Use of GPR146 as target in preparing drug for treating hypertension

Abstract

The present application relates to the technical field of biological pharmaceuticals, and in particular, to use of GPR146 as a target in preparing a drug for preventing or treating hypertension-related diseases. The present application relates to use of GPR146 in treating hypertension. The present application comprehensively applies transcriptome sequencing, a functional experiment, an animal model, and other methods, proposes the close relation between GPR146 in smooth muscle and hypertension for the first time, and deeply discusses a specific regulation mechanism of GPR146 in the occurrence and development of hypertension, providing a new insight for revealing the pathogenesis of hypertension.

Description

Application of GPR146 as a target in the preparation of a drug for treating hypertension

[0001] Cross-reference to Related Applications

[0002] This application claims priority to the Chinese patent application No. 202410538611.5, filed on April 30, 2024, entitled "Application of GPR146 as a target in the preparation of a drug for treating hypertension", the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0003] The present application relates to the field of biological medicine, in particular to the use of GPR146 as a target for preparing a drug for preventing or treating hypertension-related diseases. BACKGROUND

[0004] At present, hypertension has become a global disease with more than 1 billion people affected. Studies have shown that hypertension is the most important risk factor for cardiovascular disease, and as blood pressure rises, the risk of cardiovascular disease also increases. At the same time, hypertension is also an important cause, risk factor and one of the main causes of death of various cardiovascular and cerebrovascular diseases, and its pathogenesis is very complex.

[0005] Hydrostatic pressure refers to the pressure of blood flow per unit area perpendicular to the vessel wall, which is the basic condition for blood pressure formation. Hypertension is a disease characterized by elevated hydrostatic pressure. More and more studies suggest that elevated hydrostatic pressure can directly lead to the occurrence of various cardiovascular diseases. Endothelial cells and smooth muscle cells are the main components of the vascular wall. Existing studies have shown that high hydrostatic pressure treatment significantly affects the morphology of endothelial cells, and the endothelial cells are significantly elongated, and the actin filaments are rearranged; further induced endothelial cells to release fibroblast growth factor, promote endothelial cell proliferation, increase endothelial cell permeability, regulate extracellular matrix secretion, and ultimately lead to endothelial dysfunction. Smooth muscle cells are the most abundant and elastic cells in the vascular wall. At present, the role of hydrostatic pressure in the regulation of smooth muscle function is less studied. In smooth muscle cells, it is not yet clear how the body converts the mechanical signal of hydrostatic pressure into intracellular chemical signals and ultimately leads to vascular dysfunction.

[0006] G-protein-coupled receptors (GPCRs) are the largest superfamily of cell membrane receptors in human body, containing more than 800 members, which are expressed in various organ systems. Angiotensin type 1 receptor (AT1R) can be directly activated under mechanical stress, which is independent of angiotensin II (AngII). Further research found that the lack of surface AT1R can eliminate the myogenic vasoconstriction of mesenteric arteries. When ECs are stimulated by shear stress, bradykinin B2 receptor directly changes its conformation, and its activation contributes to vasodilation. Histamine H1 receptor in ECs can sense blood flow-mediated shear stress, promoting vasodilation. Under shear stress stimulation, GPR68 induces massive release of calcium ions, promoting ECs to release NO and cause vasodilation. Inhibition of S1PR1 can block shear stress-induced ECs rearrangement. Studies have shown that abnormal activation of GPCRs can lead to hypertension and vascular dysfunction. Angiotensin II type 1 receptor (AGTR1) and angiotensin II type 2 receptor (AGTR2) of renin-angiotensin-aldosterone system, and kinin receptors of kallikrein system regulate blood pressure by regulating sodium retention in the kidney and vascular tone. Certain short-chain fatty acids such as propionic acid and butyric acid can affect the function of intestinal cells by acting on GPCRs in the intestine, such as GPR41 and GPR43, GPR109a and OLF78, thereby affecting the vascular tone and blood pressure level in the body. In summary, GPCRs play an important role in mechanical signal transduction, vascular tone maintenance and vascular function regulation.

[0007] In order to find the GPCR that plays an important role in smooth muscle cells under high hydrostatic pressure, the expression of GPCR in smooth muscle cells after high hydrostatic pressure treatment was detected by whole transcriptome sequencing, and it was found that the expression of GPR146 was significantly increased. Subsequently, the role of GPR146 in blood pressure regulation in smooth muscle was clarified by using genetically modified mice, and finally the role of GPR146 in the treatment of hypertension was detected by using neutralizing antibody of GPR146. SUMMARY

[0008] Therefore, the technical problem to be solved by the present application is to provide an application of GPR146 as a target in the treatment of hypertension-related diseases. The present application first proposes that GPR146 is related to hypertension, and further discusses the specific regulation mechanism of GPR146 in the occurrence and development of hypertension, thereby providing new insights for revealing the pathogenesis of hypertension.

[0009] The second technical problem to be solved by the present application is to provide a related product for preventing or treating hypertension-related diseases based on detecting the expression amount of Gpr146.

[0010] To solve the above technical problems, the application discloses a use of GPR146 as a target for preparing a drug for preventing and / or treating a hypertension-related disease.

[0011] Specifically, the role of the GPR146 as a target includes:

[0012] overexpression of the GPR146 enhances microvessel contraction and weakens microvessel dilation; and / or,

[0013] knockout of the GPR146 weakens microvessel contraction and enhances microvessel dilation; and / or,

[0014] overexpression of the GPR146 promotes vascular remodeling and increases collagen content; and / or,

[0015] knockout of the GPR146 inhibits vascular remodeling and reduces collagen content; and / or,

[0016] treatment with a GPR146 monoclonal antibody can treat the blood pressure increase caused by angiotensin; and / or,

[0017] treatment with a GPR146 monoclonal antibody can inhibit or reduce the vascular remodeling and collagen content caused by angiotensin.

[0018] The GPR146 is highly expressed in a hypertension human blood vessel model.

[0019] Specifically, the role of the GPR146 as a target includes:

[0020] overexpression of the GPR146 enhances microvessel contraction and weakens microvessel dilation in mice; and / or,

[0021] knockout of the GPR146 weakens microvessel contraction and enhances microvessel dilation in mice; and / or,

[0022] overexpression of the GPR146 promotes vascular remodeling and increases collagen content in mice; and / or,

[0023] knockout of the GPR146 inhibits vascular remodeling and reduces collagen content in mice; and / or,

[0024] treatment with a GPR146 monoclonal antibody can treat the blood pressure increase caused by angiotensin in mice; and / or,

[0025] treatment with a GPR146 monoclonal antibody can inhibit or reduce the vascular remodeling and collagen content caused by angiotensin in mice.

[0026] The application further discloses a use of GPR146 for preparing a product with at least one of the following (1)-(5) effects:

[0027] (1) a product for monitoring the treatment status of a population with hypertension-related diseases;

[0028] (2) a product for monitoring the prognosis of a population with hypertension-related diseases;

[0029] (3) a product for preventing and / or treating hypertension-related diseases;

[0030] (4) a product for screening a target associated with hypertension-related diseases;

[0031] (5) a product for screening a drug for hypertension-related diseases.

[0032] Specifically, the product comprises a reagent, a kit or a drug.

[0033] Specifically, the use, the role of GPR146 as a target point comprises:

[0034] the GPR146 is dominantly expressed in the cell membrane of HASMCs of a human; and / or,

[0035] the GPR146 is highly expressed in a hypertensive mouse model; and / or,

[0036] the GPR146 is highly expressed in a hypertensive human blood vessel model.

[0037] The application also discloses a use of an inhibitor for inhibiting the expression of GPR146 in the preparation of a drug for preventing and / or treating hypertension.

[0038] Specifically, the inhibitor can inhibit the increase of the synthesis phenotype of SMCs; the inhibitor can treat hypertension by promoting the transformation of SMCs from the synthesis phenotype to the contraction phenotype.

[0039] The application also discloses a drug for preventing and / or treating hypertension-related diseases, wherein an effective component of the drug comprises a substance for inhibiting the expression of GPR146 or knocking down the expression of GPR146.

