Use of AP2A1 gene in preparing products for preventing and treating hypertension

Abstract

The present invention discloses the use of the AP2A1 gene in the preparation of a product for the prevention and treatment of hypertension. The present invention found that AP2A1 expression levels were significantly elevated in hypertensive rat models. Further, inhibiting AP2A1 expression in these rat models significantly reduced abdominal aorta systolic and diastolic blood pressures, mean arterial pressure, vascular wall thickness, and pulse wave velocity, thereby improving the hypertensive phenotype. This indicates that AP2A1 has a role in regulating hypertension and can serve as a target, which is of great significance for the future development of drugs and the prevention and treatment of hypertension.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to the use of the AP2A1 gene in preparing a product for preventing and treating hypertension. Background Art

[0002] Hypertension is a major chronic disease and the leading risk factor for death in patients with cardiovascular and other diseases. It is characterized by a high prevalence and a large number of patients. Despite the continuous updates to hypertension prevention and treatment guidelines and the continuous improvement of treatment methods, even patients with well-controlled blood pressure still experience a ceiling effect, meaning that they will eventually develop and die from target organ damage such as the heart, brain, and kidneys. Therefore, the prevention and control of hypertension requires the development of safer and more effective prevention and treatment strategies.

[0003] The mediator of intracellular membrane transport is a heterotetrameric protein complex called the adaptor-related protein (AP) complex. The AP complex plays an important role in vesicle formation. Currently, there are five known AP complexes: AP-1, AP-2, AP-3, AP-4, and AP-5. AP-2 has two α isoforms (αA and αC); the gene encoding the αA subunit of AP-2 is named AP2A1. The AP2A1 gene is located on chromosome 19q13.3 and consists of 24 coding exons and 23 intervening introns. Currently, only two alternative transcripts have been annotated for this gene. Their sequences differ by only 66 nucleotides in the coding region; therefore, the two mRNA molecules share the same untranslated region (UTR), translation start codon, and stop codon. Existing studies have shown that in fibroblasts, overexpression of full-length IL-33 (FLIL33) can lead to increased AP2A1 expression and induce Smad3 phosphorylation; si-AP2A1 eliminates the stimulatory effect of FLIL33 overexpression on Smad3 phosphorylation.

[0004] However, the effect of AP2A1 on hypertension has not been reported. Summary of the Invention

[0005] In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide a use of the AP2A1 gene in preparing a product for preventing and treating hypertension, in order to solve the above-mentioned problems.

[0006] To achieve the above objectives, the present invention specifically adopts the following technical solutions.

[0007] The first aspect of the present invention protects the use of AP2A1 as a target in screening products for preventing and / or treating hypertension.

[0008] The second aspect of the present invention protects the use of a substance that inhibits AP2A1 in the preparation of a product for preventing and / or treating hypertension.

[0009] In certain embodiments, the substance that inhibits AP2A1 can inhibit the transcription or translation of the AP2A1 gene, or can inhibit the expression or activity of the AP2A1 protein.

[0010] In certain embodiments, the substance that inhibits AP2A1 is the only active ingredient or one of the active ingredients of the product.

[0011] In certain embodiments, the substance that inhibits AP2A1 is selected from nucleic acid molecules, small molecule chemical drugs, antibody drugs, polypeptides, proteins, nucleic acid constructs, lentiviruses, adeno-associated viruses, and CRISPR / Cas9 genome editing systems.

[0012] Preferably, the nucleic acid molecule is any one or more of the following: antisense oligonucleotide, double-stranded RNA or shRNA.

[0013] Preferably, the substance that inhibits AP2A1 is shRNA, which contains a nucleotide sequence that can hybridize with the AP2A1 gene. The shRNA includes a sense chain segment and an antisense chain segment, and a stem-loop structure connecting the sense chain segment and the antisense chain segment. The sequences of the sense chain segment and the antisense chain segment are complementary, and the sequence of the sense chain segment is identical to the target sequence in the AP2A1 gene.

[0014] Preferably, the substance that inhibits AP2A1 is a double-stranded RNA, which comprises a nucleotide sequence that can hybridize with the AP2A1 gene. The double-stranded RNA comprises a first strand and a second strand, the first strand and the second strand are complementary to each other to form an RNA dimer, and the sequence of the first strand is identical to the target sequence in the AP2A1 gene.

[0015] Preferably, the substance that inhibits AP2A1 is a nucleic acid construct.

[0016] Preferably, the substance that inhibits AP2A1 is an adeno-associated virus or a lentivirus.

[0017] A third aspect of the present invention provides a nucleic acid molecule for interfering with AP2A1 gene expression, comprising a double-stranded RNA or shRNA, wherein the double-stranded RNA comprises a nucleotide sequence capable of hybridizing with the AP2A1 gene, the double-stranded RNA comprises a first strand and a second strand, the first strand and the second strand are complementary to each other to form an RNA dimer, and the sequence of the first strand is identical to a target sequence in the AP2A1 gene; the shRNA comprises a nucleotide sequence capable of hybridizing with the AP2A1 gene, the shRNA comprises a sense strand segment and an antisense strand segment, and a stem-loop structure connecting the sense strand segment and the antisense strand segment, the sequences of the sense strand segment and the antisense strand segment are complementary, and the sequence of the sense strand segment is identical to the target sequence in the AP2A1 gene;

[0018] The nucleotide sequence encoding the first strand in the double-stranded RNA comprises SEQ ID No. 1 to SEQ ID No. 3;

[0019] The shRNA encoding nucleotide sequence is shown in SEQ ID NO. 4-5, or as shown in SEQ ID NO. 6-7, or as shown in SEQ ID NO. 8-9;

[0020] The coding sequence of the double-stranded RNA or shRNA comprises the sequences shown in SEQ ID No. 1 to SEQ ID No. 3.

