Application of membrane-bound O-acyltransferase domain 7 in preparation of medicine for treating cardiac hypertrophy

Abstract

The invention discloses application of a membrane-bound O-acyltransferase structural domain 7 (Mboat7) in preparation of a medicine for treating myocardial hypertrophy, and belongs to the technical field of biology. Research results show that in a mouse cardiac hypertrophy model, compared with a control group mouse, heart weight / body weight, lung weight / body weight, cardiac muscle cell cross sectional area and fibrosis of a cardiac muscle cell specific Mboat7 gene overexpressed mouse are all remarkably reduced, and the cardiac systolic function is remarkably improved; the overexpressed Mboat7 gene can relieve isolated cell myocardial hypertrophy induced by angiotensin II. Particularly, overexpression of the Mboat7 gene can improve damage of mitochondria in myocardial tissue of a cardiac hypertrophy mouse. The results show that the Mboat7 gene overexpression relieves the disease progress of the pathological cardiac hypertrophy, and the Mboat7 or the expression promoter thereof can be used for preparing the medicine for preventing, relieving and / or treating the pathological cardiac hypertrophy.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biotechnology, and relates to the application of a gene, in particular to the application of a membrane bound O-acyltransferase domain containing 7 (Mboat7) or a gene thereof in the preparation of a drug for treating myocardial hypertrophy. BACKGROUND

[0002] Heart failure (HF) is the end-stage of various cardiovascular diseases caused by multiple etiological factors, and is also the main cause of death. Clinically, it is mainly manifested as reduced left ventricular pump function, insufficient peripheral tissue perfusion and reduced exercise tolerance. In particular, increased cardiac pressure afterload caused by aortic constriction and hypertension is an important inducement for myocardial hypertrophy, myocardial fibrosis and other structural remodeling changes of the heart, and eventually evolves into chronic heart failure. For many years, scholars in various countries have made unremitting efforts in the field of treating myocardial remodeling and heart failure, and have made some progress, but the overall efficacy is still unsatisfactory, and there has been no fundamental change. The treatment means is still limited, and the prognosis of patients is still poor. Heart 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 myocardial hypertrophy. At present, the effect of drug treatment strategies for myocardial hypertrophy is not good. Therefore, it is of great theoretical and practical significance to study the mechanism of the occurrence and development of myocardial hypertrophy and heart failure, to find specific factors and signal transduction pathways involved in myocardial hypertrophy, and to explore drugs targeting these new therapeutic targets.

[0003] Although the mechanisms of myocardial remodeling and heart failure caused by different primary diseases are different, abnormal myocardial metabolism and energy utilization disorder are their common mechanisms. The main function of mitochondria is to regulate energy metabolism, oxygen free radicals, maintain calcium homeostasis and mediate cell hypertrophy. Damage to the ultrastructure of mitochondria and impairment of oxidative phosphorylation both destroy the energy supply mechanism of mitochondria, affecting myocardial energy metabolism and function. Membrane bound O-acyltransferase domain 7 (Mboat7), also known as lysophosphatidyl inositol acyltransferase 1, is an enzyme mainly involved in remodeling of arachidonic acid and phosphatidylinositol (PI) acyl chains. MBOAT7 is anchored to the inner membrane, i.e. endoplasmic reticulum, mitochondria and mitochondria-associated membrane, through 11 transmembrane domains. MBOAT7 has been identified as an important genetic determinant of several human metabolic diseases, including fatty liver disease associated with cognitive development and metabolic dysfunction, but the regulatory role of Mboat7 in myocardial hypertrophy is not clear. SUMMARY

[0004] The application is based on the finding that Mboat7 gene plays a role in heart diseases, overexpression of Mboat7 can alleviate the disease process of pathological cardiac hypertrophy, and specifically, overexpression of Mboat7 can significantly improve myocardial hypertrophy and fibrosis, improve the systolic function of the heart, and reduce the mitochondrial damage of myocardial tissue. Based on this, the application provides a new application of Mboat7 as a drug target in the preparation of a drug for protecting the systolic function of the heart, resisting cardiac fibrosis, and / or preventing, alleviating, and / or treating myocardial hypertrophy and / or pressure overload heart failure, and further provides an application of Mboat7 or an expression promoter thereof in the preparation of a drug for protecting the systolic function of the heart, resisting cardiac fibrosis, and / or preventing, alleviating, and / or treating myocardial hypertrophy and / or pressure overload heart failure, so as to find a new therapeutic target and therapeutic drug for the treatment of clinical pathological myocardial hypertrophy and pressure overload heart failure patients.

