Use of ndrg4 as a drug target in the preparation of medicaments

Abstract

The application belongs to the technical field of gene application and function, and particularly relates to application of NDRG4 as a drug target in preparation of a drug, in particular, application of NDRG4 as a drug target in preparation of a drug for preventing and / or relieving and / or treating myocardial hypertrophy, myocardial fibrosis and heart failure. The application finds a new function of NDRG4 gene, that is, the NDRG4 gene has the effects of inhibiting myocardial hypertrophy and myocardial fibrosis and improving heart function. Based on the effects of the NDRG4 gene, the NDRG4 gene can be used for screening / preparing a drug for protecting heart function, resisting myocardial fibrosis and / or preventing, relieving and / or treating myocardial hypertrophy and related diseases, and an effective new way for treating myocardial hypertrophy is provided.

Description

Technical Field

[0001] This invention belongs to the field of gene application and function technology, specifically involving the application of NDRG4 as a drug target in drug preparation. Background Technology

[0002] Myocardial hypertrophy is an adaptive remodeling process in which the heart, under pathological stimuli such as chronic stress overload, excessive activation of neurohormones, or genetic mutations, undergoes compensatory enlargement of cardiomyocytes accompanied by interstitial fibrosis. It is mainly characterized by increased cardiomyocyte volume, increased protein synthesis, and increased extracellular matrix. Although compensatory myocardial hypertrophy can enhance myocardial contractility and maintain normal cardiac output, long-term continuous stimulation can lead to pathological myocardial hypertrophy and myocardial remodeling, further triggering heart failure and malignant arrhythmias, and even sudden death. Pathological myocardial hypertrophy is considered an early stage of chronic heart failure, ultimately leading to unfavorable clinical outcomes. Current treatments for myocardial hypertrophy, such as ACEIs and β-receptor antagonists, mainly slow disease progression but cannot reverse existing myocardial fibrosis and ventricular wall thickening. Therefore, exploring the mechanisms of myocardial hypertrophy and finding new therapeutic targets for its prevention and treatment is of great scientific significance. Myc downstream regulatory gene 4 (NDRG4) is a member of the N-Myc downstream regulatory gene family and is significantly expressed primarily in the heart and brain. The NDRG family, including NDRG1, NDRG2, NDRG3, and NDRG4, encodes proteins that play crucial roles in growth and development, cell proliferation, tumorigenesis, stress response, and endocrine and apoptosis regulation. Previous studies have found that NDRG4 plays a key role in regulating cardiomyocyte proliferation during early cardiac development. Its dysfunction can lead to significant inhibition of cardiomyocyte proliferation and reduced cell volume, resulting in incomplete development and functional impairment of early embryonic cardiac tissue. This developmental abnormality manifests as disordered cardiac circulation, reduced circulatory efficiency, and weakened myocardial contractility. Furthermore, NDRG4 dysfunction is associated with QT interval prolongation, which may increase the risk of sudden cardiac death and ventricular arrhythmias. However, to date, there are no reports on the role of NDRG4 in pathological myocardial hypertrophy, requiring further investigation. Summary of the Invention

[0003] The purpose of this invention is to overcome the shortcomings of existing technologies. Addressing the deficiencies and limitations of current clinical techniques for the prevention and treatment of hypertrophic cardiomyopathy, this invention aims to determine the relationship between NDRG4 gene expression and hypertrophic cardiomyopathy, providing a target gene for the treatment of hypertrophic cardiomyopathy, and thus applying the NDRG4 gene to the prevention or treatment of hypertrophic cardiomyopathy. This invention also provides an application of NDRG4 in the screening / preparation of cardioprotective drugs, specifically the function and application of NDRG4 in the treatment of hypertrophic cardiomyopathy, and more specifically, the application of NDRG4 as a target gene in the preparation of drugs for anti-myocardial fibrosis and / or prevention, relief, and / or treatment of hypertrophic cardiomyopathy.

