Application of GPR146 as a target in the preparation of drugs for the treatment of hypertension

Abstract

The present invention relates to the field of biomedical technology, and specifically to a use of GPR146 as a target for preparing a method for preventing or treating hypertension-related diseases. The present invention relates to the use of GPR146 in treating hypertension. The present invention comprehensively applies transcriptome sequencing, functional experiments, and animal models, and for the first time proposes that GPR146 in smooth muscle is closely related to hypertension, and deeply explores the specific regulatory mechanism of GPR146 in the occurrence and development of hypertension, providing new insights into the pathogenesis of hypertension.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the use of GPR146 as a target for preparing a drug for preventing or treating hypertension-related diseases. Background Art

[0002] At present, hypertension has become a global disease that affects more than 1 billion people. Studies have shown that hypertension is the most important risk factor for cardiovascular disease. As blood pressure increases, the risk of cardiovascular disease also increases accordingly. At the same time, hypertension is also an important cause, risk factor and major cause of death for a variety of cardiovascular and cerebrovascular diseases, and its pathogenesis is very complex.

[0003] Hydrostatic pressure refers to the pressure of blood flow perpendicular to the blood vessel wall per unit area. It is the basic condition for the formation of blood pressure. Hypertension is a disease characterized by increased hydrostatic pressure. More and more studies suggest that increased hydrostatic pressure can directly lead to the occurrence of a variety of cardiovascular diseases. Endothelial cells and smooth muscle cells are the main components of the blood vessel wall. Existing studies have shown that high hydrostatic pressure treatment significantly affects the morphology of endothelial cells, with endothelial cells significantly elongated and actin filaments rearranged; it can further induce 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 blood vessel wall. At present, there is little research on the role of hydrostatic pressure in the regulation of smooth muscle function. In smooth muscle cells, how the body converts the mechanical signal of hydrostatic pressure into intracellular chemical signals to cause vascular dysfunction is still unclear.

[0004] G-protein-coupled receptors (GPCRs) are the largest cell membrane receptor superfamily in the human body, containing more than 800 members and expressed in various organ systems. Angiotensin type I receptor (AT1R) can be directly activated under mechanical stress, and this effect is independent of angiotensin II (AngII). Further in-depth studies have found that the lack of surface AT1R can eliminate myogenic vasoconstriction in mesenteric arteries. When ECs are stimulated by shear force, bradykinin B2 receptors directly change their conformation, and its activation contributes to vasodilation. Histamine H1 receptors in ECs can sense blood flow-mediated shear force and promote vasodilation. Under shear force stimulation, GPR68 induces a large amount of calcium ion release, promotes ECs to release NO and causes vasodilation. Inhibition of S1PR1 can block shear force-induced ECs rearrangement. Studies have shown that abnormal activation of GPCRs may lead to hypertension and vascular dysfunction. Angiotensin II type 1 receptor (AGTR1) and angiotensin II type 2 receptor (AGTR2) of the renin-angiotensin-aldosterone system, as well as kinin receptors of the kallikrein system, regulate blood pressure by regulating sodium retention in the kidney and vascular tension. Certain short-chain fatty acids such as propionate and butyrate can affect the function of intestinal cells by acting on GPCRs in the intestine, such as GPR41 and GPR43, GPR109a and OLF78, thereby affecting vascular tension and blood pressure levels in the body. In short, GPCRs play an important role in mechanical signal transduction, maintenance of vascular tension and regulation of vascular function.

[0005] In order to find the GPCRs that play an important role in smooth muscle cells under high hydrostatic pressure, whole transcriptome sequencing was used to detect the expression of GPCRs in smooth muscle cells after high hydrostatic pressure treatment. It was found that the expression of GPR146 was significantly increased. Subsequently, genetically modified mice were used to clarify the role of GPR146 in smooth muscle in blood pressure regulation. Finally, neutralizing antibodies against GPR146 were used to detect its role in the treatment of hypertension. Summary of the invention

[0006] Therefore, the technical problem to be solved by the present invention is to provide an application of GPR146 as a target in the treatment of hypertension-related diseases. The present invention proposes for the first time that GPR146 is related to hypertension, and deeply explores the specific regulatory mechanism of GPR146 in the occurrence and development of hypertension, providing new insights into the pathogenesis of hypertension.

[0007] The second technical problem to be solved by the present invention is to provide a product for preventing or treating hypertension-related diseases based on detecting the expression level of Gpr146.

[0008] In order to solve the above technical problems, the present invention discloses a use of GPR146 as a target for preparing a drug for preventing and / or treating hypertension-related diseases.

[0009] Specifically, the use, the role of GPR146 as a target includes:

[0010] Overexpression of GPR146 enhances microvascular contraction and weakens microvascular relaxation in mice; and / or,

[0011] Knockout of GPR146 attenuates microvascular contraction and enhances microvascular relaxation; and / or,

[0012] Overexpression of GPR146 promotes vascular remodeling and increases collagen content in mice; and / or,

[0013] Knockout of GPR146 inhibits vascular remodeling and reduces collagen content in mice; and / or,

[0014] GPR146 monoclonal antibody treatment can treat angiotensin-induced hypertension in mice; and / or,

[0015] Treatment with GPR146 monoclonal antibody can inhibit or reduce vascular remodeling and collagen content in mice induced by angiotensin.

[0016] The present invention also discloses a use of GPR146 for preparing a product having at least one of the following effects (1) to (5):

[0017] (1) Products for monitoring the treatment status of people with hypertension-related diseases;

[0018] (2) Products for prognosis monitoring of populations with hypertension-related diseases;

[0019] (3) Products for preventing and / or treating hypertension-related diseases;

[0020] (4) Products that screen for targets associated with hypertension-related diseases;

[0021] (5) Screening products for drugs for hypertension-related diseases.

[0022] Specifically, the product includes a reagent, a kit or a medicine.

[0023] Specifically, the use, the role of GPR146 as a target includes:

[0024] The GPR146 is predominantly expressed in the cell membrane of human HASMCs; and / or,

[0025] The GPR146 is highly expressed in a hypertensive mouse model; and / or,

[0026] The GPR146 is highly expressed in a hypertensive human vascular model.

[0027] The present invention also discloses use of an inhibitor for inhibiting the expression of GPR146 in preparing a drug for preventing and / or treating hypertension.

[0028] Specifically, the inhibitor can inhibit the increase of the synthetic phenotype of SMCs; the inhibitor treats hypertension by promoting the transformation of SMCs from a synthetic phenotype to a contractile phenotype.

[0029] The present invention also discloses a drug for preventing and / or treating hypertension-related diseases, wherein the active ingredient of the drug includes a substance that inhibits or knocks down the expression of GPR146;

[0030] Preferably, the substance includes chemical small molecule drugs, nucleic acid drugs and / or antibody drugs.

[0031] Specifically, the drug for preventing and / or treating hypertension-related diseases:

[0032] The drug is a drug capable of reducing the expression of GPR146; and / or,

[0033] The drug inhibits the phenotypic transformation of aortic smooth muscle by inhibiting the expression of GPR146; and / or,

[0034] The drug reduces blood pressure levels by inhibiting the expression of GPR146.

[0035] The present invention also discloses the use of a reagent for determining the expression amount of GPR146 in preparing a detection product for preventing and treating hypertension-related diseases.

