoms, reducing the scope of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the development of the disease, improving or alleviating the state of the disease, and alleviating symptoms (whether partially or completely), whether detectable or undetectable. In addition, "treatment" can also refer to prolonging survival compared to the expected survival (if not receiving treatment).

[0042] The reagent for knocking out the Sam68 gene or inhibiting the expression of the Sam68 gene in the embodiments of the present application can destroy the expression of the Sam68 gene by any mechanism, such as by inhibiting the expression of Sam68 at the RNA or protein level, such as knocking out the Sam68 gene, reducing or inhibiting the transcription of the gene, and / or reducing or inhibiting the translation of the mRNA product of the gene. The function and role of Sam68 in pathological myocardial hypertrophy were identified through experiments, and then a reagent for knocking out the Sam68 gene or inhibiting the expression of the Sam68 gene was proposed for the preparation of a drug for preventing and / or treating pathological myocardial hypertrophy.

[0043] In the early stages of myocardial hypertrophy, it is a compensatory mechanism. The heart increases myocardial contractility through myocardial hypertrophy to maintain the heart's pumping function. For example, in patients with hypertension, myocardial hypertrophy enables the heart to overcome increased peripheral resistance within a certain period of time and maintain cardiac output close to normal levels. At this time, the structure and function of the heart can still maintain a relatively stable state. As the disease progresses, myocardial hypertrophy will gradually undergo adverse changes. Excessive hypertrophy of the myocardium will cause relative ischemia and hypoxia of myocardial cells. Because the volume of myocardial cells increases, and the growth of capillaries cannot match it, the diffusion distance of oxygen and nutrients increases. At the same time, during the process of myocardial cell hypertrophy, some signal pathways and molecular mechanisms in myocardial cells will be disordered. For example, the calcium regulation mechanism in myocardial cells is abnormal, affecting the contraction and relaxation function of the myocardium. In addition, the extracellular matrix of myocardial cells will also be reconstructed, collagen fibers will increase, and the compliance of the myocardium will decrease, leading to diastolic dysfunction of the heart. The combined effect of these factors causes the heart's pumping function to gradually decline, and eventually leads to heart failure. Heart failure refers to a pathological and physiological state in which the contraction and relaxation of the heart are impaired and cannot meet the metabolic needs of the body. In the examples of the present application, reagents for knocking out the Sam68 gene or inhibiting the expression of the Sam68 gene are used to prepare drugs for preventing and / or treating pathological myocardial hypertrophy, thereby effectively protecting heart function, curbing or alleviating myocardial hypertrophy and heart failure.

[0044] In some embodiments, the reagent for knocking out the Sam68 gene or inhibiting the expression of the Sam68 gene is a gene editing system. It is well known to those skilled in the art to use a gene editing system to knock down or knock out a target gene (eg, the Sam68 gene).

[0045] In some embodiments, the agent for knocking out the Sam68 gene or inhibiting the expression of the Sam68 gene is a reverse mimetic peptide.

[0046] Retro-inverso peptidomimetics is a peptide compound that enhances its stability and biological activity by reversing the N-terminal and C-terminal sequences of a peptide and possibly introducing D-amino acids. In some embodiments, the retro-inverso peptidomimetics include at least one of YB-0158, ICG-001, CWP232228, and CWP232291.

[0047] The structural formula of YB-0158 is as follows:

[0048]

[0049] The natural bioactive compound UCS15A competitively binds to the P4 / P5 proline domain of Sam68, inhibits its binding to the SH3 region of Src, reduces the phosphorylation of the tyrosine site of Sam68, and inhibits the binding of Sam68 to proteins containing the SH2 domain. YB-0158 was screened by simulating the interaction mode between UCS15A and Sam68.

[0050] The embodiments of the present application also provide a drug for preventing and / or treating pathological myocardial hypertrophy, which includes an agent for knocking out the Sam68 gene or inhibiting the expression of the Sam68 gene.

