Application of GRHPR in preparation of medicine for preventing and / or treating cardiac hypertrophy

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of GRHPR in preparation of medicines for preventing and / or treating myocardial hypertrophy, and GRHPR genes are derived from human, mice and rats. By constructing GRHPR overexpression plasmids and transfecting NRCM primary cardiac muscle cells, it is found that the GRHPR overexpression plasmids can significantly inhibit PE-induced cardiac hypertrophy, and the mRNA levels of cardiac hypertrophy marker genes Nppa and Nppb are significantly reduced. The result shows that the overexpression of the GRHPR gene can inhibit cardiac hypertrophy so as to improve the cardiac function. The result has important theoretical guidance significance and clinical application value for accurate treatment of cardiac hypertrophy. The medicine for preventing and / or treating cardiac hypertrophy is prepared or screened by taking the GRHPR as a target, and the application has important significance in prevention and treatment of cardiac hypertrophy.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to the application of GRHPR in the preparation of drugs for preventing and / or treating myocardial hypertrophy. Background Art

[0002] Myocardial hypertrophy is an important pathological feature of cardiovascular diseases, which can be caused by factors such as hypertension, heart valve disease, gene mutation, etc. If not treated in time, it may develop into heart failure. Currently, the clinical treatment methods for myocardial hypertrophy mainly include β-blockers, ACEI / ARB drugs and exercise intervention, but there is still a lack of effective specific targeted treatment strategies. Therefore, developing new treatment methods has important clinical value. GRHPR (glycine reductase / hydroxypyruvate reductase) is a key metabolic enzyme, mainly involved in the regulation of amino acid metabolism and redox homeostasis. Existing studies have found that GRHPR plays an important role in certain metabolic diseases (such as primary hyperoxaluria), but its function in the cardiovascular system is still unclear. Summary of the Invention

[0003] The purpose of the present invention is to provide the application of GRHPR in the preparation of drugs for preventing and / or treating myocardial hypertrophy. Overexpression of the GRHPR gene can inhibit myocardial hypertrophy and thus improve cardiac function.

[0004] The present invention provides the application of GRHPR protein and / or GRHPR gene in the preparation of drugs for preventing and / or treating myocardial hypertrophy. The sources of the GRHPR gene include humans, mice and rats;

[0005] The ID of the human GRHPR gene is 9380; the ID of the mouse GRHPR gene is 76238; the ID of the rat GRHPR gene is 680021.

[0006] As a preferred solution, it includes using GRHPR protein and / or GRHPR gene as the action target in the preparation and screening of drugs for preventing and / or treating myocardial hypertrophy.

[0007] The present invention also provides a primer pair for amplifying the GRHPR gene. The sources of the GRHPR gene include humans, mice and rats;

[0008] The ID of the human GRHPR gene is 9380; the ID of the mouse GRHPR gene is 76238; the ID of the rat GRHPR gene is 680021.

[0009] The present invention also provides a biological material containing the GRHPR gene, including a recombinant expression vector containing the GRHPR gene and / or a recombinant microorganism containing the GRHPR gene; the sources of the GRHPR gene include humans, mice, and rats;

[0010] The ID of the human GRHPR gene is 9380; the ID of the mouse GRHPR gene is 76238; the ID of the rat GRHPR gene is 680021.

[0011] As a preferred embodiment, the basic backbone of the recombinant expression vector containing the GRHPR gene includes the Fugw overexpression vector.

[0012] The present invention also provides the use of the primer pair or the biological material in the preparation of a drug for preventing and / or treating myocardial hypertrophy.

[0013] The present invention also provides a drug for preventing and / or treating myocardial hypertrophy, including one or more of a nucleic acid molecule of the GRHPR gene, a protein encoded by the GRHPR gene, a polypeptide encoded by the GRHPR gene, a recombinant expression vector containing the GRHPR gene, and a small molecule agonist related to the GRHPR gene; the sources of the GRHPR gene include humans, mice, and rats;

[0014] The ID of the human GRHPR gene is 9380; the ID of the mouse GRHPR gene is 76238; the ID of the rat GRHPR gene is 680021.

[0015] As a preferred embodiment, the drug for preventing and / or treating myocardial hypertrophy further includes a pharmaceutically acceptable excipient or carrier.

[0016] As a preferred embodiment, the preparation form of the drug for preventing and / or treating myocardial hypertrophy includes tablets, pills, powders, or injections.

[0017] The present invention also provides a method for screening a drug for preventing and / or treating myocardial hypertrophy, including the following steps: mixing a candidate drug with a myocardial hypertrophy cell model or a myocardial hypertrophy animal model, and detecting the expression of the GRHPR gene; the sources of the GRHPR gene include humans, mice, and rats;

[0018] The ID of the human GRHPR gene is 9380; the ID of the mouse GRHPR gene is 76238; the ID of the rat GRHPR gene is 680021.