[0004] The first aspect of the present invention provides the use of NDRG4 as a drug target in the preparation of drugs for the prevention and / or relief and / or treatment of heart disease.

[0005] Preferably, the heart disease is myocardial hypertrophy.

[0006] Preferably, the heart disease is myocardial fibrosis.

[0007] Preferably, the heart disease is heart failure.

[0008] A second aspect of the present invention provides the use of reagents that enhance NDRG4 expression in the preparation of preventive and / or alleviating and / or therapeutic agents for heart disease.

[0009] Preferably, the reagent includes reagents that increase the NDRG4 gene, reagents that activate the NDRG4 gene, or reagents that enhance the expression of the NDRG4 gene.

[0010] Preferably, the heart disease is myocardial hypertrophy.

[0011] Preferably, the heart disease is myocardial fibrosis.

[0012] Preferably, the heart disease is heart failure.

[0013] A third aspect of the present invention provides an agent for preventing and / or alleviating and / or treating heart disease, the agent comprising an agent for increasing the NDRG4 gene, an agent for activating the NDRG4 gene, or an agent for increasing the expression of the NDRG4 gene, the agent being a drug, a pharmaceutical composition, a health product, a food, or an additive.

[0014] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention detected the expression levels of NDRG4 protein and ANP at different time points after AB surgery in wild-type (C57) mice, and detected the changes in NDRG4 protein expression under PE / ISO stimulation in primary neonatal cardiomyocytes (NRCM) of SD rats. The results showed that the expression level of NDRG4 protein in vivo increased after AB surgery, and exhibited a change of first increasing and then decreasing over time; in vitro NDRG4 protein expression increased under PE / ISO stimulation, with the most significant increase at 12 h. This suggests that NDRG4 may be involved in the development and progression of myocardial hypertrophy.

[0015] 2. This invention uses wild-type mice and heart-specific NDRG4 knockout mice as experimental subjects. The surgical group developed a myocardial hypertrophy model through AB surgery, while the sham-operated group did not receive AB surgery. Results showed that compared to the sham-operated group, AB surgery significantly worsened cardiac function and significantly promoted myocardial hypertrophy and fibrosis. Compared to wild-type + AB group mice, the AB + NDRG4-KO group mice showed significantly worsened cardiac function and accelerated the progression of myocardial hypertrophy and fibrosis. This indicates that NDRG4 knockout significantly worsens pressure-overload-induced myocardial hypertrophy.

[0016] 3. This invention constructs a recombinant adenovirus vector (AAV9-NDRG4) overexpressing NDRG4, infects wild-type mice, and establishes a myocardial hypertrophy model by performing an ablation procedure in the surgical group, while the sham-operated group does not receive the ablation procedure. Myocardial hypertrophy, fibrosis, and cardiac function are measured in each group. Results show that, compared with the control group (AAV9-GFP), NDRG4 overexpression significantly improved cardiac function and delayed the progression of myocardial hypertrophy and fibrosis. This indicates that NDRG4 can inhibit myocardial hypertrophy and protect cardiac function.

[0017] 4. The results above indicate that in the aortic coarctation-induced myocardial hypertrophy model, NDRG4 knockout promotes myocardial hypertrophy and fibrosis, leading to worsening cardiac function, while promoting NDRG4 overexpression protects the heart and improves cardiac function. Therefore, the NDRG4 gene plays a protective role against myocardial hypertrophy and related diseases.

[0018] Therefore, this invention discovers a novel function of the NDRG4 gene: it inhibits myocardial hypertrophy and myocardial fibrosis, and improves cardiac function. Based on the function of the NDRG4 gene, it can be used to screen / prepare drugs that protect cardiac function, resist myocardial fibrosis, and / or prevent, alleviate, and / or treat myocardial hypertrophy and related diseases, providing an effective new approach for the treatment of myocardial hypertrophy. Attached Figure Description

[0019] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention and do not constitute a limitation thereof.