[0021] The fourth aspect of the present invention protects an AP2A1 gene interfering nucleic acid construct, which can express the nucleic acid molecule as described above.

[0022] The fifth aspect of the present invention protects an AP2A1 gene interfering lentivirus or an interfering adeno-associated virus, which is formed by viral packaging of the interfering nucleic acid construct as described above with the assistance of a lentiviral helper plasmid and a host cell; or, the interfering nucleic acid construct as described above is formed by viral packaging with the assistance of an adeno-associated virus helper plasmid and a host cell.

[0023] The sixth aspect of the present invention protects a composition for preventing and / or treating hypertension, comprising the nucleic acid molecule as described above; and / or the interfering nucleic acid construct as described above; and / or the interfering lentivirus or interfering adeno-associated virus as described above.

[0024] The seventh aspect of the present invention protects the use of the nucleic acid molecule as described above, or the interfering nucleic acid construct as described above, or the interfering lentivirus or interfering adeno-associated virus as described above, or the composition as described above, wherein the use is for preparing a drug for treating hypertension, or for preparing a kit for reducing the expression of the AP2A1 gene in cells.

[0025] The eighth aspect of the present invention protects a method for screening drugs for preventing and / or treating hypertension, the method comprising: using AP2A1 as a drug target, and searching for substances that can inhibit or block the expression and / or function of AP2A1 as candidate drugs.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] 1) The present application found that compared with WKY rats, the expression level of AP2A1 in the abdominal aorta of SHR rats was significantly increased. Further interference with AP2A1 expression was found to reduce blood pressure. Therefore, AP2A1 can be used as a drug target for screening hypertension.

[0028] 2) The present invention designed a suitable target sequence for the AP2A1 gene, screened out shRNA that effectively silenced AP2A1, constructed a nucleic acid construct containing the shRNA, and packaged it into a lentivirus, which can effectively inhibit the expression of AP2A1 in A7R5 cells.

[0029] 3) The present application further discovered that when a nucleic acid construct containing shRNA is packaged into an adeno-associated virus and administered to SHR rats, it was found that the systolic blood pressure, diastolic blood pressure, mean arterial pressure, and pulse wave propagation velocity of the abdominal aorta can be significantly reduced; at the same time, the thickness of the rat's blood vessel wall can be increased, thereby having the effect of preventing and / or treating hypertension.

[0030] 4) This application discovered that AP2A1 has the function of regulating hypertension and can be used as a target, which is of great significance for the future drug development and prevention and treatment of hypertension. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This figure shows the expression of AP2A1 in the abdominal aorta of spontaneously hypertensive rats in Example 1 of the present invention.

[0032] Figure 2 This figure shows the effect of sh-AP2A1 lentivirus on AP2A1 protein expression in A7R5 cells in Example 2 of the present invention.

[0033] Figure 3 Shown is a map of the pAAV-CMV-WPRE-luc-U6-spgRNA vector in Example 3 of the present invention.

[0034] Figure 4 The graphs show the effects of sh-AP2A1 adeno-associated virus injection on blood pressure and body weight in rats in Example 3 of the present invention. A represents systolic blood pressure, B represents diastolic blood pressure, C represents mean arterial pressure, and D represents body weight.

[0035] Figure 5Graphs showing the effects of sh-AP2A1 adeno-associated virus injection into rats in Example 3 of the present invention on abdominal aorta pulse velocity and vessel wall thickness. Figure A shows an ultrasound image of the abdominal aorta, Figure B shows a statistical graph of abdominal aorta pulse velocity, and Figure C shows abdominal aorta vessel wall thickness.

[0036] Figure 6 The graph shows the effect of the sh-AP2A1 adeno-associated virus injected into rats on the pathological morphology of the abdominal aorta in Example 3 of the present invention. DETAILED DESCRIPTION

[0037] The inventors accidentally discovered that AP2A1 is expressed at elevated levels in the abdominal aorta of spontaneously hypertensive rats, suggesting that it may serve as a screening target for hypertension drugs. Subsequently, the inventors discovered that inhibiting AP2A1 expression significantly reduces systolic blood pressure, diastolic blood pressure, mean arterial pressure, pulse wave velocity, and abdominal aorta wall thickness, improving hypertension symptoms and achieving the effect of preventing or treating hypertension. Therefore, AP2A1 can be used as a target, which is of great significance for the future development of drugs and prevention and treatment of hypertension.

[0038] The first aspect of the present application protects the use of AP2A1 as a target in screening products for preventing and / or treating hypertension.

[0039] In the first aspect of the present application, the product uses AP2A1 as a drug target to screen drugs or drug preparations to find drugs that can inhibit AP2A1 gene expression as candidate drugs for preventing or treating hypertension.

[0040] In the first aspect of the present application, the product can inhibit or block the expression and / or function of AP2A1 or AP2A1, thereby reducing the systolic pressure of the abdominal aorta, reducing the diastolic pressure, reducing the mean arterial pressure, reducing the pulse wave propagation velocity, and increasing the vascular wall thickness of rats, thereby preventing and / or treating hypertension.

[0041] In the first aspect of the present application, the products include medicines, health products and foods.

[0042] The second aspect of the present application protects the use of a substance that inhibits AP2A1 in preparing a product for preventing and / or treating hypertension.

[0043] In the second aspect of the present application, the products include medicines, health products and foods.

[0044] In the second aspect of the present application, the substance that inhibits AP2A1 generally includes substances that can inhibit the transcription or translation of the AP2A1 gene, or inhibit the expression or activity of the AP2A1 protein. For example, the substance that inhibits AP2A1 can partially inhibit, i.e., reduce the expression and / or function of AP2A1, or completely inhibit, i.e., substantially eliminate the expression and / or function of AP2A1. For example, the substance that inhibits AP2A1 can include nucleic acid molecules, small molecule drugs, antibody drugs, polypeptides, proteins, nucleic acid constructs, lentiviruses, adeno-associated viruses, CRISPR / Cas9 genome editing systems, etc. The nucleic acid molecule can be selected from antisense oligonucleotides, double-stranded RNA, or shRNA. In a specific embodiment of the present application, the substance that inhibits AP2A1 is double-stranded RNA or shRNA.