[0040] Optionally, the substance comprises a chemical small-molecule drug, a nucleic acid drug and / or an antibody drug.

[0041] Specifically, the drug for preventing and / or treating hypertension-related diseases:

[0042] the drug can reduce the expression of GPR146; and / or,

[0043] the drug can inhibit the phenotype transformation of aortic smooth muscle by inhibiting the expression of GPR146; and / or,

[0044] the drug can reduce the blood pressure level by inhibiting the expression of GPR146.

[0045] The application also discloses application of a reagent for determining GPR146 expression in preparation of a detection product for preventing and treating hypertension-related diseases.

[0046] The application also provides a transgenic mouse model in a research model or treatment of a pathogenesis of hypertension.

[0047] The application relates to application of GPR146 in treatment of hypertension, and comprehensively applies whole-transcriptome sequencing, functional experiments and animal models to first propose that smooth muscle GPR146 is related to hypertension, and further discuss a specific regulation mechanism of the GPR146 gene in occurrence and development of hypertension, thereby providing new insights into a pathogenesis of hypertension.

[0048] The application provides a method for preventing and / or treating hypertension, which comprises administering an inhibitor for inhibiting GPR146 expression to a subject in need.

[0049] In some embodiments, the inhibitor can inhibit an increase in a synthetic phenotype of SMCs; the inhibitor can treat hypertension by promoting transformation of SMCs from a synthetic phenotype to a contractile phenotype.

[0050] In some embodiments, the inhibitor for inhibiting GPR146 expression is a drug capable of reducing GPR146 expression; and / or,

[0051] The inhibitor for inhibiting GPR146 expression can inhibit phenotype transformation of aortic smooth muscle by inhibiting GPR146 expression; and / or,

[0052] The inhibitor for inhibiting GPR146 expression can reduce a blood pressure level by inhibiting GPR146 expression.

[0053] The application finds that high expression of GPR146 can induce phenotype transformation of SMCs, and then cause vascular remodeling and blood pressure increase, and then first finds GPR146 which can directly promote increase in blood pressure. The GPR146 can provide a new molecular target for preventing or treating hypertension, and guide screening and research and development of related drugs.

[0054] The application also finds that GPR146 can play an important role in a pathogenesis of hypertension by regulating phenotype transformation of VSMCs. GPR146 has great potential as a new target for treating hypertension, and in the future, a drug or a gene therapy strategy for GPR146 can be developed to prevent and treat hypertension, thereby providing a potential target for clinical diagnosis and treatment of hypertension.

[0055] The application studies and verifies that a GPR146 gene-specific knock-in mouse is similar to an early pathological process of a primary hypertension patient, thereby providing a new research tool for a pathogenesis of hypertension and drug treatment.

[0056] The present application uses SMC-specific deletion and overexpression mice to clarify whether GPR146 is involved in blood pressure and vascular remodeling regulation and the potential mechanism, and to verify the specific role of GPR146 gene in the occurrence and development of hypertension. BRIEF DESCRIPTION OF DRAWINGS

[0057] In order to make the content of the present application more easily understood, the present application will be further described in detail below according to specific embodiments of the present application and in conjunction with the accompanying drawings, wherein,

[0058] Figure 1 is the result of whole transcriptome sequencing (RNA-seq) after high hydrostatic pressure (200mmHg) treatment;

[0059] Figure 2 is the result of western blot detection of GPR146 protein and qPCR detection of GPR146 RNA expression level in HASMCs, WKY and SHR rats, AngII-induced mouse hypertension and its control model under different hydrostatic pressure culture (100mmHg, 200mmHg);

[0060] Figure 3 is the result of immunofluorescence and immunohistochemical detection of GPR146 expression changes in blood vessels of different hypertension model animals;

[0061] Figure 4 is the result of blood pressure monitoring of conditional knock-in GPR146 mice based on implantable telemetry blood pressure technology and vascular tension detection by vascular tension meter;

[0062] Figure 5 is the result of pathological detection of H&E staining and Masson staining of conditional knock-in GPR146 mice under the stimulation of AngII in various levels of blood vessels;

[0063] Figure 6 is the result of blood pressure monitoring of conditional knock-in GPR146 mice based on implantable telemetry blood pressure technology and vascular tension detection by vascular tension meter;

[0064] Figure 7 is the result of pathological detection of H&E staining and Masson staining of conditional knock-in GPR146 mice under the stimulation of AngII in various levels of blood vessels;

[0065] Figure 8 is the result of constructing GPR146 overexpression plasmid and control plasmid, constructing GPR146 knockdown siRNA and corresponding control siRNA, and expressing the result of transfecting overexpression plasmid and siRNA in HASMCs, respectively;

[0066] Figure 9 is the result of increasing blood pressure in mice by using AngII treatment for 5 days, tail vein injection of GPR146 neutralizing antibody and control antibody. DETAILED DESCRIPTION

[0067] The experimental methods in the following examples of the present application, unless otherwise specified, are generally conventional methods in the art, according to the conditions recommended by the reagent manufacturers.

[0068] The experimental supplies involved in the following example schemes of the present application mainly include the following categories.

[0069] 1. Experimental animals and cells

[0070] (1) Experimental animals and feeding

[0071] 8-10 weeks old, 20-30 g in weight, and genetic background of C57BL / 6 mice (WT, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.). The mice used in the study were all specific pathogen free (SPF) mice.

[0072] GPR146 gene mice in C57BL / 6 background were constructed using CRISPR / Cas9 technology (purchased from Jisui Yaoke Biotechnology (Beijing) Co., Ltd.).

[0073] All animal experiments were approved by the Animal Protection and Use Committee of the National Center for Cardiovascular Disease, Fu Wai Hospital, Chinese Academy of Medical Sciences. All mice in the experiment were raised in a sterile environment, with no more than 6 mice per cage, 12 hours of light and dark alternation, noise less than 60 dB, temperature control at 20-26℃, humidity maintained at 50%-60%, and sufficient food and water provided. The bedding of the cages was changed every week to ensure environmental hygiene. All animal experiments were in accordance with the relevant standards and ethical regulations for experimental animals and have been approved by the Animal Care and Use Committee of the Animal Center.

[0074] (2) Experimental cells

[0075] The human aortic smooth muscle cell line (Human Aortic Smooth Muscle Cells, HASMCs) was purchased from the Cell Resource Center of Basic Medical Sciences, Peking Union Medical College, Chinese Academy of Medical Sciences.

[0076] In the following examples of the present application, Figures 3, 4, 5, 6, 7, and 9 are the results of experiments using experimental animals; Figures 1 and 8 are the results of experiments using cells; and Figure 2 includes both animal experiment results and cell experiment results.

[0077] 2. Cell culture related reagents

[0078] SMC culture medium was purchased from SciencCell Company, DMEM high glucose medium and trypsin were purchased from Sigma Company of the United States. Fetal bovine serum (FBS) was purchased from Hyclone Company of the United States. Penicillin and streptomycin were purchased from North China Pharmaceutical Co., Ltd.