[0020] In the attached diagram: Figure 1 The study investigated the changes in protein expression levels of ANP and NDRG4 in cardiac tissue at different time points after AB surgery in WT mice. The results showed that NDRG4 expression increased after AB surgery but decreased with increasing AB time; ANP expression increased with increasing AB time. Figure 2 The results showed that NDRG4 protein expression levels increased under PE and ISO stimulation, with the most significant increase occurring at 12 h. Figure 3 The study investigated the differences in NDRG4 and ANP protein expression levels between normal human heart tissue and heart tissue from heart failure patients. The results showed that compared with normal human heart tissue, NDRG4 expression was significantly reduced and ANP expression was significantly increased in heart tissue from heart failure patients. Figure 4 The results showed that NDRG4 was highly expressed in the brain, followed by the heart. Figure 5 The heart-specific NDRG4 gene knockout mouse was successfully constructed. Figure 6 This is a bar chart showing the cardiac function results of WT and NDRG4-KO mice 4 weeks after AB surgery via echocardiography. The results show that cardiac function deteriorated significantly after NDRG4 knockout, with significantly increased LVEDd and LVEDs, and significantly decreased EF. LVEDd is the left ventricular end-diastolic diameter, LVEDs is the left ventricular end-systolic diameter, and EF is the ejection fraction. Figure 7 This is a statistical bar chart of heart weight to body weight ratio (HW / BW) and heart weight to tibia length ratio (HW / TL) 4 weeks after AB surgery in WT and NDRG4-KO mice. The results show that HW / BW and HW / TL are significantly increased after NDRG4 knockout. Figure 8 The images show HE staining and Sirius red staining of heart tissue in WT and NDRG4-KO mice 4 weeks after AB surgery, as well as a bar chart of cardiomyocyte cross-sectional area and fibrosis area. The results show that NDRG4 knockout promotes cardiomyocyte hypertrophy and fibrosis. Figure 9Four weeks after AB surgery in WT and NDRG4-KO mice, the mRNA levels of myocardial hypertrophy markers ANP, BNP, β-MHC and myocardial fibrosis markers CollagenI and CollagenIII in cardiac tissue were detected. The results showed that NDRG4 knockout significantly promoted the expression of markers related to myocardial hypertrophy and fibrosis. Figure 10 This is a strategy that utilizes AAV9 to construct NDRG4 overexpressing mice; Figure 11 The NDRG4 overexpression mouse was successfully constructed. Figure 12 This is a bar chart showing the cardiac function results of AAV9-GFP and AAV9-NDRG4 mice 4 weeks after AB surgery. The results show that NDRG4 overexpression improved cardiac function, as evidenced by a significant decrease in LVEDd and LVEDs, while a significant increase in EF. LVEDd is the left ventricular end-diastolic diameter, LVEDs is the left ventricular end-systolic diameter, and EF is the ejection fraction. Figure 13 This is a bar chart showing the heart weight to body weight ratio (HW / BW) and heart weight to tibia length ratio (HW / TL) of AAV9-GFP and AAV9-NDRG4 mice 4 weeks after AB surgery. The results show that HW / BW and HW / TL decreased significantly after NDRG4 overexpression. Figure 14 The images show HE staining and Sirius red staining of heart tissue from AAV9-GFP and AAV9-NDRG4 mice 4 weeks after AB surgery, as well as a bar chart of cardiomyocyte cross-sectional area and fibrosis area. The results show that NDRG4 overexpression inhibits cardiomyocyte hypertrophy and interstitial fibrosis. Figure 15 The results showed that NDRG4 overexpression significantly inhibited the expression of myocardial hypertrophy markers (ANP, BNP, and β-MHC) and myocardial fibrosis markers (CollagenI and CollagenII) in the cardiac tissue of AAV9-GFP and AAV9-NDRG4 mice 4 weeks after AB surgery. Detailed Implementation

[0021] The following is in conjunction with the appendix Figures 1-15 The preferred embodiments of the present invention will be described herein. It should be understood that the preferred embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0022] The first aspect of this invention provides the use of NDRG4 as a drug target in the preparation of drugs for the prevention and / or relief and / or treatment of myocardial hypertrophy, myocardial fibrosis, and heart failure.