[0051] In some embodiments, the agent for knocking out the Sam68 gene or inhibiting the expression of the Sam68 gene is selected from a gene editing system or a reverse analog peptide.

[0052] In some embodiments, the reverse peptidomimetic comprises at least one of YB-0158, ICG-001, CWP232228, and CWP232291.

[0053] The present application also provides a method for screening drugs for preventing and / or treating pathological myocardial hypertrophy, comprising the step of screening Sam68 inhibitors, wherein the Sam68 inhibitors can inhibit or reduce the expression of the Sam68 gene, or inhibit or block the activity of the Sam68 protein.

[0054] In some embodiments, the step of screening Sam68 inhibitors includes: detecting whether the test agent can inhibit Sam68 gene expression or Sam68 protein activity; and selecting the test agent that can inhibit Sam68 gene expression or Sam68 protein activity as a candidate drug.

[0055] The following is further described by means of specific examples.

[0056] First, the method for constructing and breeding the experimental animal model in the examples will be described.

[0057] Experimental animals and feeding Experimental animals: 8-10 weeks old, weighing 22-25g, male C57BL / 6 wild-type mice (WT), cardiomyocyte-specific Sam68 gene knockout mice (Sam68 CM- / - , genotype Sam68 flox / flox : Myh6-Cre ESR, tamoxifen-induced Cre activity), and control transgenic mice (Sam68 CM+ / + , genotype Myh6-CreESR) were used as experimental subjects.

[0058] Acquisition of cardiomyocyte-specific Sam68 gene knockout mice: Sam68 CM- / - Mice were induced by flox / flox Mice (purchased from JAX Lab, mouse number #: 037100, with loxp sites outside exons 5-8 of Sam68) and Myh6-CreESR (purchased from Saiye Bio, mouse number #: C001009) were bred, and the offspring were hybridized again to obtain the genotype Sam68 flox / flox :Myh6-Cre ESR mice were used as experimental mice. In order to induce specific knockout of Sam68 in cardiomyocytes, when the mice were 6 weeks old, 20 mg / kg / D of tamoxifen was intraperitoneally injected for 5 consecutive days to obtain Sam68 CM- / - mice; and Sam68 CM+ / + The same drugs and the same dosage were given.

[0059] Acquisition of mice overexpressing Sam68 gene in cardiomyocytes: The full length of Sam68 was amplified from mouse heart cDNA, and then the constructed plasmid was packaged into the AAV9 adenovirus vector carrying the cardiac troponin T (cTnT) promoter to specifically induce overexpression of GFP (AAV9-cTnT-GFP) and Sam68-GFP (AAV9-cTnT-Sam68-GFP) in cardiomyocytes. C57BL / 6J male mice aged 8-10 weeks were injected with AAV9-cTnT-GFP or AAV9-cTnT-Sam68-GFP virus solution (5×10 11 / mice), and the overexpression efficiency was determined by detecting GFP expression 3 weeks later.

[0060] Mouse breeding environment: All experimental mice were raised in the SPF-level experimental animal center of Southern University of Science and Technology. Breeding conditions: room temperature between 22-24°C, humidity between 40-70%, alternating light and dark lighting time of 12h, free access to water and food.

[0061] Example 1

[0062] This example analyzes the difference in expression of Sam68 between normal people and patients with cardiomyopathy, and the specific steps are as follows:

[0063] Two cardiac RNA sequencing result data sets were selected from the Gene Expression Omnibus (GEO), GSE116250 (containing cardiac RNA sequencing results of 14 normal subjects, 37 patients with dilated cardiomyopathy and 13 patients with ischemic heart disease) and GSE160997 (containing cardiac RNA sequencing results of 5 healthy subjects and 37 patients with hypertrophic cardiomyopathy). The relative expression levels of the Sam68 gene in the two data sets were analyzed by Deseq2. The results are as follows: Figure 1 As shown, NF (non-failure) is a normal person, DCM (Dilated cardiomyopathy) is a patient with dilated cardiomyopathy, ICM (Ischemic cardiomyopathy) is a patient with ischemic heart disease, HCM (hypertrophic cardiomyopathy) is a patient with hypertrophic cardiomyopathy, KHBRDS1 is another name for Sam68, *: p < 0.05 vs healthy individual heart. Figure 1 Middle A and Figure 1 As can be seen from Figure B, the transcription level of Sam68 is significantly upregulated in dilated cardiomyopathy and hypertrophic heart disease; at the same time, compared with dilated cardiomyopathy, the transcription level of Sam68 is further upregulated in ischemic cardiomyopathy.

[0064] Example 2

[0065] This example analyzes the expression of Sam68 in a mouse model of myocardial hypertrophy induced by angiotensin and aortic arch constriction surgery, and the specific steps are as follows:

[0066] (1) Angiotensin II (AngII) was used to induce a myocardial hypertrophy mouse model.

[0067] Several male C57BL / 6 wild-type mice (8 weeks old) were selected, weighed, and then anesthetized with isoflurane (adjusted to 3-4% induction concentration) provided by an anesthesia machine. Depilatory cream was evenly applied to the hair on the upper back of the mouse, and the residual depilatory cream and hair were wiped off with wet gauze.

[0068] A 0.5-1 cm incision was made on the back of the mouse, and the skin layer was gently separated with a needle holder. The micropump pre-filled with angiotensin was slowly and completely placed under the skin layer from the incision, and then the skin incision was sutured. A control group was set up for subcutaneous surgery on the back with a micropump pre-filled with saline. The mice that woke up after the operation were placed in a cage with autoclaved bedding, feed and water, and continued to be raised and observed in the breeding room.

[0069] Mice were killed by overdose of CO2. The chest was immediately opened, the right atrial appendage was cut open, and a syringe filled with 5-10 mL of cold PBS buffer was inserted into the left ventricle for cardiac perfusion. The heart was then cut out with ophthalmic scissors, quickly placed on sterile gauze, and the liquid in the heart cavity and on the surface was absorbed with sterile absorbent paper, weighed and recorded, and a portion of the heart was placed in the corresponding formalin fixative for subsequent tissue staining; the other portion of the heart was placed in the corresponding cryotube, quickly placed in a liquid nitrogen tank, and stored in a -80°C refrigerator for molecular biology testing.

[0070] (2) A mouse model of myocardial hypertrophy was established by aortic arch constriction (TAC).

[0071] Select several male C57BL / 6 wild-type mice (8 weeks old), weigh the mice first, and then use an anesthesia machine to provide isoflurane (induction concentration adjusted to 3-4%) to perform gas anesthesia on the mice. Use depilatory cream to remove the hair on the left chest, left chest and left forelimb armpit of the mouse, and wipe the surgical area with wet gauze to remove mouse hair. Use a rubber band to fix the mouse's upper incisors on a foam board, and quickly insert the endotracheal tube into the mouse's trachea, then lie on the surgical foam board, and then connect the endotracheal tube to the ventilator to fix the mouse.

[0072] The mouse limbs were fixed with medical tape, and then the mouse chest skin was disinfected with iodine. Use ophthalmic scissors to cut the upper left chest skin for about 1 cm, separate the muscles and soft tissues in turn, open the chest cavity at the level of the 2nd-3rd ribs, free the descending branch of the aortic arch, pass the 7-0 non-absorbable surgical suture through the blood vessel, and place a 27G (23-25g) flat-head syringe needle above the blood vessel, ligate the blood vessel and the needle together, and then withdraw the needle to achieve the desired vascular constriction. After the ligation is completed, suture in sequence, close the chest cavity, insert a syringe into the chest cavity from the suture and extract 1mL of gas to restore the negative pressure in the chest cavity, and quickly suture the skin incision after the syringe is pulled out. When the mouse has a strong reaction to the toe clip, remove the endotracheal tube, and place it on a heating pad until the mouse wakes up. At the same time, a control group (sham group, Sham) was set up. After freeing the descending branch of the aorta, only threading was performed without ligation. The rest of the steps were the same as the TAC surgery myocardial hypertrophy model group. The mice that recovered after surgery were placed in a cage filled with autoclaved bedding, and the feed and jelly were placed on a flat plate at the bottom of the cage. The mice continued to be fed and observed in the feeding room and switched to normal feeding on the third day after surgery.