[0045] In the second aspect of this application, the substance that inhibits AP2A1 is used alone or in combination with other drugs. That is, the substance that inhibits AP2A1 can be the sole active ingredient or one of the active ingredients of the product. The form of the product is not particularly limited and can be in the form of a common solid, liquid, gel, semi-fluid, aerosol, or the like.

[0046] In the second aspect of the present application, the hypertension refers to at least one of the following symptoms:

[0047] 1) Decreased vascular function;

[0048] 2) thickening of blood vessel walls;

[0049] 3) Vascular pathological changes.

[0050] In the second aspect of this application, the reduced vascular function refers to a reduction in vascular pulse wave propagation velocity. The product contains a substance that inhibits AP2A1, which can restore the vascular pulse wave propagation velocity of the target vessel to a level close to or equal to that of healthy blood vessels. For example, based on a healthy blood vessel, the product can restore the vascular pulse wave propagation velocity of the target vessel to at least 70%, 80%, 90%, or 100% of that of a healthy vessel.

[0051] In the second aspect of this application, the vascular wall thickening refers to thickening of the abdominal aorta. The product contains a substance that inhibits AP2A1, which can reduce the thickness of the target vascular wall to near or near that of a healthy blood vessel. For example, based on a healthy blood vessel as a benchmark, the product can reduce the thickness of the target vascular wall to no more than 130%, 120%, 110%, or 100% of the thickness of a healthy blood vessel.

[0052] The third aspect of the present application protects a nucleic acid molecule that interferes with AP2A1 gene expression, wherein the nucleic acid molecule comprises double-stranded RNA or shRNA, wherein the double-stranded RNA comprises a nucleotide sequence that can hybridize with the AP2A1 gene, and the shRNA comprises a nucleotide sequence that can hybridize with the AP2A1 gene.

[0053] In the third aspect of the present application, the double-stranded RNA comprises a first strand and a second strand, the first strand and the second strand complement each other to form an RNA dimer, and the sequence of the first strand is identical to the target sequence in the AP2A1 gene; the nucleotide sequence encoding the first strand in the double-stranded RNA comprises SEQ ID No. 1 to SEQ ID No. 3.

[0054] GCAAAGAGGCTGAGATCAA(SEQ ID No.1)

[0055] CCTAGAGACGGCTGACTAT(SEQ ID No.2)

[0056] GCAGATGAACTGCTGAATA(SEQ ID No.3)

[0057] Preferably, the double-stranded RNA is a small interfering RNA (siRNA)

[0058] In the third aspect of the present application, the shRNA comprises a nucleotide sequence capable of hybridizing to the AP2A1 gene, the shRNA comprising a sense strand segment and an antisense strand segment, and a stem-loop structure connecting the sense strand segment and the antisense strand segment, wherein the sequences of the sense strand segment and the antisense strand segment are complementary, and the sequence of the sense strand segment is identical to the target sequence in the AP2A1 gene; the encoding nucleotide sequence of the shRNA is as shown in SEQ ID NOs. 4-5, or as shown in SEQ ID NOs. 6-7, or as shown in SEQ ID NOs. 8-9. The shRNA can be converted into siRNA after enzymatic processing to specifically silence AP2A1 expression.

[0059] CcggGCAAAGAGGCTGAGATCAATTCAAGAGATTGATCTCAGCCTCTTTTGCTTTTTTg(SEQ IDNo.4)

[0060] aattcaaaaaaGCAAAGAGGCTGAGATCAATCTCTTGAATTGATCTCAGCCTCTTTGC(SEQ IDNo.5)

[0061] CcggCCTAGAGACGGCTGACTATTTCAAGAGAATAGTCAGCCGTCTCTAGGTTTTTTg(SEQ IDNo.6)

[0062] aattcaaaaaaCCTAGAGACGGCTGACTATTCTCTTGAAATAGTCAGCCGTCTCTAGG(SEQ IDNo.7)

[0063] CcggGCAGATGAACTGCTGAATATTCAAGAGATATTCAGCAGTTCATCTGCTTTTTTg(SEQ IDNo.8)

[0064] aattcaaaaaaGCAGATGAACTGCTGAATATCTCTTGAATATTCAGCAGTTCATCTGC(SEQ IDNo.9)

[0065] A fourth aspect of the present invention provides an AP2A1 gene interfering nucleic acid construct capable of expressing the nucleic acid molecule described above. The interfering nucleic acid construct can be obtained by cloning a gene fragment encoding the AP2A1 gene siRNA described above into a known vector, such as an AP2A1 gene interfering lentiviral vector or an AP2A1 gene interfering adeno-associated viral vector. The starting plasmid for the AP2A1 gene interfering lentiviral vector is pSLenti-U6-shRNA-CMV-mCherry-F2A-Puro-WPRE. The starting plasmid for the AP2A1 gene interfering adeno-associated viral vector is pAAV-CMV-WPRE-luc-U6-spgRNA.

[0066] The fifth aspect of the present invention protects an AP2A1 gene interfering lentivirus or an interfering adeno-associated virus, which is formed by viral packaging of the interfering nucleic acid construct as described above with the assistance of a lentiviral helper plasmid and a host cell; or, the interfering nucleic acid construct as described above is formed by viral packaging with the assistance of an adeno-associated virus helper plasmid and a host cell.

[0067] In a fifth aspect, the lentiviral helper plasmid is selected from the group consisting of psPAX2 and pMD2.G. The interfering lentivirus is obtained by co-transfecting 293T cells with an interfering nucleic acid construct with the aid of psPAX2 and pMD2.G plasmids. This virus can infect cells and produce small interfering RNA targeting the AP2A1 gene, thereby preparing a drug for treating hypertension.