[0079] 3. Experimental reagents and instruments

[0080] Main reagents

[0081] Angiotensin II (AngII, Cat# ab120183; purchased from Abeam, UK and stored at -20℃); Dimethylsulfoxide (DMSO, Cat# D2660; purchased from Sigma, USA); Bovine serum albumin (BSA, Cat# A3059; purchased from Sigma, USA); Dulbecco's Modified Eagle Medium (DMEM, Cat# C11995500BT; purchased from Gibco, USA); Fetal bovine serum (FBS, Cat# 10091-148; purchased from Gibco, USA); Phosphate buffered saline (PBS, Cat# 20012017; purchased from Thermo scientific, USA); TRIzol reagent (Cat# 10296010; purchased from Thermo Fisher Scientific, USA); Protein loading marker (Cat# 26616, 26619; purchased from Thermo Fisher Scientific, USA); Penicillin, streptomycin (Cat# 15070063; purchased from Thermo Fisher Scientific, USA); Blocking reagent (Cat# P1622-500; purchased from Proliferate, Beijing, China); Methanol (purchased from Beijing Chemical Plant, China); Glycine (Cat# 56-40-6; purchased from Sigma-Aldrich, USA); Ammonium persulfate (Cat# A82100; purchased from Shanghai Jituo Biotech Co., Ltd.); TEMED (Cat# 1610800; purchased from BioRad, USA); DAB substrate (Cat# ZLI-1097; purchased from Zhongshanjinqiao, China); Ponceau S (Cat# 2611-82-7; purchased from Sigma-Aldrich, USA); Nitrocellulose membrane (Cat# 101; purchased from Millpore, USA); Sodium chloride (powder; purchased from Modern Oriental Technology Co., Ltd.); 4% paraformaldehyde (Cat# P0099; purchased from Biyun Tian Biotech Co., Ltd.); Chloroform (Cat# C44710; purchased from Shanghai Jituo Biotech Co., Ltd.); Isopropyl alcohol (Cat# 67-63-0; purchased from Sigma-Aldrich, USA); Absolute ethanol (Cat# 64-17-5; purchased from Beijing Chemical Plant, China); Phosphate buffered saline (PBS, Cat# P5439; purchased from Sigma-Aldrich, USA); Sodium dodecyl sulfate (SDS, Cat# 151-21-3; purchased from Sigma-Aldrich, USA); Acrylamide (Cat# 01696;DAPI staining solution (product number: D9542; purchased from Sigma-Aldrich, USA); Green Supermix (product number: 170880; purchased from Bio-Rad, USA); Triton X-100 (product number: P0095; purchased from Biyun Tian Biotechnology Co., Ltd., China); Tris-HCl (pH 6.0) (product number: ST768; purchased from Biyun Tian Biotechnology Co., Ltd., China); Tris-HCl (pH 8.0) (product number: ST780; purchased from Biyun Tian Biotechnology Co., Ltd., China); Tween-20 (product number: 9005-64-5; purchased from Biyun Tian Biotechnology Co., Ltd., China); Blocking goat serum working solution (product number: ZLI-9002; purchased from Beijing Zhongshanjinqiao Biotechnology Co., Ltd., China); EDTA antigen repair solution (50x) (product number: P0085; purchased from Biyun Tian Biotechnology Co., Ltd., China); Membrane regeneration solution (product number: P1651; purchased from Beijing Puli Lei Gene Technology Co., Ltd., China).

[0082] Main instruments

[0083] The main equipment involved in the examples of the present application is shown in Table 1 below.

[0084] Table 1 Main instrument equipment

[0085] Main antibodies

[0086] GPR146 antibody (product number: ab117104); β-actin antibody (product number: ab8226); Collagen I antibody (product number: ab34710); calponin antibody (product number: ab46794); α-SMA antibody (product number: ab7817, ab5694); PCNA (product number: ab18197); p-ERK antibody (product number: ab65142); t-ERK antibody (product number: ab214036).

[0087] Main kits

[0088] BCA kit (purchased from Thermo Fisher Scientific, USA); High-sig ECL Western Blotting Substrate (purchased from Tanon, China); High-purity total RNA rapid extraction kit (catalog number: RP1202, purchased from Wuxi Biotek, China); Reverse transcription kit (catalog number: K1622, purchased from Thermo scientific, USA).

[0089] Other materials and chemical reagents

[0090] Cell culture dishes, T25 cell culture bottles, T75 cell culture bottles, 6-well plates, 96-well enzyme-labeled plates, centrifuge tubes, pipettes, pipettes, etc. were purchased from Corning, USA.

[0091] Protease inhibitor (catalog number: B14001), phosphatase inhibitor (catalog number: B15001) were purchased from Bimake, Shanghai, China.

[0092] In the following example scheme of the present application, statistical analysis was performed using GraphPad Prism 8.0 software. Unless otherwise specified, numerical values are expressed as mean ± standard deviation. All data were subjected to normality test and homogeneity of variance test. If the data pass the above tests, t-test is used for comparison between two groups. If the data do not pass the above tests, Mann-Whitney U test is used for comparison between groups. P<0.05 is considered to be significantly different between groups, which is statistically significant.

[0093] Example 1 RNA-seq sequencing

[0094] Cell preparation: Cells before P8 generation were plated for use.

[0095] RNA extraction: Add TRIzol to the culture plate directly to lyse the cells. For cell suspension, collect the cells by centrifugation, and then add TRIzol to lyse the cells. Incubate the homogenate at room temperature for 10 minutes to completely separate the nucleic acid protein complex. If the sample contains more protein, fat, polysaccharide or extracellular material, centrifuge at 10,000 x g for 10 minutes at 2-8°C, take the supernatant for the next step, add chloroform and shake vigorously, and then incubate at room temperature for 2-3 minutes. Centrifuge at 10,000 x g for 15 minutes at 4°C, and the sample is divided into three layers: yellow organic phase, colorless water phase and middle layer. Transfer the water phase to a new centrifuge tube, and add isopropanol to precipitate RNA. Add 0.5 ml of isopropanol for every 1 ml of TRIzol, and incubate at room temperature for 10 minutes. Centrifuge at 10,000 x g for 10 minutes at 4°C, and a gelatinous precipitate can be seen on the side and bottom of the tube. Wash the RNA precipitate with 75% ethanol, and then centrifuge at 4°C for 5 minutes. Dry the RNA precipitate at room temperature, add an appropriate amount of RNase-free water to dissolve the RNA, detect the RNA concentration, and send the sample.

[0096] Library construction and analysis: Library preparation. Including steps such as RNA fragmentation, primer addition, end repair, adapter addition, PCR amplification, etc. to ultimately form a library that can be used for high-throughput sequencing. Library quality control. The concentration and insert size of the library are detected by Qubit, Agilent 2100, etc. to ensure the quality of the library. Sequencing. The library is sequenced on high-throughput sequencing platforms such as Illumina to obtain a large amount of sequence read data. Data analysis. Including alignment, assembly, filtering, normalization, etc. of sequencing data, ultimately for quantifying gene expression.

[0097] In this embodiment, the experimental results involved are shown in Figure 1. The results show that the expression of GPR146 in smooth muscle cells under high hydrostatic pressure is significantly increased.

[0098] Example 2 Hydrostatic pressure cell culture

[0099] Cell preparation

[0100] 1) HASMCs were cultured in a special medium containing 2% FBS and cell growth factors, and placed in a 37°C, 5% carbon dioxide, humidified cell culture box.

[0101] 2) When the cell density reaches 80%-90%, plate. Generally, plate at a ratio of 1:3. Plant the cells in a 6-well plate, gently shake the culture plate to distribute the cells evenly, and carefully place it in the incubator for incubation.

[0102] Hydrostatic pressure culture

[0103] 1) Cells with good adhesion and growth density at 60%-70% were placed in a 37°C pressure incubator with pre-UV sterilization, and the cells were marked differently according to different pressure chambers.

[0104] 2) Check the airtightness of different pressure chambers.

[0105] 3) Adjust the air valve according to the pre-designed gas flow rate, and inject binary gas containing 95% oxygen and 5% carbon dioxide mixture into different pressure chambers.

[0106] 4) Observe the pressure change, and make the instruments of the two pressure culture chambers show 100mmHg and 200mmHg respectively.

[0107] 5) Collect cells after 24h.

[0108] In this example, the experimental results are shown in Figures 1 and 2.

[0109] As shown in Figure 1, the expression of GPR146 in smooth muscle cells under high hydrostatic pressure was significantly increased.

[0110] As shown in Figure 2, the expression of GPR146 in smooth muscle cells under high hydrostatic pressure (200mmHg) was significantly increased.

[0111] Example 3 Construction of a hypertensive animal model

[0112] In this example, a mouse AngII osmotic pump was implanted subcutaneously to induce a mouse hypertensive model.

[0113] Dissolve AngII (angiotensin II) powder: The required dose of AngII for each mouse is 1000ng / kg / min, calculate the required amount of AngII powder, and prepare unopened sterile saline. Weigh the required amount of AngII powder with an intelligent electronic scale, add it to an appropriate amount of saline, and mix slowly to ensure complete dissolution.