[0005] The application uses mice with myocardial cell-specific Mboat7 gene overexpression mediated by adeno-associated virus 9 (AAV9) (AAV9-Mboat7) and control group mice (AAV9-null) as experimental objects, and studies the function of Mboat7 gene by inducing a myocardial hypertrophy animal model through aortic arch constriction (TAC), and the results show that in the myocardial hypertrophy model of mice, the expression of Mboat7 is significantly up-regulated compared with the normal group; compared with wild-type mice in the same litter, the heart weight / body weight, lung weight / body weight, myocardial cell cross-sectional area, and fibrosis of the myocardial cell-specific Mboat7 gene overexpression mice are significantly reduced, and the systolic function of the heart is obviously improved. At the same time, overexpression of Mboat7 gene can alleviate the hypertrophy of isolated myocardial cells induced by angiotensin II. In addition, transmission electron microscope images show that overexpression of Mboat7 gene can reduce the mitochondrial damage in the left ventricular myocardial tissue. These results show that myocardial cell-specific overexpression of Mboat7 gene can alleviate the disease process of pathological cardiac hypertrophy.

[0006] Therefore, Mboat7 gene can be used as a drug target for screening drugs and / or biological reagents for preventing, alleviating, and / or treating myocardial hypertrophy, and achieving the purpose of preventing, alleviating, and / or treating pathological myocardial hypertrophy through genetic engineering technology. In addition, small molecule compound activators can be designed with Mboat7 as a target, and through screening, molecules that can specifically activate Mboat7 are found, thereby providing new therapeutic molecules for the treatment of myocardial hypertrophy.

[0007] In a first aspect, the present application provides an application of Mboat7 in screening drugs, wherein the drugs include one or more of the following: drugs for protecting cardiac contractile function, drugs for resisting cardiac fibrosis, drugs for preventing, alleviating and / or treating cardiac hypertrophy, and drugs for preventing, alleviating and / or treating pressure overload heart failure. The screening includes screening molecules capable of specifically activating Mboat7 and screening substances capable of promoting expression of Mboat7.

[0008] NCBI Gene ID of human Mboat7 is 79143, and the sequence number is NM_001146056.3.

[0009] In a second aspect, the present application provides an application of Mboat7 in preparing drugs, wherein the drugs include one or more of the following: drugs for protecting cardiac contractile function, drugs for resisting cardiac fibrosis, drugs for preventing, alleviating and / or treating cardiac hypertrophy, and drugs for preventing, alleviating and / or treating pressure overload heart failure. The application includes Mboat7 as an effective component of the drugs, or substances capable of promoting expression of Mboat7 as an effective component of the drugs.

[0010] In a third aspect, the present application provides an application of a biomaterial expressing Mboat7 in preparing drugs. The drugs include one or more of the following: drugs for protecting cardiac contractile function, drugs for resisting cardiac fibrosis, drugs for preventing, alleviating and / or treating cardiac hypertrophy, and drugs for preventing, alleviating and / or treating pressure overload heart failure.

[0011] The biomaterial expressing Mboat7 includes any one of the following 1) to 4): 1) a nucleic acid molecule encoding Mboat7 protein; 2) an expression cassette containing the nucleic acid molecule of 1); 3) a recombinant vector containing the nucleic acid molecule of 1), or an expression cassette of 2); the recombinant vector includes an adenovirus vector, an adeno-associated virus vector, a lentivirus vector, etc. 4) a transgenic cell line containing the nucleic acid molecule of 1), an expression cassette of 2), or a recombinant vector of 3).

[0012] In a fourth aspect, the present application provides an application of an expression promoter of Mboat7 in preparing drugs, wherein the drugs include one or more of the following: drugs for protecting cardiac contractile function, drugs for resisting cardiac fibrosis, drugs for preventing, alleviating and / or treating cardiac hypertrophy, and drugs for preventing, alleviating and / or treating pressure overload heart failure. The expression promoter of Mboat7 includes one or more of the following: proteins, polynucleotides, and small molecule compounds.

[0013] Further, the Mboat7 or the Mboat7 expression promoter allows entry into cells by one or more of the following methods: direct naked DNA injection, liposome-encapsulated DNA direct injection, gold-coated DNA gene gun bombardment, plasmid DNA-carrying reproduction-deficient bacteria, replication-deficient adenovirus-carrying target DNA, PEG-modified protein drug injection, liposome-encapsulated protein intravenous injection, protein microsphere preparation subcutaneous injection.

[0014] Further, the Mboat7 expression promoter is a recombinant plasmid containing the Mboat7 gene and capable of normally expressing the Mboat7 protein.

[0015] Further, the Mboat7 expression promoter is an adenovirus Ad Mboat7 carrying the Mboat7 sequence. Further, the adenovirus Ad

[0016] The myocardial hypertrophy or heart failure described above includes myocardial hypertrophy or heart failure caused by hypertension, aortic stenosis, and mitral valve insufficiency.

[0017] The present application has the following advantages and effects: (1) A Mboat7 myocardial cell-specific overexpression mouse model is constructed, which helps to further study the role of Mboat7 in myocardial remodeling and heart failure, and provides a new research direction for the treatment of related diseases.

[0018] (2) A new function of the Mboat7 gene is discovered, i.e., Mboat7 gene overexpression has the effect of improving myocardial hypertrophy.

[0019] (3) Based on the role of Mboat7 in improving myocardial hypertrophy and / or heart failure, it provides a target for preparing a drug for preventing, alleviating, and / or treating myocardial hypertrophy.