[0068] In a fifth aspect, the adeno-associated virus helper plasmid is selected from the group consisting of pAAV-RC and pHelper plasmids. The interfering adeno-associated virus is obtained by co-transfecting AAV293 cells with an interfering nucleic acid construct with the aid of pAAV-RC and pHelper plasmids. The virus can infect cells and produce small interfering RNA targeting the AP2A1 gene, thereby preparing a drug for treating hypertension.

[0069] The sixth aspect of the present invention protects a composition for preventing and / or treating hypertension, comprising the nucleic acid molecule as described above; and / or the interfering nucleic acid construct as described above; and / or the interfering lentivirus or interfering adeno-associated virus as described above.

[0070] In the sixth aspect of the present application, the composition further comprises a pharmaceutically acceptable excipient. Pharmaceutically acceptable excipients are those that do not produce adverse, allergic, or other undesirable reactions when properly administered to an animal or human. The pharmaceutically acceptable excipient should be compatible with the substance that inhibits AP2A1, i.e., capable of being blended with the substance without significantly reducing the effectiveness of the substance that inhibits AP2A1 under normal circumstances. The pharmaceutically acceptable excipient is selected from one or more of a carrier, a diluent, a binder, a lubricant, and a wetting agent. Specific examples of substances that can serve as pharmaceutically acceptable carriers, diluents, binders, lubricants, and wetting agents include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium methylcellulose, ethylcellulose, and methylcellulose; tragacanth powder; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and cocoa butter; polyols such as propylene glycol, glycerol, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers such as Tween; wetting agents such as sodium lauryl sulfate; colorants; flavorings; tableting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline solutions; and phosphate buffers. These substances are used as needed to help stabilize the formulation or to enhance the activity or its bioavailability or to create an acceptable taste or flavor when taken orally.

[0071] In the sixth aspect of the present application, the composition is in the form of one or more of a solution, an injection, a spray, nasal drops, an aerosol, a powder spray, a tablet, a capsule, and a granule. The above-mentioned various dosage forms can be prepared according to conventional methods in the pharmaceutical field. Preferably, the composition is an injection.

[0072] In the sixth aspect of this application, the composition can be introduced into the body, such as into muscle, intradermal, subcutaneous, intravenous, or mucosal tissue, by injection, spray, nasal drip, eye drop, infiltration, absorption, or physical or chemical methods; or can be mixed with or encapsulated in other substances and then introduced into the body. Preferably, the composition is administered intraperitoneally. The composition can also be used in combination with other treatments, including surgery, radiotherapy, chemotherapy, and targeted therapy.

[0073] The seventh aspect of the present invention protects the use of the nucleic acid molecule as described above, or the interfering nucleic acid construct as described above, or the interfering lentivirus or interfering adeno-associated virus as described above, or the composition as described above, wherein the use is for preparing a drug for treating hypertension, or for preparing a kit for reducing the expression of the AP2A1 gene in cells.

[0074] The eighth aspect of the present application protects an in vitro method for screening drugs for preventing and / or treating hypertension, the method comprising: using AP2A1 as a drug target, and searching for substances that can inhibit or block the expression and / or function of AP2A1 as candidate drugs.

[0075] In a ninth aspect of the present application, the method comprises: applying a drug to be selected to cells in vitro, and detecting the content of AP2A1 in the cells after co-culturing. The cells may be derived from mammals.

[0076] Experimenters can determine whether a drug has therapeutic significance by measuring the level of AP2A1 after co-culture. Generally speaking, drugs that can reduce AP2A1 levels by 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% compared to the control group can be considered therapeutically significant.

[0077] In the ninth aspect of the present application, the candidate drug is determined to be a drug with therapeutic significance if it can reduce AP2A1 in cells by at least 50%.

[0078] As used herein, treatment refers to slowing, interrupting, arresting, controlling, stopping, alleviating, or reversing the progression or severity of a sign, symptom, disorder, condition, or disease after the disease has begun to develop, but does not necessarily involve the complete elimination of all disease-associated signs, symptoms, conditions, or disorders.

[0079] Prevention as described herein refers to all actions to suppress symptoms or delay specific symptoms by administering the products described herein.

[0080] The following describes the implementation of the present invention through specific embodiments. People skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0081] Before further describing the specific embodiments of the present invention, it should be understood that the scope of the present invention is not limited to the specific embodiments described below. It should also be understood that the terminology used in the examples is intended to describe specific embodiments and is not intended to limit the scope of the present invention. The experimental procedures in the following examples, where specific conditions are not specified, were generally performed under conventional conditions or according to the conditions recommended by the respective manufacturers.

[0082] When the embodiments provide numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the understanding of the prior art by those skilled in the art and the description of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention may also be used to implement the present invention.

[0083] In the following examples of this application, the main experimental instruments are as follows:

[0084] Pipette (Raining, USA); electronic balance scale (Shanghai Ohaus Instrument Co., Ltd.); non-invasive rat tail sphygmomanometer (Kent Co., Ltd., USA); small animal ultrasound imaging system Vevo2100 (Fujifilm Investment Co., Ltd.); inhalation small animal anesthesia machine (Shenzhen Ruiwode Life Science Technology Co., Ltd.); pathological slicer (Leica, Germany); paraffin embedding machine (Hubei Xiaogan Yaguang Medical Electronic Technology Co., Ltd.).