[0036] The present invention also provides a transgenic mouse model for research or treatment of the pathogenesis of hypertension.

[0037] The present invention relates to the application of GPR146 in treating hypertension. The present invention comprehensively applies methods such as whole transcriptome sequencing, functional experiments and animal models, and for the first time proposes that smooth muscle GPR146 is associated with hypertension, and deeply explores the specific regulatory mechanism of the GPR146 gene in the occurrence and development of hypertension, providing new insights into revealing the pathogenesis of hypertension.

[0038] The present invention found that high expression of GPR146 can induce SMCs phenotypic transformation, leading to vascular remodeling and increased blood pressure, and thus discovered for the first time that GPR146 can directly promote increased hypertension. The GPR146 can provide a new molecular target for the prevention or treatment of hypertension, and guide the screening and development of related drugs.

[0039] The present invention also found that GPR146 may play an important role in the pathogenesis of hypertension by regulating the phenotypic transformation of VSMCs. GPR146 has great potential as a new target for the treatment of hypertension. In the future, drugs or gene therapy strategies targeting GPR146 may be developed to prevent and treat hypertension, providing potential targets for clinical diagnosis and treatment of hypertension.

[0040] The present invention studies and verifies that the early pathological process of GPR146 gene-specific knock-in mice is similar to that of patients with essential hypertension, which provides a new research tool for the pathogenesis and drug treatment of hypertension.

[0041] The present invention uses SMC-specific deletion and overexpression mice to clarify whether GPR146 is involved in the regulation of blood pressure and vascular remodeling and the underlying mechanism, and verifies the specific role of the GPR146 gene in the occurrence and development of hypertension. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0043] Figure 1 The whole transcriptome sequencing (RNA-seq) results after high hydrostatic pressure (200 mmHg) treatment;

[0044] Figure 2 Western blot was used to detect GPR146 protein in HASMCs cultured at different hydrostatic pressures (100 mmHg, 200 mmHg), WKY and SHR rats, AngII-induced mice with hypertension and their control models, and qPCR was used to detect GPR146 RNA expression levels;

[0045] Figure 3 Immunofluorescence and immunohistochemistry were used to detect the expression of GPR146 in blood vessels of different hypertensive model animals;

[0046] Figure 4 Conditional knock-in GPR146 mice were used to monitor blood pressure using implantable telemetry and vascular tension using a vascular tension meter.

[0047] Figure 5 H&E staining and Masson staining were used to detect the pathological results of blood vessels at all levels in conditional knock-in GPR146 mice under AngII stimulation;

[0048] Figure 6 Conditional knockout of GPR146 mice were used to monitor blood pressure using implantable telemetry and vascular tension using a vascular tension meter.

[0049] Figure 7 H&E staining and Masson staining were used to detect the pathological results of blood vessels at all levels in conditional knockout GPR146 mice under AngII stimulation;

[0050] Figure 8 To construct GPR146 overexpression plasmid and control plasmid, siRNA for knockdown of GPR146 and corresponding control siRNA were constructed, and the expression results of overexpression plasmid and siRNA were transfected in HASMCs respectively;

[0051] Fig. 9 The mice were treated with AngII for 5 days and the GPR146 neutralizing antibody and control antibody were injected into the tail vein to increase the blood pressure of the mice. DETAILED DESCRIPTION

[0052] The experimental methods in the following examples of the present invention without specifying specific conditions are generally conventional methods in the art and are carried out according to the conditions recommended by the reagent manufacturers.

[0053] In the following embodiments of the present invention, the experimental supplies involved mainly include the following categories.

[0054] 1. Experimental animals and cells

[0055] (1) Experimental animals and their breeding

[0056] Mice of 8-10 weeks old, weighing 20-30 g, with a genetic background of C57BL / 6 (WT, purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.) were used in the study. All mice used in the study were specific pathogen-free (SPF) mice.

[0057] CRISPR / Cas9 technology was used to construct GPR146 gene mice in the C57BL / 6 background (purchased from Jicui Pharmaceutical Biotechnology (Beijing) Co., Ltd.).

[0058] All animal experiments were approved by the Animal Care and Use Committee of the National Center for Cardiovascular Diseases, Fuwai Hospital, Chinese Academy of Medical Sciences. All mice were kept in a sterile environment, with no more than 6 mice per cage, 12-hour light and dark alternation, noise below 60 dB, temperature controlled at 20-26°C, humidity maintained at 50%-60%, and adequate food and water provided. The bedding of the cages was changed weekly to ensure environmental hygiene. All animal experiments complied with the relevant standards and ethical regulations for experimental animals and were approved by the Animal Care and Use Committee of the Animal Center.

[0059] (2) Experimental cells

[0060] Human aortic smooth muscle cells (HASMCs) were purchased from the Cell Resource Center of the Basic Medical College, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College.

[0061] In the following embodiments of the present invention, Figure 3 , 4 , 5, 6, 7, and 9 are the results of experiments using experimental animals; Figure 1 , 8 The experimental results of cells used in the experiment; Figure 2 It includes both animal and cell experiment results.

[0062] 2. Cell culture related reagents

[0063] SMC culture medium was purchased from SciencCell, DMEM high glucose culture medium and trypsin were purchased from Sigma, USA. Fetal bovine serum (FBS) was purchased from Hyclone, USA. Penicillin and streptomycin were purchased from North China Pharmaceutical Co., Ltd.