[0073] Mice were killed by overdose of CO2. The chest was immediately opened, the right atrial appendage was cut open, and a syringe filled with 5-10 mL of cold PBS buffer was inserted into the left ventricle for cardiac perfusion. The heart was then cut out with ophthalmic scissors, quickly placed on sterile gauze, and the liquid in the heart cavity and on the surface was absorbed with sterile absorbent paper, weighed and recorded, and a portion of the heart was placed in the corresponding formalin fixative for subsequent tissue staining; the other portion of the heart was placed in the corresponding cryotube, quickly placed in a liquid nitrogen tank, and stored in a -80°C refrigerator for molecular biology testing.

[0074] (3) Detect the expression level of Sam68 in the heart of the myocardial hypertrophy mouse model constructed by (1) and (2).

[0075] Protein was extracted from the collected hearts and subjected to SDS-PAGE immunoblotting (Western blot). The expression of Sam68 was determined using Abcam #ab76471, an antibody that specifically recognizes Sam68 protein, and an antibody against β-actin, an internal reference. The test results are shown in Figure 2. Figure 2 As shown in A, Saline represents the control group in (1) in which a micropump pre-filled with saline solution was surgically implanted subcutaneously in the back, AngII represents the myocardial hypertrophy mouse model group induced by angiotensin (AngII) in (1), Sham represents the control group (sham operation group) in (2), and TAC represents the myocardial hypertrophy mouse model group constructed by aortic arch constriction surgery in (2). Figure 2As shown in A, the expression level of Sam68 gene in the heart of the mouse model of cardiac hypertrophy induced by AngII and the mouse model of cardiac hypertrophy constructed by TAC was significantly upregulated. At the same time, the expression of Sam68 gene in cardiomyocytes was analyzed by immunofluorescence co-staining (mean fluorescence intensity). Figure 2 As shown in B (*: p < 0.05 vs sham-operated mouse heart), Figure 2 As can be seen in B, the Sam68 gene is mainly expressed in the nucleus of cardiomyocytes, and the expression of the Sam68 gene is significantly upregulated in the cardiomyocytes of the myocardial hypertrophy mouse model constructed by TAC.

[0076] Example 3

[0077] This example analyzes the effect of specific knockout of Sam68 gene in cardiomyocytes on the symptoms of myocardial hypertrophy in an AngII-induced myocardial hypertrophy mouse model. The specific steps are:

[0078] (1) Using the same method as in Example 2, myocardial cell-specific Sam68 gene knockout mice (Sam68 CM- / - ) and control transgenic mice (Sam68 CM+ / + ) A myocardial hypertrophy mouse model was constructed, including an experimental group induced by AngII and a control group using normal saline. The constructed myocardial hypertrophy mouse model was raised under the same conditions for four weeks and then samples were collected.