[0020] (4) A comprehensive study is conducted using various experimental techniques: through echocardiography, Western blotting, immunofluorescence, qRT-PCR, transmission electron microscopy, and other techniques, the role of Mboat7 in heart disease is comprehensively and systematically studied, which provides rich experimental data and methods for research in related fields.

[0021] (5) The expression promoter of Mboat7 can be used to prepare a drug for preventing, alleviating, and / or treating myocardial hypertrophy and / or heart failure. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1Specific overexpression of Mboat7 in mouse heart by tail vein injection of adeno-associated virus 9 (AAV9). A: schematic diagram of plasmid structure in AAV9 vector containing full-length Mboat7 transcript (NCBI Gene ID: 77582; NM_029934.4) and cTNT promoter; B-F: Western blot detection of Mboat7 protein levels in various organs of mice injected with Mboat7 overexpression and control empty AAV9, 4 weeks later, and quantitative analysis of Mboat7 protein levels (p values are shown in the figure).

[0023] Figure 2 Western blot detection of Mboat7 protein levels in the hearts of normal people and patients with clinical heart failure and quantitative analysis results, GADPH as internal reference (p values are shown in the figure). A: western blot figure, B: protein quantitative analysis results.

[0024] Figure 3 Mboat7 expression in the hearts of wild-type mice in the sham operation (Sham) group and the aortic arch constriction (TAC) group. A: WGA / Mboat7 / DAPI tissue immunofluorescence co-staining figure, the cross-sectional area of cardiomyocytes in the heart significantly increased 4 weeks after TAC, and the expression of Mboat7 was up-regulated. B: Western blot detection of Mboat7 protein levels in the hearts of C57BL / 6 mice 2 weeks, 4 weeks, and 8 weeks after sham operation and aortic arch constriction (TAC) modeling, with GAPDH as an internal reference. C: protein quantitative results in Figure B (p values are shown in the figure). The expression of Mboat7 in the heart with myocardial hypertrophy was up-regulated in a time-dependent manner at 2 weeks and 4 weeks.

[0025] Figure 4 Heart gross figure of AAV9-Mboat7 and AAV9-mull mice after 4 weeks of sham operation or TAC modeling, and statistical figure of heart weight / body weight (HW / BW), heart weight / tibia length (HW / TL), and lung weight / body weight (LW / BW) (p values are shown in the figure). A: heart gross figure; B-D are the statistical figures of heart weight / body weight (HW / BW), heart weight / tibia length (HW / TL), and lung weight / body weight (LW / BW), respectively.

[0026] Figure 5 WGA staining figure of heart tissue and statistical quantitative results of cross-sectional area of cardiomyocytes of AAV9-Mboat7 and AAV9-mull mice after 4 weeks of sham operation or TAC modeling (p values are shown in the figure). A: WGA staining figure; B: statistical quantitative results of cross-sectional area of cardiomyocytes.

[0027] Figure 6Figure 7 is a Masson staining chart and statistical quantification of left ventricular collagen deposition of heart tissue paraffin sections of AAV9-Mboat7 and AAV9-mull mice after sham operation or TAC modeling for 4 weeks (p values are shown in the figure). A: Masson staining chart; B: statistical quantification of left ventricular collagen deposition results.

[0028] Figure 7 Figure 8 is a statistical chart of M-mode echocardiogram and cardiac function quantitative analysis results, including ejection fraction (EF) and fractional shortening (FS), of AAV9-Mboat7 and AAV9-mull mice after sham operation or TAC modeling for 4 weeks (p values are shown in the figure). A: M-mode echocardiogram; B: ejection fraction (EF) statistical chart; C: fractional shortening (FS) statistical chart.

[0029] Figure 8 Figure 9 is a mitochondrial morphology analysis of AAV9-Mboat7 and AAV9-mull mice after sham operation or TAC modeling for 4 weeks. A: Transmission electron microscope images of left ventricular tissue of mice in each group, with the magnified part mainly showing mitochondrial morphology; B: statistical quantification chart of the proportion of complete mitochondrial cristae under electron microscopy (p values are shown in the figure).

[0030] Figure 9 Figure 10 is the expression of Mboat7 in myocardial cells after control group (PBS) or Ang II stimulation. A: Cell immunofluorescence co-staining chart of phalloidin / Mboat7 / DAPI, the volume of primary rat myocardial cells after Ang II stimulation increased, and the expression of Mboat7 was up-regulated. B: Western blotting detected the protein level of Mboat7 in the control group and after 24 hours of Ang II stimulation in vitro myocardial cells, with GAPDH as the internal reference. C: Protein quantification results in Figure B (p values are shown in the figure).

[0031] Figure 10 Figure 11 is the overexpression of Mboat7 in primary rat myocardial cells by transfection of adenovirus (Ad). A: Schematic diagram of plasmid structure in Mboat7 overexpression adenovirus vector, which contains the full-length Mboat7 transcript (NCBI Gene ID: 308309; NM_001134978.2); B-C: Western blotting detected the protein level of Mboat7 and its quantitative analysis after Mboat7 overexpression and control empty adenovirus transfection of neonatal rat myocardial cells (p values are shown in the figure).