[0064] 3. Experimental related reagents and instruments

[0065] Main reagents

[0066] Angiotensin II (AngII, Catalog No.: ab120183; purchased from Abcam, UK, and stored at -20°C); Dimethyl sulfoxide (DMSO, Catalog No.: D2660; purchased from Sigma, USA); Bovine serum albumin (BSA, Catalog No.: A3059; purchased from Sigma, USA); Dulbecco's Modified Eagle Medium (DMEM, Catalog No.: C11995500BT; purchased from Gibco, Massachusetts, USA); Fetal bovine serum (FBS, Catalog No.: 10091-148; purchased from Gibco, USA); Phosphate buffered saline for cell culture (PBS, Catalog No.: 20012017; purchased from Thermo scientific, USA); TRIzol reagent (Catalog No.: 10296010; purchased from Thermo Fisher Scientific, USA) Scientific Company); protein loading marker (Cat. No.: 26616, 26619; purchased from Thermo Fisher Scientific, USA); penicillin, streptomycin (Cat. No.: 15070063; purchased from Thermo Fisher Scientific, USA Scientific Company); skim milk for blocking (item number: P1622-500; purchased from Beijing Pulilai Company); methanol (purchased from Beijing Chemical Plant, China); glycine (item number: 56-40-6; purchased from Sigma-Aldrich Company, USA); ammonium persulfate (item number: A82100; purchased from Shanghai Jizhi Biochemical Technology Co., Ltd., China); TEMED (item number: 1610800; purchased from BioRad Company, USA); DAB color developing solution (item number: ZLI-1097; purchased from Zhongshan Jinqiao Company, China); Ponceau powder (item number: 2611-82-7; purchased from Sigma-Aldrich, USA); nitrocellulose membrane (item number: 101; purchased from Millpo, USA re Company); sodium chloride (powder; purchased from China Modern Oriental Technology Co., Ltd.); 4% paraformaldehyde (catalog number: P0099; purchased from China Biyuntian Biotechnology Co., Ltd.); chloroform (catalog number: C44710; purchased from Shanghai Jizhi Biochemical Technology Co., Ltd., China); isopropanol (catalog number: 67-63-0; purchased from Sigma-Aldrich Company, USA); anhydrous ethanol (catalog number: 64-17-5; purchased from Beijing Chemical Plant, China); phosphate buffered saline (PBS, catalog number: P5439; purchased from Sigma-Aldrich Company, USA); sodium dodecyl sulfate (SDS, catalog number: 151-21-3; purchased from Sigma-Aldrich Company, USA); acrylamide (catalog number: 01696;Purchased from Sigma-Aldrich, USA); DAPI staining solution (Cat. No.: D9542; purchased from Sigma-Aldrich, USA); Green Supermix (Cat. No. 170880; purchased from Bio-Rad, USA); immunostaining permeabilization solution (Triton X-100) (Cat. No. P0095; purchased from Bioyuntian Biotechnology Co., Ltd., China); Tris-HCl (pH 6.0) (Cat. No. ST768; purchased from Bioyuntian Biotechnology Co., Ltd., China); Tris-HCl (pH 8.0) (Cat. No. ST780; purchased from Bioyuntian Biotechnology Co., Ltd., China); Tween-20 (Cat. No. 9005-64-5; purchased from Bioyuntian Biotechnology Co., Ltd., China); blocking goat serum working solution (Cat. No. ZLI-9002; purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., China); EDTA antigen retrieval solution (50×) (Cat. No. P0085; purchased from Bioyuntian Biotechnology Co., Ltd., China); membrane regeneration solution (Cat. No. P1651; purchased from Beijing Prilai Gene Technology Co., Ltd., China).

[0067] Main instruments

[0068] The main equipment involved in the following embodiments of the present invention are shown in Table 1 below.

[0069] Table 1 Main instruments and equipment

[0070]

[0071]

[0072] Primary Antibodies

[0073] GPR146 antibody (Cat. No.: ab117104); β-actin antibody (Cat. No.: ab8226); Collagen I antibody (Cat. No.: ab34710); calponin antibody (Cat. No.: ab46794); α-SMA antibody (Cat. No.: ab7817, ab5694); PCNA (Cat. No.: ab18197); p-ERK antibody (Cat. No.: ab65142); t-ERK antibody (Cat. No.: ab214036).

[0074] Main kit

[0075] 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 Biotech Biotechnology Co., Ltd., China); reverse transcription kit (catalog number: K1622; purchased from Thermo scientific, USA).

[0076] Other materials and chemicals

[0077] Cell culture dishes, T25 cell culture flasks, T75 cell culture flasks, 6-well plates, 96-well ELISA plates, centrifuge tubes, pipettes, and transfer pipettes were all purchased from Corning, USA.

[0078] Protease inhibitors (Cat. No. B14001) and phosphatase inhibitors (Cat. No. B15001) were purchased from Shanghai Bimake Company, China.

[0079] In the following embodiment scheme of the present invention, GraphPad Prism 8.0 software was used for statistical analysis. Unless otherwise specified, the values ​​are expressed as mean ± standard deviation. All data were tested for normality and homogeneity of variance. If the data passed the above test, the t test was used for comparison between the two groups. If the data did not pass the above test, the Mann-Whitney U test was used for inter-group comparison. P < 0.05 was considered to be significantly different between the groups and statistically significant.

[0080] Example 1 RNA-seq sequencing

[0081] Cell preparation: Cells before P8 were plated for later use.

[0082] RNA extraction: Add TRIzol directly to the culture plate to lyse the cells. Cell suspensions need to be centrifuged to collect cells and then add TRIzol for lysis. Place the homogenized sample at room temperature for 10 minutes to allow the nucleic acid-protein complex to be completely separated. If the sample contains more protein, fat, polysaccharide or extracellular material, centrifuge at 10,000×g for 10 minutes at 2-8°C, take the supernatant for the next step, add chloroform and shake vigorously, and place at room temperature for 2-3 minutes. Centrifuge at 10,000×g for 15 minutes at 4°C. The sample is divided into three layers: yellow organic phase, colorless aqueous phase and middle layer. Transfer the aqueous phase to a new centrifuge tube and add isopropanol to precipitate RNA. Add 0.5ml of isopropanol for every 1ml of TRIzol used and place at room temperature for 10 minutes. Centrifuge at 10,000×g for 10 minutes at 4°C. At this time, 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. Place the dried 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.

[0083] Library construction and analysis: Library preparation. Including RNA fragmentation, adding primers, end repair, adding adapters, PCR amplification and other steps, finally forming a library that can be used for high-throughput sequencing. Library quality control. The concentration and insert size of the library are tested by Qubit, Agilent 2100 and other equipment 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 length data. Data analysis. Including alignment, assembly, filtering, normalization, etc. of sequencing data, which is ultimately used to quantify gene expression levels.

[0084] In this embodiment, the experimental results involved are as follows Figure 1 The results showed that the expression of GPR146 in smooth muscle cells was significantly increased under high hydrostatic pressure.

[0085] Example 2 Hydrostatic Cell Culture

[0086] Cell preparation

[0087] 1) HASMCs were cultured in a special culture medium containing 2% FBS and cell growth factors in a humidified cell culture incubator at 37°C and 5% carbon dioxide.

[0088] 2) When the cell density reaches 80%-90%, plate the cells. Generally, plate the cells at a ratio of 1:3. Plant the cells in a 6-well plate, gently shake the culture plate horizontally to make the cells evenly distributed, and carefully place it in an incubator for static culture.

[0089] Hydrostatic culture

[0090] 1) Place cells that are well attached and have a growth density of 60%-70% in a 37°C pressure incubator that has been sterilized with ultraviolet light in advance, and mark the cells differently according to different pressure chambers.

[0091] 2) Check the air tightness of different pressure chambers.

[0092] 3) Adjust the gas valve and inject a binary gas containing 95% oxygen and 5% carbon dioxide into different pressure chambers according to the pre-designed gas flow rate.

[0093] 4) Observe the pressure changes and make the instruments of the two pressure culture chambers display stable 100 mmHg and 200 mmHg respectively.

[0094] 5) Harvest cells after 24 hours.

[0095] In this embodiment, the experimental results involved are as follows Figure 1 , Figure 2 shown.

[0096] like Figure 1 The results show that the expression of GPR146 in smooth muscle cells was significantly increased under high hydrostatic pressure.

[0097] like Figure 2 The results show that the expression of GPR146 in the blood vessels of animals treated with high hydrostatic pressure (200 mmHg) in vitro or in different hypertension models was significantly increased.

[0098] Example 3 Construction of Hypertension Animal Model

[0099] In this example, subcutaneous implantation of mouse AngII osmotic pump was performed to induce a mouse hypertension model.

[0100] Dissolve AngII angiotensin II powder: The AngII dose required for each mouse is 1000ng / kg / min. Calculate the required amount of AngII powder and prepare unopened sterile saline. Weigh the required AngII powder with a smart electronic scale, add it to an appropriate amount of saline, and slowly shake to mix and fully dissolve it.