[0019] Beneficial effects: The present invention provides the use of GRHPR in the preparation of drugs for preventing and / or treating myocardial hypertrophy. The sources of the GRHPR gene include humans, mice, and rats. The ID of the human GRHPR gene is 9380; the ID of the mouse GRHPR gene is 76238; the ID of the rat GRHPR gene is 680021. By constructing a GRHPR overexpression plasmid and transfecting NRCM, the present invention found that it can significantly inhibit PE-induced myocardial hypertrophy, as manifested by a significant down-regulation of the mRNA levels of the myocardial hypertrophy marker genes Nppa and Nppb. It is indicated that overexpression of the GRHPR gene can inhibit myocardial hypertrophy and thus improve cardiac function. This result has important theoretical guiding significance and clinical application value for the precise treatment of myocardial hypertrophy. The present invention uses the GRHPR gene as a target to prepare or screen drugs for preventing and / or treating myocardial hypertrophy, which is of great significance in the prevention and treatment of myocardial hypertrophy.

[0020] The plasmid construction of the present invention is based on the Fugw overexpression vector and is transfected into cardiomyocytes. The overexpression plasmid does not participate in the occurrence of any diseases, and plasmid transfection will not integrate into the host genome, reducing the risk of potential gene mutations. Among them, the Fugw overexpression vector can stably and efficiently express the target gene sequence in the heart tissue, thereby effectively improving the myocardial hypertrophy phenotype. Brief description of the drawings

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments.

[0022] Figure 1 For the Western blot detection in Example 1 of the protein expression of GRHPR 4 weeks after aortic arch constriction; where n = 6, Sham is the sham operation; TAC is the aortic arch constriction operation; ***, P < 0.01;

[0023] Figure 2 For the gray-scale statistical chart of the Western blot detection in Example 1 of the protein expression of GRHPR 4 weeks after aortic arch constriction; where n = 6, Sham is the sham operation; TAC is the aortic arch constriction operation; ***, P < 0.01;

[0024] Figure 3 For the RT-qPCR detection in Example 2 of the overexpression efficiency of the transfected GRHPR OE plasmid; where n = 6, Fugw is the control plasmid; GRHPR OE is the GRHPR overexpression plasmid; ***, P < 0.01;

[0025] Figure 4For the immunofluorescence staining in Example 3 to detect the size of cardiomyocyte area in the PE model of NRCM after transfection with GRHPR OE plasmid; where n = 6, Fugw is the control plasmid; GRHPR OE is the GRHPR overexpression plasmid; the scale bar length in the figure is 100 μm;

[0026] Figure 5 Statistical chart of the size of cardiomyocyte area in the PE model of NRCM after transfection with GRHPR OE plasmid detected by immunofluorescence staining in Example 3; where n = 6, Fugw is the control plasmid; GRHPR OE is the GRHPR overexpression plasmid; ***, P < 0.01;

[0027] Figure 6 For the RT-qPCR in Example 3 to detect the mRNA levels of hypertrophic factors Nppa and Nppb; where n = 6, Fugw is the control plasmid; GRHPR OE is the GRHPR overexpression plasmid; ***, P < 0.01. Detailed implementation mode

[0028] The present invention provides the application of GRHPR protein and / or GRHPR gene in the preparation of drugs for preventing and / or treating myocardial hypertrophy. The sources of the GRHPR gene include human, mouse and rat; the ID of the human GRHP R gene is 9380; the ID of the mouse GRHPR gene is 76238; the ID of the rat GRHPR gene is 680021. The myocardial hypertrophy described in the present invention includes pathological myocardial hypertrophy at the animal level or cardiomyocyte hypertrophy at the cell level, and the pathological myocardial hypertrophy includes cardiomyocyte hypertrophy induced by phenylephrine (PE).

[0029]

[0030]

[0031]

[0032] As a specific embodiment, the amino acid sequence of human GRHPR protein is shown in SEQ ID NO.4: MRPVRLMKVFVTRRIPAEGRVALARAADCEVEQWDSDEPIPAKELERG VAGAHGLLCLLSDHVDKRILDAAGANLKVISTMSVGIDHLALDEIKKRGIRVGYTPDVLTDTTAELAVSLLLTTCRRLPEAIEEVKNGGWTSWKPLWLCGYGLTQSTVGIIGLGRIGQAIARRLKPFGVQRFLYTGRQPRPEEAAEFQAEFVSTPELAAQSDFIVVACSLTPATEGLCNKDFFQKMKETAVFINISRGDVVNQDDLYQALASGKIAAAGLDVTSPEPLPTNHPLLTLKNCVILPHIGSATHRTRNTMSLLAANNLLAGLRGEPMPSELKL.