[0114] Inject ALZET 2004 osmotic pump: Wear gloves and a mask, open the package of ALZET 2004 osmotic pump. Wipe the surface of the ALZET 2004 osmotic pump with disinfectant alcohol to ensure that the osmotic pump is sterile. Remove the ALZET 2004 osmotic pump with tweezers, and inject the dissolved AngII drug into the injection hole of the ALZET 2004 osmotic pump with a sterile syringe. Inject slowly to avoid the generation of bubbles. Invert the osmotic pump in a centrifuge tube containing sterile saline, and incubate in a 37°C water bath overnight.

[0115] Preparation of ALZET 2004 osmotic pump: Take out the ALZET 2004 osmotic pump filled with the drug and wipe the outside with alcohol cotton ball for thorough disinfection.

[0116] Implantation of mice: ALZET 2004 osmotic pump is implanted into the subcutaneous adipose tissue of mice. Sterile ophthalmic scissors, forceps, hemostats, suture needles, needle holders, sutures, etc. that have been autoclaved are prepared. The mice are anesthetized with 1% sodium pentobarbital intraperitoneal injection before the operation. The mice are placed on a sterile operating table, their limbs are fixed with adhesive tape, and the surgical site is cleaned with disinfectant alcohol. A small incision is made on the back of the mouse near the neck, the subcutaneous tissue of the mouse is gently separated, the ALZET 2004 osmotic pump is inserted, and the incision is carefully sutured.

[0117] Disinfection and observation: After the operation is completed, the surgical site is wiped with a sterile cotton ball to ensure aseptic conditions. The behavior and physiological condition of the mice are observed, and after 30 minutes, the mice are left alone after observing that they have no obvious discomfort.

[0118] The state of the mice is observed twice a day in the morning and afternoon. After 28 days of pump implantation, the intervention is completed, and all mice are taken for sampling. During the induction of the model, if a mouse dies, immediate necropsy is required, and the cause of death is observed and recorded by exploratory laparotomy.

[0119] Blood vessel sampling: Prepare the materials and instruments required for perfusion (sterile syringes, needles, surgical instruments, physiological saline, etc.). Anesthetize the mice with 500 mg / ml sodium pentobarbital. The limbs of the anesthetized mouse are fixed to the dissection plate with adhesive tape, and 75% disinfectant alcohol is sprayed on the skin of the mouse's abdominal cavity for disinfection. The skin of the chest is cut with surgical scissors, the sternum is cut, the chest cavity is opened, and the heart is exposed. Carefully cut the right atrial appendage, and blood will flow out at this time. Use a needle to puncture the left ventricle of the heart, connect the needle to the syringe, and slowly perfuse physiological saline into the left ventricle to push the blood out of the aorta, while expelling air and coagulated blood from the blood. The color of the mouse's liver, spleen, and other organs gradually lightens during perfusion. Replace the perfusion solution with pre-cooled 4% paraformaldehyde (PFA) solution and slowly perfuse for about 2 minutes to fix the sample.

[0120] Carefully cut the lung, thymus tissue with surgical scissors, cut the diaphragm, and use a sterile cotton swab to help remove the liver, spleen, and gastrointestinal tissues. Use ophthalmic scissors to carefully separate the aortic tissue, renal artery, superior mesenteric artery, and their secondary branches by tightly attaching to the sternum. Place the separated heart and vascular tissue in a silicone dish containing sterile physiological saline, carefully remove the surrounding excess adipose tissue under a body microscope, and rinse off the blood clots attached to the surface, then place it in 4% PFA for fixation.

[0121] Vessel fixation: Place the vessels in 4% PFA fixative solution and fix at room temperature for 12 hours. After fixation, rinse the tissues in water for 30 minutes. Place the tissues in a Leica automatic tissue dehydrator for dehydration and clearing, then immerse the tissues in molten paraffin, usually 56-60°C paraffin, for 1 hour until the tissues are completely immersed in paraffin. Place the paraffin-impregnated tissues in a tissue embedding machine and heat in 60°C paraffin to gradually impregnate the tissues. After the paraffin solidifies, perform paraffin sectioning, and cut the embedded tissues into 5-micron sections on a rotary microtome, keeping the blade clean and cooling it before each section. Attach the sections to glass slides and store in a slide box for subsequent staining and other experiments.

[0122] In this embodiment, the experimental results involved are shown in Figures 3, 4, 5, 6, 7, and 9.

[0123] Example 4 Tissue Staining

[0124] Hematoxylin-eosin staining (HE staining)

[0125] Remove the paraffin-embedded tissue sections and deparaffinize and clear the tissue sections with xylene. Dehydrate with anhydrous ethanol, 95% ethanol, 70% ethanol, and distilled water, immersing the tissue sections in each concentration of ethanol for 2-3 minutes and then in distilled water for 1-2 minutes. Stain the tissue sections by immersing them in hematoxylin solution for 5 minutes, then rinse the tissue sections in distilled water for 30 seconds; stain the tissue sections by immersing them in eosin solution for 3 minutes, then rinse the tissue sections in distilled water for 30 seconds. Dehydrate and clear with 95% ethanol, anhydrous ethanol, and xylene. Immerse the tissue sections in each concentration of ethanol for 2-3 minutes and then in xylene for 2-3 minutes. Mount the tissue sections with mounting medium, then observe and photograph them under a microscope.

[0126] Immunofluorescence staining

[0127] Antigen retrieval: Dilute 50x EDTA antigen retrieval solution with deionized water to make 1x EDTA antigen retrieval solution. Place the antigen retrieval solution in a boiling pot, then place the slides in the boiling antigen retrieval solution for 2 minutes, then cool the antigen retrieval solution with running water, and take out the slides and wash them with TBS for 3 times, 3 minutes each time. Endogenous enzyme removal: Block the activity of endogenous peroxidase by incubating with 3% H2O2 at room temperature for 10 minutes, then wash the slides with TBS for 3 times, 3 minutes each time. Blocking: Circle the tissue blocks on the slides with an immunohistochemical pen, and make sure not to touch the tissue to avoid damaging the tissue structure and affecting the subsequent staining. Block the slides with 5% goat serum containing 0.3% Triton X-100 at room temperature for 1 hour. Wash the slides with TBS for 3 times, 3 minutes each time. Primary antibody incubation: Carefully suck the diluted primary antibody with a pipette and gently drop it on the tissue blocks, and incubate overnight at 4°C. The concentration of the primary antibody is generally 1: 100-1: 500. The next day, warm the slides in a 37°C incubator for 30 minutes, then wash them with PBS for 3 times, 3 minutes each time. Secondary antibody incubation: Incubate the diluted fluorescently labeled anti-rabbit or anti-mouse secondary antibody (1: 1000) slowly on a shaker for 1 hour, and wash with PBS for 3 times, 3 minutes each time. DAPI restain the cell nucleus and mount the slides, and then image the slides using a laser scanning confocal microscope after mounting.

[0128] MASSON staining

[0129] Antigen retrieval: Dilute 50x EDTA antigen retrieval solution with deionized water to make 1x EDTA antigen retrieval solution. Place the antigen retrieval solution in a boiling pot, then place the slides in the boiling antigen retrieval solution for 2 minutes, then cool the antigen retrieval solution with running water, and take out the slides and wash them with TBS for 3 times, 3 minutes each time. Endogenous enzyme removal: Block the activity of endogenous peroxidase by incubating with 3% H2O2 at room temperature for 10 minutes, then wash the slides with TBS for 3 times, 3 minutes each time. Blocking: Circle the tissue blocks on the slides with an immunohistochemical pen, and make sure not to touch the tissue to avoid damaging the tissue structure and affecting the subsequent staining. Block the slides with 5% goat serum containing 0.3% Triton X-100 at room temperature for 1 hour. Wash the slides with TBS for 3 times, 3 minutes each time. Primary antibody incubation: Carefully suck the diluted primary antibody with a pipette and gently drop it on the tissue blocks, and incubate overnight at 4°C. The concentration of the primary antibody is generally 1: 100-1: 500. The next day, warm the slides in a 37°C incubator for 30 minutes, then wash them with PBS for 3 times, 3 minutes each time. Secondary antibody incubation: Incubate the diluted fluorescently labeled anti-rabbit or anti-mouse secondary antibody (1: 1000) slowly on a shaker for 1 hour, and wash with PBS for 3 times, 3 minutes each time. DAPI restain the cell nucleus and mount the slides, and then image the slides using a laser scanning confocal microscope after mounting.