[0032] Figure 11Effect of Mboat7 overexpression (Ad-Mboat7) on Ang II-stimulated hypertrophy of primary rat cardiomyocytes. A: Primary rat cardiomyocytes cultured in vitro were infected with adenovirus Ad-null and Ad-Mboat7, and subjected to Ang II stimulation for 48 hours before being subjected to phalloidin immunofluorescence staining. B: Quantitative analysis of cardiomyocyte area by Image J software. The column chart shows the results of distribution of cardiomyocyte area in each group (p values are shown in the chart). DETAILED DESCRIPTION

[0033] The application will be further described in detail below with reference to the examples and drawings, but the embodiments of the application are not limited thereto.

[0034] Experimental animals and feeding of experimental animals: 8-10-week-old male mice weighing 25.5±2.0 g were selected as experimental objects, and AAV9-mediated Mboat7 gene overexpression mice and control AAV9-null mice were used as experimental objects. Feeding environment: all experimental mice were fed in the Specific Pathogen Free (SPF) level experimental animal center of the Institute of Cardiovascular Disease, Wuhan University. Feeding conditions: the room temperature was between 22-24℃, the humidity was between 50-70%, the light and dark alternating lighting time was 12 hours, and the mice were allowed to drink water and eat freely.

[0035] Example 1: Construction of Mboat7 gene overexpression (AAV9-Mboat7) mouse specific to cardiomyocytes Wz Biosciences Inc (Shandong, China) was commissioned to construct an adeno-associated virus 9 (AAV9-Mboat7) carrying a mouse Mboat7 gene under the control of a cTnT promoter. The construction process was as follows: mouse Mboat7 gene (NCBI Gene ID: 77582; NM_029934.4) was synthesized into pCDNA3.1-P2A-GFP vector to obtain pCDNA3.1-Mboat7-P2A-GFP, GCCACC was added before the ATG of the mouse Mboat7 gene, and 3×flag tag was added at the C-terminus, and SfaAI / MluI enzyme digestion site was selected. pCDNA3.1-Mboat7-P2A-GFP and pAV-cTNT-P2A-GFP were digested by SfaAI / MluI enzyme, ligated, transformed, and verified by sequencing, and the mouse Mboat7 gene was subcloned into the pAV-cTNT-P2A-GFP vector to obtain the adeno-associated virus plasmid pAV-cTNT-Mboat7-P2A-GFP (pAV-cTNT-Mboat7-P2A-GFP). Figure 1A); pAV-cTNT-Mboat7-P2A-GFP was transfected into HEK293T cells by an adeno-associated virus / AAV helper-free system (AAV Helper-Free System), and the virus was collected after 72 h of culture to obtain AAV9-Mboat7.

[0036] Eight to ten-week-old C57BL / 6J mice (purchased from Beijing Huafukang Biotechnology Co., Ltd.) were used, and AAV9-Mboat7 (titer: 4.79 x 10 13 vg / mL) and control empty AAV9-null (titer: 6.99 x 10 13 vg / mL) were diluted with normal saline according to the product instructions provided by the company. AAV9-Mboat7 was injected through the tail vein to achieve specific overexpression of Mboat7 in myocardial cells, and AAV9-null was injected as a control. Four weeks after virus injection, Western blot was used to verify the specific overexpression of Mboat7 in the mouse heart (B-F), and then the mice were subjected to TAC surgery or sham operation (see Example 3 for details of TAC surgery or sham operation). Figure 1 B-F), and then the mice were subjected to TAC surgery or sham operation (see Example 3 for details of TAC surgery or sham operation).

[0037] Example 2: Expression of Mboat7 in normal human and clinical heart failure patient hearts Normal human hearts (individuals donated for non-cardiogenic death) and clinical heart failure patient hearts (recipients replaced or heart biopsy tissues from patients undergoing heart transplantation surgery) were selected, and Western blot was performed on the extracted proteins of the hearts to determine the expression of Mboat7 (proteintech, 26646-1-AP), with GAPDH (Servicebio, GB15004) as an internal reference. The detection results are shown in Figure 2 The expression of Mboat7 in the hearts of clinical heart failure patients was significantly up-regulated.

[0038] Example 3: Expression of Mboat7 in wild-type mouse sham operation (Sham) group and aortic arch constriction (TAC) group hearts The mouse myocardial hypertrophy model was established by aortic arch constriction surgery (TAC), and the model operation procedure was as follows: 1. Preoperative preparation (1) Anesthesia: First, weigh the mouse, calculate the amount of anesthetic (3% sodium pentobarbital) needed according to 90 mg / kg body weight, inject it intraperitoneally, and record the injection time point. The success criteria for anesthesia are no obvious reaction when the tail and toes are clamped and the mouse is in good condition (usually no obvious reaction after injection for about 10 min, and the mouse reacts to toe clamping about 50 min after anesthesia, and the optimal surgery time is about 30 min after anesthesia).