[0033] As a specific embodiment, the amino acid sequence of mouse GRHPR protein is shown in SEQ ID NO.5: MKPARLMKVFVTGPLPAEGRAALAQAADCEVEQWNSDDPIPRKDLE QGVVGAHGLLCRLSDRVDKKLLDAAGANLRVISTLSVGVDHLALDEIKKRGIRVGYTPGVLTDATAELAVSLLLTTCRRLPEAIEEVKNGGWSSWSPLWMCGYGLSQSTVGIVGLGRIGQAIARRLKPFGVQRFLYTGRQPRPQEAAEFQAEFVPIAQLAAESDFIVVSCSLTPDTMGLCSKDFFQKMKNTAIFINISRGDVVNQEDLYQALASGQIAAAGLDVTTPEPLPPSHPLLTLKNCVILPHIGSATYKTRNTMSLLAANNLLAGLRGEAMPSELKL.

[0034] As a specific embodiment, the amino acid sequence of the GRHPR protein of a rat is shown in SEQ ID NO.6: MFAAAGLVKPARLMKVFVTGPLPAQGRAALAQATDCEVEQWNSDD PIPSKDLEQGVAGAYGLLCRLSDRVDKKLLDAAGANLRVISTLSVGVDHLALDEIKKRGIRVGYTPGVLTDATAELAVSLLLTTCRRLPEAIEEVKNGGWSSWSPLWMCGYGLSESTVGIVGLGRIGQAIARRLKPFGVQRFLYTGRQPRPQEAAEFQAEFVPIAQLAAESDFIVVSCSLTPATRGLCNKDFFQKMKNTAVFINISRGDVVNQEDLYQALASGQIAAAGLDVTTPEPLPPSHPLLTLKNCVILPHIGSATYKTRNTMSLLAANNLLAGLRGEPMPSELKL。

[0035] The application of the GRHPR protein and / or GRHPR gene of the present invention in the preparation of a drug for preventing and / or treating myocardial hypertrophy includes using the GRHPR protein and / or GRHPR gene as a target for the preparation and screening of a drug for preventing and / or treating myocardial hypertrophy.

[0036] The present invention also provides a primer pair for amplifying the GRHPR gene, and the sources of the GRHPR gene include human, mouse and rat; the ID of the human GRHPR gene is 9380; the ID of the mouse GRHPR gene is 76238; the ID of the rat GRHPR gene is 680021.

[0037] The present invention also provides a biological material containing the GRHPR gene, including a recombinant expression vector containing the GRHPR gene and / or a recombinant microorganism containing the GRHPR gene; the sources of the GRHPR gene include human, mouse and rat; the ID of the human GRHPR gene is 9380; the ID of the mouse GRHPR gene is 76238; the ID of the rat GRHPR gene is 680021. The basic backbone of the recombinant expression vector containing the GRHPR gene of the present invention includes the Fugw overexpression vector. As a specific embodiment, the recombinant expression vector containing the GRHPR gene includes inserting the GRHPR gene into the Fugw overexpression vector to obtain the GRHPR OE recombinant expression vector. As a specific embodiment, the amplified microorganism of the recombinant vector containing the GRHPR gene includes TransStbl3 containing the GRHPR gene.

[0038] As a specific embodiment, the GRHPR OE recombinant expression vector is based on the Fugw overexpression vector as the backbone and is transfected into cardiomyocytes. The GRHPR OE recombinant expression plasmid is not involved in the occurrence of any diseases, and plasmid transfection will not integrate into the host genome, reducing the risk of potential gene mutations. Among them, the Fugw overexpression vector can stably and highly express the target gene sequence in the heart tissue. As a specific embodiment, transferring the GRHPR OE recombinant expression vector into phenylephrine (PE)-induced myocardial hypertrophic cells can treat and improve myocardial hypertrophy; specifically, GRHPR overexpression can inhibit the increase in cardiomyocyte area induced by phenylephrine and inhibit the mRNA expression of hypertrophic factors Nppa and Nppb.

[0039] The present invention also provides the use of the primer pair or the biological material described above in the preparation of a drug for preventing and / or treating myocardial hypertrophy.

[0040] The present invention also provides a drug for preventing and / or treating myocardial hypertrophy, including one or more of the nucleic acid molecule of the GRHPR gene, the protein encoded by the GRHPR gene, the polypeptide encoded by the GRHPR gene, the recombinant expression vector containing the GRHPR gene, and the small molecule agonist related to the GRHPR gene; the sources of the GRHPR gene include humans, mice, and rats; the ID of the human GRHPR gene is 9380; the ID of the mouse GRHPR gene is 76238; the ID of the rat GRHPR gene is 680021.

[0041] The drug for preventing and / or treating myocardial hypertrophy according to the present invention further includes a pharmaceutically acceptable excipient or carrier.