[0085] In the following examples of this application, the statistical method is as follows:

[0086] SPSS 26.0 software (SPSS / PC+) was used for statistical analysis. The Shapiro-Wilk test was used to test the normality of the data. Data are expressed as mean ± standard deviation. When comparing the differences between three or more groups, for data that conformed to the normal distribution and had homogeneous variance, ANOVA multiple group comparisons were performed with the Bonferroni test; for data that conformed to the normal distribution but had unequal variances, ANOVA multiple group comparisons were performed with the Kruskal-Wallis test. For data that did not conform to the normal distribution, the rank sum test was used. P < 0.05 was considered statistically significant. All data analyses were performed in SPSS 26.0.

[0087] Example 1

[0088] In this Example 1, the expression of AP2A1 in different rats was studied, including the following:

[0089] 1.1 Experimental Materials

[0090] Six WKY rats and six SHR rats were anesthetized and sacrificed. The abdominal aorta was removed from the abdomen and rinsed with pre-cooled saline to remove residual blood. The abdominal aorta was then divided into three equal sections, one of which was placed in 4% paraformaldehyde, and the other two were quickly stored in liquid nitrogen for later use. The abdominal aorta was first soaked in 4% paraformaldehyde for 24 hours and then placed in an embedding cassette, clearly labeled with the organ number and group. The tissue was dehydrated and waxed. The waxed tissue was placed in an embedding machine, and liquid wax was poured into the embedding cassette. Before the liquid wax solidified, the tissue was placed in the embedding cassette and cooled on an ice table. After the paraffin solidified, the wax block was removed and trimmed to obtain a tissue wax block.

[0091] 1.2 Immunohistochemistry

[0092] After sectioning and dewaxing, tissue wax blocks were fixed with 100°C citrate fixative for 10 min, cooled, and returned to room temperature. Endogenous peroxidase was added for 10 min, and the sections were washed three times with PBS for 5 min each. Blocking solution was used for 1 h at room temperature, and the primary antibody (AP2A1, SAB Co., Ltd., Catalog No. 43215) was incubated. The next day, secondary antibodies (biotinylated goat anti-mouse, rabbit, or mouse / rabbit IgG, Fuzhou Maixin Biotechnology Development Co., Ltd., Catalog No. KIT-9730) were used for 1 h, followed by streptomycin treatment for 1 h and three 5-min washes with PBS. After staining with DAB colorimetric reagent, the sections were stained with hematoxylin, blued with ultrapure water for 5 min, dried with a hair dryer, and mounted with neutral gum. The slides were imaged at 400x magnification using an intelligent automated optical microscope, and six fields of view were randomly selected for each sample.

[0093] The expression of AP2A1 in the abdominal aorta of SHR rats and WKY rats was detected by immunohistochemistry. Figure 1 .

[0094] from Figure 1 It can be seen that AP2A1 expression is increased in the abdominal aorta of SHR rats and may be a new target for the development of hypertension.

[0095] Example 2 Construction and screening of AP2A1-specific shRNA-expressing lentivirus

[0096] In this Example 2, based on the design of siRNA that can effectively silence the AP2A1 gene, a lentiviral expression vector was used to express the siRNA to inhibit the replication of AP2A1, and its inhibitory effect was tested through cell experiments.

[0097] siRNA targets were designed based on the Rat Ap2a1 gene transcript. Single-stranded primers were synthesized based on the siRNA target and annealed to form double-stranded oligoDNA. This was then ligated into a double-digested, linearized RNA interference vector, replacing the original ccdB toxic gene, to generate an AP2A1-specific shRNA-expressing lentivirus. The vector was transformed into competent Escherichia coli cells, and transformants were screened by colony PCR. Positive clones were verified by sequencing. After sequencing, the correct clones were purified and purified. Lentivirus was then co-transfected with the lentiviral packaging plasmid psPAX2 and the lentiviral envelope protein plasmid pMD2.G into 293T cells to produce lentivirus. The viral titer was determined, and lentiviral particles of appropriate titer were used to infect A7R5 cells. Western blot analysis was performed to determine the optimal interference fragment. Construction of the AP2A1-specific shRNA-expressing lentivirus was commissioned by Yuanhe Biotechnology.

[0098] 2.1. Obtaining sh-AP2A1 Lentivirus

[0099] The specific experimental steps are as follows:

[0100] 2.1.1. Interference target design and primer synthesis

[0101] Targeting rat AP2A1 (Gene ID: 308578), the siRNA target sequences sh-AP2A1-1, sh-AP2A1-2, and sh-AP2A1-3 were designed based on the Ap2a1 gene sequence (NM_001395092.1) from GenBank and general shRNA design principles. A control sequence, GL428NC2, was also designed, with the interfering sequence bases randomly shuffled.

[0102] Design and synthesize corresponding oligo primers based on the siRNA target. siRNA target sequences are shown in Table 1, and primer sequences are shown in Table 2.

[0103] Table 1 siRNA target sequences

[0104]

[0105]

[0106] Table 2 Primer fragments

[0107]

[0108] 2.1.2. Primer annealing to form double-stranded fragments with sticky ends

[0109] Dissolve the oligo primers synthesized in step 2.1.1 to 20 μM using oligo annealing buffer, then take 30 μL of each dissolved oligo primer and mix them to obtain an oligo mixture.

[0110] The oligo mixture was then heated in a water bath at 95°C for 5 minutes, and then the water bath was opened and allowed to cool naturally to room temperature to anneal and form double-stranded oligo fragments.

[0111] Take 1 μL of the double-stranded oligo fragment for the subsequent ligation reaction in step 2.1.4 and store the rest at -20°C.

[0112] Table 3 Double-stranded oligoDNA

[0113]

[0114]

[0115] 2.1.3 Preparation of linearized interference vector

[0116] The expression vector pSLenti-U6-shRNA-CMV-mCherry-F2A-Puro-WPRE (provided by Heyuan Biotechnology, number GL428) was digested with AgeI (2448) and EcoRI (2804) restriction enzymes. The enzyme digestion reaction system is shown in Table 4.