[0101] Injection into the ALZET 2004 osmotic pump: Wear gloves and a mask and open the package of the ALZET 2004 osmotic pump. Wipe the surface of the ALZET 2004 osmotic pump with disinfectant alcohol to ensure that the osmotic pump is sterile. Take out 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 into a centrifuge tube filled with sterile saline and incubate it in a 37°C water bath overnight.

[0102] Prepare the ALZET 2004 osmotic pump: Take out the ALZET 2004 osmotic pump filled with medication and wipe the outside with an alcohol cotton ball for thorough disinfection.

[0103] Implantation in mice: ALZET 2004 osmotic pumps were implanted into the subcutaneous fat tissue of mice. Prepare sterile ophthalmic scissors, forceps, hemostats, suture needles, needle holders, sutures, etc. that have been autoclaved. Anesthetize mice with 1% sodium pentobarbital intraperitoneally before the operation. Place the mice on a sterile operating table, fix the limbs of the mice with tape, and clean the surgical site with disinfectant alcohol. Make a small incision on the back of the mouse near the neck, gently separate the subcutaneous tissue of the mouse, insert the ALZET 2004 osmotic pump, and carefully suture the incision.

[0104] Disinfection and observation: After the operation is completed, wipe the surgical site with a sterile cotton ball to ensure sterility. Observe the behavior and physiological condition of the mouse. After 30 minutes, observe that the mouse has no obvious discomfort before leaving.

[0105] The status of the mice was observed twice a day in the morning and afternoon. After 28 days of implantation, the intervention was terminated and all mice were sampled. During the model induction period, if a mouse died, an autopsy was performed immediately, and the cause of death was investigated by laparotomy, observed, and recorded.

[0106] Blood vessel sampling: Prepare the materials and instruments required for perfusion (sterile syringes, needles, surgical instruments, saline, etc.). Anesthetize the mouse with 500mg / ml sodium pentobarbital. Fix the limbs of the anesthetized mouse to the dissection board with tape, and spray 75% disinfectant alcohol on the mouse abdominal skin for disinfection. Use surgical scissors to cut the skin of the chest, cut the sternum, open the chest cavity, and expose the heart. Carefully cut the right atrial appendage, and blood will flow out. Use a needle to puncture the left ventricle of the heart. Connect the needle to the syringe and slowly perfuse saline into the left ventricle to push the blood out of the aorta, while expelling the air and coagulation in the blood. During perfusion, the color of the liver and spleen of the mouse gradually becomes lighter. Replace the perfusion fluid with a pre-cooled 4% paraformaldehyde (PFA) solution, and slowly perfuse for about 2 minutes to fix the sample.

[0107] Use surgical scissors to carefully cut off the lung and thymus tissue, cut open the diaphragm, and use sterile cotton swabs to help remove the liver, spleen, gastrointestinal tissue, and other tissues. Use ophthalmic scissors to carefully separate the aortic tissue, renal artery, superior mesenteric artery and its secondary branches close to the sternum. Place the separated heart and vascular tissue in a silicone dish filled with sterile saline, carefully peel off the surrounding excess fat tissue under a stereo microscope, rinse off the blood clots attached to the surface, and then place it in 4% PFA for fixation.

[0108] Vascular fixation: Place the blood vessels in 4% PFA fixative and fix at room temperature for 12 hours. After fixation, rinse the tissue with clean water for 30 minutes. Place the tissue in a Leica fully automatic tissue dehydrator for dehydration and transparency, and then immerse the tissue in molten paraffin, usually 56-60°C paraffin, for 1 hour until the tissue is completely immersed in paraffin. Place the paraffin-infiltrated tissue in a tissue embedding machine and heat it in 60°C paraffin to allow the tissue to be gradually infiltrated. After the paraffin solidifies, perform paraffin sectioning and cut the embedded tissue into 5-micron slices on a rotary slicer. Keep the blade clean during slicing and cool it before each slicing. Attach the slices to a glass slide and store them in a slice box for subsequent staining and other experiments.

[0109] In this embodiment, the experimental results involved are as follows Figure 3 , Figure 4 , Figure 5 , Figure 6 , Figure 7 , Fig. 9 shown.

[0110] Example 4 Tissue staining

[0111] Hematoxylin-eosin staining (HE staining)

[0112] Take out the paraffin-embedded tissue sections, remove the paraffin with xylene and make the tissue sections transparent. Dehydrate with anhydrous ethanol, 95% ethanol, 70% ethanol and distilled water respectively. Soak the tissue sections in each concentration of ethanol for 2-3 minutes, and then soak in distilled water for 1-2 minutes. Soak the tissue sections in hematoxylin solution for 5 minutes and wash the tissue sections with distilled water for 30 seconds; soak the tissue sections in eosin solution for 3 minutes and wash the tissue sections with distilled water for 30 seconds. Dehydrate and make transparent with 95% ethanol, anhydrous ethanol and xylene respectively. Soak the tissue sections in each concentration of ethanol for 2-3 minutes, and then soak in xylene for 2-3 minutes. Seal the tissue sections with sealing media, and then observe and photograph them under a microscope.

[0113] Immunofluorescence staining

[0114] Bake the slices at 68°C for 1 hour on a baking machine, and then dewax the slices to water. Antigen repair: dilute 50× EDTA antigen repair solution with deionized water to 1× EDTA antigen repair solution, put the antigen repair solution into a pressure cooker and boil it, then put the slices neatly on the slice rack and put them into the boiling antigen repair solution for 2 minutes, then cool the antigen repair solution with running water, take out the slices and wash them with TBS 3 times, 3 minutes each time. Removal of endogenous enzymes: After incubating with 3% H2O2 at room temperature for 10 minutes to block the activity of endogenous peroxidase, wash the slices with TBS 3 times, 3 minutes each time. Blocking: Use an immunohistochemistry pen to circle the tissue block on the slide, and be careful not to touch the tissue to avoid damaging the tissue structure and affecting subsequent staining. Block the slices with 5% goat serum containing 0.3% Triton X-100 at room temperature for 1 hour. Wash the slices with TBS 3 times, 3 minutes each time. Primary antibody incubation: Use a pipette to carefully draw the diluted primary antibody and gently drop it on the tissue block, and incubate it at 4°C overnight. The primary antibody concentration is generally 1:100-1:500. The next day, the slices were rewarmed in a 37°C incubator for 30 minutes, and then washed with PBS three times, 3 minutes each time. Secondary antibody incubation: The diluted fluorescently labeled anti-rabbit or anti-mouse secondary antibody (1:1000) was slowly incubated on a shaker for 1 hour, and then washed with PBS three times, 3 minutes each time. DAPI counterstained the cell nucleus and sealed the slices. After sealing, the slices were imaged using a laser scanning confocal microscope.

[0115] Masson staining

[0116] The slices were baked at 68°C for 1 hour on a slide baker, and then the slices were dewaxed and watered in a conventional manner. The tissue slices were stained in a hematoxylin solution for 5 minutes, and the tissue slices were washed with distilled water for 30 seconds; the water on the slides was removed, and the slices were stained with Ponceau red for 5-10 minutes, and the Ponceau red stain was rinsed with running water for about 30 seconds, and phosphomolybdic acid was added for 2-5 minutes, and the slices were observed under a microscope for dedifferentiation; the phosphomolybdic acid was poured off, and aniline blue dye solution was added for 2 minutes, and the slices were rinsed with running water; 95% ethanol, anhydrous ethanol, and xylene were used for dehydration and transparency, respectively. The tissue slices were soaked in each concentration of ethanol for 2-3 minutes, and then soaked in xylene for 2-3 minutes. The tissue slices were sealed with a sealing agent, and then observed and photographed under a microscope.