[0039] (2) Operative area preparation: The skin of the left chest, left side chest and left forelimb under the axilla of the mouse was depilated. After shaving, the surgical area was wiped with a wet gauze to remove the mouse hair without affecting the surgical field.

[0040] (3) Tracheal intubation: The mouse's upper incisors were fixed on the inclined surface of the V-shaped plate with an elastic band, and the tracheal tube was quickly inserted into the trachea through the glottis. Then the mouse was placed on a heating pad in a right lateral position (the heating pad was preheated). Then the tracheal tube was connected with the respirator, and the mouse was fixed. If the mouse's chest fluctuation was consistent with the respirator frequency, it indicated that the tracheal intubation was successful.

[0041] 2. Aortic constriction (TAC) The mouse was taken in a right lateral position, and the left forelimb was placed above the right forelimb, and the two forelimbs were fixed with medical tape. A cotton swab was placed under the right chest to elevate the chest. The skin of the surgical area was disinfected with iodine and alcohol with a volume fraction of 75%. The left chest skin was pinched with an ophthalmic forceps, and the right hand held an ophthalmic scissors to cut the skin about 1 cm. The muscles and soft tissues were separated in turn, and the chest cavity was opened at the level of the 2-3 ribs. The left lung was slightly probed with a cotton swab, and the descending branch of the aortic arch was isolated. A 7-0 surgical suture was passed through the blood vessel, and a 26G (25.0-27.5g mouse) or 27G (23.5-25.0g mouse) syringe needle was placed parallel above the blood vessel. The blood vessel and needle were ligated together, and then the needle was withdrawn to achieve the corresponding degree of vascular constriction. After ligation, it was sutured in turn, the chest cavity was closed, and a syringe was inserted into the chest cavity from the suture to extract 1cc gas to restore the negative pressure in the chest cavity. After the syringe was pulled out, the skin incision was quickly sutured. Sham group: after the descending aorta was isolated, only the thread was passed without ligation, and the rest of the steps were the same as the myocardial hypertrophy model group.

[0042] 3. Postoperative care After TAC, when the mouse appeared spontaneous breathing and the toe appeared strong reaction, the tracheal tube was pulled out, and the mouse was placed in a feeding cage containing high-pressure sterilized bedding, feed and drinking water, and continued to be fed and observed in the feeding room.

[0043] 4. The proteins of the mouse heart after sham operation and TAC for 2 weeks, 4 weeks and 8 weeks were extracted respectively, and Western blot was performed to determine the expression of Mboat7, and GAPDH was used as an internal reference. WGA / Mboat7 / DAPI immunofluorescence co-staining of heart tissue sections was performed 4 weeks after TAC (paraffin section and fluorescence staining process are described in Example 4). The detection results are shown in Table 1. Figure 3 As shown in Table 1, the expression of Mboat7 in the heart with myocardial hypertrophy was time-dependent up-regulated at 2 weeks and 4 weeks.

[0044] Example 4: AAV9-Mboat7 mouse myocardial hypertrophy model pathology detection 1. Tissue collection (1) Preparation: Prepare the urine cup with 20 mL of 10% formaldehyde solution in advance, and label it (mouse number, group, type of surgery, and date of tissue collection). Place a culture dish filled with 10% KCl solution at the collection site. Turn on the analytical balance and zero it for use. Weigh the mouse again after it is killed.

[0045] (2) Tissue collection: Hold the blood vessel pedicle under the auricle with an ophthalmic curved forceps, cut off the heart, and quickly place it in a 10% KCl solution. After the heart stops beating in diastole, place it on sterile gauze, gently squeeze the liquid in the heart cavity, and then weigh and record the surface liquid. Place the heart in the corresponding urine cup and fix it for 48 hours for pathological examination.

[0046] (3) Related measurements and calculations: Remove the mouse heart and lung, trim the filter paper, weigh and record. Cut the skin at the tibia of the mouse hind limb, measure and record the tibia length. Calculate the ratio of heart weight to body weight (HW / BW), lung weight to body weight (LW / BW), and heart weight to tibia length (HW / TL).

[0047] 2. Pathological examination 2.1 Preparation of paraffin specimen sections The main operation procedures include: trimming the heart → processing the embedding frame → water flushing → dehydration → transparency → wax immersion → embedding → sectioning → spreading → drying or baking for standby.

[0048] 2.2 Wheat germ agglutinin (WGA) staining Main steps: Place the paraffin specimen section baked at 60°C for 30 min in xylene for 5 min x 3 times → 100% ethanol for 5 min x 2 times → 95% ethanol for 5 min → 70% ethanol for 5 min → distilled water rinse for 5 min x 2 times → PBS rinse for 5 min → discard PBS, add trypsin working solution (DIG-3008, Fuzhou Mayxin) 37°C for 20 min in the dark → PBS rinse for 5 min x 3 times → remove the section, wipe off the liquid around the tissue with filter paper (do not dry the tissue), place it flat in a wet box with a histological pen → add WGA-Alexa Flour 488 working solution (10 μg / mL) 37°C for 2 h in the dark → discard the staining solution, PBS rinse for 5 min x 3 times → DAPI mounting → observe under a fluorescence microscope and take photos.