[0079] Prepare a centrifuge tube with 20mL formalin in advance and mark it. Open the analytical balance and adjust it to zero for later use. Use excess CO2 to kill the mouse and weigh it, then quickly use PBS buffer to perfuse the heart, and immediately inject 100uL of 10% KCl solution into the left ventricle to keep the heart in diastole, then use absorbent paper to absorb the heart cavity and surface liquid and weigh and record the weight. Then transfer the heart to the corresponding formalin centrifuge tube and fix it for 24 hours for pathological detection. Cut the skin at the tibia of the mouse hind limb, measure and record the tibia length, and calculate the heart / body weight ratio (HW / BW) and the heart / tibia length ratio (HW / TL). After trimming the heart, perform tissue gradient dehydration, transparency and wax immersion, then paraffin embedding of the tissue, and then tissue sectioning and spreading, wait for the sections to dry and bake for later use. During the process, retain part of the heart tissue and freeze it directly, and then use it to detect the expression of cardiac function molecular indicators ANP, BNP, and Sam68.

[0080] (2) The heart / body weight ratio and heart / tibia length ratio of mice in different groups are as follows: Figure 3 As shown. Figure 3 As can be seen, using Sam68 CM- / -The heart / body weight ratio (HW / BW) and heart / tibia length ratio (HW / TL) of the myocardial hypertrophy mouse model induced by AngII were lower than those of the model induced by Sam68. CM+ / + The mouse model of myocardial hypertrophy was established by inducing AngII in mice, which further demonstrated that specific knockout of the Sam68 gene in cardiomyocytes can reduce the degree of myocardial hypertrophy in mice induced by AngII.

[0081] (3) Staining analysis.

[0082] Hematoxylin-eosin (HE) staining: Dewax the baked sections with xylene, rehydrate with alcohol in decreasing gradients, then stain with hematoxylin for 5 minutes, wash with water for 1 minute, separate with 1% hydrochloric acid alcohol for 1-3 seconds, wash with water for 1 minute, stain with eosin for 3-5 minutes, wash to remove floating color, dehydrate with alcohol in increasing gradients, seal with neutral resin immediately after soaking in xylene, blow dry in a fume hood and take photos under a microscope.

[0083] Wheat germ agglutinin (WGA) staining: The baked paraffin specimen sections were placed in xylene for dewaxing, and rehydrated with gradient alcohol, washed twice with PBS buffer, and then the pancreatic enzyme working solution was added. After incubation at 37°C for 20 minutes, the liquid around the tissue was wiped dry with filter paper, and a circle was drawn with a histochemical pen and WGA-AlexaFlour488 working solution (10μg / mL) was added. After incubation at 37°C in the dark for 2 hours, the staining solution was discarded, and the sections were rinsed with PBS buffer for 3 times. The sections were sealed with an anti-fluorescence quencher containing DAPI, and photographed using a fluorescence microscope. Finally, the cell area was calculated using ImageJ.

[0084] Sirius red staining: The baked paraffin specimen sections were placed in xylene for dewaxing, and rehydrated with gradient alcohol, and then placed in saturated Sirius red staining solution for staining for 8 minutes. After staining, they were rinsed with anhydrous alcohol for several minutes, dried and transparentized in xylene for 5 minutes, and then sealed with neutral resin and photographed under a microscope. Finally, ImageJ was used to calculate the collagen ratio, collagen ratio = collagen area / (total area-blank area).

[0085] The staining analysis results are as follows Figure 4 (Hematoxylin-eosin (HE) staining) and Figure 5 (Wheat germ agglutinin staining and Sirius red staining). Figure 4 It can be seen that using Sam68 CM- / - The myocardial cell size of the mouse model of cardiac hypertrophy induced by AngII was significantly smaller than that of the mouse model induced by Sam68. CM+ / + The myocardial hypertrophy mouse model was established by Ang II induction in mice. Figure 5 It can be seen that using Sam68 CM- / - The size of cardiomyocytes and the level of cardiac fibrosis in the mouse model of cardiac hypertrophy induced by AngII were significantly smaller than those induced by Sam68. CM+ / + The mouse model of myocardial hypertrophy was established by AngII-induced myocardial hypertrophy in mice, which further demonstrated that specific knockout of Sam68 could reduce the size of myocardial cells and the area of ​​collagen deposition activated by AngII in the mouse model of myocardial hypertrophy induced by AngII.