[0023] A second aspect of the present invention provides the use of reagents that enhance NDRG4 expression in the preparation of preventive and / or alleviating and / or therapeutic agents for heart disease.

[0024] Preferably, the reagent includes reagents that increase the NDRG4 gene, reagents that activate the NDRG4 gene, or reagents that enhance the expression of the NDRG4 gene.

[0025] The heart diseases mentioned include myocardial hypertrophy, myocardial fibrosis, and heart failure.

[0026] The administration method of the reagent that enhances NDRG4 expression is any one of the following: direct naked DNA injection, liposome-encapsulated DNA direct injection, gold-coated DNA gene gun bombardment, reproduction-deficient bacteria carrying plasmid DNA, replication-deficient adenovirus carrying target DNA, PEG-modified protein drug injection, liposome-encapsulated protein intravenous injection, or protein microsphere formulation subcutaneous injection.

[0027] A third aspect of the present invention provides an agent for preventing and / or alleviating and / or treating heart disease, the agent comprising an agent for increasing the NDRG4 gene, an agent for activating the NDRG4 gene, or an agent for increasing the expression of the NDRG4 gene, the agent being a drug, a pharmaceutical composition, a health product, a food, or an additive.

[0028] Animal experiments: 1. Origin and construction of laboratory mice: (1) SPF grade C57BL / 6 strain mice (abbreviated as C57): purchased from Hubei Beinte Biotechnology Co., Ltd., and raised at Hubei Provincial Institute of Cardiovascular Diseases after passing quarantine.

[0029] (2) Acquisition of C57BL / 6J background NDRG4 deletion mice (KO): Purchased from Cyagen Biotechnology Co., Ltd., and raised at Hubei Provincial Institute of Cardiovascular Diseases after passing quarantine.

[0030] (3) Acquisition of C57BL / 6J background NDRG4 overexpression mice (AAV9-NDRG4): C57 mice were selected for this experiment. When the mice were 5-6 weeks old, NDRG4 overexpression mice were constructed by injecting AAV9-NDRG4 into the tail vein.

[0031] I. Detection of NDRG4 protein expression changes in a myocardial hypertrophy model 1. In vivo and in vitro models of myocardial hypertrophy (1) Grouping of experimental animals: Eight-week-old male C57 mice weighing 23.5-27.5g were selected. The surgical group underwent aortic arch coarctation (AB). Specific methods: ① Anesthesia: The mice were weighed and anesthetized with 3% sodium pentobarbital; ② Skin preparation: The hair in the surgical area was removed; ③ Tracheal intubation; ④ Aortic arch descending branch ligation surgery: The mice were placed in the right lateral decubitus position, the skin was disinfected, the chest cavity was opened, the descending aortic arch was freed, and a 7-0 surgical suture was used to ligate the blood vessel. After that, the chest cavity was closed and the skin was sutured. Sham group mice: After freeing the descending aortic branch, only the suture was passed through without ligation, and the remaining steps were the same as AB surgery.

[0032] (2) Mouse heart sampling: Mice were weighed at 1, 2, 4 and 8 weeks after AB surgery, and were euthanized by cervical severing. The chest cavity was opened, the heart was removed, weighed, and flash-frozen in liquid nitrogen at -80°C for use in molecular biology experiments.