[0042] The dosage form of the drug for preventing and / or treating myocardial hypertrophy according to the present invention includes tablets, pills, powders, or injections.

[0043] The present invention also provides a method for screening a drug for preventing and / or treating myocardial hypertrophy, including the following steps: mixing a candidate drug with a myocardial hypertrophy cell model or a myocardial hypertrophy animal model, and detecting the expression of the GRHPR gene; the sources of the GRHPR gene include humans, mice, and rats; the ID of the human GRHPR gene is 9380; the ID of the mouse GRHPR gene is 76238; the ID of the rat GRHPR gene is 680021.

[0044] In order to further illustrate the present invention, the following examples are used to describe in detail the application of GRHPR provided by the present invention in the preparation of a drug for preventing and / or treating myocardial hypertrophy, but they cannot be understood as limiting the protection scope of the present invention.

[0045] Unless otherwise specified, the present invention has no special requirements for the preparation raw materials, and commercially available products well-known to those skilled in the art can be used.

[0046] Example 1 Establishment of aortic coarctation (TAC) model and detection of GRHPR protein expression in the heart

[0047] 1) Establishment of aortic coarctation (TAC) model: C57BL / 6 mice at 8 weeks of age and weighing about 25 g were selected and randomly divided into a sham operation group (Sham group) and an aortic coarctation group (TAC group).

[0048] Aortic coarctation group (TAC group): Anesthetize by intraperitoneal injection of Pentobarbital Sodium. Shave the hair on the neck and chest, and disinfect the surgical area. Perform tracheotomy, insert a tracheal intubation, connect to a small animal ventilator, and set the breathing parameters (tidal volume 10 mL / kg, respiratory rate 100 - 120 times / minute).

[0049] Incise the skin along the midline of the sternum, separate the parasternal muscles, and cut open the sternum to expose the thoracic cavity. Carefully separate the thymus and adipose tissue to expose the aortic arch and its branches (innominate artery, left common carotid artery, left subclavian artery). Place a 7-0 suture distal to the left subclavian artery (aortic isthmus). Wrap the suture around the aortic arch and tie it parallel to a needle with a diameter of 27G, then remove the needle to form a stenosis, ensuring that the degree of stenosis is consistent.

[0050] Suture the sternum, muscles, and skin layer by layer. Remove the tracheal intubation, place the animal on a warming pad to recover until fully awake. Observe the animal's status daily, including activity, diet, breathing, etc. Four weeks after the operation, anesthetize the mice again, take heart tissue after thoracotomy, and perform western blot detection.

[0051] Sham operation group (Sham group): Only thread the suture without ligation during the operation, and the remaining operations are the same as those in the aortic coarctation group (TAC group).

[0052] 2) Detection of GRHPR expression in the heart by Western blot: 5 mg of heart tissue (TAC group and Sham group) was taken respectively, added with 300 μL of protein lysate (containing protease inhibitor) for homogenization. After lysing on ice for 30 minutes, the supernatant was collected by centrifugation at 4°C (12,000 rpm, 15 minutes) to obtain protein samples. The protein concentration was measured using the BCA method. After adjusting the protein sample to 0.5 mg / mL, 5×SDS loading buffer was added, and the protein was denatured by boiling for 10 minutes. Subsequently, the 0.5 mg / mL protein sample was separated by SDS-PAGE gel electrophoresis and transferred to a PVDF membrane. After the transfer was completed, it was blocked with 5% skim milk at room temperature for 1 hour, and then primary antibodies (anti-GRHPR antibody and anti-Tubulin antibody) were added respectively and incubated overnight at 4°C. The next day, the membrane was washed 3 times with TBST, 10 minutes each time, and the corresponding HRP-labeled secondary antibody was added and incubated at room temperature for 1 hour. After washing the membrane again, ECL chemiluminescence reagent (manufacturer: Tianneng; product number: 180-501) was used for development, and the signal was collected by an imaging system. Finally, software such as ImageJ was used to analyze the gray values of the bands of GRHPR and Tubulin, and the expression of GRHPR was normalized and quantified with Tubulin as an internal reference.

[0053] The results are as Figure 1 and Figure 2 shown. A total of 12 mouse hearts were detected for the expression of GRHPR protein. The specific data of the Sham group were: 0.810959, 1.063619, 0.982131, 0.981024, 1.118386, 1.043881; the specific data of the TAC group were: 0.693626, 0.70655, 0.696381, 0.738502, 0.643794, 0.754011. It can be seen that in the mouse model of pathological myocardial hypertrophy induced by aortic constriction (TAC), it was found that the expression of GRHPR protein was significantly decreased during the progression of pathological myocardial hypertrophy.