[0130] SIRIUS RED staining

[0131] Antigen retrieval: Dilute 50x EDTA antigen retrieval solution with deionized water to make 1x EDTA antigen retrieval solution. Place the antigen retrieval solution in a boiling pot, then place the slides in the boiling antigen retrieval solution for 2 minutes, then cool the antigen retrieval solution with running water, and take out the slides and wash them with TBS for 3 times, 3 minutes each time. Endogenous enzyme removal: Block the activity of endogenous peroxidase by incubating with 3% H2O2 at room temperature for 10 minutes, then wash the slides with TBS for 3 times, 3 minutes each time. Blocking: Circle the tissue blocks on the slides with an immunohistochemical pen, and make sure not to touch the tissue to avoid damaging the tissue structure and affecting the subsequent staining. Block the slides with 5% goat serum containing 0.3% Triton X-100 at room temperature for 1 hour. Wash the slides with TBS for 3 times, 3 minutes each time. Primary antibody incubation: Carefully suck the diluted primary antibody with a pipette and gently drop it on the tissue blocks, and incubate overnight at 4°C. The concentration of the primary antibody is generally 1: 100-1: 500. The next day, warm the slides in a 37°C incubator for 30 minutes, then wash them with PBS for 3 times, 3 minutes each time. Secondary antibody incubation: Incubate the diluted fluorescently labeled anti-rabbit or anti-mouse secondary antibody (1: 1000) slowly on a shaker for 1 hour, and wash with PBS for 3 times, 3 minutes each time. DAPI restain the cell nucleus and mount the slides, and then image the slides using a laser scanning confocal microscope after mounting.

[0132] In this embodiment, the experimental results of H&E staining, immunofluorescence staining, MASSON staining and related experiments are shown in Figures 3, 5 and 7, respectively.

[0133] As shown in Figure 3, the expression of GPR146 in the blood vessels of different hypertension model animals was detected by immunofluorescence and immunohistochemistry. The results showed that, compared with the normal blood pressure group, the GPR146 staining in the left renal artery and mesenteric artery of different hypertension model mice (spontaneous hypertension rats / AngII-induced hypertensive mice) was significantly increased, indicating that the expression of GPR146 in the blood vessels of hypertensive animals was significantly increased. The immunohistochemical results showed that the positive staining of GPR146 in the smooth muscle layer of the internal mammary artery of hypertensive patients (HTN) was significantly higher than that of the normal blood pressure group (NTN), indicating that the expression of GPR146 in the blood vessels of hypertensive patients was significantly increased. The immunohistochemical results showed that the positive staining of GPR146 in the 3rd branch of the blood vessels of normal mice was significantly higher than that in the 1st and 2nd branches, indicating that the expression of GPR146 in the 3rd branch of the blood vessels was significantly increased.

[0134] As shown in Figure 5, the pathological detection results of the blood vessels of the conditional knock-in GPR146 mice under the stimulation of AngII were detected by H&E staining and Masson staining. The results showed that, compared with the control group, the Gpr146 Cre / + The ratio of media / tube of the left renal artery and mesenteric artery of the mice was significantly increased; the collagen staining of the blood vessels was deepened, indicating that the collagen deposition was significantly increased; the Gpr146 Cre / + The heart hypertrophy of the mice was more severe compared with the control group, and the collagen staining of the heart was increased, indicating that the collagen deposition in the heart was more severe.

[0135] As shown in Figure 7, the pathological detection results of the blood vessels of the conditional knock-in GPR146 mice under the stimulation of AngII were detected by H&E staining and Masson staining. The results showed that, compared with the control group, the Gpr146

[0136] Example 5: Detection of vascular tension function

[0137] The liquid preparation system of 500 ml in this embodiment is as follows:

[0138] 10×kreb B solution: NaHCO3 10.5 g;

[0139] 10×kreb A solution: NaCl 34.615 g, KCl 1.768 g, KH2PO4 0.812 g, MgSO4.7H2O 1.469 g, CaCl2 1.407 g.

[0140] The liquid required for the experiment is (1L for example): 100ml A + 100ml B + 2.18g glucose, make up to 1L, pass binary gas half an hour in advance.

[0141] The instruments required for the experiment are: silica gel tray, microscissors, stereoscope, ice box, acupuncture needle, tungsten wire.

[0142] Take the material: anesthetize the mouse or cause death by cervical dislocation; take the blood vessels of the corresponding part of the mouse (superior mesenteric artery or its branches, aorta, renal artery, etc.) and place them on the ice box to keep the blood vessels active.

[0143] Go to the machine, the specific operation steps are as follows:

[0144] 1) Separate the fat around the blood vessels of the corresponding part to avoid pulling and damaging the blood vessels;

[0145] 2) Use tungsten wire to pass through the lumen of the corresponding blood vessel and fix it in the corresponding channel of the machine;

[0146] 3) Fix the blood vessels in other channels in the same way;

[0147] 4) After fixing the blood vessels in all channels, change the liquid and balance for 15 minutes, change the liquid;

[0148] 5) Slowly adjust the diastolic pressure (1mN for branches, 3mN for superior mesenteric artery, and 5mN for aorta);

[0149] 6) Change the liquid after the diastolic pressure is stable, and balance for 15 minutes;

[0150] 7) Change the liquid and give high K (4M) after it is stable;

[0151] 8) After stabilization, wash three times, balance for 15 minutes, record the balance value, and change the liquid;

[0152] 9) After stabilization, give phenylephrine (PE) from low concentration to high concentration (10 -9 ~ 10 -5 mol / L), record the corresponding values after giving different concentrations;

[0153] 10) Give acetylcholine (Ach) from low concentration to high concentration (10 -9 ~ 10 -5 mol / L), record the corresponding values after giving different concentrations;

[0154] 11) Wash, change the liquid, balance for 15 minutes, change the liquid;

[0155] 12) Give the highest concentration in 9, record the corresponding values;

[0156] 13) After stabilization, sodium nitroprusside (SNP) was administered from low to high concentration (10 -9 ~10 -5 mol / L) and the corresponding values were recorded after stabilization at different concentrations;

[0157] 14) Data analysis.

[0158] In this example, the vascular tension function mainly detects the contraction and relaxation function of the blood vessels, and the experimental results are shown in the blood vessel function part of FIG. 4 and FIG. 6.

[0159] As shown in FIG. 4, the results of changes in blood pressure and vascular function of conditional knock-in GPR146 mice compared with controls under basal and AngII stimulation; the results show that, whether in the basal state (the upper 4 figures) or in the AngII induced state (the lower 4 figures), smooth muscle specific knock-in GPR146 can significantly increase blood pressure, promote PE induced vasoconstriction, and weaken SNP induced vasodilation.

[0160] As shown in FIG. 6, the results of changes in blood pressure and vascular function of conditional knock-in GPR146 mice compared with controls under basal and AngII stimulation; the results show that, whether in the basal state (the upper 4 figures) or in the AngII induced state (the lower 4 figures), smooth muscle specific knock-in GPR146 can significantly increase blood pressure, promote PE induced vasoconstriction, and weaken SNP induced vasodilation.

[0161] Example 6 Implant telemetry blood pressure monitoring

[0162] One day before surgery:

[0163] 1) High pressure sterilization and disinfection of surgical instruments and related supplies;

[0164] 2) Detect the power of the implant and disinfect it;

[0165] 3) Start fasting half a day (can fast the night before surgery, but give water) anesthesia and implantation process.