[0117] Table 4 Enzyme digestion reaction system

[0118] Reagents capacity Expression vector pSLenti-U6-shRNA-CMV-mCherry-F2A-Puro-WPRE 2 μg 10× reaction buffer 5μL Restriction enzymes 1 μL Deionized water Make up to 50 μL

[0119] The enzyme digestion reaction system in Table 4 was incubated in a 37°C water bath for more than 2 h to obtain the enzyme digestion product.

[0120] The digestion product was subjected to agarose gel electrophoresis to detect the digestion effect, and the interference target vector band was cut out from the gel after agarose gel electrophoresis and recovered using TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.3.0. The specific steps were referred to the kit instructions to obtain the linearized interference vector.

[0121] 2.1.4 Ligation reaction

[0122] The double-stranded oligo fragment obtained in step 2.1.2 and the linearized interference vector obtained in step 2.1.3 were ligated using T4 DNA ligase. The reaction system is shown in Table 5. The ligation was carried out at 16°C overnight to obtain a ligation product.

[0123] Table 5 Ligation reaction system

[0124] Reagents Volume (μL) 10mM double-stranded oligo fragment obtained in step 2.1.2 1 40 ng / μL of the linearized interference vector obtained in step 2.1.3 3 10×T4 DNA ligase Buffer 2 T4 DNA ligase 1 <![CDATA[dd H2O]]> Up to 20

[0125] 2.1.5 Transformation of Competent Cells

[0126] Transfect the ligation products obtained in step 2.1.4 into DH5α competent cells respectively. Refer to the "Succinct Molecular Biology Experiment Guide" for transfection methods, then spread them on plates and incubate at 37°C overnight.

[0127] 2.1.6. Identification of positive transformants by colony PCR

[0128] The transformants grown on the plate were picked and resuspended in 10 μL LB culture medium. 1 μL was taken as a template for colony PCR identification to determine whether the constructed recombinant lentiviral vector was correct.

[0129] Positive clones obtained from colony identification were sent to a sequencing company for sequencing verification. The sequencing results were compared and analyzed using Vector NTI software.

[0130] The correct positive clones were verified by sequencing, and plasmid miniprep was performed to obtain three AP2A1-specific shRNA-expressing lentiviral vectors.

[0131] 2.1.7 Lentiviral packaging

[0132] Co-transfect 293T cells with the three AP2A1-specific shRNA-expressing lentiviral vectors obtained in step 2.1.6, along with the lentiviral packaging helper plasmid psPAX2 and the lentiviral envelope protein plasmid pMD2.G, to produce lentivirus. Lentiviruses sh-AP2A1-1, sh-AP2A1-2, and sh-AP2A1-3 were obtained. Viral titers were determined.

[0133] The viral titer of lentivirus sh-AP2A1-3 was 3.10×10 8 TU / mL; the viral titer of lentivirus sh-AP2A1-2 was 2.68×10 8 TU / mL; the viral titer of lentivirus sh-AP2A1-1 was 3.03×10 8 TU / mL.

[0134] At the same time, the control sequence GL428NC2 was used to obtain the control group lentivirus (sh-Ctrl) using the same method.

[0135] 2.2 Effect of sh-AP2A1 lentivirus on AP2A1 protein expression in A7R5 cells

[0136] The lentivirus sh-AP2A1-1, sh-AP2A1-2, and sh-AP2A1-3 prepared in step 2.1 were transfected into A7R5 cells and verified by Western blot.

[0137] A7R5 cells were transfected with sh-AP2A1 lentivirus according to a specific system, digested, and harvested. Lysis buffer was then added for cell lysis, with shaking once every 5 minutes for 5 seconds each time, for a total of three times. The cells were centrifuged at 12,000 rpm for 20 minutes at 4°C. After BCA protein denaturation, SDS-PAGE gel electrophoresis and transfer to the membrane were performed. The cells were blocked with milk blocking buffer for 2 hours and then incubated with primary antibody (AP2A1, SAB Co., Ltd., Catalog No.: 43215). The next day, secondary antibody (goat anti-rabbit IgG secondary antibody, SAB Co., Ltd., Catalog No.: L3012) was incubated for imaging.

[0138] from Figure 2 It can be seen that compared with the lentivirus sh-Ctrl group, the grayscale of lentivirus shAP2A1-1, lentivirus sh-AP2A1-2 and lentivirus sh-AP2A1-3 was reduced, and the reduction of the lentivirus obtained by the sh-AP2A1-3 sequence was the most obvious, indicating that the shRNA of the present application has the best interference effect on AP2A1, and it is used as a subsequent experiment.

[0139] Example 3 In vivo experiment

[0140] In this Example 3, an adeno-associated virus sh-AP2A1 was constructed and injected into rats via the tail vein to conduct in vivo experimental studies, including the following:

[0141] 3.1 Experimental Materials and Grouping

[0142] 3.1.1 Construction of adeno-associated virus sh-AP2A1

[0143] 1) Construction of recombinant expression plasmid rAAV-shRNA

[0144] The sh-AP2A1-3 sequence and the control sh-Ctrl sequence in Example 1 were used for AAV packaging. Yuanhe Bio was commissioned to carry out the AAV packaging.

[0145] The coding gene of the shRNA fragment (i.e., sh-AP2A1-3) was cloned into the pAAV-CMV-WPRE-luc-U6-spgRNA vector (Heyuan Biotechnology Co., Ltd., number H4681). After sequencing and confirmation, it was double-digested with EcoRI and HindIII and cloned into the pAAV-MCS plasmid (Stratagene Co., Ltd.). After positive recombinant clones were identified, they were named recombinant expression plasmid rAAV-shRNA. The mass spectrometry spectrum is shown in Figure 3 .

[0146] 2) Packaging of recombinant adeno-associated virus

[0147] AAV293 cells (human embryonic kidney cells) were inoculated into a 10 cm diameter culture dish and transfected when the confluence reached 70-80%.