[0117] Sirius red staining

[0118] Bake the slices at 68℃ for 1 hour on a slide oven, then dewax the slices to water. Dewax the slices to water. Stain the nuclei with hematoxylin for 5 minutes. Dye with Sirius red staining solution at room temperature and away from light for 1 hour. Rinse with anhydrous ethanol (do not use water) to remove the staining solution on the surface of the slices. Seal the slices with neutral gum, then observe and take pictures with a microscope.

[0119] In this embodiment, the experimental results of H&E staining, immunofluorescence staining, MASSON staining, and Sirius red staining are shown in the attached Figure 3 , Figure 5 and Figure 7 shown.

[0120] like Figure 3 As shown, immunofluorescence and immunohistochemistry were used to detect the changes in GPR146 expression in the blood vessels of different hypertensive model animals. The results showed that compared with the normotensive group, the staining of GPR146 in the left renal artery and mesenteric artery in the blood vessels of different hypertensive model mice (spontaneously hypertensive 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 immunohistochemistry 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 normotensive group (NTN), indicating that the expression of GPR146 in the blood vessels of human hypertensive patients was significantly increased; the immunohistochemistry results showed that the positive staining of GPR146 in the 3rd order branches of the blood vessels of normal mice was significantly higher than that of the 1st and 2nd orders, indicating that the expression of GPR146 in the 3rd order branches of the blood vessels was significantly increased.

[0121] like Figure 5 The results of H&E staining and Masson staining were used to detect the pathological changes of blood vessels at all levels in mice with conditional knock-in of GPR146 under AngII stimulation. Cre / + The media / lumen ratio of the left renal artery and mesenteric artery of mice increased significantly; the vascular collagen staining deepened, indicating that collagen deposition increased significantly; Gpr146 Cre / + Compared with the control group, the mice had aggravated cardiac hypertrophy and increased cardiac collagen staining, indicating that the deposition of cardiac collagen was aggravated.

[0122] like Figure 7 The results of H&E staining and Masson staining were used to detect the pathological examination results of blood vessels at all levels in GPR146 knockout mice under AngII stimulation. The results showed that compared with the control group, the media / lumen ratio of the left renal artery and mesenteric artery of Gpr146 knockout mice was significantly reduced; vascular collagen staining was reduced, indicating that collagen deposition was significantly alleviated and the degree of myocardial hypertrophy in mice was reduced; cardiac collagen staining was reduced, indicating that cardiac collagen deposition was reduced.

[0123] Example 5 Vascular tension function detection

[0124] The system of 500ml of liquid preparation in this embodiment is as follows:

[0125] 10×kreb B solution: NaHCO3 10.5g;

[0126] 10×kreb A solution: NaCl 34.615g, KCl 1.768g, KH2PO4 0.812g, MgSO4.7H2O1.469g, CaCl2 1.407g.

[0127] The liquid required for the experiment is (taking 1L as an example): 100ml A + 100ml B + 2.18g glucose, make up to 1L, and pass binary gas half an hour in advance.

[0128] Instruments required for the experiment: silicone plate, microscissors, stereoscope, ice box, acupuncture needle, tungsten wire.

[0129] Sampling: Mice were anesthetized or killed by cervical dislocation; blood vessels from corresponding parts of the mice (superior mesenteric A or its branches, aorta, renal artery, etc.) were taken and placed on an ice box to maintain vascular activity.

[0130] On the machine, the specific steps are as follows:

[0131] 1) Separate the fat around the blood vessels in the corresponding area and avoid pulling to avoid damaging the blood vessels;

[0132] 2) Pass the tungsten wire through the lumen of the corresponding blood vessel and fix it in the corresponding channel of the machine;

[0133] 3) Use the same method to fix the blood vessels in other channels;

[0134] 4) After all the blood vessels in the channels are fixed, change the medium, balance for 15 minutes, and then change the medium;

[0135] 5) Slowly adjust the tension (1mN for branches, 3mN for superior mesenteric artery, 5mN for aorta);

[0136] 6) After the tension is stable, change the solution and balance for 15 minutes;

[0137] 7) Change the solution and give high K (4M) after stabilization;

[0138] 8) After stabilization, wash three times, balance for 15 minutes, record the balance value, and change the solution;

[0139] 9) After stabilization, phenylephrine (PE) was administered, from low concentration to high concentration (10 -9 ~10 -5 mol / L), and record the corresponding values ​​after different concentrations stabilize;

[0140] 10) Give acetylcholine (Ach) from low concentration to high concentration (10 -9 ~10 -5 mol / L), and record the corresponding values ​​after different concentrations stabilize;

[0141] 11) Wash, change the solution, balance for 15 min, and change the solution;

[0142] 12) Give the highest concentration in 9 and record the corresponding value;

[0143] 13) After stabilization, sodium nitroprusside (SNP) was administered, from low concentration to high concentration (10 -9 ~10 -5 mol / L), and record the corresponding values ​​after different concentrations stabilize;

[0144] 14) Data analysis.

[0145] In this embodiment, the vascular tension function mainly detects the contraction and relaxation function of the blood vessels. The experimental results are shown in the attached Figure 4 and attached Figure 6 Partial results of vascular function.

[0146] like Figure 4 These are the changes in blood pressure and vascular function in mice with conditional knock-in of GPR146 compared with controls under basal and AngII stimulation; the results show that smooth muscle-specific knock-in of GPR146 can significantly increase blood pressure, promote PE-induced vasoconstriction, and weaken SNP-induced vasodilation, regardless of the basal state (4 figures above) or AngII-induced state (4 figures below).

[0147] like Figure 6 The results of the conditional knockout of GPR146 mice in the basal state and under AngII stimulation compared with the control group showed that smooth muscle-specific knockout of GPR146 significantly reduced hypertension, weakened PE-induced vasoconstriction, and enhanced SNP-induced vasodilation, regardless of the basal state (4 figures above) or AngII-induced state (4 figures below).

[0148] Example 6 Implant Telemetry Blood Pressure Monitoring

[0149] The day before surgery:

[0150] 1) Autoclave and disinfection of surgical instruments and related supplies;

[0151] 2) Check the implant's charge level and disinfect it;

[0152] 3) Start fasting half a day in advance (you can fast the night before the operation, but you must drink water) for the anesthesia and implantation process.