[0166] Operation:

[0167] 1) Fix the mouse on the mouse board under the stereoscope, fully expose the surgical field;

[0168] 2) Cut the skin with ophthalmic scissors, carefully separate the thymus and meat tissue with microforceps, and fully expose the carotid artery;

[0169] 3) Carefully pass 3 pieces of 5-0 silk braided thread through the carotid artery, 1 piece is ligated at the distal end of the carotid artery to block the blood supply, 1 piece is passed through the incision edge of the proximal end, and the third piece is passed through the middle of the other two threads, and the knot is pretreated;

[0170] 4) Take hemostatic forceps to pull the two lines in front and back to block the blood supply, and add a small amount of physiological saline to keep the blood vessels moist;

[0171] 5) Take the hook needle with the left hand, and hold the implant with the right hand;

[0172] 6) When the implant is inserted, do not release the blood flow at the proximal end at this time, so as not to cause the implant to be impacted out due to excessive blood flow. Tie the middle line, and then slowly release the blood supply at the proximal end to insert the implant into the corresponding position;

[0173] 7) At this time, tie the two lines at the proximal end to fix the implant;

[0174] 8) Use the hemostatic forceps to bluntly separate, so as to better put the implant into the mouse body;

[0175] 9) After blunt separation, inject about 500ul of physiological saline to maintain the balance of the mouse body fluid;

[0176] 10) Restore the position of the mouse thymus, and suture the wound with a 4-0 suture needle;

[0177] 11) Put the mouse on the heating pad to make the mouse wake up faster.

[0178] Postoperative recovery:

[0179] 1) Isolated in a disinfected clean cage to ensure the ambient temperature;

[0180] 2) Observe the recovery state of the mouse every day;

[0181] 3) Data collection can be started 5 days after the operation.

[0182] In this example, the blood pressure changes of the mouse were mainly detected, and the experimental results are shown in the blood pressure monitoring part of FIG. 4 and FIG. 6.

[0183] As shown in FIG. 4, the blood pressure changes of the smooth muscle-specific knock-in GPR146 mouse under the basic and AngII stimulation are shown. The results show that the smooth muscle-specific knock-in GPR146 can significantly increase the blood pressure under the basic state and the AngII-induced state.

[0184] As shown in FIG. 6, the blood pressure changes of the smooth muscle-specific knock-in GPR146 mouse under the basic and AngII stimulation are shown. It can be seen that the smooth muscle-specific knock-in GPR146 significantly reduces the high blood pressure under the basic state and the AngII-induced state.

[0185] Example 7 Subculture of Human Aortic Smooth Muscle Cells

[0186] Human aortic smooth muscle cells were cultured in SMCM medium containing 2% FBS, 100 U / ml penicillin / streptomycin and smooth muscle cell growth factor at 37 °C in a humidified cell culture incubator with 5% carbon dioxide. When the cell density reached 80-90%, the cells were passaged. The liquid in the culture medium was discarded and the cells were gently washed with sterile PBS for 3 times. The culture dish was added with 0.25% trypsin for digestion, and the culture dish was gently shaken to make the trypsin fully contact with the cells. The digestion time was generally 2 minutes, and the state was observed under a microscope. When most of the cells were rounded, the digestion was terminated by adding the culture medium containing 10% FBS. The cells were gently blown off from the culture dish with a pipette, and the cell suspension was placed in a sterile centrifuge tube and centrifuged at 1000 rpm for 5 minutes. The centrifuge tube was taken out, the supernatant was discarded, and the culture medium containing SMCM was added. The cells were gently blown apart with a pipette, and then the cells were sucked into the culture dish with a pipette. The required culture medium was supplemented in the culture dish, and the cells were generally passaged at a ratio of 1:3. The culture dish was gently shaken to make the cells evenly distributed, and then carefully placed in the incubator for culture.

[0187] The cell experiments involved in the experimental results were the cells cultured in this example, and the experimental results involved were shown in FIG. 1, FIG. 2 and FIG. 8.

[0188] Example 8 Extraction and quantification of protein

[0189] In this example, the total protein was extracted by lysing the tissues and cells with RIPA lysis buffer (R0010, Solarbio, Beijing, China) added with protease inhibitor (B14001, bimake) and phosphatase inhibitor (B15001, bimake). The protein concentration was determined by BCA protein assay kit (23227, Thermo scientific, MA, USA). The proteins were separated by 8-12% sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membrane (1620113, Bio-Rad, USA). The membrane was blocked with 5% skim milk in TBST (50 mM Tris-HCl, 150 mM NaCl and 0.1% Tween 20) at room temperature for 1 hour. Then, the membrane was incubated with the primary antibody at 4 °C overnight. After washing with TBST for 3 times, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody (1:10000) at room temperature for 1 hour. Finally, the chemiluminescence was performed using ECL reagent.

[0190] Cell protein extraction

[0191] Discard the supernatant of the cells in the 6-well plate, gently wash twice with pre-cooled PBS, and then add 100 μl of RIPA lysis buffer to which protease inhibitors and phosphatase inhibitors have been added in advance. After adding the lysis buffer, gently shake the 6-well plate to ensure that the lysis buffer is in full contact with the cells, and keep the temperature low to prevent protein degradation. Carefully scrape the cells with a sterile cell scraper, and then use a pipette to transfer the lysed cell solution to a sterile 1.5 ml EP tube. Incubate for 30 minutes in an ice box. During the lysis process, use a pipette to beat the cells to ensure that the cells are fully lysed. Place the lysed cells in a centrifuge and centrifuge at 4°C and 12000 rpm for 10 minutes. Then, aspirate the supernatant from the EP tube and transfer it to a new sterile EP tube for subsequent experimental operations or storage at -80°C.

[0192] Protein quantification

[0193] Protein quantification was performed using a BCA kit. Prepare the standard: dilute the 5 mg / ml protein standard with PBS to 0.5 mg / ml. Prepare the working solution: mix the A and B solutions in the kit at a ratio of 50:1, for example, take 10 ml of A solution in a clean centrifuge tube, add 200 μl of B solution, and then gently shake to mix well. Prepare a clean 96-well plate, and use a pipette to add 0, 1, 2, 4, 8, 12, 16, and 20 μl of standard solution to one column of the 96-well plate, respectively, and then add sterile PBS to each well to make the total volume of each well 20 μl. Therefore, from top to bottom, the concentration of the standard in each well is 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / ml, respectively. In another column of wells, add an appropriate volume of sample and supplement with PBS to 10 μl. Then, add 100 μl of BCA mixed working solution to each well of all the above wells, mix gently, and place in a 37°C incubator for 30 minutes. Take out the 96-well plate and use a microplate reader to measure the OD value at 562 nm. According to the results, draw a standard curve and calculate the concentration of the measured protein. Add 1 / 5 volume of 5x SDS-loading buffer to the protein solution, mix well, heat at 100°C in a metal bath for 10 minutes, and then store in a -80°C freezer for later use.

[0194] In this example, protein extraction and quantification are mainly for WB experiments to provide protein extraction and quantification, and the experimental results are shown in FIG. 2 and FIG. 8.

[0195] Example 9 Immunoblotting

[0196] The glue system of this embodiment is as follows: prepare 10% AP (ready to use): weigh 0.1 g of ammonium persulfate with an electronic scale, put it into a clean EP tube, add 1 ml of deionized water, vortex and shake to dissolve thoroughly. Prepare 10% SDS: weigh 10.0 g of SDS with an electronic scale, then add 100 ml of deionized water to dissolve thoroughly, and store at room temperature after preparation.

[0197] In this embodiment, the corresponding concentrated glue (see Table 2 below) and separation glue (see Table 3 below) are prepared according to the glue preparation scheme.

[0198] Table 2 5% concentrated glue (12 ml)

[0199] Table 3 separation glue system

[0200] Wash the glass plate clean, carefully add the prepared separation glue between the two glass plates, the volume of the separation glue is generally 7-8 ml, and the volume of the concentrated glue is 3-4 ml. After adding the separation glue, press the line with anhydrous ethanol on the upper layer, and pay attention not to leak. After standing for 3-4 minutes, discard the anhydrous ethanol on the upper layer, add the concentrated glue, and insert a 1 cm comb, and stand for 3 minutes.