[0054] Example 2 Construction and efficiency verification of GRHPR overexpression plasmid (GRHPR OE)

[0055] 1) After reverse transcribing the total RNA of rat heart tissue, PCR amplification was performed according to the GRHPR specific primers, and the cDNA sequence of GRHPR was obtained by recovery; among them, the primers for PCR amplification: upstream primer (as shown in SEQ ID NO.7): 5’-GCTGCAGGTCGACTCTAGAGGCCACCTTAATGTTT GCAGCTGCGGGT-3’, downstream primer (as shown in SEQ ID NO.8): 5’-GATAAGCTTG ATATCGAATTCTACAGCTTGAGTTCACTGGGCA-3’.

[0056] 50 μL PCR amplification reaction system: upstream primer (10 μM) 1.5 μL (final concentration 0.3 μM), downstream primer (10 μM) 1.5 μL (final concentration 0.3 μM), 10×KOD plus buffer 5 μL, 2 mM dNTP 5 μL (final concentration 0.2 mM), 25 mM MgSO4 2 μL (final concentration 1.0 mM), KOD enzyme 5 μL (KOD plus, product number KOD-201), cDNA template (500 ng / μL) 1 μL, made up to 50 μL with enzyme-free water.

[0057] PCR reaction procedure: pre-denaturation at 94 °C for 2 min; denaturation at 94 °C for 15 s, annealing at 60 °C for 30 s, extension at 68 °C for 60 s, 35 cycles; keep at 4 °C. The PCR product was subjected to gel recovery using Tiangen Biotech agarose gel DNA recovery kit (product number DP219) to obtain the GRHPR amplification product. The specific method was as follows: The PCR amplification product was detected by 1% agarose gel electrophoresis. Dissolve 2 g of agarose (Bio-West, product number BYR0100) in 200 mL of 1×TAE buffer, heat to melt and add 20 μL of GelRed nucleic acid dye (TIANGEN, product number RT211), pour the gel and insert a comb to form sample wells. After the gel was completely solidified, place it in the electrophoresis tank and cover it with 1×TAE buffer. Mix 100 μL of the PCR product with 20 μL of 6×DNA Loading Buffer and load the sample, with a loading volume of 10 μL per well. Electrophoresis was carried out at a voltage of 120 V for 30 minutes, and the target band was observed using an ultraviolet gel imaging system and cut out for gel recovery.

[0058] 2) The overexpression vector (overexpression vector Fugw, purchased from Shanghai Lianmai Biotechnology Co., Ltd., product number LM1997) and the GRHPR amplification product were subjected to homologous recombination by the DNA seamless cloning method. ClonExpress MultiS One Step Cloning Kit (Vazyme, product number C113-01 / 02) was used for homologous recombination. Each 20 μL reaction system included: 100 ng of the enzymatically digested and linearized Fugw vector (BamHI (Bamhi: Thermo product number FD0054), EcorI (Ecori: Thermo, product number FD0274)), 20 ng of the GRHPR amplification product, 4 μL of 5×CE MultiS Buffer, 2 μL of Exnase MultiS, and sterile ddH2O was added to make up to 20 μL. After mixing, it was placed in a PCR instrument and reacted at 37 °C for 30 min, and immediately placed on ice for cooling after completion.

[0059] 3) 100 μL of competent cells (competent cells TransStbl3, Tsingke Biological, product number TSC-C06) were thawed on ice, 10 μL of the GRHPR OE recombinant product obtained in step 2) was added, flicked gently to mix evenly, and placed on ice for 25 min; heat shock at 42 °C for 45 s, then placed on ice for 2 min; then 500 μL of medium (antibiotic-free LB medium) was added, mixed evenly and cultured at 37 °C at 180 rpm for 1 h to resuscitate the bacteria. It was spread on a culture plate resistant to ampicillin (0.1 g / mL) and cultured overnight. After overnight culture, single colonies were picked and cultured in a shaker, and sent for Sanger sequencing. TransStbl3 containing the GRHPR OE overexpression plasmid was obtained.

[0060] 4) Neonatal rat cardiomyocytes (NRCM) were seeded at 2.0×10 5Seed cells at a density of

[0061] 5) After 72 hours of transfection, discard the culture medium, wash with PBS, and then add Trizol lysis buffer to extract total RNA from the heart tissue. Use a reverse transcription kit (Thermo Scientific, K1622) to obtain cDNA. Detect the expression level of GRHPR by qPCR, and use the -ΔCT method to calculate and analyze the expression changes of GRHPR.