[0049] 2.3 WGA / Mboat7 / DAPI fluorescence co-staining (1) Paraffin section deparaffinization: Place the section in xylene for 5-10 minutes at room temperature, repeat 2 times. Through gradient ethanol (100%, 95%, 70%) for 5 minutes each, finally put into distilled water for 5 minutes.

[0050] (2) Antigen retrieval: Place the sections in antigen retrieval solution (citrate buffer) and microwave or water bath for 15-30 minutes.

[0051] (3) Blocking: Place the sections in blocking solution and incubate at room temperature for 30 minutes to block non-specific binding sites.

[0052] (4) Primary antibody incubation: Dilute the primary antibody in antibody diluent (blocking solution or PBS) and place the sections in the primary antibody. Incubate at 4°C overnight.

[0053] (5) Washing: Wash the sections with washing buffer for 3 times, 5 minutes each time, to remove unbound primary antibody.

[0054] (6) Secondary antibody incubation: Dilute the secondary antibody in antibody diluent and place the sections in the secondary antibody. Incubate at 37°C for 1-2 hours.

[0055] (7) Washing: Repeat the washing step in step (5).

[0056] (8) DAPI mounting: Place the sections on a glass slide, add DAPI, and cover with a coverslip.

[0057] (9) Microscopic observation: Use a fluorescence microscope to observe the sections and record the fluorescence signal.

[0058] 2.4 Masson's trichrome staining The main steps are: 55°C baking for 30 min → 2 min of xylene, 3 times → 100% alcohol for 1 min → 95% alcohol for 1 min → 70% alcohol for 1 min → 10 min of running water → double distilled water for 1 min → Weigert's iron hematoxylin staining for 5 min → 5 min of tap water washing → remove residual liquid → 1% hydrochloric acid alcohol differentiation for 4 s → 5 min of tap water washing to return blue → 10 min of staining with acidic magenta liquid → 5 min of distilled water washing → about 5 min of phosphomolybdic acid aqueous solution treatment → 5 min of aniline blue liquid restaining → 1% glacial acetic acid treatment for 1 min → 70% alcohol once → 90% alcohol once → 100% alcohol for 30 s, 3 times → 2 min of xylene, 3 times → immediately cover the coverslip with xylene before it dries, and take a photo under a microscope.

[0059] The results of the phenotype of AAV9-Mboat7 group mice and AAV9-null mice after TAC modeling are shown in Table 2. Figures 4-6The heart gross phenotype, the heart of the Sham group had no obvious difference, the heart of the TAC group was larger than that of the Sham group, and the heart of the AAV9-Mboat7 group was significantly smaller than that of the AAV9-null control group; in addition, the differences in HW / BW, LW / BW and HW / TL between the AAV9-Mboat7 mice and the AAV9-null mice in the Sham group were not statistically significant; the HW / BW, LW / BW and HW / TL of the mice 4 weeks after TAC were higher than those of the Sham group; 4 weeks after TAC, the HW / BW, LW / BW and HW / TL of the AAV9-Mboat7 mice were lower than those of the AAV9-null mice Figure 4 WGA staining sections can be observed: after TAC, the cell hypertrophy of the AAV9-Mboat7 group is significantly reduced compared with the AAV9-null group, and the difference is statistically significant Figure 5 After Masson staining, it is found that the collagen content in the myocardial interstitium of the TAC group is increased compared with the Sham group, the collagen around the arterial blood vessels is increased more obviously, the collagen is thickened, and the arrangement is disordered into a network; the collagen content in the myocardial interstitium and the collagen content around the blood vessels of the AAV9-Mboat7 mice after TAC are reduced compared with the AAV9-null mice Figure 6 The above results show that after TAC modeling, the mice have obvious myocardial hypertrophy and myocardial fibrosis, and Mboat7 overexpression can significantly improve myocardial hypertrophy and fibrosis.

[0060] Example 5: Detection of mouse heart function by echocardiography 1. Preparation (1) Anesthesia machine preparation: first connect the oxygen cylinder to the gas inlet interface of the anesthesia machine, then unscrew the drug addition port seal cover on the anesthesia machine, quickly add isoflurane to the safe scale, and then tighten the seal cover. Unscrew the total valve on the oxygen cylinder, adjust the rotation knob of the flow control valve, and maintain the outlet gas pressure at 0.2-0.3 mPa.

[0061] (2) Preparation of mice to be tested: after the mice to be tested are anesthetized with isoflurane, the left chest area is shaved, and the head of the treated mouse is inserted into the anesthesia conduit sleeve head. Maintain the mouse in a stable anesthetic state with 1.5-2.0% isoflurane.

[0062] 2. Heart function detection The mouse is placed in a left lateral position or a supine position, and the ultrasonic coupling agent is evenly applied to the shaved area. A high-frequency ultrasonic diagnostic instrument is used, with a frequency of 15 MHz. The standard left ventricular papillary muscle short-axis section is selected, and the left ventricular end-diastolic diameter, left ventricular end-systolic diameter, left ventricular ejection fraction and short-axis shortening rate are measured.