[0201] Electrophoresis

[0202] Prepare 1×SDS-PAGE electrophoresis buffer (ready to use): weigh 28.8 g of glycine, 6 g of Tris-base, and 2 g of SDS with an electronic scale, add 2 L of deionized water to dissolve thoroughly. Loading: in the wells of the SDS-PAGE gel, add the same mass of protein, generally 25-50 μg. The lanes with large volume difference or empty lanes are supplemented with loading buffer. Add 5 μl of marker to the first column, and supplement the volume with loading buffer. Connect the current, set the starting voltage to 90 V, when the sample moves from the concentrated glue to the separation glue, adjust the voltage to 120 V, and cut off the power when the target protein moves to the appropriate position.

[0203] Electrotransfer

[0204] Prepare 1×electrotransfer liquid: weigh 28.8 g of glycine, 6 g of Tris-base, and 400 ml of methanol with an electronic scale, add deionized water to make up to 2 L, and dissolve thoroughly. Carefully take the SDS-PAGE gel from the glass plate, handle gently, and pay attention not to damage the SDS-PAGE gel. Place the gel and NC membrane with sandwich method, i.e. black clip-sponge-filter paper-gel-NC membrane-filter paper-sponge-white clip. Place the clip with the gel and NC membrane in the electrotransfer tank, and add enough electrotransfer liquid. Place the electrotransfer tank in an ice box, and add enough ice to maintain a low temperature. Generally, the protein is electrotransferred at 200 mA for 2 hours.

[0205] Seal

[0206] Seal milk powder solution was configured according to the following Table 4: 2.5 g of skim milk powder was weighed with an electronic balance and added to a 50 ml clean centrifuge tube, then 50 ml of 1x TBST solution (Table 4) was added, vortexed and shaken to mix well.

[0207] Table 4 Configuration of 1x TBST Buffer (1L)

[0208] After the electrotransfer was completed, the NC membrane was removed, the blank part of the edge beyond the glue was trimmed off, and the NC membrane was immersed in 1x Lysol red dye for 2 minutes. Whether there was a band was observed to determine whether the protein was successfully transferred to the NC membrane. If the membrane transfer was successful, continue to wash with TBST solution for 3 times, 5 minutes each time. Then the NC membrane was placed in the skim milk powder blocking solution and incubated on a shaker at room temperature for 1 hour.

[0209] Primary antibody

[0210] After blocking, the NC membrane was gently washed with 1x TBST, and the primary antibody was diluted according to the instructions. The NC membrane was immersed in the primary antibody and incubated overnight in a 4℃ chromatography cabinet shaker.

[0211] Secondary antibody

[0212] The overnight NC membrane was washed with 1x TBST for 3 times, 10 minutes each time. The labeled secondary antibody was prepared with TBST solution containing 5% skim milk powder, and the general dilution concentration was 1:5000-1:10000. The secondary antibody was incubated at room temperature for 1 hour.

[0213] Development

[0214] After incubation of the secondary antibody, the NC membrane was washed with 1x TBST for 3 times, 10 minutes each time. The membrane was placed in the exposure machine and exposed to light liquid for exposure imaging.

[0215] The results of the immunoblotting technology experiment of this embodiment are shown in Figures 2 and 8.

[0216] As shown in Figure 2, the expression level of GPR146 protein was detected in different hydrostatic pressure cultured (100 mmHg, 200 mmHg) HASMCs, WKY and SHR rats, AngII induced mouse hypertension and its control model. The results showed that the high hydrostatic pressure (200 mmHg) treatment significantly increased the expression of GPR146 protein and RNA; the GPR146 protein of SHR rats was higher than that of WKY group; after AII treatment, the expression of GPR146 increased.

[0217] According to the results shown in FIG. 8, overexpression and knockout of GPR146 at the cellular level can change the expression of smooth muscle phenotype transformation marker proteins. Overexpression of GPR146 in smooth muscle cells increases the expression of PCNA, p-ERK and COL1, and decreases the expression of a-SMA. Knockdown of GPR146 in smooth muscle cells decreases the expression of PCNA, p-ERK and COL1, and increases the expression of a-SMA.

[0218] Example 10 quantitative reverse transcription polymerase chain reaction

[0219] Total RNA extraction: the treated cells were cultured in a 6-well plate, the culture medium was discarded, the cells were washed with pre-cooled PBS for 3 times, the PBS was discarded, 500 μl of TRIzol was added to each well, the cells were scraped on ice with a clean cell scraper, and then the lysis solution was added to a 1.5 ml clean EP tube and lysed on ice for 5 minutes. 200 μl of chloroform was added to each EP tube, and the mixture was inverted to mix well and placed on ice for 10 minutes to completely separate the nuclear protein complex. The EP tube was placed in a pre-cooled 4°C centrifuge at 12000 rpm for 10 minutes. The EP tube was taken out, and the liquid in the EP tube was divided into 3 layers: the upper layer was the aqueous phase, the middle layer was the protein layer, and the lower layer was the organic phase, and the RNA was present in the upper aqueous phase. The upper aqueous phase was carefully aspirated into a new clean EP tube with a pipette, being careful not to aspirate the middle protein layer. 500 μl of isopropanol (isopropanol was pre-cooled on ice) was added. After standing for 10 minutes, centrifugation was performed at 4°C and 13000 rpm for 10 minutes. The supernatant was discarded, 1 ml of 75% ethanol was added, and the RNA precipitate was carefully washed and mixed, then centrifuged at 4°C and 12000 rpm for 10 minutes. The supernatant was discarded, and the RNA precipitate was air-dried at room temperature for 5-10 minutes. An appropriate amount of DEPC water (usually 20-50 μl) was added to the EP tube to dissolve the RNA precipitate, which was gently blown with a gun head to promote its complete dissolution. The concentration and purity of the RNA were determined by spectrophotometry. DEPC water was used as a control for measurement, and the measured RNA concentration was recorded for subsequent use. The purity of the RNA was determined by the ratio of the optical density values (OD 260 / 280) at 260 nm and 280 nm. Generally, when the ratio of OD 260 / 280 is 1.8-2.0, the purity of the RNA is very high.

[0220] Reverse transcription: reverse transcription kit TaKaRa was used for reverse transcription. First, the genomic DNA was removed, and the proportion was prepared according to Table 5, and then it was placed in a PCR instrument, and the parameters were set as follows: 42°C, 2 minutes.

[0221] Table 5 reverse transcription system

[0222] The RNA was reverse transcribed into cDNA, and the proportion was prepared according to Table 6.

[0223] Table 6 Reverse transcription system

[0224] Put into PCR instrument, set parameters: 37℃, 15 minutes; 85℃, 5 seconds, get cDNA product, can be stored at -20℃ for standby.

[0225] RT-qPCR: qRT-PCR was performed using Select Master Mix, and the reaction solution system was prepared according to the following Table 7 scheme (20 μl).

[0226] Table 7 qRT-PCR system

[0227] The PCR Forward Primer and the PCR Reverse Primer used in the present application were synthesized by Shanghai Yunxu Biological Co., Ltd.

[0228] The reaction mixture was sucked into a 96-well enzyme-free PCR plate, covered with an enzyme-free PCR sealing film, and then carefully scraped flat with a PCR scraper. The PCR plate was placed in a centrifuge for instantaneous centrifugation for 3 minutes to make the reaction solution all located at the bottom of the hole. When loading, 3 replicate wells were set in each well. The 96-well plate was placed in a VII7 PCR instrument, and the reaction conditions were set as follows: 95℃, 10 minutes (pre-denaturation); 95℃, 15 seconds; 60℃, 30 seconds; 72℃, 30 seconds (40 cycles); 95℃, 15 seconds; 60℃, 1 minute; 95℃, 15 seconds (melting curve). The internal reference was GAPDH, and the expression of the target gene in the control group was calculated as 1, and the expression of the target gene in the experimental group was calculated by 2 -ΔΔCt .

[0229] The experimental results of the present example are shown in the PCR results in Figure 2. The results show that the expression of GPR146 is significantly increased by giving high hydrostatic pressure (200 mmHg) treatment.