[0062] qPCR reaction system (10 μL): 5 μL of SYBR Green, 0.5 μL each of upstream primer F and downstream primer R (10 μM), 3 μL of ddH2O, and 1 μL of cDNA dilution;

[0063] The sequences of upstream primer F and downstream primer R for qPCR of GRHPR gene are as follows: Upstream primer F (SEQ ID NO.9): 5’-GTTTGCAGCTGCGGGTCTTG-3’; Downstream primer R (SEQ ID NO.10): 5’-GGATGGGATCATCCGAATTCCA-3’. The internal reference gene is 18s, and the sequences of upstream primer F and downstream primer R for qPCR are as follows: Upstream primer F (SEQ ID NO.11): 5’-TCAAGAACGAAAGTCGGAGG-3’; Downstream primer R (SEQ ID NO.12): 5’-GGACATCTAAGGGCATCAC-3’; The PCR reaction program is as follows: Pre-denaturation at 95 °C for 30 sec; Denaturation at 95 °C for 15 sec, annealing and extension at 60 °C for 30 sec, for 40 cycles.

[0064] The results are as follows Figure 3 As shown, the expression of GRHPR was detected in 12 cell well plates. The specific data are as follows: in the Fugw group: 0.746562, 1.268684, 0.998845, 1.030492, 1.108288, 0.925518; in the GRHPR OE group: 7.276947, 7.826316, 7.201679, 8.10231, 7.353002, 7.908112. The results indicate that the constructed GRHPR OE expression plasmid can successfully induce overexpression of GRHPR.

[0065] Example 3 Protective effect of overexpressing GRHPR on phenylephrine (PE)-induced myocardial hypertrophy

[0066] 1) Establishment of the phenylephrine (PE) model: Transfect the GRHPR OE plasmid or the Fugw control plasmid into NRCM (cardiomyocytes). The steps are the same as those in step 4) of Example 2. Eight hours after transfection with the GRHPR OE plasmid or the Fugw control plasmid, the transfected NRCM (cardiomyocytes) are treated with the PE working solution or phosphate buffer (PBS) respectively. Detection is carried out 48 hours after treatment. (After treating cardiomyocytes with the PE working solution for 48 hours, pathological hypertrophy of cardiomyocytes can be induced.)

[0067] Preparation of the PE working solution: Weigh 1 mg of PE (sigma; product number PHR1017), add it to 500 μL of starvation medium (DMEM containing 1% penicillin-streptomycin), and then dilute it 100× to 100 μM to obtain the PE working solution.

[0068] The experimental groups are divided into 4 groups. The specific grouping information is as follows:

[0069] Group 1 Phosphate buffer (PBS) + control plasmid (Fugw), transfect the control plasmid into NRCM and treat it with PBS. The number of samples is 6 wells. Specifically, transfect the Fugw control plasmid into NRCM (cardiomyocytes). The steps are the same as those in step 4) of Example 2, that is, add 200 μL of the transfection complex to about 38,000 NRCM per well. Eight hours after transfection, the NRCM transfected with Fugw is obtained; treat the NRCM transfected with Fugw with PBS, add 200 μL of DMEM medium containing PBS (that is, 200 μL of DMEM plus 2 μL of PBS), and carry out detection 48 hours after treatment.

[0070] Group 2: Phosphate-buffered saline (PBS) + GRHPR overexpression plasmid (GRHPR OE). NRCMs were transfected with the overexpression plasmid and treated with PBS, with 6 wells of samples. Specifically, NRCMs (cardiomyocytes) were transfected with the GRHPR OE plasmid. The procedure was the same as step 4) in Example 2, that is, 200 μL of the transfection complex was added to approximately 38,000 NRCMs per well. After 8 hours of transfection, NRCMs transfected with GRHPR OE were obtained. The NRCMs transfected with GRHPR OE were treated with PBS, and 200 μL of DMEM medium containing PBS (that is, 2 μL of PBS was added to 200 μL of DMEM) was added. Detection was performed after 48 hours of treatment.

[0071] Group 3: Phenylephrine (PE) + control plasmid (Fugw). NRCMs were transfected with the control plasmid and treated with PE, with 6 wells of samples. Specifically, NRCMs (cardiomyocytes) were transfected with the Fugw control plasmid. The procedure was the same as step 4) in Example 2, that is, 200 μL of the transfection complex was added to approximately 38,000 NRCMs per well. After 8 hours of transfection, NRCMs transfected with Fugw were obtained. The NRCMs transfected with Fugw were treated with PE, and 200 μL of DMEM medium containing PE (the final concentration of PE dissolved in DMEM was 100 μM, that is, 2 μL of 10 mM PE stock solution was contained in 200 μL of DMEM) was added. Detection was performed after 48 hours of treatment.

[0072] Group 4: Phenylephrine (PE) + GRHPR overexpression plasmid (GRHPR OE). NRCMs were transfected with the overexpression plasmid and treated with PE, with 6 wells of samples. Specifically, NRCMs (cardiomyocytes) were transfected with the GRHPR OE plasmid. The procedure was the same as step 4) in Example 2, that is, 200 μL of the transfection complex was added to approximately 38,000 NRCMs per well. After 8 hours of transfection, NRCMs transfected with GRHPR OE were obtained. The NRCMs transfected with GRHPR OE were treated with PE, and 200 μL of DMEM medium containing PE (the final concentration of PE dissolved in DMEM was 100 μM, that is, 2 μL of 10 mM PE stock solution was contained in 200 μL of DMEM) was added. Detection was performed after 48 hours of treatment.