[0153] Surgical procedures:

[0154] 1) Fix the mouse on a killing board and place it under a stereoscope to fully expose the surgical field;

[0155] 2) Cut the skin with ophthalmic scissors, carefully separate the thymus and muscle tissue with micro forceps, and fully expose the carotid artery;

[0156] 3) Carefully pass three 5-0 silk braided sutures through the carotid artery, tie one at the distal end of the carotid artery to block the blood supply, pass one through the edge of the incision at the proximal end, and pass the third through the middle of the other two sutures, pre-knotted;

[0157] 4) Use hemostatic forceps to pull the front and back threads to block the blood supply, and drip a small amount of normal saline to keep the blood vessels moist;

[0158] 5) Take the hook needle with your left hand and hold the implant with the needle holder in your right hand;

[0159] 6) When the implant is inserted, do not release the blood flow at the proximal end to avoid excessive blood flow that will knock the implant out. Tie a knot in the middle line, then slowly release the blood supply at the proximal end and insert the implant into the corresponding position;

[0160] 7) At this time, tie the two lines at the proximal end to fix the implant;

[0161] 8) Use hemostats to perform blunt separation to better place the implant into the mouse;

[0162] 9) Inject about 500ul of normal saline into the mouse during blunt separation to maintain the body fluid balance of the mouse;

[0163] 10) Restore the thymus of the mouse and suture the wound with a 4-0 suture needle;

[0164] 11) Place the mouse on a heating pad to help it wake up faster.

[0165] Postoperative recovery:

[0166] 1) Keep the animals individually in sterilized, clean cages and keep the ambient temperature constant;

[0167] 2) Observe the recovery status of mice every day;

[0168] 3) Data collection can begin 5 days after surgery.

[0169] In this example, the blood pressure changes of mice were mainly detected. The experimental results are shown in the attached Figure 4 and attached Figure 6 Some results of blood pressure monitoring.

[0170] like Figure 4 These are the results of blood pressure changes in mice with smooth muscle-specific knock-in of GPR146 under basal and AngII stimulation; the results showed that smooth muscle-specific knock-in of GPR146 could significantly increase blood pressure regardless of basal or AngII-induced states.

[0171] like Figure 6 The results show the changes in blood pressure of mice with smooth muscle-specific knockout of GPR146 under basal and AngII stimulation. It can be seen that smooth muscle-specific knockout of GPR146 significantly reduces hypertension, regardless of basal or AngII-induced states.

[0172] Example 7 Subculture of human aortic smooth muscle cells

[0173] Human aortic smooth muscle cells were cultured in SMCM medium containing 2% FBS, 100U / ml penicillin / streptomycin and smooth muscle cell growth factor, and placed in a humidified cell culture incubator at 37°C and 5% carbon dioxide. When the cell density reaches 80%-90%, the cells were subcultured. The liquid in the culture medium was discarded and the cells were gently washed 3 times with sterile PBS. 0.25% pancreatin was added to the culture dish for digestion, and the culture dish was gently shaken to allow the pancreatin to fully contact the cells. The general digestion time is 2 minutes, and the state is observed under a microscope. When most of the cells become round in shape, the culture medium containing 10% FBS is added to terminate the digestion. The cells were gently blown off the culture dish with a pipette, and the cell suspension was placed in a sterile centrifuge tube and centrifuged at 1000rpm in a centrifuge for 5 minutes. Take out the centrifuge tube, discard the supernatant, add the culture medium containing SMCM, use a pipette to gently blow the cells apart, then use a pipette to suck the cells into the culture dish, and add the required culture medium to the culture dish, generally at a ratio of 1:3 for subculturing. Gently shake the culture dish horizontally to evenly distribute the cells, and carefully place it in the incubator for static culture.

[0174] The cell experiments involved in the experimental results are all cells cultured in this embodiment, and the experimental results involved are Figure 1 , Figure 2 , Figure 8 .

[0175] Example 8 Extraction and Quantification of Protein

[0176] In this embodiment, RIPA lysis buffer (R0010, Solarbio, Beijing, China) with added protease inhibitors (B14001, bimake) and phosphatase inhibitors (B15001, bimake) was used to lyse tissues and cells, and total protein was extracted. Protein concentration was determined using a BCA protein assay kit (23227, Thermo scientific, MA, USA). Proteins were separated by 8%-12% dodecyl-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane (1620113, Bio-Rad, USA). The membrane was blocked with 5% skim milk powder in TBST (50mM Tris-HCl, 150mM NaCl and 0.1% Tween 20) for 1 hour at room temperature. Subsequently, the membrane was incubated with a primary antibody at 4°C overnight. After washing 3 times with TBST, it was incubated with a secondary antibody (1:10000) conjugated to horseradish peroxidase at room temperature for 1 hour. Finally, chemiluminescence was performed using an ECL reagent.

[0177] Cell protein extraction

[0178] Discard the supernatant of the cells in the 6-well plate, gently rinse twice with pre-cooled PBS, aspirate the PBS, and add 100μl RIPA lysis buffer, to which protease inhibitors and phosphatase inhibitors are added in advance. After adding the lysis buffer, gently shake the 6-well plate to allow the lysis buffer to fully contact the cells and keep the temperature low to prevent protein degradation. Carefully scrape the cells with a sterile cell scraper after autoclaving, then use a pipette to add the lysed cell solution to a sterile 1.5ml EP tube and lyse in an ice box for 30 minutes. During the lysis process, the cells can be blown with a pipette to make the cells lyse more fully. Put the lysed cells into a centrifuge at 4℃ and 12000rpm for 10 minutes. Then aspirate the supernatant in the EP tube and transfer it to a new sterile EP tube for subsequent experimental operations or freeze at -80℃.

[0179] Protein quantification

[0180] Use BCA kit for protein quantification. Prepare standard: dilute the protein standard with a concentration of 5mg / ml to 0.5mg / ml with PBS. Prepare working solution: mix solution A and solution B in the kit in a ratio of 50:1, for example, take 10ml of solution A in a 15ml clean centrifuge tube, add 200μl of solution B, and then shake gently to mix evenly. Prepare a clean 96-well plate, use a pipette to take 0, 1, 2, 4, 8, 12, 16, 20μl of standard solution and add it to one column of the 96-well plate, then add sterile PBS to each well so that the total volume of each well is 20μl, so 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, 0.5mg / ml respectively. In the wells of another column, add an appropriate volume of sample and make up the volume to 10μl with PBS. Then, add 100 μl of BCA mixed working solution to each well, mix gently, and place in an incubator at 37°C for 30 minutes. Take out the 96-well plate and measure the OD value at 562nm with an enzyme marker. Draw a standard curve and calculate the concentration of the measured protein based on the results. Add 1 / 5 volume of 5×SDS-loading buffer to the protein solution, mix well, heat in a 100°C metal bath for 10 minutes, and then freeze in a -80°C refrigerator for later use.

[0181] In this embodiment, protein extraction and quantification are mainly used for WB experiments. The experimental results are shown in the attached Figure 2 and attached Figure 8 .

[0182] Example 9: Western Blotting

[0183] The adhesive system of this embodiment is as follows: Prepare 10% AP (prepared and used immediately): weigh 0.1g of ammonium persulfate with an electronic scale, put it into a clean EP tube, add 1ml of deionized water, vortex and shake to fully dissolve it. Prepare 10% SDS: weigh 10.0g of SDS with an electronic scale, add 100ml of deionized water to fully dissolve it, and store it at room temperature after preparation.

[0184] In this embodiment, the corresponding concentrated gel (see Table 2 below) and separated gel (see Table 3 below) are prepared according to the gel preparation scheme.