[0086] (4) RNA and total protein were extracted from the frozen cardiac tissues in (1), and the expression levels of the cardiac function molecular indicators ANP, BNP, and Sam68 genes were detected by real-time fluorescence PCR and SDS-PAGE protein immunoblotting, respectively. The results of real-time fluorescence PCR and immunoblotting are shown in Figure 6 As shown in A and B, *: p < 0.05. Figure 6 Middle A and Figure 6 As can be seen in B, using Sam68 CM- / - The mRNA transcription levels of cardiac function molecular markers ANP and BNP and the expression level of Sam68 gene in the myocardial hypertrophy mouse model induced by AngII were significantly lower than those in the model induced by Sam68. CM+ / + The myocardial hypertrophy mouse model was constructed by AngII-induced mice, which further demonstrated that myocardial cell-specific knockout of the Sam68 gene could significantly reduce the mRNA transcription levels of the molecular markers ANP and BNP and the expression level of the Sam68 gene in the cardiac cells of the AngII-induced myocardial hypertrophy mouse model.

[0087] Based on the results of (1)-(3) in Example 3, it can be concluded that Sam68 gene knockout significantly inhibited myocardial hypertrophy and fibrosis.

[0088] Example 4

[0089] This example analyzes the effect of specific knockout of the Sam68 gene in cardiomyocytes on the establishment of a myocardial hypertrophy mouse model by aortic arch coarctation (TAC), and the specific steps are as follows:

[0090] (1) Using the same method as in Example 2, myocardial cell-specific Sam68 gene knockout mice (Sam68 CM- / - ) and control transgenic mice (Sam68 CM+ / + ), a mouse model of myocardial hypertrophy was established by aortic arch constriction (TAC), including an experimental group and a sham operation group. The established mouse model of myocardial hypertrophy (mice) was raised under the same conditions for four weeks and then the samples were collected.

[0091] (2) The cardiac function of the mice in different groups in (1) was tested by ultrasound at 2 weeks and 8 weeks of feeding. First, connect the oxygen cylinder and the air inlet interface on the anesthesia machine, then unscrew the sealing cap of the drug adding port on the anesthesia machine, quickly add isoflurane to the safety mark and tighten the sealing cap. Unscrew the main valve on the oxygen cylinder and adjust the knob of the flow control valve. After the mice to be tested were quickly anesthetized with isoflurane, the left chest area was shaved and a depilatory cream was applied. Then, the remaining depilatory cream and hair were wiped clean with wet gauze. The treated mouse head was inserted into the anesthetic catheter sleeve, and the mouse was maintained in a stable anesthesia state with 1-2% isoflurane. The mice were placed in a supine position, and an ultrasonic coupling agent was evenly applied to the shaved area. A high-frequency ultrasound diagnostic instrument with a frequency of 15MHz was used to select the standard left ventricular papillary muscle short-axis section to measure the left ventricular end-diastolic posterior wall thickness (LVPW; d), left ventricular end-systolic posterior wall thickness (LVPW; s), ejection fraction (EF) and short-axis shortening rate (FS). Test results such as Figure 7 As shown, *: p < 0.05. Figure 7 The results of A and B can be seen to be similar to Sam68 CM+ / + Compared with the sham-operated group, Sam68 CM+ / + Mice 4 weeks after TAC showed increased diastolic and end-systolic ventricular cavity size, and decreased ejection fraction (EF) and fractional shortening (FS). CM- / - Compared with the sham-operated group, Sam68 CM- / - Four weeks after TAC surgery, the size of the heart chambers of mice decreased, and the cardiac function indicators EF and FS were significantly improved. This indicates that myocardial cell-specific knockout of the Sam68 gene can improve the cardiac function of the myocardial hypertrophy mouse model established by TAC surgery.