[0063] Figure 7These are the cardiac function test results after TAC (transcutaneous cardiac arrest) in AAV9-Mboat7 and AAV9-null mice. Compared with the Sham group mice, the mice in the AAV9-Mboat7 group showed weakened cardiac function 4 weeks after TAC, mainly manifested as a decrease in ejection fraction and fractional shortening, indicators reflecting cardiac function. Four weeks after TAC, the cardiac dysfunction in the AAV9-Mboat7 mice was alleviated compared to the AAV9-null mice.

[0064] Example 6: Observation of mitochondrial morphology in mouse cardiac cardiomyocytes using transmission electron microscopy 1. Preliminary work: Prepare EP tubes containing 100 μL of tissue electron microscopy fixative (G1102, Servicebio) and label them (mouse number, group, surgery type, and sampling date). Place a culture dish containing 10 mL of tissue electron microscopy fixative at the sampling site. Weigh and euthanize the mice.

[0065] 2. Sample Collection: Using ophthalmic curved forceps, grasp the vascular pedicle below the atrial appendage, cut off the heart, quickly place it on sterile gauze, gently squeeze out the fluid inside the heart chambers, pat dry the surface fluid, immerse the heart in tissue electron microscopy fixation solution, and use ophthalmic scissors to cut approximately 1.0 mm from the apex of the heart. 3 Take small tissue blocks, avoiding compression of the apical tissue, and place them in labeled EP tubes. Prepare transmission electron microscopy tissue samples within 48 hours.

[0066] 3. Related measurements and calculations: Mitochondrial morphology was observed under a transmission electron microscope, and the morphology of mitochondria in myocardial tissue of each group was recorded by taking pictures. The ratio of intact mitochondrial cristae to the total mitochondrial area was calculated using ImageJ software.

[0067] Figure 8 For mitochondrial morphology analysis. The results showed that compared with the sham-operated group, the mitochondrial cristae in the apical tissue of mice 4 weeks after TAC surgery were significantly damaged, and the number of intact mitochondrial cristae was reduced; while the number of intact mitochondrial cristae was increased in mice overexpressing Mboat7, suggesting that Mboat7 overexpression can significantly reduce mitochondrial damage in the myocardial tissue of stress-loaded mice.

[0068] Example 7: Expression of Mboat7 in cardiomyocytes stimulated with either control group (PBS) or angiotensin II (Ang II). Cardiomyocytes from neonatal Sprague-Dawley rats (days 1-3) were isolated and cultured. After 48 hours of primary cardiomyocyte culture, the medium was changed (the specific procedure for primary neonatal SD rat cardiomyocyte culture is described in Example 8 below). Serum-free DMEM / F12 was added to starve the cardiomyocytes for 12 hours to synchronize them. Then, the cells were stimulated with PBS and angiotensin II (Ang II, 1 μM) for 48 hours respectively. Proteins were extracted from the cardiomyocytes and Western blot was performed to determine the expression of Mboat7, with GAPDH used as an internal control. Simultaneously, cardiomyocytes were subjected to phalloidin / Mboat7 / DAPI co-immunofluorescence staining. The results are as follows: Figure 9 As shown, Ang II stimulation increased the volume of primary rat cardiomyocytes and significantly upregulated the expression of Mboat7.

[0069] Example 8: Effect of Mboat7 overexpression (Ad-Mboat7) on Ang II-stimulated primary cardiomyocyte hypertrophy 1. Primary culture of neonatal SD rat cardiomyocytes (1) Ten newborn Sprague-Dawley mice aged 1-3 days were disinfected below the neck with 75% alcohol. The heart was removed with ophthalmic scissors and micro forceps and placed in a glass petri dish containing 10 mL of DMEM / F12 solution. The same process was repeated for another mouse.

[0070] (2) Wash the heart with DMEM / F12 medium and cut the heart into 1-2 mm pieces. 3 The fragments were transferred to a serum bottle containing a rotor, DMEM / F12 was removed, and trypsin digestion solution was added. The rotor speed was 120 r / min, and digestion was carried out for 15 min. After standing for a few seconds, the supernatant was discarded.

[0071] (3) Add trypsin digestion solution, rotate at 120 r / min, and digest for 15 min. Let stand for a few seconds, aspirate the supernatant, terminate digestion with DMEM / F12 medium containing 20% ​​fetal bovine serum, and store at 4°C. Repeat this step several times. When collecting the supernatant, try to collect as much as possible. When the tissue block turns white and becomes significantly smaller, stop digestion.

[0072] (4) Centrifuge the collected myocardial cell suspension at 1500 rpm for 8 min and discard the supernatant. Add an appropriate amount of culture medium to the centrifuge tube, gently pipette to resuspend the cells, and concentrate them into a 50 mL centrifuge tube. Filter the cell suspension through a 40 μm cell filter.

[0073] (5) Seed the cells in a 100 mm culture dish and allow them to adhere for 90 min. Then, filter the unadhered cell suspension. Add BrdU (final concentration 0.1 mM) according to the total amount of cell suspension, mix well, and then add it to a dish coated with 0.1% gelatin.