[0033] (3) Extraction and culture of NRCM: ① Take 10 newborn mice within 3 days of birth, wipe them with 75% alcohol, place them on sterile gauze in a clean bench, grab the mice, cut open the chest cavity, squeeze to make the heart beat out, quickly cut off the heart, place it in a culture dish, repeat the above steps, and take out all the mice's hearts in sequence; ② Shred the heart: gently wash the heart on an ice plate, place it in another culture dish containing 10ml DMEM / F12, and cut the heart into 1-2mm3 pieces with ophthalmic scissors; ③ Digestion: put the shredded heart tissue into a serum bottle with a magnetic rotor containing 8ml of digestive enzyme, place the serum bottle in a water bath beaker on a magnetic stirrer, stir at 37℃ and 150r / min, digest for 15min, let stand for a few seconds, and discard. ④ Add 8 ml of trypsin digestion solution to the serum bottle again, gently pipette and digest for 15 min according to the above parameters. After standing for a few seconds, collect the supernatant and place it in 20 ml of DMEM / F12 medium containing 20% ​​fetal bovine serum to stop digestion. Store at 4℃. ⑤ Repeat step 4 until the tissue block turns white and becomes smaller, then stop digestion. ⑥ Centrifuge: Centrifuge at 1500 rpm for 8 min and discard the supernatant. ⑦ Resuspend the precipitate and filter it through a 40 μm filter. Inoculate and allow the cells to adhere to the culture vessel for 90 min. ⑧ Count the cells in the supernatant (i.e., the non-adherent cells), add BrdU (bromodeoxyuridine) (final concentration 0.1 mM), inoculate, and gently shake left and right and up and down alternately to disperse the cells. ⑨ Incubate at 37℃ in a 5% carbon dioxide incubator for subsequent experiments.

[0034] (4) PE / ISO was added to the NRCM experimental group to induce myocardial hypertrophy; the Con group was treated with an equal volume of PBS, and cells with different incubation times were taken for molecular biology experiments.

[0035] 2. Detection of NDRG4 protein levels by Western blotting (WB) 2.1 Extracting protein samples: (1) Sterilize the equipment for protein extraction, including ophthalmic scissors, ophthalmic straight forceps, steel balls, etc., under high pressure.

[0036] (2) Quickly remove the protein sample to be extracted and place it on dry ice. Label the EP tubes and put 4 steel balls into each EP tube, then pre-cool them on dry ice. Quickly cut the sample into small pieces with ophthalmic scissors and put them into the corresponding EP tubes. Weigh and record the weight of the sample.

[0037] (3) Add the appropriate amount of RIPA lysis buffer (10 mg / 100 μl) according to the sample weight. To avoid the lysis buffer solidifying and freezing the steel beads due to excessively low temperature, shake well quickly after adding the lysis buffer. After pre-cooling the grinding box with liquid nitrogen for 5-8 minutes, place the sample symmetrically in the grinding box and put it into the grinder. Set the grinding parameters: frequency 30 Hz / s, time 90 s.

[0038] (4) After grinding is complete, remove the steel ball and rinse it with anhydrous ethanol. Place the sample on ice for 10 minutes to allow for complete lysis. Use an ultrasonic lysis apparatus to lyse the sample at 5 kHz / cycle, repeating 10-15 times, 1 second each time, with a 1-second interval. To avoid generating a large number of air bubbles, the probe should be inserted 2 / 3 below the liquid surface during lysis. After ultrasonic lysis, place the sample on ice for 10 minutes. Centrifuge the sample in a pre-cooled centrifuge at 4°C at 12000 rpm for 30 minutes. Carefully aspirate the supernatant and quantify it, recording the lysis volume of the sample.

[0039] 2.2 Measurement of protein concentration using the BCA method 2.3 Protein buffering and preservation: (1) Add the corresponding amounts of 4×LB and 10×DTT lysis buffer according to the measured protein concentration, and calibrate the total protein to the same concentration. LB is 1 / 4 of the final volume, and DTT is 1 / 10 of the final volume. After mixing the protein sample, centrifuge at 12000 rpm and aliquot the sample.