[0073] 2) Immunofluorescence staining: After treatment with PE or PBS for 48 h, the cells were washed 3 times with PBS to remove the culture medium. The cardiomyocytes were fixed with 4% paraformaldehyde for 15 minutes, washed 3 times with PBS for 5 minutes each time. Subsequently, they were permeabilized with 0.1% Triton X-100 for 10 minutes to increase antibody permeability. After washing with PBS, they were blocked with 5% BSA at room temperature for 1 hour to reduce non-specific binding. Then, the anti-α-actinin primary antibody was added and incubated overnight at 4°C. The next day, they were washed 3 times with PBS, and the fluorescently labeled secondary antibody (CY3-labeled anti-mouse IgG) was added and incubated for 2 hours at room temperature in the dark. After washing again, Hoechst 33342 (diluted 1:1000) was added to stain the cell nuclei and incubated for 10 minutes at room temperature in the dark. Finally, they were washed 3 times with PBS, observed and images were collected under a fluorescence microscope. The results are shown in Figure 4 . The α-actinin signal was shown as red fluorescence, and the cell nuclei were shown as blue fluorescence. The cell area could be further quantified by Image J image analysis software. The specific data were the normalized cell area, and the results are shown in Figure 5 .

[0074] The specific data were as follows: the first group of PBS + Fugw group: 1.028621, 0.989993, 0.972739, 0.925029, 1.123271, 0.960347; the second group of PBS + GRHPR OE group: 1.337775, 1.41096, 1.21938, 1.212219, 1.188744, 1.241195; the third group of PE + Fugw group: 1.757947, 1.854799, 1.823542, 1.842836, 1.988717, 1.971513; the fourth group of PE + GRHPR OE group: 1.578433, 1.537035, 1.478342, 1.426421, 1.42131, 1.519991.

[0075] It can be seen from Figure 4 and Figure 5 that after treatment with the PE model, the pathological hypertrophy of NRCM was significantly increased compared with that of the control group; while under the PE model, overexpression of GRHPR could protect against the pathological myocardial hypertrophy induced by the PE experiment.

[0076] 3) RT-qPCR detection of the mRNA levels of hypertrophic factors Nppa and Nppb: After treatment with PE or PBS for 48 h, the cells were washed 3 times with PBS to remove the culture medium. Trizol lysis solution was added to extract the total RNA in the heart tissue, and cDNA was obtained using a reverse transcription kit. The expression levels of Nppa and Nppb were detected by qPCR, and 2 -ΔCTThe expression changes of Nppa and Nppb were calculated and analyzed by the 2-ΔΔCt method. The sequences of the forward primer F and the reverse primer R for the qPCR reaction were as follows: Nppa forward primer F (SEQ ID NO.13): 5’-GAAGATGCCGGTAGAAGATGAG-3’; reverse primer R (SEQ ID NO.14): 5’-AGAGCCCTCAGTTTGCTTTTC-3’. Nppb forward primer F (SEQ ID NO.15): 5’-GGTGCTGCCCCAGATGATT-3’; reverse primer R (SEQ ID NO.16): 5’-CTGGAGACTGGCTAGGACTTC-3’. The internal reference gene was 18s, and the sequences of the forward primer F and the reverse primer R for the qPCR reaction were as follows: forward primer F (SEQ ID NO.17): 5’-TCAAGAACGAAAGTCGGAGG-3’; reverse primer R (SEQ ID NO.18): 5’-GGACATCTAAGGGCATCAC-3’; The PCR reaction procedure was as follows: pre-denaturation at 95°C for 30 sec; denaturation at 95°C for 15 sec, annealing and extension at 60°C for 30 sec, for 40 cycles. After completion, the 2-ΔΔCt method was used to calculate and analyze the expression changes of Nppa and Nppb. The results are as -ΔCT shown. A total of 12 cell well plates were detected for the expression of Nppa and Nppb mRNA. Figure 6 Specific data for Nppa: The first group, PBS + Fugw group: 0.83268, 0.963151, 1.050323, 1.249052, 1.306614, 0.727406; The second group, PBS + GRHPR OE group: 0.705068, 0.914359, 1.25774, 1.357388, 0.901771, 0.853128; The third group, PE + Fugw group: 4.245202, 5.048425, 5.281081, 5.52446, 6.003623, 5.172398; The fourth group, PE + GRHPR OE group: 2.869568, 2.76223, 2.595178, 3.330718, 2.705384, 2.595178;

[0077]