[0092] Example 5

[0093] This example analyzes the effect of overexpression of the Sam68 gene in cardiomyocytes on the myocardial hypertrophy mouse model established by aortic arch coarctation (TAC), and the specific steps are as follows:

[0094] (1) Using the same method as in Example 2, mice with Sam68 gene overexpression in myocardial cells (AAV9-cTnT-Sam68-GFP) and mice with Sam68 gene overexpression in the control group (AAV9-cTnT-GFP) were selected to construct a myocardial hypertrophy mouse model by aortic arch coarctation (TAC), including an experimental group and a sham operation group. The constructed myocardial hypertrophy mouse model (mice) was raised under the same conditions for four weeks and then the samples were collected.

[0095] (2) The method in Example 3 was used to measure the expression levels of the cardiac function molecular indicators ANP, BNP and Sam68 gene of different mice in (1). The results are as follows: Figure 8As shown. AAV9-GFP represents the control group mice that only overexpressed GFP (green fluorescent protein), and AAV9-Sam68 represents mice that expressed the Sam68 gene, *: p < 0.05. Figure 8 As can be seen from Figures A and B, TAC surgery can significantly upregulate the transcription level and protein expression level of the molecular markers ANP and BNP in the heart of the mouse model of cardiac hypertrophy. Overexpression of the Sam68 gene in cardiomyocytes can further increase the mRNA transcription level and protein expression level of ANP and BNP in the heart of the TAC-induced cardiac hypertrophy mouse model.

[0096] (3) The method in Example 3 was used to measure the heart / body weight ratio (HW / BW) and heart / tibia length ratio (HW / TL) of different mice in (1). The results are as follows: Fig. 9 As shown, AAV9-GFP represents the control group mice that only overexpressed GFP (green fluorescent protein), and AAV9-Sam68 represents mice that express the Sam68 gene, *: p < 0.05. Fig. 9 As can be seen from A and B, 4 weeks after TAC surgery, the heart / body weight ratio (HW / BW) and heart / tibia length ratio (HW / TL) of the myocardial hypertrophy mouse model in which myocardial cells overexpressed the Sam68 gene were higher than those of the control group, indicating that the degree of myocardial hypertrophy was increased.

[0097] (4) The method in Example 4 was used to test the cardiac function of different mice in (1). The results are as follows: Fig.10 As shown, AAV9-GFP represents the control group mice that only overexpressed GFP (green fluorescent protein), and AAV9-Sam68 represents mice that express the Sam68 gene, *: p < 0.05. Fig.10 As can be seen in Figures AD, 4 weeks after TAC surgery, the left ventricular internal diameter at end-systole (LVID; S) and left ventricular internal diameter at end-diastole (LVID; D) of the heart of the myocardial hypertrophy mouse model in which the cardiomyocytes overexpressed the Sam68 gene were significantly increased and higher than those of the control group. The cardiac function (EF and FS values) of the myocardial hypertrophy mouse model in which the cardiomyocytes overexpressed the Sam68 gene was significantly lower than that of the control group.

[0098] Combining the results of (2)-(4), it can be seen that overexpression of the Sam68 gene in cardiomyocytes promotes myocardial hypertrophy and fibrosis and reduces cardiac contractile function.

[0099] Example 6

[0100] Based on the results of Example 1 to Example 5, it can be seen that the Sam68 gene has a significant effect in the myocardial hypertrophy mouse model, and the regulation of Sam68 gene expression (knockout and overexpression) has a significant effect on the degree of myocardial hypertrophy, degree of fibrosis and cardiac function in the myocardial hypertrophy mouse model. Based on this, this example applies Sam68 inhibitor to the myocardial hypertrophy mouse model to analyze its effect. The specific steps are:

[0101] (1) Fig.11 As shown in A, the same method as in Example 2 (1) was used to induce cardiac hypertrophy in mice using AngII, and then the cardiac hypertrophy mouse model was treated with Sam68 inhibitor YB-0158. The experimental group was treated with intraperitoneal injection at a dose of 5 mg / kg / day, and the control group was treated with a solvent (Veh) without YB-0158 by intraperitoneal injection. After 5 consecutive days of treatment, the mice were cultured for 1 week, and finally killed by overdose of CO2.