[0040] (2) The sample was denatured in a 100°C water bath for 10 min and then stored in a -80°C refrigerator after cooling.

[0041] 2.4 Protein Gel Electrophoresis: Place the pre-prepared SDS-PAGE gel plate in the electrophoresis tank, and add appropriate amounts of external and internal electrophoresis solutions. Take 50 μg of each protein sample and load them sequentially into the SDS-PAGE gel lanes. After loading, perform electrophoresis at a constant voltage of 120 V. Electrophoresis is considered complete when the bromophenol blue dye reaches the bottom of the separating gel, and then turn off the power.

[0042] 2.5 Transfer: (1) Cut the PVDF membrane to 8×5.9cm and make a notch in one corner to mark the direction. Before use, soak it in methanol for 15s and then place it in the transfer solution for later use.

[0043] (2) Position the clamps left and right, with the negative electrode facing right, the white side being the positive electrode, and the black side being the negative electrode. Place 4-5 sheets of filter paper and 1-2 sheets of sponge on each clamp, which have been pre-soaked in transfer buffer.

[0044] (3) Remove the gel from the gel plate, wet the gel with transfer buffer, spread the gel evenly on filter paper, remove any air bubbles, cover it with the PVDF membrane, aligning the marked area with one corner of the high molecular weight marker, remove any air bubbles, cover with the left side of the filter paper and sponge, and cover with the clamp. Place the clamp into the transfer tank, ensuring the negative electrode of the transfer tank is placed together with the negative electrode of the clamp, and fill with transfer buffer to submerge the gel.

[0045] (4) Connect the power supply to the transfer tank and transfer at 25V for 2 hours. Stain the gel with Coomassie Brilliant Blue solution to determine whether the protein transfer is complete. After the transfer is complete, remove the PVDF membrane.

[0046] Figure 1 and Figure 2 The experimental results showed that, compared with the control group, the expression level of NDRG4 protein in the myocardial hypertrophy model showed a change of first increasing and then decreasing, which suggests that NDRG4 is likely involved in the mechanism of myocardial hypertrophy.

[0047] II. Effects of heart-specific NDRG4 gene knockout on AB-induced myocardial hypertrophy 1. Experimental Groups: WT+sham, WT+AB, NDRG4-KO+sham, NDRG4-KO+AB 2. Mouse heart sampling: Four weeks after AB, mice were euthanized by cervical dislocation, and the heart was removed. The heart was quickly placed on sterile gauze, and the fluid inside the heart chambers was gently squeezed out. After drying the surface fluid, the heart was weighed, and a portion was flash-frozen in liquid nitrogen at -80°C for subsequent molecular biological assays; the other portion was fixed in 10% neutral formalin for subsequent pathological analysis. The skin at the tibia of the hind limb was cut open, and the tibia length was measured and recorded. The heart weight to body weight ratio (HW / BW) and the ratio of heart weight to tibia length (HW / TL) were calculated to evaluate the degree of increase in heart weight.

[0048] 3. Echocardiography of cardiac function: Mice were anesthetized with isoflurane and shaved. The mice were placed in the left lateral decubitus or supine position, and ultrasound 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 short-axis section of the left ventricular papillary muscle and measure the left ventricular end-diastolic diameter (LVEDd), left ventricular end-systolic diameter (LVEDs), and ejection fraction (EF).

[0049] 4. Pathological examination: (1) Preparation of paraffin specimen sections: The heart was removed from formalin, a gross photograph was taken, then it was dehydrated, embedded, sectioned, and dried for later use. (2) HE hematoxylin-eosin staining: Baking at 55℃ for 30 min → xylene for 5 min, 3 times → 100% alcohol for 1 min → 95% alcohol for 1 min → 70% alcohol for 1 min → double distilled water for 1 min → hematoxylin solution for 5 min → wash with water for 1 min → 1% hydrochloric acid alcohol for 1-3 s → wash with water for 1 min → Scott's solution for 1 min → wash with water for 1 min → eosin solution for 3-5 min → wash away excess stain with distilled water → 70% alcohol for 1 s → 95% alcohol for 1 s → 100% alcohol for 30 s, 3 times → xylene for 2 min, 3 times → mount the slide immediately before the xylene dries → dry in a fume hood and take pictures under a microscope. Select at least 3 cells with clear boundaries and nuclei roughly located in the center for each image, and circle the cell area using Image-ProPlus 6.0 software.