[0185] Table 2 5% concentrated gel (12ml)

[0186] <![CDATA[ddH2O]]> 8.2ml 30% acrylamide solution 2.0ml 1M Tris-Cl, pH 6.8 1.5ml 10% SDS 0.12ml 10% AP 0.12ml TEMED 0.012ml

[0187] Table 3 Separation gel system

[0188] Reagents 8% separation gel (30ml) 10% separation gel (30ml) 12% separation gel (30ml) <![CDATA[ddH2O]]> 13.8ml 11.8ml 9.9ml 30% Acrylamide 8.0ml 10ml 12ml 1.5M Tris-Cl 7.6ml 7.6ml 7.5ml 10% SD 0.3ml 0.3ml 0.3ml 10% AP 0.3ml 0.3ml 0.3ml TEMED 0.018ml 0.012ml 0.012ml

[0189] Wash the glass plates and carefully add the prepared separation gel between the two glass plates. Generally, the volume of separation gel is 7-8ml, and the volume of concentrated gel is 3-4ml. After adding separation gel, press the upper layer with anhydrous ethanol, and be careful not to leak. After standing for 3-4 minutes, discard the anhydrous ethanol on the upper layer, add concentrated gel, insert a 1cm comb, and stand for 3 minutes.

[0190] Electrophoresis

[0191] Prepare 1×SDS-PAGE electrophoresis buffer (prepare and use immediately): weigh 28.8g of glycine, 6g of Tris-base, and 2g of SDS on an electronic scale, and add 2L of deionized water to fully dissolve. Loading: Add an equal amount of protein to the wells of the SDS-PAGE gel, generally 25-50μg. Fill the lanes with large volume differences or empty lanes with loading buffer. Add 5μl of marker to the starting column and make up the volume with loading buffer. Turn on the current and set the starting voltage to 90V. When the sample moves down from the concentrated gel to the separation gel, adjust the voltage to 120V. When the target protein moves down to the appropriate position, cut off the power supply.

[0192] Electroporation

[0193] Prepare 1× electrotransfer solution: weigh 28.8g of glycine, 6g of Tris-base, and 400ml of methanol on an electronic scale, add deionized water to make up to 2L, and fully dissolve. Carefully remove the SDS-PAGE gel from the glass plate, operate gently, and be careful not to damage the SDS-PAGE gel. Use the sandwich method to place the gel and NC membrane, that is: black clips-sponge-filter paper-gel-NC membrane-filter paper-sponge-white clips. Put the clips with the gel and NC membrane into the electrotransfer tank and add enough electrotransfer solution. Put the electrotransfer tank in an ice box and add enough ice to maintain a low temperature. Generally, proteins are electrotransferred at 200mA for 2 hours.

[0194] Closed

[0195] Prepare skim milk powder blocking solution according to Table 4 below: weigh 2.5 g of skim milk powder using an electronic scale, add it to a 50 ml clean centrifuge tube, then add 50 ml of 1× TBST solution (Table 4), vortex and shake to make it fully uniform.

[0196] Table 4 Configuration of 1×TBST Buffer (1L)

[0197] NaCl 10g 1M Tris-HCl (pH 8.0) 20ml <![CDATA[ddH2O]]> 980ml Tween 20 50μl

[0198] Remove the NC membrane after electrotransfer, trim off the blank part of the edge beyond the gel, soak the NC membrane in 1× Ponceau red dye solution for 2 minutes, and observe whether there are bands to determine whether the protein is successfully transferred to the NC membrane. If the transfer is successful, continue to wash with TBST solution 3 times, 5 minutes each time. Then put the NC membrane in skim milk powder blocking solution and incubate it on a shaker at room temperature for 1 hour.

[0199] Primary Antibody

[0200] After blocking, the NC membrane was gently rinsed with 1×TBST, the primary antibody was diluted in proportion according to the antibody instruction manual, the NC membrane was immersed in the primary antibody, and incubated overnight in a shaker in a chromatography cabinet at 4°C.

[0201] Secondary Antibodies

[0202] The overnight NC membrane was washed 3 times with 1×TBST, 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.

[0203] development

[0204] After incubation with the secondary antibody, the NC membrane was washed three times with 1×TBST, each time for 10 minutes. The membrane was placed in an exposure machine, and the luminescent liquid was added for exposure imaging.

[0205] The results of the immunoglobulin technique experiment in this example are shown in the attached Figure 2 and attached Figure 8 .

[0206] like Figure 2 The results shown in the figure showed that the expression level of GPR146 protein was detected in HASMCs, WKY and SHR rats, AngII-induced hypertension mice and their control models cultured with different hydrostatic pressures (100mmHg, 200mmHg). The results showed that high hydrostatic pressure (200mmHg) treatment significantly increased the expression of GPR146 protein and RNA; the protein of GPR146 in SHR rats was higher than that in the WKY group; after AII treatment, the expression of GPR146 increased.

[0207] As attached Figure 8 The results shown show that according to the overexpression and knockout of GPR146 at the cellular level, the expression of smooth muscle phenotypic transformation marker proteins changed. It can be seen that when GPR146 was overexpressed in smooth muscle cells, the expression of PCNA, p-ERK, and COL1 increased, and the expression of a-SMA decreased; when GPR146 was knocked down in smooth muscle cells, the expression of PCNA, p-ERK, and COL1 decreased, and the expression of a-SMA increased.

[0208] Example 10 Quantitative reverse transcription polymerase chain reaction

[0209] Extraction of total RNA: Treat cells in a 6-well plate, discard the culture medium, wash the cells three times with pre-cooled PBS, discard the PBS, add 500μl TRIzol to each well, scrape the cells on ice with a clean cell scraper, then add the lysate to a 1.5ml clean EP tube and lyse on ice for 5 minutes. Add 200μl chloroform to each EP tube, mix thoroughly by turning upside down, and let stand on ice for 10 minutes to completely separate the nucleoprotein complex. Place the EP tube in a pre-cooled 4℃ centrifuge, centrifuge at 12000rpm for 10 minutes. Take out the EP tube, the liquid in it is divided into 3 layers, the upper layer is the aqueous phase, the middle layer is the protein layer, and the lower layer is the organic phase. RNA is present in the upper aqueous phase. Use a pipette to carefully pipette the upper aqueous phase into a new clean EP tube, taking care not to suck up the protein in the middle layer. Add 500μl isopropanol (isopropanol is pre-cooled on ice in advance). Let stand for precipitation for 10 minutes. Then centrifuge at 4°C, 13000rpm, for 10 minutes. Discard the supernatant, add 1ml of 75% ethanol, carefully wash the RNA precipitate, mix well, and centrifuge at 4°C, 12000rpm for 10 minutes. Discard the supernatant and air-dry the RNA precipitate at room temperature for 5-10 minutes. Add an appropriate amount of DEPC water (generally 20-50μl) to the EP tube to dissolve the RNA precipitate. You can gently blow with a gun tip to promote its full dissolution. Use a spectrophotometer to determine the concentration and purity of RNA. Use DEPC water as a control for measurement, and record the measured RNA concentration for subsequent use. The purity of RNA is judged based on the ratio of the optical density values ​​at 260nm and 280nm (OD 260 / 280). It is generally believed that when the ratio of OD 260 / 280 is 1.8-2.0, the RNA purity is very high.