[0102] (2) The method in Example 4 was used to detect the cardiac function of different mice in (1). The results were as follows: Fig.11 As shown in B, Saline+Veh represents the control group using the myocardial hypertrophy mouse model, AngII+Veh represents the control group of Sam68 inhibitor YB-0158, and AngII+YB-0158 represents the experimental group. Fig.11 As can be seen from B, the systolic and diastolic interventricular septal thickness (IVS; s and IVS; d) of the experimental group of mice were lower than those of the mice not treated with YB-0158, indicating that YB-0158 can reduce the myocardial hypertrophy indicators in the AngII-induced myocardial hypertrophy mouse model.

[0103] (3) The method in Example 3 was used to measure the heart / body weight ratio (HW / BW) and heart / tibia length ratio (HW / TL) of different mice in (1). The results are as follows: Fig.11 As shown in C, Saline+Veh represents the control group using the myocardial hypertrophy mouse model, AngII+Veh represents the control group of Sam68 inhibitor YB-0158, and AngII+YB-0158 represents the experimental group. Fig.11 As can be seen from Figure C, YB-0158 can reduce the heart / body weight ratio (HW / BW) and heart / tibia length ratio (HW / TL) in the AngII-induced cardiac hypertrophy mouse model.

[0104] (4) The method in Example 3 was used to measure the expression levels of the cardiac function molecular indicators ANP, BNP and Sam68 gene of different mice in (1). The results are as follows: Fig.11As shown in D. Saline+Veh is the control group using the myocardial hypertrophy mouse model, AngII+Veh is the control group of Sam68 inhibitor YB-0158, and AngII+YB-0158 is the experimental group. Fig.11 As can be seen in D, YB-0158 can reduce the mRNA transcription level and protein expression level of ANP and BNP in the heart of the AngII-induced cardiac hypertrophy mouse model.

[0105] Based on the results of (2)-(4), it can be concluded that YB-0158 has the effect of inhibiting myocardial hypertrophy.

[0106] It should be understood that the application of the present application is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to this application.

Claims

1. Use of an agent for knocking out the Sam68 gene or inhibiting the expression of the Sam68 gene in the preparation of a drug for preventing and / or treating pathological myocardial hypertrophy.

2. The use according to claim 1, characterized in that The reagent is a gene editing system.

3. The use according to claim 1, characterized in that The reagent is a reverse mimetic peptide.

4. The use according to claim 3, characterized in that The reverse analog peptide includes at least one of YB-0158, ICG-001, CWP232228, and CWP232291.

5. The use according to any one of claims 1 to 4, characterized in that The reagent knocks out the Sam68 gene, reduces or inhibits the transcription of the Sam68 gene, and / or reduces or inhibits the translation of the mRNA product of Sam68.

6. A drug for preventing and / or treating pathological myocardial hypertrophy, characterized in that: The drug includes an agent for knocking out the Sam68 gene or inhibiting the expression of the Sam68 gene.

7. The drug according to claim 6, characterized in that The reagent is a gene editing system; or The reagent is a reverse mimetic peptide.

8. A method for screening drugs for preventing and / or treating pathological myocardial hypertrophy, characterized in that: The method comprises screening for Sam68 inhibitors.

9. The method according to claim 8, characterized in that The Sam68 inhibitor can inhibit or reduce the expression of the Sam68 gene, or inhibit or block the activity of the Sam68 protein.

10. The method according to claim 8, characterized in that The step of screening Sam68 inhibitors comprises: Detect whether the test agent can inhibit Sam68 gene expression or Sam68 protein activity; The test agents that can inhibit Sam68 gene expression or Sam68 protein activity are selected as candidate drugs.

Images