[0074] (6) Gently shake to disperse the cells, do not vortex. Incubate at 37°C and 5% CO2 for 48 hours, wash once with PBS, and change the culture medium.

[0075] 2. Effects of Mboat7 overexpression on an Ang II-induced cardiomyocyte hypertrophy model We commissioned Shandong Wz Biosciences Inc. (Shandong, China) to construct an adenovirus (Ad-Mboat7) overexpressing the rat Mboat7 gene. The construction process was as follows: The rat Mboat7 gene (NCBI Gene ID: 308309; NM_001134978.2) was synthesized. A GCCACC tag was added before the rat Mboat7 gene, and a 3×flag tag was added to the C-terminus. SfaAI / MluI restriction sites were selected. Through restriction enzyme digestion, ligation, transformation, and sequencing verification, the adenovirus plasmid pADM-CMV-Mboat7-mCMV-copGFP was obtained by cloning the rat Mboat7 gene into the pADM-CMV-C-3flag-mCMV-copGFP vector. Figure 10 A) pADM-CMV-Mboat7-mCMV-copGFP was transfected into HEK293A cells using the adenovirus AdMax system. After culturing for 72 hours, the virus was collected to obtain Ad-Mboat7.

[0076] Ad-null (adenovirus containing an empty vector, used as a control) and Ad-Mboat7 (adenovirus containing a Mboat7 overexpression vector) 10 MOIs were used to infect primary rat cardiomyocytes cultured for 3 days. After 24 hours, Western blot was used to verify successful Mboat7 overexpression. Figure 10 BC), and then stimulated with 1 μM Ang II (purchased from ENZO, ALX-151-039-M025) or control PBS for 48 hours before performing immunofluorescence experiments.

[0077] Immunofluorescence staining results as follows Figure 11 The surface area of ​​cardiomyocytes infected with Ad-Mboat7 adenovirus was significantly smaller than that of the Ad-null control group, indicating that the overexpression of Mboat7 adenovirus inhibited cardiomyocyte hypertrophy.

[0078] The above embodiments are only used to help illustrate the present invention. The implementation of the present invention is not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention should be considered as equivalent substitutions and are included within the protection scope of the present invention.

Claims

1. The application of Mboat7 in drug screening, characterized by: The drugs mentioned include one or more of the following: drugs that protect cardiac contractile function, drugs that fight cardiac fibrosis, drugs that prevent, alleviate and / or treat myocardial hypertrophy, and drugs that prevent, alleviate and / or treat stress-induced heart failure. The screening includes: screening for molecules that can specifically activate Mboat7, and screening for substances that can promote Mboat7 expression.

2. The application of Mboat7 in drug preparation, characterized by: The drug includes one or more of the following: drugs that protect cardiac contractile function, drugs that fight cardiac fibrosis, drugs that prevent, alleviate and / or treat myocardial hypertrophy, and drugs that prevent, alleviate and / or treat stress-induced heart failure.

3. The application according to claim 2, characterized in that: Mboat7 can be used as an active ingredient in a drug, or substances that can promote the expression of Mboat7 can be used as active ingredients in a drug.

4. The application of biomaterials expressing Mboat7 in drug preparation, characterized by: The drug includes one or more of the following: drugs that protect cardiac contractile function, drugs that fight cardiac fibrosis, drugs that prevent, alleviate and / or treat myocardial hypertrophy, and drugs that prevent, alleviate and / or treat stress-induced heart failure.

5. The application according to claim 4, characterized in that: The biomaterials expressing Mboat7 include any one of the following 1)-4): 1) Nucleic acid molecules encoding the Mboat7 protein; 2) An expression cassette containing the nucleic acid molecule described in 1); 3) A recombinant vector containing the nucleic acid molecule described in 1), or a recombinant vector containing the expression cassette described in 2); 4) A transgenic cell line containing the nucleic acid molecule described in 1), or a transgenic cell line containing the expression cassette described in 2), or a transgenic cell line containing the recombinant vector described in 3).

6. The application of Mboat7 expression promoters in drug preparation, characterized by: The drug includes one or more of the following: drugs that protect cardiac contractile function, drugs that fight cardiac fibrosis, drugs that prevent, alleviate and / or treat myocardial hypertrophy, and drugs that prevent, alleviate and / or treat stress-induced heart failure.

7. The application according to claim 6, characterized in that: The expression promoters of Mboat7 include one or more of proteins, polynucleotides, and small molecule compounds.

8. The application according to claim 6, characterized in that: The Mboat7 expression promoter is a recombinant plasmid containing the Mboat7 gene and capable of normally expressing the Mboat7 protein.

9. The application according to claim 6, characterized in that: The expression promoter of Mboat7 is an adenovirus carrying the Mboat7 sequence.

10. The application according to claim 1, 2, 4 or 6, characterized in that: The aforementioned myocardial hypertrophy or heart failure includes: myocardial hypertrophy or heart failure caused by hypertension, aortic stenosis, or mitral regurgitation.

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