[0210] Reverse transcription: Use the reverse transcription kit TaKaRa for reverse transcription, remove the genome first, prepare according to the proportions in Table 5, then put it into the PCR instrument and set the parameters: 42°C, 2 minutes.

[0211] Table 5 Reverse transcription system

[0212]

[0213]

[0214] Reverse transcribe RNA into cDNA and prepare according to the ratio in Table 6.

[0215] Table 6 Reverse transcription system

[0216] Prime Script RT Enzyme Mix 1μl 5× Prime Buffer 4μl RT Prime Mix 1μl RNase free water 4μl

[0217] Place it in a PCR instrument and set the parameters: 37°C, 15 minutes; 85°C, 5 seconds to obtain cDNA products, which can be stored at -20°C for future use.

[0218] RT-qPCR: Select Master Mix for qRT-PCR, and the reaction solution system is prepared according to the scheme in Table 7 below (20 μl).

[0219] Table 7 qRT-PCR system

[0220] SYBR Green Master Mix 10μl PCR Forward Primer 0.5μl PCR Reverse Primer 0.5μl cDNA 4μl RNase free water 5μl

[0221] The PCR Forward Primer and PCR Reverse Primer used in this application were synthesized by Shanghai Yunxu Biotechnology Co., Ltd.

[0222] Aspirate the reaction mixture into a 96-well enzyme-free PCR plate, cover with enzyme-free PCR sealing film, and then carefully scrape it flat with a PCR scraper. Place the PCR plate in a centrifuge and centrifuge for 3 minutes so that the reaction solution is at the bottom of the well. Set up 3 replicates for each well when loading. Place the 96-well plate in the VII7 PCR instrument and set the reaction conditions: 95°C, 10 minutes (pre-denaturation); 95°C, 15 seconds; 60°C, 30 seconds; 72°C, 30 seconds (40 cycles); 95°C, 15 seconds; 60°C, 1 minute; 95°C, 15 seconds (melting curve). The internal reference is GAPDH, the expression level of the target gene in the control group is calculated as 1, and the expression level of the target gene in the experimental group is calculated as 2. -ΔΔCt Method calculation.

[0223] The experimental results of this embodiment are shown in the attached Figure 2 The results of PCR showed that high hydrostatic pressure (200 mmHg) treatment significantly increased the expression of GPR146.

[0224] Example 11 Plasmid and siRNA transfection

[0225] Cell plating

[0226] Cells are usually plated one day before transfection, and are plated after counting the cell growth rate to ensure that the cell density reaches about 80%-90% when transfected the next day.

[0227] Cell transfection

[0228] Taking lip3000 as a transfection reagent as an example, replace the cell culture medium (without double antibodies and serum) before transfection, dilute the plasmid or siRNA with opti-MEM, and dilute lip3000 with opti-MEM. After the two are prepared and let stand for 5 minutes, mix them in the same tube. Avoid rough blowing when mixing (because it may cause the liposome to fail). Let it stand for 15 minutes, add the prepared transfection solution to the cell culture plate, and gently shake it. The liquid-changing aid transfection reagent (such as lip3000) has certain cytotoxicity, so it is necessary to change the liquid in time 6 hours after the plasmid or siRNA transfection cells (replace the serum-free culture medium with complete culture medium).

[0229] Detection

[0230] 24 hours after successful transfection, the cells were removed from the incubator and the proteins were collected for subsequent related tests.

[0231] In this embodiment, the experimental results involved are shown in the attached Figure 8 The results showed that overexpression or knockout of GPR146 could alter smooth muscle phenotypic transformation.

[0232] Example 12 GPR146 neutralizing antibody reduces AII-induced blood pressure in mice

[0233] The preparation process of mouse Gpr146 monoclonal antibody is as follows.

[0234] Phase I: Antigen research. Protein localization: localized on the nuclear membrane.

[0235] Phase II: Construction of antigen expression vector and expression verification of GPR146. Protein antigen: Extracellular design was adopted, and the extracellular region of mouse GPR146 was selected as the target protein for recombinant expression. A signal peptide was added to the N-terminus to guide secretory expression. At the same time, an hFc tag was added to the rear end of the signal peptide for easy purification. Mammalian 293 suspension cells were used as the host for recombinant expression.

[0236] Phase III: Immunization and polyclonal titer assessment. Rabbits will be immunized with the recombinant antigens obtained in Phase II, including the immunization route, adjuvants, and booster strategies. After the third and fourth immunizations, rabbit sera will be analyzed to identify responding animals. Sera will be evaluated based on antigen ELISA testing.

[0237] Phase 4: Antibody purification. The rabbits with positive responses identified in Phase 3 provide peripheral blood (while the spleens of the rabbits with positive responses are subjected to differential screening, enrichment and cryopreservation of B cells for subsequent preparation and development of recombinant rabbit monoclonal antibodies) to complete the purification of rabbit polyclonal antibodies.

[0238] Phase 5: Single B cell screening and sequencing. The cryopreserved B cells identified in Phase 4 are screened and sequenced through single B cell culture testing and antibody gene sequence amplification to complete the screening of positive single B cell antibody sequences.

[0239] Phase 6: Antibody recombinant expression and identification. Based on the antibody gene sequence obtained in Phase 6, the recombinant antibody vector is constructed and expressed in the E. coli system. After the recombinant antibody is purified, it is screened and evaluated for potency using ELISA to determine the best antibody clone.

[0240] Preparation of AngII-induced hypertensive mouse model: The hypertensive mouse model constructed in Example 3 was injected with GPR146 neutralizing antibody (anti-GPR146) through the tail vein, and treated with AngII for 5 days to increase the blood pressure of mice; GPR146 neutralizing antibody and control antibody were injected through the tail vein.

[0241] In this embodiment, the experimental results are shown in the attached Fig. 9 The results showed that the GPR146 neutralizing antibody group significantly reduced AngII-induced blood pressure increase compared with the control group.

[0242] In summary, GPR146 can provide a new molecular target for the prevention or treatment of hypertension and guide the screening and development of related drugs.

[0243] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A GPR146 Use as a target for preparing drugs for preventing and / or treating hypertension; Said GPR146 The role of the target is: Overexpression GPR146 Enhance microvascular contraction and weaken microvascular relaxation in mice; and / or, Knockout GPR146 Reduce microvascular constriction and enhance microvascular relaxation; and / or, Overexpression GPR146 Promotes vascular remodeling and increases collagen content in mice; and / or, Knockout GPR146 Inhibits vascular remodeling and reduces collagen content in mice; and / or, give GPR146 Monoclonal antibody treatment for angiotensin-induced hypertension in mice; and / or, give GPR146 Monoclonal antibody treatment can inhibit or reduce vascular remodeling and collagen content in mice induced by angiotensin.

2. A GPR146 Use as a target for preparing a product having at least one of the following effects (1)-(5): (1) Products for monitoring the treatment status of people with hypertension; (2) Products for prognosis monitoring of people with hypertension; (3) Products for the prevention and / or treatment of hypertension; (4) Products that screen for targets associated with hypertension; (5) Screening of products for hypertension drugs; Said GPR146 The role of the target is: Said GPR146 Highly expressed in human vascular models of hypertension.

3. The use according to claim 2, characterized in that: The product is a reagent, a kit or a medicine.

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