[0230] Example 11 Plasmid and siRNA transfection

[0231] Cell plating

[0232] Cell plating is usually performed one day before transfection, and plating is performed after counting according to the cell growth rate, so that the cell density reaches about 80%-90% on the next day of transfection.

[0233] Cell transfection

[0234] Take lip3000 as a transfection reagent for example, replace the cell culture medium (without double antibody and serum) before transfection, dilute the plasmid or siRNA with opti-MEM, and dilute lip3000 with opti-MEM. After mixing and standing for 5 minutes, mix them in the same tube. Avoid rough blowing during mixing (because it may cause the failure of liposome). Stand for 15 minutes. Add the prepared transfection solution to the cell culture plate and shake gently to help the transfection reagent (such as lip3000). Because the transfection reagent has certain cytotoxicity, it is necessary to replace the medium (replace the serum-free medium with complete medium) after transfecting the plasmid or siRNA into the cells for 6 hours.

[0235] Detection

[0236] After successful transfection for 24 hours, the cells were taken out of the incubator, the protein was collected, and the subsequent related detection was carried out.

[0237] In this embodiment, the experimental results involved are shown in Figure 8. The results show that overexpression or knockout of GPR146 can change the smooth muscle cell type transformation.

[0238] Example 12 GPR146 neutralizing antibody reduces AII-induced blood pressure in mice

[0239] The preparation process of mouse Gpr146 monoclonal antibody is as follows.

[0240] First stage: antigen research. Protein localization: located in the nuclear membrane.

[0241] Second stage: antigen expression vector construction, expression verification GPR146. Protein antigen: adopt extracellular design, select mouse GPR146 extracellular region as the target protein for recombinant expression, add signal peptide at the N-terminus to guide secretion expression, and add hFc tag at the rear end of the signal peptide to facilitate purification, and use mammalian 293 suspension cells as the host for recombinant expression.

[0242] Third stage: immunization and polyclonal titer evaluation. Immunize rabbits with the recombinant antigen obtained in the second stage, including immunization route, adjuvant and booster immunization strategy. After the third and fourth immunization, the rabbit serum will be analyzed to determine the responding animals. The serum will be evaluated based on antigen ELISA detection.

[0243] Fourth stage: antibody purification. Positive responding rabbits determined in the third stage provide peripheral blood (at the same time, the spleen of the positive responding rabbit is subjected to differential screening of B cells to enrich the frozen storage for subsequent preparation and development of recombinant rabbit monoclonal antibodies), and the purification of rabbit polyclonal antibodies is completed.

[0244] Fifth stage: single B cell screening and sequencing. The frozen B cells determined in the fourth stage are screened and sequenced by single B cell culture detection and antibody gene sequence amplification to determine the positive single B cell antibody sequence.

[0245] Sixth stage: antibody recombinant expression and identification. Based on the antibody gene sequence obtained in the sixth stage, the recombinant antibody vector is constructed and expressed in the E. coli system. After the recombinant antibody is purified, ELISA is used for screening and titer evaluation to determine the optimal antibody clone.

[0246] AngII-induced hypertensive mouse model preparation: The hypertensive mouse model constructed in Example 3 was injected with GPR146 neutralizing antibody (anti-GPR146) via tail vein, and the mice were treated with AngII for 5 days to increase the blood pressure of the mice; the GPR146 neutralizing antibody and the control antibody were administered to the mice via tail vein injection.

[0247] In this example, the experimental results are shown in Figure 9. The results show that the GPR146 neutralizing antibody group significantly reduces the AngII-induced increase in blood pressure compared with the control group.

[0248] In summary, the GPR146 can provide a new molecular target for preventing or treating hypertension, guiding the screening and development of related drugs.

[0249] Obviously, the above examples are only examples for the purpose of clarity, and are not intended to limit the embodiments. Based on the above description, those skilled in the art can also make other different forms of changes or variations. Here, it is not necessary and impossible to exhaust all the embodiments. The obvious changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. Use of GPR146 as a target for preparing a medicament for preventing and / or treating a hypertension-related disease.

2. Use according to claim 1, characterized in that, The effects of the GPR146 as a target include: Overexpression of GPR146 enhances mouse microvascular constriction and impairs microvascular dilation; and / or, Knockout of GPR146 impairs microvascular constriction and enhances microvascular dilation; and / or, Overexpression of GPR146 promotes mouse vascular remodeling and increases collagen content; and / or, Knockout of GPR146 inhibits mouse vascular remodeling and decreases collagen content; and / or, Administration of a GPR146 monoclonal antibody treatment can treat the mouse blood pressure increase caused by angiotensin; and / or, Administration of a GPR146 monoclonal antibody treatment can inhibit or reduce the mouse vascular remodeling and collagen content caused by angiotensin.

3. Use of GPR146 as a target for preparing a product having at least one of the following (1)-(5) effects: (1) a product for monitoring the treatment condition of a hypertension-related disease population; (2) a product for monitoring the prognosis of a hypertension-related disease population; (3) a product for preventing and / or treating a hypertension-related disease; (4) a product for screening a target related to a hypertension-related disease; (5) a product for screening a hypertension-related disease medicament.

4. Use according to claim 3, characterized in that, The product includes a reagent, a kit or a medicament.

5. Use according to claim 3 or 4, characterized in that, The effects of the GPR146 as a target include: The GPR146 is predominantly expressed in the cell membrane of human HASMCs; and / or, The GPR146 is highly expressed in a hypertensive mouse model; and / or, The GPR146 is highly expressed in a hypertensive human vascular model.

6. Use of an inhibitor for inhibiting the expression of GPR146 in preparing a medicament for preventing and / or treating hypertension.

7. Use according to claim 6, characterized in that, The inhibitor can inhibit the increase of SMCs synthetic phenotype; the inhibitor treats hypertension by promoting the transformation of SMCs from a synthetic phenotype to a contractile phenotype.

8. A medicament for preventing and / or treating a hypertension-related disease, characterized by, The effective component of the medicament includes a substance for inhibiting the expression of GPR146 or knocking down the expression of GPR146; Preferably, the substance includes a chemical small molecule drug, a nucleic acid drug and / or an antibody drug.

9. The medicament for preventing and / or treating a hypertension-related disease according to claim 8, wherein: The medicament is a drug capable of reducing the expression of GPR146; and / or, The medicament inhibits the phenotype transformation of aortic smooth muscle by inhibiting the expression of GPR146; and / or, The medicament reduces the blood pressure level by inhibiting the expression of GPR146.

10. Use of a reagent for measuring the expression amount of GPR146 in preparing a detection product for preventing and treating a hypertension-related disease.

11. A method for preventing and / or treating hypertension, comprising administering an inhibitor for inhibiting the expression of GPR146 to a subject in need.

12. The method according to claim 11, wherein the inhibitor can inhibit the increase of SMCs synthetic phenotype; the inhibitor treats hypertension by promoting the transformation of SMCs from a synthetic phenotype to a contractile phenotype.

13. The method according to claim 12, wherein the inhibitor for inhibiting the expression of GPR146 is a drug capable of reducing the expression of GPR146; and / or, The inhibitor that inhibits the expression of GPR146 inhibits the phenotypic transformation of aortic smooth muscle by inhibiting the expression of GPR146; and / or, The inhibitor that inhibits the expression of GPR146 reduces the blood pressure level by inhibiting the expression of GPR146.

14. Use according to claim 1, characterized in that, The role of GPR146 as a target point includes: Overexpression of GPR146 enhances microvascular constriction and weakens microvascular dilation; and / or, Knockout of GPR146 weakens microvascular constriction and enhances microvascular dilation; and / or, Overexpression of GPR146 promotes vascular remodeling and increases collagen content; and / or, Knockout of GPR146 inhibits vascular remodeling and reduces collagen content; and / or, Treatment with GPR146 monoclonal antibodies can treat the blood pressure increase caused by angiotensin; and / or, Treatment with GPR146 monoclonal antibodies can inhibit or reduce the vascular remodeling and collagen content caused by angiotensin. GPR146 is highly expressed in a hypertensive human vascular model.

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