[0078] ​The specific data for the first group of PBS+Fugw in Nppb are: 0.823115, 0.952088, 1.038259, 1.230433, 1.282685, 0.778715; for the second group of PBS+GRHPR OE are: 0.744409, 0.90073, 1.176227, 1.332529, 0.891413, 0.843329; for the third group of PE+Fugw are: 5.873281, 6.984548, 7.230857, 7.255961, 7.643146, 7.20584; for the fourth group of PE+GRHPR OE are: 2.460867, 3.269719, 3.147413, 4.284618, 3.956341, 3.795176. The results show that by constructing the GRHPR overexpression plasmid and transfecting NRCM, it was found that it could significantly inhibit PE-induced myocardial hypertrophy, manifested as a significant decrease in the mRNA levels of the myocardial hypertrophy marker genes Nppa and Nppb.

[0079] Conclusion: Through the mouse model of pathological myocardial hypertrophy induced by transverse aortic constriction (TAC), it was found that the expression of GRHPR was significantly decreased during the progression of pathological myocardial hypertrophy.

[0080] In the model of neonatal rat cardiomyocyte (NRCM) hypertrophy induced by phenylephrine (PE), the expression level of GRHPR was significantly decreased.

[0081] By constructing the GRHPR overexpression plasmid and transfecting NRCM, it was found that it could significantly inhibit PE-induced myocardial hypertrophy, manifested as a significant decrease in the mRNA levels of the myocardial hypertrophy marker genes Nppa and Nppb.

[0082] Thus, it can be seen that by constructing the GRHPR overexpression plasmid and transfecting NRCM in the present invention, it was found that it could significantly inhibit PE-induced myocardial hypertrophy, manifested as a significant decrease in the mRNA levels of the myocardial hypertrophy marker genes Nppa and Nppb. It indicates that overexpression of the GRHPR gene can inhibit myocardial hypertrophy and thus improve cardiac function. This result has important theoretical guiding significance and clinical application value for the precise treatment of myocardial hypertrophy.

[0083] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments without creative work based on this embodiment, and these embodiments all belong to the protection scope of the present invention.

Claims

1. Use of GRHPR protein and / or GRHPR gene in the preparation of a drug for preventing and / or treating myocardial hypertrophy, characterized in that, The sources of the GRHPR gene include human, mouse and rat; The ID of the human GRHPR gene is 9380; the ID of the mouse GRHPR gene is 76238; the ID of the rat GRHPR gene is 680021.

2. The application according to claim 1, characterized in that, It includes using the GRHPR protein and / or GRHPR gene as a target for the preparation and screening of drugs for preventing and / or treating myocardial hypertrophy.

3. Primer pair for amplifying GRHPR gene, characterized in that, The sources of the GRHPR gene include human, mouse and rat; The ID of the human GRHPR gene is 9380; the ID of the mouse GRHPR gene is 76238; the ID of the rat GRHPR gene is 680021.

4. A biological material containing the GRHPR gene, characterized in that, It includes a recombinant expression vector containing the GRHPR gene and / or a recombinant microorganism containing the GRHPR gene; The sources of the GRHPR gene include human, mouse and rat; The ID of the human GRHPR gene is 9380; the ID of the mouse GRHPR gene is 76238; the ID of the rat GRHPR gene is 680021.

5. The biomaterial according to claim 4, wherein The basic backbone of the recombinant expression vector containing the GRHPR gene includes the Fugw overexpression vector.

6. Use of the primer pair according to claim 3 or the biomaterial according to claim 4 or 5 in the preparation of a drug for preventing and / or treating myocardial hypertrophy.

7. A drug for preventing and / or treating myocardial hypertrophy, characterized in that, It includes one or more of the nucleic acid molecule of the GRHPR gene, the protein encoded by the GRHPR gene, the polypeptide encoded by the GRHPR gene, the recombinant expression vector containing the GRHPR gene, and the small molecule agonist related to the GRHPR gene; The sources of the GRHPR gene include human, mouse and rat; The ID of the human GRHPR gene is 9380; the ID of the mouse GRHPR gene is 76238; the ID of the rat GRHPR gene is 680021.

8. The drug according to claim 7, characterized in that, The drug for preventing and / or treating myocardial hypertrophy further includes pharmaceutically acceptable excipients or carriers.

9. The drug according to claim 7, characterized in that, The preparation forms of the drug for preventing and / or treating myocardial hypertrophy include tablets, pills, powders or injections.

10. A method for screening a drug for preventing and / or treating myocardial hypertrophy, characterized in that, It includes the following steps: mixing a candidate drug with a myocardial hypertrophy cell model or a myocardial hypertrophy animal model, and detecting the expression of the GRHPR gene; The sources of the GRHPR gene include human, mouse and rat; The ID of the human GRHPR gene is 9380; the ID of the mouse GRHPR gene is 76238; the ID of the rat GRHPR gene is 680021.

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