[0050] (3) Staining with Sirius red (PSR): Baking at 55℃ for 30 min → xylene for 2 min, 3 times → 100% alcohol for 1 min → 95% alcohol for 1 min → 70% alcohol for 1 min → rinsing with running water for 10 min → double distilled water for 1 min → 0.2% phosphomolybdic acid for 2 min → drop 0.1% Sirius red picric acid solution onto the tissue and stain in a humidified chamber for 90 min → remove residual liquid → 0.01N hydrochloric acid for 4 s → 70% alcohol once → 90% alcohol once → 100% alcohol for 30 s, 3 times → xylene for 2 min, 3 times → immediately cover with a glass slide before the xylene dries and take a picture under a microscope.

[0051] 5. PCR detection of markers of myocardial hypertrophy and fibrosis: (1) Extract RNA, add 1 ml Trizol to each sample, and let stand for 10 min; (2) Add 200 μL of chloroform to each tube for extraction, let stand for 5 min, and then centrifuge at 12000 g for 15 min at 4 °C. (3) RNA precipitation: Aspirate the upper aqueous phase into a new EP tube, add an equal volume of isopropanol, mix well, let stand for 10 min, centrifuge at 12000g, 4℃ for 10 min; (4) After centrifugation, white flocculent precipitate (RNA) can be seen at the bottom of the tube. Remove the supernatant, rinse the RNA precipitate thoroughly with 75% ethanol, and centrifuge at 7500g, 4℃ for 5min. (5) Dissolution of RNA precipitate: Remove the supernatant, dry at room temperature for 10 min, and after the precipitate becomes colorless, dissolve the RNA precipitate in 20 μL of DEPC water; (6) Measure the RNA concentration of each sample; (7) Reverse transcription PCR; (8) RT-PCR: Using cDNA template and specific primer sequences to determine the RNA level of the indicator.

[0052] The primers used for RNA are as follows: ; Phenotypic results of WT and NDRG4-KO mouse models of myocardial hypertrophy are shown in the figure. Figure 6 , Figure 7 , Figure 8 , Figure 9 There were no statistically significant differences in LVEDd, LVEDs, and EF between WT and NDRG4-KO mice in the sham group; after AB4w, compared with WT mice, NDRG4-KO mice showed significantly increased LVEDd and LVEDs, and significantly decreased EF. Figure 6 There was no statistically significant difference in HW / BW among the sham group; after AB4w, compared with WT mice, the HW / BW and HW / TL of NDRG4-KO mice were significantly increased ( Figure 7 HE staining revealed no statistically significant difference in the cross-sectional area of ​​cardiomyocytes in the sham group, but the cross-sectional area of ​​cardiomyocytes in NDRG4-KO mice in the AB group was larger than that in WT mice. PSR staining revealed no statistically significant difference in the area of ​​fibrosis in the sham group, but the area of ​​fibrosis in NDRG4-KO mice in the AB group was larger than that in WT mice. Figure 8 RT-PCR results showed no significant difference in mRNA levels of myocardial hypertrophy markers and myocardial fibrosis markers in the sham group; in the AB group, compared with WT, the mRNA levels of myocardial hypertrophy markers ANP, BNP, β-MHC and myocardial fibrosis markers Collagen I and Collagen III in cardiomyocytes of NDRG4-KO mice were significantly increased. Figure 9 Cardiac-specific NDRG4 gene knockout significantly aggravates myocardial hypertrophy and fibrosis, and worsens cardiac function.