[0148] The recombinant expression plasmid rAAV-shRNA, pAAV-RC (Stratagene), and shuttle plasmid pHelper (Stratagene) obtained in step 3.1.1 of this example were dissolved in Opti-MEM medium at a ratio of 1:1:1 to a total volume of 500 μL and gently mixed to obtain a mixed solution A.

[0149] Dilute Obio transfection reagent in Opti-MEM medium to a total volume of 500 μL and mix gently to obtain mixed solution B.

[0150] Mixture A and mixture B were mixed to obtain mixture C, which was used to transfect AAV293 cells. 6 h after transfection, the culture medium was removed, the cells were washed once with PBS, and 10 mL of fresh complete culture medium was added for culture.

[0151] At the same time, pAAV empty virus was packaged with pAAV-MCS plasmid as a negative control (sh-Ctrl).

[0152] 3) Purification of Adeno-associated Virus sh-AP2A1

[0153] 72 hours after transfection, scrape the cells from step 2) from the culture dish and collect them into a centrifuge tube, and collect the cell supernatant into a centrifuge tube. Centrifuge the supernatant, filter it and use it for subsequent cell pellet resuspension. After the cell pellet is shaken in an oscillator, resuspend it with the supernatant to obtain a cell suspension. Take the cell suspension and freeze and thaw it repeatedly in a 37°C water bath. Centrifuge the frozen and thawed cell suspension and filter it into a centrifuge tube. Transfer the filtered liquid into an ultracentrifuge tube and centrifuge it. After ultracentrifugation, discard most of the supernatant, add nuclease digestion to remove residual plasmid DNA, and incubate at 37°C. After centrifugation, filter it into an ultracentrifuge tube. Add iodixanol gradient solution for ultracentrifugation to collect the purified adeno-associated virus sh-AP2A1.

[0154] The purified adeno-associated virus sh-AP2A1 was tested for pyrogen and microbial contamination to ensure that it was free of pyrogen, bacteria, fungi, viruses, and other microbial contamination, and that there was no wild-type virus replication. The number of AAV viral particles was determined by quantitative PCR to detect the genome copy number of the AAV vector in the genome. The titers of the adeno-associated virus were as follows: sh-AP2A1: 1.63 × 10 13 TU / mL.

[0155] 3.1.2 Animal Grouping

[0156] This experiment used 12 SPF-grade 4-week-old male spontaneously hypertensive rats (SHR) and 12 Wistar Kyoto (WKY) rats. The experimental animals were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd., and the experimental animal use license number is SCXK (Beijing) 2021-0006. The experimental animals were raised in the SPF-grade laboratory of the Medical Experimental Animal Center of Fujian University of Traditional Chinese Medicine. They were free to eat and drink water, and the room was well ventilated and well lit. The room temperature was controlled at (23±1)℃, the relative humidity was about 50-60%, and the light / dark cycle was alternating for 12 hours. The breeding environment was kept quiet and undisturbed, and adequate food and water were provided. All experimental operations were in compliance with the ethics of experimental animals and the regulations of the Medical Experimental Animal Center of Fujian University of Traditional Chinese Medicine.

[0157] Twelve 4-week-old male WKY rats were randomly divided into two groups: WKY+sh-Ctrl group and WKY+sh-AP2A1 group, with 6 rats in each group; twelve 4-week-old male SHR rats were randomly divided into SHR+sh-Ctrl group and SHR+sh-AP2A1 group, with 6 rats in each group.

[0158] Before the tail vein injection, the purified adeno-associated virus sh-AP2A1 obtained in step 3.1 was placed on ice and the recombinant adeno-associated virus sh-AP2A1 was diluted to an appropriate titer (1×10 12 Infectious units / mL), the rats were then fixed using a tail vein injection device with their tails exposed; the rat tails were wiped with an alcohol cotton ball to dilate the blood vessels; the diluted sh-Ctrl and sh-AP2A1 viruses were injected into WKY and SHR mice, respectively, and 100 μL of the diluted virus was drawn up with a 1 mL syringe; the rat tails were straightened to make the red veins clearly visible, the needle was inserted 1 / 3 of the way from the tail tip, the virus was slowly injected, and then the needle was removed, and the injection site was pressed with a cotton ball to stop bleeding; the rats were removed from the fixator and placed back in their original cages.

[0159] 3.2 Test indicators and results

[0160] 3.2.1. Effects on body weight and blood pressure

[0161] In this experiment, the tail artery systolic blood pressure (SBP), diastolic blood pressure (DBP) and mean arterial pressure (MAP) of rats in each group were monitored using the Kent non-invasive rat tail artery blood pressure detector.

[0162] The steps are as follows: Install the non-invasive sphygmomanometer and check the instrument for leaks or other abnormalities. Then use a restraint to secure it. Install an occlusion sleeve approximately 1 cm from the base of the rat's tail. Then insert the volume pressure sensor until the sensor can no longer be pushed toward the base of the rat's tail. Place the fixed restraint on a heating plate and adjust the temperature to the optimal level. Cover the restraint with a blackout cloth. Ensure that the rat is in a quiet state. After the rat rests for 5 minutes, start the measurement. Record the rat's SBP, DBP, and MAP. Weigh the rat weekly, take the average value, and return it to its original cage. Measure the blood pressure of each group of rats once a week before sampling for 10 consecutive weeks.

[0163] The blood pressure and body weight test results of the four groups of rats are shown in Figure 4 . Figure 4 In the figure, A is the change result of systolic blood pressure (SBP), B is the change result of diastolic blood pressure (DBP), C is the change result of mean arterial pressure (MAP), and D is the change result of weight.

[0164] from Figure 4 It can be seen that for SHR rats, the systolic blood pressure, diastolic blood pressure and mean arterial pressure of the SHR+sh-AP2A1 group were significantly lower than those of the SHR+sh-Ctrl group (#P<0.05); there was no significant difference in body weight.