[0053] III. Effects of NDRG4 gene overexpression on AB-induced myocardial hypertrophy 1. Constructing NDRG4 overexpression mice: WT mice aged 6-7 weeks were selected and injected with AAV9-NDRG4 via the tail vein, while the control group was injected with AAV9-GFP. Western blotting was used to verify NDRG4 expression. Results are as follows: Figure 11 As shown, this indicates that the NDRG4 overexpression mouse was successfully constructed.

[0054] 2. Experimental Groups: Phenotypic results of AAV9-GFP+sham, AAV9-GFP+AB, AAV9-NDRG4+sham, AAV9-NDRG4+ABA, and AAV9-GFP and AAV9-NDRG4 mouse models of myocardial hypertrophy are shown in the table below. Figure 12 , Figure 13 , Figure 14 , Figure 15 In groups AB, the LVEDd and LVEDs of AAV9-NDRG4 mice were significantly lower than those of AAV9-GFP mice, while EF was significantly higher. Figure 12 After AB surgery, the HW / BW and HW / TL ratios of AAV9-NDRG4 mice decreased significantly. Figure 13 HE staining revealed that the cross-sectional area of ​​cardiomyocytes in AAV9-NDRG4 mice in group AB was smaller than that in AAV9-GFP mice. PSR staining revealed that the fibrotic area in AAV9-NDRG4 mice in group AB was smaller than that in AAV9-GFP mice. Figure 14 RT-PCR results showed that after AB treatment, the mRNA levels of myocardial hypertrophy markers ANP, BNP, and β-MHC, as well as myocardial fibrosis markers Collagen I and Collagen III, in cardiomyocytes of NDRG4-KO mice were significantly lower than those in AAV9-GFP mice. Figure 9 Overexpression of the NDRG4 gene can delay myocardial hypertrophy and fibrosis and improve cardiac function.

[0055] The results above indicate that in the aortic arch coarctation-induced myocardial hypertrophy model, NDRG4 gene deficiency significantly promoted myocardial hypertrophy and fibrosis, and worsened cardiac function; while NDRG4 gene overexpression significantly inhibited myocardial hypertrophy and fibrosis, and protected cardiac function. Therefore, the NDRG4 gene has a protective effect on cardiac function and inhibits myocardial hypertrophy and fibrosis.

[0056] As described above, the basic principles, main features, and advantages of the present invention have been explained. The above embodiments and specifications are merely descriptions of preferred embodiments of the present invention, and the present invention is not limited to the above embodiments. Various changes and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit and scope of the present invention should fall within the protection scope defined by the present invention.

Claims

1. Use of NDRG4 as a drug target in the preparation of drugs for the prevention and / or relief and / or treatment of heart disease.

2. The use according to claim 1, characterized in that, The heart disease is myocardial hypertrophy.

3. The use according to claim 1, characterized in that, Heart disease is myocardial fibrosis.

4. The use according to claim 1, characterized in that, The heart disease is heart failure.

5. Application of reagents that enhance NDRG4 expression in the preparation of drugs for the prevention and / or relief and / or treatment of heart disease.

6. The application according to claim 5, characterized in that, The reagents include reagents that increase the NDRG4 gene, reagents that activate the NDRG4 gene, or reagents that enhance the expression of the NDRG4 gene.

7. The application according to claim 6, characterized in that, The heart disease mentioned is myocardial fibrosis.

8. The application according to claim 6, characterized in that, The heart disease mentioned is myocardial hypertrophy.

9. The application according to claim 6, characterized in that, The heart disease mentioned is heart failure.

10. An formulation for the prevention and / or relief and / or treatment of heart disease, characterized in that, The formulation contains reagents that increase the NDRG4 gene, reagents that activate the NDRG4 gene, or reagents that enhance the expression of the NDRG4 gene. The formulation is a drug, a pharmaceutical composition, a health product, a food, or an additive.

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