[0165] from Figure 4 It can be seen that for WKY rats, the systolic blood pressure, diastolic blood pressure and mean arterial pressure of the WKY+sh-AP2A1 group were not significantly different from those of the WKY+sh-Ctrl group; there was no significant difference in body weight.

[0166] from Figure 4 It can be seen that for SHR rats and WKY rats, the systolic blood pressure, diastolic blood pressure and mean arterial pressure of the SHR+sh-Ctrl group were significantly higher than those of the WKY+sh-Ctrl group (*P<0.05); however, there was no significant difference in body weight between SHR rats and WKY rats.

[0167] from Figure 4 It can be seen that for SHR rats and WKY rats, the systolic blood pressure, diastolic blood pressure and mean arterial pressure of the SHR+sh-AP2A1 group were significantly higher than those of the WKY+sh-AP2A1 group (P<0.05); however, there was no significant difference in body weight between SHR rats and WKY rats.

[0168] 3.2.2 Effects on Pulse Wave Velocity (PWV) and Thickness of the Abdominal Aorta

[0169] Ultra-high-resolution small animal ultrasound (Vevo 2100) was used to measure the pulse wave velocity (PWV) of the abdominal aorta in each group of rats. Before testing, the rats' abdominal hair was removed using depilatory cream and anesthetized with 2% isoflurane. The rats were then placed in a supine position on a 37°C constant-temperature heating plate. The heart rate was controlled (450-600 bp). Coupling agent was applied to the abdomen. An ultrasound probe was positioned below the sternum to acquire images of the abdominal aorta. The probe was rotated parallel to the abdominal midline to obtain cross-sectional images of the aorta. These images were analyzed using Vevo Software (Vevo LAB 1.7.1). Pulse wave velocity (PWV) was calculated according to the following formula: PWV = abdominal aorta length / (distal end conduction time - cardiac end conduction time). At least three measurements were performed for each rat, and the average value was calculated.

[0170] The abdominal aorta PWV and vascular wall thickness of rats in each group are shown in Figure 5 . Figure 5 In the figure, A is an ultrasound image of the abdominal aorta, B is a statistical graph of the pulse wave velocity (PWV) of the abdominal aorta, and C is the vascular wall thickness of the abdominal aorta.

[0171] from Figure 5 As shown in Figures A and B, compared with the WKY+sh-Ctrl group, the PWV value of the abdominal aorta of the rats in the SHR+sh-Ctrl group was significantly increased; compared with the SHR+sh-Ctrl group, the PWV of the abdominal aorta of the rats in the SHR+sh-AP2A1 group was significantly decreased.

[0172] from Figure 5 As shown in Figure C, compared with the WKY+sh-Ctrl group, the thickness of the abdominal aorta wall in the SHR+sh-Ctrl group was significantly increased (*P<0.05); compared with the SHR+sh-Ctrl group, the thickness of the abdominal aorta wall in the SHR+sh-AP2A1 group was significantly decreased (#P<0.05).

[0173] 3.3.3 Impact on the pathological morphology of the abdominal aorta

[0174] The abdominal aorta of rats was dissected and fixed in 4% paraformaldehyde for 24 h. The tissue was then dehydrated using different concentrations of ethanol, immersed in xylene to replace the ethanol, and immersed in wax. The tissue blocks were cut into 4 μm slices using a microtome, expanded in 37°C water, transferred to glass slides, and baked, dried, hydrated in xylene, and dewaxed using different concentrations of ethanol. The rehydrated sections were placed in hematoxylin solution and rinsed with tap water once. They were immersed in 1% hydrochloric acid alcohol for differentiation for 1 second, placed in water for anti-blueing, and then placed in eosin solution for staining for 2 seconds. The sections were dried and sealed, and the pathological morphological changes of the tissues were observed under a microscope.

[0175] The pathological changes of abdominal aorta in rats in each group are shown in the following table. Figure 6 .

[0176] from Figure 6 It can be seen that compared with the WKY+sh-Ctrl group, the thickness of the abdominal aorta wall of the SHR+sh-Ctrl group was significantly increased; compared with the SHR+sh-Ctrl group, the thickness of the abdominal aorta wall of the rats in the SHR+sh-AP2A1 group was significantly decreased.

[0177] Taken together, these results indicate that AP2A1 silencing can alleviate the pathological morphological changes in the abdominal aorta of spontaneously hypertensive rats.

[0178] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Anyone skilled in the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by one of ordinary skill in the art without departing from the spirit and technical principles disclosed herein are intended to be covered by the claims of the present invention.

Claims

1. Use of a substance that inhibits AP2A1 in preparing a product for preventing and / or treating hypertension; the substance that inhibits AP2A1 is a nucleic acid molecule, the nucleic acid molecule being a shRNA, the shRNA comprising a nucleotide sequence capable of hybridizing with the AP2A1 gene, the shRNA comprising a sense strand segment and an antisense strand segment, and a stem-loop structure connecting the sense strand segment and the antisense strand segment, the sequences of the sense strand segment and the antisense strand segment being complementary, and the sequence of the sense strand segment being identical to a target sequence in the AP2A1 gene; the nucleotide sequence encoding the shRNA is as shown in SEQ ID NOs. 4-5, or as shown in SEQ ID NOs. 6-7, or as shown in SEQ ID NOs. 8-9; And / or, the substance that inhibits AP2A1 is a nucleic acid construct; the nucleic acid construct can express the nucleic acid molecule; and / or, the substance that inhibits AP2A1 is an adeno-associated virus or a lentivirus; the adeno-associated virus or the lentivirus is formed by viral packaging of the nucleic acid construct.

2. The use according to claim 1, characterized in that The substance that inhibits AP2A1 is the only active ingredient or one of the active ingredients of the product.

Images